CN110760582A - The application of metformin in the treatment of KRAS mutant colorectal cancer - Google Patents

Abstract

The invention discloses a marker for determining a treatment plan for colorectal cancer. The marker is KRAS gene and/or protein. Metformin is selected for KRAS mutant colorectal cancer to treat colorectal cancer. The present invention is the first to clarify that patients with KRAS mutant colorectal cancer have more significant benefits from using metformin, and further clarify that the down-regulation of metformin excretion channel MATE1 expression is the key mechanism for KRAS mutant colorectal cancer cells to be sensitive to metformin. The concentration of intracellular metformin, thereby enhancing the effect of metformin on tumor cell proliferation. The present invention provides a new idea and method for the treatment of colorectal cancer, has far-reaching significance and is worthy of vigorous promotion.

Description

The application of metformin in the treatment of KRAS mutant colorectal cancer

technical field

The present invention relates to the technical field of treatment of colorectal cancer, more particularly, to the application of metformin in the treatment of KRAS mutant colorectal cancer.

Background technique

Colorectal cancer (CRC) is one of the most common malignant tumors. At present, oxaliplatin or irinotecan-based chemotherapy combined with anti-epidermal growth factor receptor (EGFR) monoclonal antibody can improve the median overall survival time of colorectal cancer patients to more than 2 years . However, about 1/4 of the patients in China have already developed tumor metastasis at the time of diagnosis, and the chemotherapy effect is not good. In addition, colorectal cancer is a genetically heterogeneous disease, with alterations (mutations or deletions) of APC, KRAS, TP53, BRAF, PIK3CA and other genes, as well as microsatellite instability (MSI) and chromosomal instability. , CIN) and other epigenetic changes play an important role in the process of intestinal polyps to carcinogenesis, tumor metastasis and chemotherapeutic drug resistance. Among them, the probability of KRAS gene mutation in Chinese colorectal cancer patients is as high as 30% to 50%, and a large number of clinical studies have shown that such patients fail to benefit from anti-EGFR targeted therapy, resulting in a rapid rate of colorectal cancer tumor-related mortality in my country rising trend.

Therefore, the development of therapeutic regimens and drugs targeting individual epigenetic changes in colorectal cancer patients, and the transition from traditional indiscriminate cell-killing chemotherapy drugs to multi-targeted targeted drug therapy, are the key to precise tumor therapy.

The current treatment strategies for KRAS-mutant CRC include inhibiting KRAS activation or inhibiting the activation of MEK/ERK, a pro-proliferative signaling pathway downstream of KRAS, but all of them ended in failure of phase II clinical trials. The former is because farnesyltransferase inhibitors cannot completely inhibit KRAS activation, while the latter may be related to feedback activation of the PI3K/AKT signaling pathway.

Metformin is currently the first-line drug for the treatment of type 2 diabetes, which can effectively reduce and maintain the blood sugar level and insulin level of patients, and improve insulin resistance. In recent years, many retrospective studies have shown that metformin has certain preventive and therapeutic effects on colorectal cancer, and its mechanism mainly includes direct effects on tumor cells: inhibition of MEK-ERK, PI3K-AKT and mTOR signaling pathway activation; And indirect effects on tumor cells: such as reducing and maintaining blood sugar and insulin levels, inhibiting inflammatory responses, increasing the proportion of CD8+ T cells, thereby improving the cellular immune function of tumors. However, some studies also reported that metformin could not improve the overall survival time and progression-free survival time of colorectal cancer patients.

The above studies suggest that the therapeutic effect of metformin on colorectal cancer may vary by type and individual. At present, the effective treatment type and individual of metformin in the treatment of rectal cancer have not been identified, and its mechanism has not been elucidated.

SUMMARY OF THE INVENTION

The purpose of the present invention is to overcome the deficiencies of the prior art and provide the application of metformin in the treatment of KRAS mutant colorectal cancer.

The first object of the present invention is to provide a marker for determining a treatment regimen for colorectal cancer.

The second object of the present invention is to provide the application of KRAS gene and/or protein as a marker for determining the treatment regimen of colorectal cancer.

The third object of the present invention is to provide the application of the KRAS gene mutation detection reagent in the preparation of a kit for determining a treatment plan for colorectal cancer.

The fourth object of the present invention is to provide the application of a KRAS protein detection reagent in the preparation of a kit for determining a treatment plan for colorectal cancer.

The fifth object of the present invention is to provide a kit for determining a treatment plan for colorectal cancer.

The sixth object of the present invention is to provide the application of metformin in the treatment of colorectal cancer or the preparation of medicaments for the treatment of colorectal cancer.

The seventh object of the present invention is to provide the application of the combination of MEK signaling pathway and AKT signaling pathway as a therapeutic target for colorectal cancer.

The eighth object of the present invention is to provide the combined use of an inhibitor of MEK signaling pathway and an inhibitor of AKT signaling pathway to treat KRAS mutant colorectal cancer.

The ninth object of the present invention is to provide a pharmaceutical composition for treating KRAS mutant colorectal cancer.

In order to achieve the above object, the present invention is achieved through the following technical solutions:

1. A retrospective study was carried out using a stratified Cox proportional hazards model, and for the first time it was confirmed that the benefit of metformin was more significant in patients with KRAS-mutant colorectal cancer. To provide evidence-based medicine for the clinical selective use of metformin in the treatment of colorectal cancer.

2. The KRAS (G13D) point mutation model and the KRAS knockdown model were constructed in cell experiments to verify that KRAS mutant colorectal cancer cells were sensitive to the anti-tumor effect of metformin. It is clarified that metformin inhibits the proliferation of KRAS mutant colorectal cancer cells by simultaneously inhibiting the two pathways of ERK/cyclin D1/RB and AKT/mTOR/4E-BP1. It provides evidence for the clinical efficacy of combined use of MEK and AKT inhibitors to enhance the treatment of KRAS mutant colorectal cancer cells, and also provides experimental evidence for metformin as an optional drug.

3. For the first time, it is clear that the down-regulation of metformin excretion channel MATE1 is the key mechanism of KRAS-mutant colorectal cancer cells being sensitive to metformin. It is clarified that the mutant KRAS protein promotes the methylation of the CpG island of the MATE1 promoter by up-regulating the methyltransferase DNMT1 and down-regulating the demethylase TET1/2, which reduces the transcription level of MATE1.

4. Clinical specimens and cell experimental evidence suggest that metformin can be selectively used in the treatment of colorectal cancer by detecting KRAS genotype.

Therefore the present invention claims the following content:

A marker for determining the treatment plan of colorectal cancer, the marker is KRAS gene and/or protein, and KRAS mutant colorectal cancer selects metformin to treat colorectal cancer.

Use of KRAS gene and/or protein as a marker for determining treatment regimens for colorectal cancer.

Specifically, metformin is used for KRAS mutant colorectal cancer treatment of colorectal cancer, and the KRAS mutant colorectal cancer described here is continuously activated and highly expressed after KRAS gene mutation

The application of the KRAS gene mutation detection reagent in the preparation of a kit for determining a treatment plan for colorectal cancer also falls within the protection scope of the present invention.

The application of the KRAS protein detection reagent in the preparation of a kit for determining a treatment plan for colorectal cancer also falls within the protection scope of the present invention.

A kit for determining a treatment plan for colorectal cancer, the kit includes a KRAS mutant colorectal cancer detection reagent.

The application of metformin in the treatment of colorectal cancer or the preparation of a drug for the treatment of colorectal cancer, wherein the colorectal cancer is KRAS mutant colorectal cancer.

The above KRAS mutant colorectal cancer does not limit the type of KRAS mutation, and the current experimental data support the common mutation of codon12.13.

Since MEK and AKT are important signaling pathways that regulate cell proliferation downstream of KRAS, the clinical phase II trial of MEK inhibitor or AKT inhibitor alone failed. Metformin inhibits both MEK and AKT signaling pathways, so it can effectively inhibit KRAS-mutant colorectal cancer. Cell Proliferation.

Therefore the present invention further claims the following content:

The combination of MEK signaling pathway and AKT signaling pathway as a therapeutic target for colorectal cancer;

The inhibitor of MEK signaling pathway and the inhibitor of AKT signaling pathway are used in combination in preparation for the treatment of KRAS mutant colorectal cancer;

A pharmaceutical composition for treating KRAS mutant colorectal cancer, characterized by comprising an inhibitor of MEK signaling pathway and an inhibitor of AKT signaling pathway.

Preferably, the above-mentioned treatment of colorectal cancer is to promote the G1 arrest phase of colorectal cancer cells, inhibit the proliferation of colorectal cancer cells, inhibit tumor growth, inhibit tumor weight gain, prolong the overall survival time of patients and/or prolong the progression-free time of chemotherapy .

More preferably, the progression-free time of extended chemotherapy is the progression-free time of extended first-line chemotherapy.

The above-mentioned inhibitor is any substance that can reduce the corresponding protein, gene or signaling pathway.

Compared with the prior art, the present invention has the following beneficial effects:

The present invention is the first to clarify that patients with KRAS mutant colorectal cancer have more significant benefits from using metformin, and further clarify that the down-regulation of metformin excretion channel MATE1 expression is the key mechanism for KRAS mutant colorectal cancer cells to be sensitive to metformin. The concentration of intracellular metformin, thereby enhancing the effect of metformin on tumor cell proliferation. The present invention provides a new idea and method for the treatment of colorectal cancer, has far-reaching significance and is worthy of vigorous promotion.

Description of drawings

Figure 1 is a schematic diagram of clinical patient inclusion grouping.

Figure 2 shows the use of metformin to improve the overall survival time of colorectal cancer patients with diabetes.

Figure 3 shows that metformin improves overall survival and progression-free survival during first-line chemotherapy in patients with KRAS-mutant colorectal cancer.

Figure 4 is a schematic diagram of intracellular gene modification in SW48 KRAS(G13D) cell line.

Figure 5 shows the sequence alignment of KRAS exon2 GGC>GAC mutant positive clones.

Figure 6 shows that metformin inhibits the growth of KRAS mutant colorectal cancer cells.

Figure 7 shows that metformin inhibits KRAS mutant PDX tumor growth.

Figure 8 shows that metformin promotes G1 phase arrest of KRAS mutant colorectal cancer cells and inhibits tumor cell proliferation.

Figure 9 shows the mechanism by which metformin promotes G1 phase arrest of KRAS mutant colorectal cancer cells and inhibits tumor cell proliferation.

Figure 10 shows that KRAS mutant colon cancer cells are more sensitive to metformin than KRAS wild type colon cancer cells.

Figure 11 shows the accumulation of metformin concentrations in KRAS mutant colorectal cancer cells and PDX tumor tissues.

Figure 12 shows that KRAS mutation down-regulates MATE1, thereby increasing the intracellular concentration of metformin and enhancing the effect of metformin on the proliferation of colorectal cancer cells.

Figure 13 shows that KRAS mutation regulates MATE1 methylation and then downregulates MATE1 expression.

Figure 14 shows the amplification sequence and amplification primers for the CpG island of the MATE1 promoter after the genomic DNA sample has been modified with bisulfite.

Figure 15 shows the expression of DNMT/TET in SW48 KRAS(G13D) cells and KRAS knockdown Lovo cells

Figure 16 shows that KRAS mutation downregulates MATE1 expression by regulating DMNT1/TET.

Detailed ways

The present invention will be further elaborated below with reference to the accompanying drawings and specific embodiments of the specification, and the embodiments are only used to explain the present invention, but not to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents, etc. used are commercially available reagents and materials unless otherwise specified.

1. Experimental materials

fresh tumor tissue

Colorectal cancer patients diagnosed and treated by Sun Yat-sen University Cancer Center, after surgical resection of the in situ tumor, took about 5 × 5 × 5 mm of cancer tissue. The collection of cases strictly followed the operation procedures of Sun Yat-sen University Cancer Center, approved by the hospital and obtained the consent of the patients themselves and their families.

laboratory animals

4-6 weeks old male nude mice (BALB/c nude mice), weighing 14-18 g, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd., production license number: SCXK (Beijing) 2016-0011, raised in Zhongshan In the specific pathogen free (SPF) environment of the University (North Campus of Laboratory Animal Center), the license number of the experimental unit: SYXK (Guangdong) 2017-0081. Do the experiment after passing the quarantine. In the process of raising animals, five basic welfares of experimental animals are guaranteed, and the experimental process follows the 3R principles of Replacement, Reduction and Refinement.

cell line

SW48 was purchased from Shenzhen Huatuo Biotechnology Co., Ltd., CaCO2, HCT-116 and LoVo were donated by the Institute of Gastroenterology, The Sixth Affiliated Hospital of Sun Yat-sen University, and SW480 and SW620 cells were cell lines preserved in the laboratory. The STR identification of these 6 colorectal cancer cell lines was completed by Guangzhou Saiku Biotechnology Co., Ltd., 100% matched with the information provided by ATCC, and there was no other cell contamination and STR changes. The KRAS genotypes of the above cells were queried from the ATCC official website and verified by PCR sequencing.

2. Experimental method

1. Filming and tumor pathological grading and quantification of proliferating cells

1) Using an automatic digital slide scanning system (Axio Scan Z1) to photograph whole tissue samples (10× and 20×);

2) After the images are exported, the pathologist will classify the tumor into well differentiated, moderately differentiated, poorly differentiated and undifferentiated;

3) Ki67-stained tumor tissue images were quantitatively analyzed for Ki67(+) nuclei using the IHC Profiler plug-in of ImageJ, and recorded as proliferating cells;

2. Cell viability assay (CCK-8 method)

Cells were seeded at a concentration of 5000 cells/well (48-well plate, 200 μl per well volume). The cells were treated in different ways according to the experimental requirements, and 10 μl of CCK8 solution was added after a certain period of time. Incubate in an incubator for 1 h, pipette 200 μl of the supernatant into a 96-well microtiter plate, and measure the OD ₄₅₀ with a microplate reader.

Example 1 The effect of metformin on the prognosis of patients with metastatic colorectal cancer

1. Grouping and clinical characteristics of metastatic colorectal cancer patients with type 2 diabetes mellitus

1. Experimental sample

Among 4751 patients with metastatic colorectal cancer (mCRC) admitted to Sun Yat-sen University Cancer Center from 2004 to 2016, 282 patients with type 2 diabetes (T2DM) before diagnosis were included. Divided into metformin group (metformin use, n=109), insulin or insulin-releasing (n=141) and other anti-diabetic drugs use (n=22) and Untreated group (without anti-diabetic treatments, n=32). The patient groupings are shown in Figure 1.

2. Experimental method

General clinical characteristics of patients, such as gender, age, body mass index (BMI), and clinical characteristics (such as primary tumor site, pathological grade, metastatic site, first-line chemotherapy regimen, and KRAS genotype) was used to exclude confounding factors, and then to clarify the overall survival (OS) and progression-free survival (PFS) of first-line chemotherapy in colorectal cancer patients with type 2 diabetes treated with metformin. influences. The basic clinical characteristics of the patients are listed in Table 1.

Table 1 Basic clinical information of colorectal cancer patients with type 2 diabetes mellitus:

The distribution of individual clinical characteristics between the metformin group and the non-metformin group was analyzed. Continuous variables (age, BMI) were analyzed by univariate method, and categorical variables (gender, primary tumor site, pathological grade, KRAS genotype, metastatic site, first-line chemotherapy regimen, and age group, BMI group) were analyzed by chi-square test.

3. Experimental results

Statistical analysis of the 180 patients showed that there were no significant differences in gender, age, BMI, primary tumor site, pathological grade, metastatic site, and KRAS genotype between the metformin group and the non-metformin group (P>0.05). See Table 2.

Table 2 Distribution of clinical characteristics of 180 patients with metastatic colorectal cancer with type 2 diabetes with clear Kras genotype in metformin group and other hypoglycemic drug groups

2. Metformin improves overall survival time and progression-free survival time of first-line chemotherapy in metastatic colorectal cancer patients with type 2 diabetes mellitus

Kaplan-Meier survival curve analysis was used to determine whether metformin improved the prognosis of metastatic colorectal cancer patients with type 2 diabetes in this center compared with other antidiabetic drug groups.

1. Experimental method

GraphPad Prism 7 was used to make Kaplan-Meier survival curves, and Log-rank (Mantel-Cox) statistical analysis was performed; in addition, the adjusted hazard ratio (stratified HR) was used to calculate the difference in OS between different hypoglycemic drugs and no hypoglycemic treatment group. and PFS effects.

2. Experimental results

As shown in - Figure 2, compared with the untreated diabetes group, the median survival time of the untreated group with diabetes was reduced by 11.2 months (P=0.007), and the median survival time of the metformin group was prolonged by 11.3 months (P=0.022) , other antidiabetic drugs did not improve significantly. The results showed that in 282 patients with metastatic colorectal cancer with type 2 diabetes diagnosed by Sun Yat-sen University Cancer Center from 2004 to 2016, metformin could significantly prolong the overall survival time. In addition, the adjusted hazard ratio statistics (see Table 3) showed that hypoglycemic therapy improved the prognosis (HR=0.547, 95%CI: 0.327-0.913); the use of metformin alone or in combination could improve the prognosis of patients with mCRC, while the other There was no statistical difference in the effect of sugar drugs, thus indicating that in addition to lowering blood sugar, the improvement effect of metformin on the prognosis of mCRC may also be related to other factors.

Table 3. Effects of different hypoglycemic drugs on OS and PFS compared with no hypoglycemic treatment group.

3. The effect of metformin on patient death or tumor progression is in line with the equal proportional hazards assumption

1. Experimental method

A proportional hazard assumption, which assumes that the effect of metformin use on patient death or tumor progression cannot change over time, should be a fixed value. We used the Kolmogorov-Smirnov test and Cramer von Mises test for hypothesis testing.

2. Experimental results

The results in Table 4 indicate that the effect of metformin on patient death or tumor progression conformed to the assumption of equal proportional hazards (P>0.05).

Table 4 Equal-hazards assumption for the effect of metformin on patient death or tumor progression

4. Patients with KRAS-mutant metastatic colorectal cancer benefit more from metformin

The clinical characteristics collected above were stratified using a stratified Cox proportional hazards model to explore individual factors influencing the effectiveness of metformin.

1. Experimental method

After the equal proportional hazards assumption, we further hypothesized that gender, age, BMI, primary tumor site, pathological grade, metastatic site, and KRAS genotype may act as confounding factors to influence the antitumor effect of metformin. Therefore, we performed hierarchical regression analysis on the above clinical characteristics to identify individual differences that interact with metformin.

2. Experimental results

As shown in Table 5, after incorporating the above clinical characteristics as confounding factors into the equation of the proportional hazards regression model, we can find that the use of metformin compared with other antidiabetic drugs reduces the hazard ratio (HR) of death to 0.746, The 95% confidence interval (CI) was 0.496-1.121, the interval spanned 1, and there was no statistical difference (P>0.05); the HR for reducing tumor progression during first-line chemotherapy was 0.737, and the 95% CI was 0.501-1.086. There was no statistical difference (P>0.05), indicating that there are individual differences in whether the use of metformin can benefit patients with metastatic colorectal cancer compared with other hypoglycemic drugs.

Further hierarchical regression analysis of the above clinical characteristics showed that for patients with KRAS-mutant metastatic colorectal cancer, the use of metformin was more effective than other antidiabetic drugs in reducing the risk of death (HR=0.272, 95%CI=0.120- 0.617), and also reduced the risk of tumor progression during first-line chemotherapy (HR=0.405, 95% CI 0.212-0.774). In addition, using R-based EmpowerStats software for interaction test, we found that KRAS mutation significantly enhanced the effect of metformin in reducing the risk of death (P _interaction <0.001) and the effect of reducing the risk of tumor progression during first-line chemotherapy (P _interaction= 0.02). In addition, Kaplan-Meier survival curves were used to analyze KRAS wild-type and KRAS-mutant mCRC patients, respectively. Metformin significantly prolonged the overall survival time (P<0.001) and the progression-free survival time of first-line chemotherapy (P<0.001) in KRAS-mutant mCRC patients (P<0.001). P<0.01) (Fig. 3A-B), and was ineffective in KRAS wild-type mCRC patients (Fig. 3C-D).

Table 5. Analysis of proportional hazards regression models using metformin with overall survival and progression-free survival and their interaction with each clinical feature:

5. Metformin inhibits the proliferation of KRAS mutant colorectal cancer cells

The effect of this clinical feature on the anti-tumor therapy of metformin was verified from the immunohistochemical and cytological experiments of colorectal cancer tissue sections.

1. Experimental method

(1) In order to further clarify the effect of metformin on KRAS-mutant colorectal cancer cells, the pathological sections (including primary and metastatic lesions) of 28 patients diagnosed with metastatic colorectal cancer who had taken metformin before tumor resection were collected. , DNA was extracted from paraffin tissue for KRAS genotype identification.

The location of tumor cells (nuclear hyperstaining, heterogeneity, destruction of glandular epithelial structure) was determined by hematoxylin-eosin stain (H&E), and cells in the proliferative phase were marked with Ki67 staining.

①Hematoxylin-eosin staining

1) Make the fixed tumor tissue into paraffin sections;

2) Dewaxing the sections with xylene;

3) The slices were placed in methanol for 2 min;

4) Hematoxylin staining for 3min;

5) Separation solution: 70% alcohol + 10ml glacial acetic acid (a few minutes until the color is appropriate)

6) The running water returns to blue for 5min;

7) Eosin staining for 1 min;

8) 75%, 80%, 90%, 95% absolute ethanol for 30sec each;

9) 2-3 cylinders of 100% alcohol, 30sec per cylinder;

10) 3 cylinders of 100% xylene, 10 minutes for the first cylinder, and 5 minutes for the second and third cylinders.

11) Neutral gum sealant.

②Ki67 staining

1) Baked sheet: 65℃, 2h;

2) Xylene dewaxing: 30min×3 times, room temperature;

3) Absolute ethanol: 10min×1 time, room temperature;

4) Hydration: 100%, 95%, 90%, 80%, 70% ethanol for 5min each;

5) Washing with dH ₂ O: 5min×1 time;

6) PBST washing: 5min×3 times;

7) Antigen retrieval: 1L of 10 mmol/L citrate buffer (PH 6.0) was boiled in a pressure cooker, put the slice rack into it and fully immerse it, cover the pot tightly, wait for the gas to be released, and control the time for 2 minutes, and cool the buffer naturally to Remove the slices after room temperature;

8) Washing with dH ₂ O: 2min×2 times;

9) PBST washing: 5min×3 times;

10) Blocking: use immunohistochemical strokes to circle, add 3% H2O2, room temperature for 30min;

11) PBST washing: 5min×3 times;

12) Blocking: goat serum at room temperature for 1 h, then washed with PBST for 5 min × 3 times;

13) Primary antibody incubation: Dilute Ki67 with antibody diluent at 1:400, cover the tissue, place the slices in a humid box to prevent dry slices, overnight at 4°C;

14) PBST washing: 5min×3 times;

15) Secondary antibody: Immunohistochemical Pika and Rabbit Universal Detection Kit Solution A, incubated at room temperature for 30 minutes;

16) PBST washing: 5min×3 times;

17) DAB color development: The solution B of the immunohistochemical pika and rabbit universal detection kit was diluted with solution C at a ratio of 1:50, and the reaction was stopped in tap water and then washed with water for 30 minutes;

18) Hematoxylin counterstaining: 1min, differentiate with ethanol hydrochloride (1:1000) for 10s to remove non-specific staining; continue washing with tap water for 20min after stopping the reaction;

19) Dehydration: 70%, 80%, 90%, 95%, 100% ethanol for 5 minutes each;

20) Xylene dehydration and transparency: 5min×2 times, and the film is sealed with neutral gum.

(2) KRAS wild-type colorectal cancer cell lines SW48 and CaCO2, KRAS G13D mutant colorectal cancer cell lines HCT-116 and LoVo, and KRAS G12V mutant colorectal cancer cell lines SW480 were treated with gradient concentrations of metformin in vitro and SW620. In addition, the KRAS wild-type colorectal cancer cell line SW48 transfected with KRAS G12V, KRAS G13D and KRAS G12D plasmids was treated with graded concentrations of metformin.

(3) The KRAS (G13D) mutant SW48 cell line constructed by the CRISPR/Cas9 system was treated with a gradient concentration of metformin in vitro, and it was positively verified that metformin inhibited the activity of KRAS mutant colorectal cancer cells.

①KRAS G13D point mutation CRISPR/Cas9 plasmid construction

1) sgRNA (sequence of guide RNA) design: Zhang Lab, the website provided by MIT http://crispr.mit.edu for the first 250 bp of the start codon ATG of exon 2 of KRAS gene on human chromosome 12 Design. Take the sgRNA with low off-target efficiency with a score of >85, use the CRISPR RGEN tool Cas-OFFinder website http://www.rgenome.net/cas-offinder/, set the number of mismatched bases ≤ 2, and do not use mismatched bases sgRNA to avoid non-specific cleavage. Our chosen sgRNA: 5'-GCATTTTTCTTAAGCGTCGA-3'.

2) gRNA synthesis and recombinant vector connection:

A. Chemical synthesis of gRNA oligo (purified by PAGE), the underline is the BbsⅠ restriction enzyme ligation site, the 3'-end of the reverse sequence is not cytosine and a cytosine base needs to be added, as follows:

Target sequence(PAM): GCATTTTTCTTAAGCGTCGA(TGG);

Forward: CACC GCATTTTTCTTAAGCGTCGA;

Reverse: AAAC TCGACGCTTAAGAAAAATG C;

B. The oligo per OD was dissolved using ddH2O ( ₁₀ μL/1 nmol ratio) to a final concentration of 100 μM. Double-stranded nucleotides were synthesized from oligo by the following reaction:

C. Restriction: using BbsI restriction endonuclease, the plasmid pSpCas9(BB)-2A-puro(PX459)V2.0 was digested with agarose electrophoresis and gel recovery and purification. The digestion conditions are as follows:

D. Ligation: T4 ligase was used to connect PX459 and gRNA, then agarose electrophoresis identification and gel recovery and purification, recorded as PX459/hKRAS gRNA. The connection conditions are as follows:

3) Construction of KRAS G13D point mutation donor plasmid:

A. Gene extraction: First, use the genomic DNA extraction kit to extract the genomic DNA of LoVo, and then amplify the DNA sequence of about 3000bp including sgRNA and exon2 by high-fidelity PCR, run electrophoresis identification and gel recovery and purification. PCR reaction system and conditions are as follows:

B. Construction of T vector: the DNA purified in the previous step is added to the pGM-T vector after reaction with A, and then transferred into competent bacteria for amplification, and single clones are selected for sequencing verification. Verify that the sequence is correct and denote it as pGM-T/KRAS-homology. The addition of A and the ligation reaction conditions are as follows:

C. TGG>TGA point mutation of PAM of sgRNA: use high-fidelity PCR to amplify the sequence of sgRNA PAM before TGG, the primers are hKRAS-Left arm-F and hKRAS-TGG mut-Left arm-R, and the upstream introduced The 15bp sequence is homologous to the 3' end of pGM-T, and a point mutation sequence CCA>TCA is introduced at the 5' end of the downstream primer; the primers for the other DNA fragment are hKRAS-TGG mut-Right arm-F and hKRAS-Right arm- R, the 5' end of the upstream primer introduces a 15 bp homologous sequence to the 3' end of the previous DNA fragment (including ACC>ACT), and the 3' end introduces a sequence homologous to the 5' end of pGM-T. The PCR reaction system of PrimeSTAR Max Premix is the same as above, and the reaction conditions are changed to 15s for extension time. After the two PCR products were purified by gel recovery, they were ligated with the pGM-T vector using seamless cloning, followed by gel recovery and purification, transformation, amplification and single-clone sequencing. The seamless cloning reaction system and reaction conditions are as follows:

D. Connect the sequence for homologous recombination to the donor vector pDONR 221: through primer design, attB1 was introduced at the 5' and 3' ends, respectively, and the fragment was connected to pDONR 221 by BP reaction to form an Entry Clone, Denoted as pDONR 221/hKRAS-homology plasmid. The primer design is shown in Table 6.

The primers used in Table 6 are self-named and their corresponding sequences:

The pDONR 221 ligation reaction system and conditions are as follows:

②Construct SW48KRAS(G13D) cell line

1) Passage SW48 into a 3cm ³ dish, when the cell confluence reaches 80%-90%, replace the fresh culture medium 2h in advance. 1.5μg of PX459/hKRAS gRNA and 1.5μg of pDONR 221/hKRAS-homology plasmid were respectively taken and added to 150μL opti-MEM and 9μL of P3000; another 3.5μL of Lipo3000 was added to 150μL of opti-MEM, and incubated at room temperature for 5 minutes. Then, the plasmid premix was added to Lipo3000 premix, incubated at room temperature for 15 min, and then added dropwise to the SW48 cell culture medium.

2) 12h after transfection, replace the fresh culture medium, and add 1 μM Scr7 to inhibit the NHEJ reaction and improve the efficiency of homologous recombination (reported in the literature that it can be increased by 4-5 times). 24h after transfection, 1 μM puromycin was added to screen the transfected positive cells.

3) 72h after transfection, replace with fresh culture medium and completely remove puromycin. If the cells are 70%-90% confluent and have good viability, proceed to passage.

4) After passage, 1×10 ⁶ cells were taken to extract genomic DNA, and hKRAS-Homology-F and hKRAS-Homology-R were used for PCR amplification. The steps and reaction conditions were the same as above. After ligation of pGM-T only and transformation into DH5α for amplification, about 30 clones were plated and picked for sequencing. Homologous recombination is successful if there are sgRNA PAM TGG>TGA and KRAS exon2 GGC>GAC mutations in the sequencing results. Figure 4 shows the pattern of intracellular gene modification.

5) Under the condition that the cell viability of the SW48 mixed cell line is good, digest and resuspend the cells, count 200 cells, adjust the concentration to 1 cell/200 μL, and plant them in a 96-well plate, about 1 cell per 200 μl of culture medium. cell.

6) Observe the next day, pay attention to the humidity of the incubator, and prevent the evaporation of the culture medium in the 96-well plate. After single cells grow into clones, pick 20-50 clones for passage.

7) After the monoclonal cell line is amplified, the operation is the same as above, and one bacterial clone is taken from each cell clone for sequencing. The presence of KRAS exon2 GGC>GAC mutant is a positive clone. Preserved species, recorded as SW48KRAS (G13D) cell line. The positive sequence alignment is shown in Figure 5.

(4) Using gradient concentrations of metformin to treat lentiviral shRNA in vitro to construct a KRAS knockdown LoVo cell line, and reversely verify that metformin inhibits the activity of KRAS mutant colorectal cancer cells.

①Construction of KRAS stably knocked down LoVo cell line

1) KRAS interfering lentivirus (sh-KRAS) was constructed and packaged by Shanghai Jikai Gene Co., Ltd., and a total of 2 targets were designed, see Table 7 for details.

Table 7 sh-KRAS target sequence information:

2) Passage LoVo into a 6-well plate (about 1×10 ⁵ /well), when the cell confluency does not exceed 50% after adherence, replace the fresh culture medium 2 hours in advance, and add lentiviral infection enhancer 10 μg/ mL polybrene. The multiplicity of infection (MOI) of LoVo is about 20. According to the formula: the amount of virus added to each well (μl)=MOI×number of cells/titer (TU/ml)×1000 to add lentivirus. The medium was changed for 16-18 h, and 1 μM puromycin was routinely added to screen the cell lines with stable expression of KRAS interfering RNA.

2. Experimental results

Immunohistochemical results showed that there were 18 cases of KRAS wild-type mCRC and 10 cases of KRAS mutant mCRC. As shown in Figure 3C, the proportion of Ki67(+) cells in KRAS mutant colorectal cancer tissues was significantly lower than that of KRAS wild type, with a statistically significant difference (P<0.01).

Cell experiments showed that metformin had inhibitory effects on KRAS G13D, G12D and G12V mutant colorectal cancer cells, but had no effect on KRAS wild-type colon cancer cells (Figure 6).

In conclusion, the results showed that the overall survival time and progression-free survival time of KRAS-mutant colorectal cancer patients treated with metformin were longer than those of KRAS wild-type, and the same results were verified in tumor tissue sections and cell viability experiments. .

Example 2 Validation of the therapeutic effect of metformin on KRAS mutant tumors in a tumor animal model

First, the construction of the PDX model

Using the PDX model: Take the tumor tissues of clinical KRAS wild-type and mutant colorectal cancer patients, and culture the tumor cells by digestion method. After identifying the KRAS mutation, the tumor cells are planted in the axilla area of nude mice.

1. Experimental method

1) Collection of tumor specimens from patients: For specimens after surgical resection, take the tumor tissue with no necrosis at the edge, soak it in 5% fetal bovine serum, PRMI1640 medium containing 1× penicillin and streptomycin double antibody, and transport at 4°C;

2) Cut the tumor tissue into small pieces of about 2 × 2 × 2 mm in the ultra-clean workbench, wash it with the above-mentioned culture medium 3 times, and remove the congestion;

3) Use 4.8% chloral hydrate to anesthetize 4-6 week old BALB/C nude mice. After anesthesia, make a small incision of about 3 mm in the skin of the armpit, use forceps to perform blunt dissection, and bury the trimmed tumor tissue. Subcutaneously, 6-0 sutures are used to close the incision, and double antibody is used to prevent wound infection;

4) Take 50 mg of tumor tissue to extract genomic DNA for KRAS genotype identification, and store the remaining tissue at -80°C for future use;

5) About 4 to 6 weeks after the transplanted tumor site, the raised nodules were about 1 cm ³ in size, then the tumor tissue was taken out and trimmed into 2×2×2 mm tissue blocks for passage. The passaging operation is the same as above;

6) After about 2 to 3 generations, the nude mice were divided into 4 groups: KRAS wild-type tumor control group, KRAS wild-type tumor metformin group, KRAS mutant tumor control group, KRAS mutant tumor metformin group, 10 mice each;

7) When the transplanted tumor grows to 100-200 mm ³ , intragastric administration is performed at 9 am every day for 28 consecutive days. The control group was given normal saline by gavage, and the metformin group was given metformin solution by gavage (100 mg/kg body weight of nude mice every day, dissolved in normal saline). The size of the transplanted tumor was measured daily during the period, volume (mm ³ )=[length×width ² ]/2;

8) Nude mice were sacrificed after 30 days, and tumor tissues were taken for photography, measurement, embedding and sectioning.

2. The therapeutic effect of metformin on KRAS mutant tumors

1. Experimental method

Metformin 200 mg/kg (equivalent to human 1000 mg) was dissolved in water, and metformin was given to KRAS wild-type and mutant tumor animals respectively to measure the tumor size. After 30 days, the tumor tissue was sacrificed and weighed.

2. Experimental results

Metformin significantly inhibited the size and weight of KRAS mutant tumors relative to KRAS wild-type tumor models. Metformin has a good therapeutic effect on KRAS-mutant colorectal cancer, suggesting that clinical KRAS-mutant patients can choose to use metformin, which provides a basis for drug development in KRAS-mutant patients (Figure 7).

Example 3 Metformin inhibits the proliferation of KRAS mutant colorectal cancer cells

1. The effect of metformin on apoptosis of colorectal cancer cells

The effect of metformin on colorectal cancer cell apoptosis was detected by Annexin V/PI double staining on KRAS wild-type cells SW48 and KRAS(G13D) mutant cells LoVo.

1. Experimental method

1) The cells were seeded in a 6-well plate, adhered for 12 h, starved overnight, treated with drugs for 24 h, digested with EDTA-free trypsin, and washed once with PBS without fixation.

2) Add 300 μl of Binding Buffer to resuspend the cells, add 3 μl of Annexin V-FITC and 3 μl of Propidium Iodide (PI), mix well, incubate at room temperature for 30 minutes in the dark, and perform flow cytometry on the machine.

3) Flow cytometry antibody: Apoptosis kit (A211-02) was purchased from KGI company; Cell cycle PI single staining detection kit (558662) was purchased from BD company.

2. Experimental results

Using Annexin V/PI double staining to detect cell apoptosis, the results showed that 2.5mM, 5mM and 10mM metformin did not promote the apoptosis of KRAS wild-type colorectal cancer cells SW48 and KRAS mutant colorectal cancer cells LoVo.

The effect of metformin on the proliferation of colorectal cancer cells

Edu was used to detect the proportion of proliferating cells, plate colony formation assay and PI/Rnase single staining to detect cell cycle distribution to clarify the effect of metformin on the proliferation of colorectal cancer cells.

1. Experimental method

(1) Proliferation detection (Edu method)

1) EdU-treated cells: Seed the cells in a petri dish (sterilized coverslips have been placed), adhere to the wall for 12 hours, starve overnight, add drugs for the corresponding time, and add EdU (final concentration 10 μM) in the last 2-6 hours (EdU treatment time depends on cell growth rate).

2) Fixation: add 1 mL/well of 4% paraformaldehyde, fix at room temperature for 15 minutes.

3) Wash the slides: wash twice with 1 mL/well of 3% BSA (dissolved in PBS).

4) Membrane rupture: 0.5% TritonX-100, 1 mL/well, room temperature for 20 minutes.

5) Development: same as step 3.

6) Add ClickiT reaction mixture: 50 μL/sheet, protected from light, room temperature for 30 minutes.

7) Wash the slides: 1 mL/well of 3% BSA (dissolved in PBS), washed twice, and washed once with PBS.

8) Nuclei staining: 50 μL DAPI (1:3000, PBS configuration) was stained in the dark for 10 minutes.

9) Washing: wash twice with PBS.

10) Dry in the dark, add anti-quenching agent, and seal with nail polish.

11) Observe and analyze the results under an automatic upright fluorescence microscope, and take pictures and record (100×, 200×, 400×, save the original image).

(2) Plate clone formation

1) Cell inoculation: Aspirate and discard the culture medium, collect the cells, suspend the cells in the treated conditioned medium, count 3 times to take the average value, and adjust the concentration of the cell suspension to 1×10 ³ /ml. 2.5ml of treatment conditioned medium was added to each well of the 6-well culture plate, and 0.5ml of cell suspension was added to each well (ie, 500 cells per well), with a final volume of 3ml. When inoculating, pay attention to shaking the culture plate several times in a cross direction to distribute the cells as evenly as possible.

2) Cell culture: The cells were cultured under standard conditions for 2-3 weeks, and the colony formation was observed, and the conditioned medium was replaced about every 3 days.

3) Clonal staining: Terminate the culture when the cells form clones visible to the naked eye (the number of cloned cells per well is about 50-150). Discard the culture medium, carefully wash twice with PBS, add 1.5 ml of 4% paraformaldehyde to each well and fix at room temperature for 15 minutes; discard the fixative, slowly rinse with running water, add 1.5 ml of crystal violet working solution to each well, and let stand at room temperature for staining For 30 min, the dyeing solution was slowly washed with running water and dried in a fume hood.

4) Counting of clones: place the culture plate in a gel imaging system, and under the condition of visible light, count the number of clones with the random software, scan and save the image. Colony formation rate (%)=number of clones/number of cells inoculated×100%. Each group of cell samples was inoculated into 3 replicate wells, and the experiment was repeated 3 times independently.

(3) Cell cycle detection

1) The cells were seeded in a 6-well plate, adhered for 12 hours, starved overnight, treated with drugs for 24 hours, digested and washed three times with PBS, centrifuged to discard the supernatant, and added dropwise with -20°C pre-cooled 70% ethanol, Vortex to mix cells and fix overnight at -20°C.

2) Centrifuge horizontally at 2000 rpm for 10 min, discard the supernatant, and wash with PBS 2-3 times to remove residual ethanol, and centrifuge for 5 min each time.

3) The cells were resuspended with 500 μL of PI/RNase staining solution, incubated in the dark for 15 min, and detected by flow cytometer within 2 h, with an excitation wavelength of 488 nm.

4) Use FlowJo 7.6 software to perform graph analysis.

2. Experimental results

Metformin reduced the proportion of EdU-positive proliferating cells in KRAS mutant colorectal cancer cells LoVo and inhibited LoVo clonogenicity, but had no significant effect on KRAS wild-type colorectal cancer cells SW48 (Figure 8a-b).

The flow cytometry results of the cell cycle showed that metformin concentration-dependently increased the proportion of KRAS mutant colorectal cancer cells in LoVo G1 phase and decreased the proportion of cells in S phase, but had no significant effect on KRAS wild-type colorectal cancer cells SW48 (Fig. 8c).

Lentiviral shRNA was used to construct a KRAS protein knockdown LoVo cell line. The results showed that interfering with the expression of KRAS (G13D) could down-regulate the inhibitory effect of metformin on LoVo cell viability and the inhibitory effect of metformin on the transformation of LoVo cells from G1 phase to S phase (Fig. 8d), KRAS(G13D) mutation constructed using the CRISPR/Cas9 system enhanced the anti-proliferation effect of metformin in tumor cells, cell cycle (Fig. 8e).

3. Mechanism of metformin inhibiting the proliferation of KRAS-mutated colorectal cancer cells

The changes of metformin on proliferation signaling pathways MEK/ERK/cyclin D1/RB and PI3K/AKT/mTOR/4E-BP1-related molecules were detected by Western blot, and the mechanism by which metformin inhibited KRAS mutation and affected the proliferation of colorectal cancer cells was clarified.

1. Experimental method

①In vitro cell experiments

1) The cells were seeded in a petri dish, cultured to 60% confluence, and treated with drugs for 24 hours.

2) Collect protein, wash 3 times with PBS, add 100 μl 1×SDS buffer (100 mmol/l Tris-Cl pH6.8, 2% SDS, 10% glycerol), at this time, the cell lysate is viscous.

3) Collect the cell lysate in a 0.5ml centrifuge tube with a cell scraper, and cook at 100°C for 30min.

4) BCA kit method (BIO-RAD company) was used for protein quantification of total cell protein extracts.

5) Treat the sample according to the ratio of 9 μl of denatured protein sample: 1 μl of denaturation buffer (β-mercaptoethanol, 0.4% bromophenol blue), boil at 100° C. for 30 min, and set aside.

6) Take the prepared SDS-PAGE gel (10% separating gel and 5% stacking gel), add 40ug/well of sample, electrophoresis at 80V for 30min, and electrophoresis at 120V for 90min.

7) Transfer the membrane at a constant current of 300 mA for 180 min, and transfer the protein on the gel to the PVDF membrane.

8) Block the membrane with 7% skim milk for 90 min, add the corresponding primary antibody and shake overnight at 4°C.

9) The next day, the membrane was washed 3 times with TBST for 10 min each time, and then the corresponding secondary antibody was added to incubate at 4°C for 4 h.

10) After washing the membrane 3 times with TBST, add ECL for exposure.

②The tumor tissue of PDX animal was taken, homogenized to extract protein, and WB was performed.

2. Experimental results

The phosphorylation levels of ERK, RB, AMPK, AKT, mTOR and 4E-BP1 were inhibited in KRAS mutant colorectal cancer cells LoVo treated with metformin for 24 h, but not in KRAS wild-type colorectal cancer cells SW48 (Fig. 9A). -C). This indicates that metformin can inhibit the proliferation of KRAS mutant colorectal cancer cells by simultaneously inhibiting the ERK and AKT signaling pathways.

In the KRAS(G13D) mutant SW48 cell line constructed using the CRISPR/Cas9 system, the changes in cyclinD1/RB and AKT/mTOR/4E-BP1 signaling pathways (Fig. 9D) were consistent with KRAS mutant colorectal cancer cells LoVo. Lentiviral shRNA was used to construct KRAS protein knockdown LoVo cell line, and the results showed that interfering with the expression of KRAS (G13D) could down-regulate the effect of metformin on the inhibition of LoVo RB protein and 4E-BP1 phosphorylation (Figure 9E).

In PDX tumor tissues, metformin inhibited ERK and AKT signaling pathways in KRAS-mutant colorectal cancer tissues (Fig. 9F).

In conclusion, metformin promotes G1 phase arrest in KRAS-mutant colorectal cancer cells and inhibits tumor cell proliferation instead of inducing apoptosis. Metformin can simultaneously inhibit ERK and AKT, down-regulate the phosphorylation of RB and 4E-BP1 and inhibit KRAS. Mutant colorectal cancer cells.

It is the first time to verify the effect and mechanism of metformin on KRAS-mutant colorectal cancer cells, and it also enhances the effect of combined inhibition of ERK/cyclin D1/RB and AKT/mTOR/4E-BP1 pathways in the treatment of KRAS-mutant colorectal cancer cells Evidence provided.

Example 4 KRAS mutation enhances the sensitivity of colorectal cancer cells to metformin

1. The effect of metformin on inhibiting the proliferation of colorectal cancer cells is related to the intracellular concentration

Lansoprazole was used to inhibit the absorption of metformin by cells, and cell activity was detected by CCK8, which confirmed that the effect of metformin on inhibiting the proliferation of colorectal cancer cells was related to the intracellular concentration.

The KRAS(G13D) mutant cells constructed by the CRISPR/Cas9 system and the LoVo cell line with stable KRAS knockdown were used to verify the increased concentration of metformin in KRAS mutant cells.

1. Experimental method

HCT-116 and LoVo were simultaneously treated with 5 μM and 10 μM lansoprazole, and cells were treated with metformin for 48 h, and cell viability was detected by CCK8.

KRAS(G13D) mutant cells constructed by CRISPR/Cas9 system and LoVo cell line with stable KRAS knockdown were treated with metformin, cell lysates at different time points were collected, and intracellular metformin concentration was detected by mass spectrometry.

The PDX model uses mass spectrometry to detect metformin concentrations in KRAS wild-type and mutant tumor tissues.

2. Experimental results

The CCK8 results showed that the IC50 of metformin against KRAS wild-type colorectal cancer cells SW48 was 90.83 mM (95% CI 68.60-127.60) and CaCO2 was 88.12 mM (95% CI 74.71-106.92), while for KRAS mutant Type colorectal cancer cells HCT-116 had an IC50 of 23.71 mM (95% confidence interval 17.22-33.54) and a LoVo of 8.18 mM (95% confidence interval 6.52-10.13) (Figure 10). The IC50s of HCT-116 and LoVo were increased by more _than 1.2-2.5-fold following inhibition of metformin uptake by cells with lansoprazole (Fig. 11a-b).

The mass spectrometry results showed that the intracellular metformin concentration in the KRAS(G13D) mutant cells constructed by the CRISPR/Cas9 system was significantly increased compared with the control cells (Fig. 11c); while the intracellular metformin concentration in the LoVo cell line with stable KRAS knockdown was significantly lower than that in LoVo (Fig. 11c). Figure 11d).

Mass spectrometry results showed that metformin-treated KRAS-mutant tumor tissues increased metformin concentrations compared with KRAS wild-type tumor tissues (Fig. 11f).

2. RNA levels of metformin channel protein in cells

By analyzing the TCGA-COAD database, the RNA levels of metformin channel proteins in cells were detected, and the differential expression between KRAS mutant colorectal cancer cell lines HCT-116 and LoVo, and KRAS wild-type colorectal cancer cells SW48 and CaCO2 were screened.

1. Experimental method

(1) Real-time quantitative PCR (real-time quantitative PCR, RT-qPCR)

1) RNA extraction

For specific steps, please refer to the instruction manual of the RNA extraction kit (CW0581) of Kangwei Century Biotechnology Co., Ltd.

2) reverse transcription

RNA concentrations were measured with a Nanodrop UV spectrophotometer. 500ng NA was reverse transcribed into cDNA as follows:

3) RT-qPCR

Green I染料。引物利用PubMed的primer-blast程序设计合成，由Lifetechnology公司合成，引物序列如下表：The instrument is a lightcycler fluorescence quantitative PCR instrument or a CFX 96 fluorescence quantitative PCR instrument of BIO-RAD. Reagents for Takara

Green I dye. The primers were designed and synthesized using the primer-blast program of PubMed, and were synthesized by Lifetechnology Company. The primer sequences were as follows:

The fluorescent quantitative PCR reaction procedure is as follows:

1) Three-step method (high efficiency, but poor specificity):

①Pre-denaturation: 95℃ for 30s, 1 cycle;

②PCR reaction: denaturation at 95°C for 5s, annealing at 55°C for 30s, extension at 72°C for 45s, 35 cycles;

③ Melting curve analysis: 95°C for 0s, 65°C for 15s, 95°C for 0s, 1 cycle.

2) Two-step method (high specificity, but low efficiency):

①Pre-denaturation: 95℃ for 30s, 1 cycle;

②PCR reaction: denaturation at 95°C for 5s, annealing at 60°C and extension for 30s, 40 cycles;

③ Melting curve analysis: 95℃ for 0s, 65℃ for 20s, 95℃ for 0s, 1 cycle.

Premix Ex TaqⅡ试剂说明书)。(cited from

Instructions for Premix Ex TaqⅡ Reagent).

After PCR, refer to the melting curve to determine the specificity of the primers, and calibrate with the standard curve and internal reference of different genes according to the Cp value obtained by the reaction.

2. Experimental results

Analysis in the TCGA-COAD database and screening of RNA levels of metformin channels in cell lines revealed that KRAS mutant colorectal cancer cells (HCT-116 and LoVo) had reduced MATE1 (SLC47A1) expression compared with KRAS wild-type colorectal cancer cells (Fig. 12A). The expression of MATE1 (SLC47A1) in the KRAS(G13D) mutant SW48 cell line was decreased (Fig. 12B), and the expression of sh-KRAS-LoVo MATE1 (SLC47A1) was increased (Fig. 12C).

3. The expression level of MATE1

Differentially expressed MATE1 was screened and then validated on clinical specimens by immunohistochemistry and by intracellular gene overexpression or knockdown experiments.

1. Experimental method

Immunohistochemistry (p-RB marker) was the same as in Example 1.

2. Experimental results

Immunohistochemical results of clinical specimens showed that compared with KRAS wild-type colorectal cancer, MATE1 protein level was decreased in KRAS mutant colorectal cancer; The proliferation indicator p-RB was expressed directly (Figure 12D).

Overexpressing MATE1 on LoVo for forward experiments or interfering with MATE1 on SW48 for reverse experiments proved that down-regulation of MATE1 expression promoted the effect of metformin on inhibiting the proliferation of colorectal cancer cells (Figure 12E-F).

The expression of MATE1 and transcription factor Sp1 was detected by Western blot, and the results showed that the expression of MATE1 in colorectal cancer cells was not correlated with the expression of Sp1.

4. In vivo verification of the role of low-expression MATE1 in the tumor-inhibiting effect of metformin

The role of MATE1 in the tumor-inhibiting effect of metformin was verified by cell-derived xenograft (CDX) experiments.

1. Experimental method

In the cell-derived xenograft model (CDX) experiment, we first established the KRAS ^G13D SW48 cell line using CRISPR/Cas9; knocked out MATE1 by shRNA lentiviral transduction in SW48 to construct the sh-MATE1-SW48 cell line; KRAS ^G13D Lentiviral infection of SW48 cells overexpressed MATE1. 1×10 ⁶ cells suspended in basement membrane matrix (100 μl high-concentration basement membrane matrix and 100 μl PBS)) were injected subcutaneously into BALB/c nude mice. CDX mice were randomly divided into metformin-treated and control groups. Metformin 200 mg/kg (equivalent to 1000 mg for humans) was dissolved in water, and the animals were given to four groups of animals to drink. The tumor size was measured. After 30 days, the tumor tissue was sacrificed and weighed.

2. Experimental results

The results showed that metformin treatment in SW48 xenograft model had no obvious antitumor effect compared with control, but after knockdown of MATE1, metformin treatment significantly inhibited SW48+sh-MATE1 tumor growth. In contrast, after overexpression of MATE1, metformin was ineffective in KRAS ^G13D SW48 xenografts compared to KRAS ^G13D SW48 tumors (Figure 12G-L).

5. Differences in methylation levels of CpG islands in the promoter of MATE1 gene

Analysis and correlation statistics by mRNA-seq and MethyArray 450K data in TCGA-COAD database indicated that MATE1 expression in colorectal cancer cells was negatively correlated with MATE1 promoter methylation ( FIG. 13A ).

Using the method of bisulfite sequencing to detect the difference in the methylation level of the MATE1 gene promoter CpG island between KRAS mutant colorectal cancer cell lines HCT-116 and LoVo, and KRAS wild-type colorectal cancer cells SW48 and CaCO2 .

1. Experimental method

(1) DNA bisulfite sequencing (bisulfite sequencing PCR, BSP)

1) Modification of DNA with sodium bisulfite:

A. Extract genomic DNA, then carry out 5mC>U sodium bisulfite reaction according to the following reaction system and reaction conditions:

B. DNA purification after bisulfite treatment, see the product specification of DNA Bisulfite Conversion Kit (DP215).

2) PCR amplification and T vector ligation:

A. After the genomic DNA sample was modified with bisulfite, the CpG island of the MATE1 promoter was amplified. The sequence and amplification primers were marked as shown in Figure 14 (the primer sequences are framed, and the CpG sites are underlined).

The PCR reaction system and conditions are as follows:

B. The gel is recovered and purified, then the blunt-ended PCR product is added with A reaction, connected to the T vector pGM-T, transformed into competent bacteria and amplified, and 5 single clones are picked for sequencing. The specific steps are the same as those in Example 3.

3) Analysis of methylation sites and methylation levels: The sequencing results were directly analyzed using the website http://quma.cdb.riken.jp/QUantification tool for Methylation Analysis (QUMA), and a dot plot was selected, each dot Represents 1 CpG site, white is unmethylated, black is methylated, a total of 34 CpG sites.

(2) Quantification of hydroxymethylated DNA

1) Hydroxymethylated DNA immunoprecipitation (Hydroxymethylated DNA immunoprecipitation, hMeDIP):

A. Extract genomic DNA, use Sonics ultrasonic disruptor with 25% energy, 10s/pulse×4 pulses, and sonicate at a frequency of 40s each time. The genomic DNA can be broken into fragments of about 400bp.

B. Use Abcam's hMeDIP ChIP kit to enrich DNA fragments containing 5hmC. For specific steps, refer to the instruction manual of Abcam's hMeDIP ChIP Kit (ab117134).

2) RT-qPCR for semi-quantitative analysis: RT-qPCR primers are as follows, RT-qPCR reaction system and conditions are as described above. Unenriched DNA was used as a correction.

2. Experimental results

By bisulfite sequencing PCR, it was found that the methylation level of MATE1 promoter was higher in KRAS mutant colorectal cancer cells HCT-116 and LoVo compared with KRAS wild-type colorectal cancer cells SW48 and CaCO2 (Fig. 13B) .

6. KRAS mutation regulates MATE1 expression by regulating the methylation level of MATE1 promoter

Lentiviral shRNA was used to construct KRAS knockdown LoVo cell line, and KRAS (G13D) mutant SW48 cell line constructed by CRISPR/Cas9 system was used to verify that KRAS mutation regulates MATE1 expression by regulating the methylation level of MATE1 promoter. . MATE1 methylation levels were detected in PDX KRAS mutant and wild-type tumor tissues.

1. Experimental method

The methylation level detection method is the same as above.

2. Experimental results

The KRAS(G13D) mutant SW48 cell line constructed using the CRISPR/Cas9 system had increased methylation levels of the MATE1 promoter (Fig. 13C); KRAS knockdown LoVo cell lines were constructed using lentiviral shRNA, and the methylation level of the MATE1 promoter was increased. The methylation level was decreased (FIG. 13D-E); the methylation level of MATE1 was significantly increased in PDX KRAS mutant tumor tissues compared with wild type (FIG. 13F).

7. Differential expression and validation of methyltransferases and demethylases

Detection of RNA levels in cell lines, screening of differentially expressed methyltransferases and demethylases, and construction of KRAS knockdown LoVo cell lines in clinical samples and lentiviral shRNA, and KRAS (G13D) mutants constructed by CRISPR/Cas9 system Validated in SW48 cell line.

1. Experimental method

The RNA detection method is the same as above, and the primers are as follows

2. Experimental results

The differential expression of methyltransferase and demethylase in cell lines was detected, and the results indicated that methyltransferases DNMT1 and DNMT3A were up-regulated in KRAS-mutant colorectal cancer cells, and demethylase TET1/2 was up-regulated in KRAS-mutant colorectal cancer cells. Downregulated in rectal cancer cells (Figure 15a). In the KRAS knockdown LoVo cell line constructed using lentiviral shRNA, TET1/2 was upregulated (Fig. 15b).

At the same time, the expression of DNMT1 and TET1 in 59 colon cancer samples was verified by histochemical staining, and the proportion of cells with strong nuclear staining was calculated using ImageJ software. The results showed that the protein level of DNMT1 in KRAS mutant colon cancer tissues was higher than that of KRAS wild type (P=0.0003), while the expression of TET1 had no significant difference between the two groups (P=0.2989). The results of Spearman rank correlation analysis showed that the proportion of DNMT1 strongly positive cells was significantly negatively correlated with the expression of MATE1 (r=-0.66 P<0.0001, n=59) (Fig. 16A). Increased DNMT1 expression and decreased TET1 expression in KRAS-mutant PDX tumor tissues (Figure 16B)

On the KRAS(G13D) mutant SW48 cell line constructed by CRISPR/Cas9 system, the results showed that the expression of DNMT1 was increased and the expression of TET1/2 was decreased (Fig. 16C). In the KRAS knockdown LoVo cell line constructed by lentiviral shRNA, the expression of DNMT1 was decreased. , TET1/2 expression increased (Fig. 16D).

Using the methylase inhibitor azacitidine in LoVo cells and KRAS(G13D) mutant SW48 cell line can upregulate the expression of MATE1 in KRAS mutant colorectal cancer cells, thereby inhibiting the anti-proliferative effect of metformin (Figure 16E). -F). Interfering with TET1/2 in KRAS knockdown LoVo cells re-downregulated MATE1 expression and promoted the effect of metformin on tumor cell proliferation inhibition (Fig. 16G).

In conclusion, KRAS mutation down-regulates MATE1 to increase the intracellular concentration of metformin and enhance the effect of metformin on inhibiting the proliferation of colorectal cancer cells. Clinically, metformin can be selectively used in the treatment of colorectal cancer by detecting the KRAS exon 2 genotype.

Claims (9)

1. A marker for determining a treatment regimen for colorectal cancer, wherein said marker is KRAS gene and/or protein.

Use of KRAS gene and/or protein as a marker for determining a colorectal cancer treatment regimen.

Use of a reagent for detecting a mutation in the KRAS gene in the manufacture of a kit for determining a treatment for colorectal cancer.

Use of a reagent for detecting KRAS protein in the manufacture of a kit for determining a treatment for colorectal cancer.

5. A kit for determining a treatment regimen for colorectal cancer, the kit comprising KRAS mutant detection reagents, wherein the KRAS mutant colorectal cancer is treated with metformin.

6. Use of metformin in the treatment of colorectal cancer or in the manufacture of a medicament for the treatment of colorectal cancer, wherein the colorectal cancer is KRAS mutant colorectal cancer.

Use of a combination of the MEK signaling pathway and the AKT signaling pathway as a target for the treatment of colorectal cancer.

Treating KRAS mutant colorectal cancer with an inhibitor of the MEK signaling pathway in combination with an inhibitor of the AKT signaling pathway.

9. A pharmaceutical composition for treating KRAS mutant colorectal cancer comprising an inhibitor of the MEK signaling pathway and an inhibitor of the AKT signaling pathway.

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