WO2021004372A1 - 二甲双胍在治疗kras突变型结直肠癌中的应用 - Google Patents

二甲双胍在治疗kras突变型结直肠癌中的应用 Download PDF

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WO2021004372A1
WO2021004372A1 PCT/CN2020/099974 CN2020099974W WO2021004372A1 WO 2021004372 A1 WO2021004372 A1 WO 2021004372A1 CN 2020099974 W CN2020099974 W CN 2020099974W WO 2021004372 A1 WO2021004372 A1 WO 2021004372A1
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colorectal cancer
kras
metformin
cells
tumor
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高国全
周倜
杨霞
谢晋烨
夏良平
何文卓
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中山大学
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Definitions

  • the present invention relates to the technical field of treatment of colorectal cancer, and more specifically, to the application of metformin in the treatment of KRAS mutant colorectal cancer.
  • Colorectal cancer is one of the most common malignant tumors.
  • chemotherapy based on oxaliplatin or irinotecan, combined with anti-epidermal growth factor receptor (EGFR) monoclonal antibody can increase the median overall survival time of patients with colorectal cancer to 2 years the above.
  • EGFR anti-epidermal growth factor receptor
  • colorectal cancer is a genetically heterogeneous disease.
  • MSI microsatellite instability
  • chromosomal instability chromosomal instability
  • CIN chromosomal instability
  • the current treatment strategies for the presence of KRAS mutant CRC include inhibiting KRAS activation or inhibiting the activation of MEK/ERK downstream of the pro-proliferation signaling pathway of KRAS, but they all ended in the failure of phase II clinical trials.
  • the former is because farnesyltransferase inhibitors cannot completely inhibit KRAS activation, and 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 patient's blood sugar and insulin levels, and improve insulin resistance.
  • metformin has a certain preventive and therapeutic effect on colorectal cancer, and its mechanism mainly includes direct effects on tumor cells: inhibiting the activation of MEK-ERK, PI3K-AKT and mTOR signaling pathways; And the indirect effects on tumor cells: such as reducing and maintaining blood sugar and insulin levels, inhibiting inflammation, increasing the proportion of CD8+ T cells, thereby improving the cellular immune function of tumors.
  • metformin cannot improve the overall survival time and progression-free survival time of patients with colorectal cancer.
  • the purpose of the present invention is to overcome the shortcomings 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 the treatment plan for colorectal cancer.
  • the second objective of the present invention is to provide the application of KRAS gene and/or protein as a marker for determining a treatment plan for 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 the treatment plan of colorectal cancer.
  • the fourth object of the present invention is to provide the application of the KRAS protein detection reagent in the preparation of a kit for determining the treatment plan of 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 drugs for the treatment of colorectal cancer.
  • the seventh objective of the present invention is to provide the application of the combination of MEK signaling pathway and AKT signaling pathway as a treatment target for colorectal cancer.
  • the eighth object of the present invention is to provide an inhibitor of the MEK signaling pathway and an inhibitor of the AKT signaling pathway to be used in combination 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.
  • the KRAS (G13D) point mutation model and KRAS knockdown model were constructed in cell experiments to verify that KRAS mutant colorectal cancer cells are sensitive to the anti-tumor effect of metformin. Clarify that metformin inhibits both ERK/cyclin D1/RB and AKT/mTOR/4E-BP1 pathways to inhibit the proliferation of KRAS mutant colorectal cancer cells. It provides evidence for the clinically 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 alternative drug.
  • mutant KRAS protein promotes 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.
  • a marker for determining a treatment plan for colorectal cancer is KRAS gene and/or protein, and KRAS mutant colorectal cancer selects metformin to treat colorectal cancer.
  • KRAS gene and/or protein as a marker for determining the treatment plan for colorectal cancer.
  • KRAS mutant colorectal cancer is treated with metformin for the treatment of colorectal cancer.
  • the KRAS mutant colorectal here is the continuous activation of KRAS gene mutation and high expression
  • the application of the KRAS gene mutation detection reagent in the preparation of a kit for determining the treatment plan of colorectal cancer also belongs to the protection scope of the present invention.
  • the application of the KRAS protein detection reagent in the preparation of a kit for determining the treatment plan of colorectal cancer also belongs to the protection scope of the present invention.
  • kits for determining a treatment plan for colorectal cancer comprising a KRAS mutant colorectal cancer detection reagent.
  • metformin in the treatment of colorectal cancer or the preparation of a medicine for the treatment of colorectal cancer, the colorectal cancer being KRAS mutant colorectal cancer.
  • KRAS mutant colorectal cancer does not limit the types of KRAS mutations, and the current experimental data support codon 12.13 common mutations.
  • MEK and AKT are important signal pathways downstream of KRAS that regulate cell proliferation, the 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.
  • Inhibitors of the MEK signaling pathway and AKT signaling pathway are used in combination to treat KRAS mutant colorectal cancer
  • a pharmaceutical composition for treating KRAS mutant colorectal cancer which is characterized by comprising an inhibitor of the MEK signal pathway and an inhibitor of the AKT signal pathway.
  • the above-mentioned treatment of colorectal cancer is to promote the G1 stagnation phase of colorectal cancer cells, inhibit the proliferation of colorectal cancer cells, inhibit tumor growth, inhibit tumor weight gain, prolong the patient's overall survival time and/or prolong the progression-free time of chemotherapy .
  • the extending the progression-free time of chemotherapy is extending the progression-free time of first-line chemotherapy.
  • the above-mentioned inhibitor is any substance that can reduce the corresponding protein, gene or signal pathway.
  • 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 the expression of the metformin excretion channel MATE1 is the key mechanism for KRAS mutant colorectal cancer cells to be sensitive to metformin. It increases tumors by reducing the transcription level of MATE1 The concentration of metformin in the cell, thereby enhancing the effect of metformin on inhibiting tumor cell proliferation.
  • the invention provides a new idea and method for the treatment of colorectal cancer, which has far-reaching significance and is worthy of vigorous promotion.
  • Figure 1 is a schematic diagram of clinical patients included in the group.
  • 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 the overall survival time of patients with KRAS mutant colorectal cancer and the progression-free survival time during first-line chemotherapy.
  • Figure 4 is a schematic diagram of gene modification in the SW48KRAS (G13D) cell line.
  • Figure 5 shows the sequence alignment of KRAS exon2 GGC>GAC mutants that are positive clones.
  • Figure 6 shows that metformin inhibits the growth of KRAS mutant colorectal cancer cells.
  • Figure 7 shows that metformin inhibits the growth of KRAS mutant PDX tumors.
  • Figure 8 shows that metformin promotes G1 arrest of KRAS mutant colorectal cancer cells and inhibits tumor cell proliferation.
  • Figure 9 shows the mechanism by which metformin promotes G1 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 concentration in KRAS mutant colorectal cancer cells and PDX tumor tissues.
  • Figure 12 shows that KRAS mutation down-regulates MATE1 to increase the intracellular concentration of metformin and enhance the effect of metformin on the proliferation of colorectal cancer cells.
  • Figure 13 shows that KRAS mutation regulates MATE1 methylation and then down-regulates MATE1 expression.
  • Figure 14 shows the amplified sequence and primers of the CpG island of the MATE1 promoter after genomic DNA samples have been modified with bisulfite.
  • Figure 15 shows the expression of DNMT/TET in SW48KRAS (G13D) cells and KRAS knockdown Lovo cells
  • Figure 16 shows that KRAS mutation down-regulates MATE1 expression by regulating DMNT1/TET.
  • 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 are cell lines preserved in the laboratory.
  • the STR identification of these six colorectal cancer cell lines was completed by Guangzhou Saiku Biotechnology Co., Ltd., and 100% matched the information provided by ATCC, without other cell contamination or STR changes.
  • the KRAS genotypes of the above cells were queried from the ATCC official website and verified by PCR sequencing.
  • the pathologist After exporting the image, the pathologist will classify the tumor pathologically, which is divided into well differentiated, moderately differentiated, poorly differentiated and undifferentiated;
  • Ki67-stained tumor tissue image uses the IHC Profiler plug-in of ImageJ to quantitatively analyze the nuclei of Ki67(+) and record them as proliferating cells;
  • the cells were seeded at a concentration of 5000 cells/well (48-well plate, 200 ⁇ l volume per well). Treat the cells in different ways according to experimental requirements, and add 10 ⁇ l of CCK8 solution after a certain period of time. Incubate in an incubator for 1 hour, draw 200 ⁇ l of supernatant into a 96-well microplate, and measure OD 450 with a microplate reader.
  • Example 1 The effect of metformin on the prognosis of patients with metastatic colorectal cancer
  • Collect the general clinical characteristics of patients such as gender, age, body mass index (BMI); and clinical characteristics that have been reported to affect the prognosis of mCRC (such as tumor primary site, pathological grade, metastasis site, first-line chemotherapy regimen, and KRAS genotype) is used to eliminate confounding factors and clarify the overall survival (OS) of colorectal cancer patients with type 2 diabetes and the progression-free survival (PFS) of first-line chemotherapy. influences.
  • OS overall survival
  • PFS progression-free survival
  • Table 2 The distribution of clinical characteristics of 180 patients with metastatic colorectal cancer with type 2 diabetes with a clear Kras genotype in the metformin group and other hypoglycemic drug groups
  • Metformin improves the overall survival time of patients with metastatic colorectal cancer and type 2 diabetes and the progression-free survival time of first-line chemotherapy
  • metformin improves the prognosis of patients with metastatic colorectal cancer with type 2 diabetes in the center compared with the group taking other hypoglycemic drugs.
  • GraphPad Prism 7 was used as the Kaplan-Meier survival curve, and Log-rank (Mantel-Cox) statistical analysis was performed; in addition, the corrected risk ratio (stratified HR) was used to calculate the difference between different hypoglycemic drugs and the non-hyperglycemic treatment group. And PFS.
  • Table 3 The effect of different hypoglycemic drugs on OS and PFS compared with non-hyperglycemic treatment group.
  • a proportional hazard assumption is made, that is, it is assumed that the effect of metformin on patient death or tumor progression cannot change over time, and should be a fixed value.
  • the stratified Cox proportional hazard model was used to stratify the collected clinical features to explore the individual factors that affect the effectiveness of metformin.
  • metformin reduces the hazard ratio (HR) of 0.746.
  • the 95% confidence interval (confidence interval, CI) was 0.496-1.121, and the interval crossed 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 is 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.
  • Table 5 uses the proportional hazard regression model of metformin and overall survival time and progression-free survival time and the analysis of the interaction with each clinical feature:
  • Hematoxylin-eosin stain was used to determine the location of tumor cells (deep nuclear staining, heterogeneity, glandular epithelial structure destruction), and Ki67 staining was used to mark cells in the proliferation stage.
  • Blocking Use an immunohistochemical pen to draw a circle, add 3% H2O2, and room temperature for 30 minutes;
  • KRAS wild-type colorectal cancer cell lines SW48 and CaCO2 In vitro treatment of KRAS wild-type colorectal cancer cell lines SW48 and CaCO2 with gradient concentrations of metformin, and KRAS G13D mutant colorectal cancer cell lines HCT-116 and LoVo, and KRAS G12V mutant colorectal cancer cell lines SW480 And SW620.
  • KRAS wild-type colorectal cancer cell line SW48 transfected with KRAS G12V, KRAS G13D and KRAS G12D plasmids was treated with gradient concentrations of metformin.
  • sgRNA sequence of guide RNA design: Zhang Lab, the website http://crispr.mit.edu provided by MIT, for the base 250bp before the ATG start codon of the KRAS gene on human chromosome 12 Design. Take the sgRNA with a low off-target efficiency with a score of >85, use CRISPR RGEN tool Cas-OFFinder website http://www.rgenome.net/cas-offinder/, set the number of mismatch bases ⁇ 2, do not use mismatch SgRNA to avoid non-specific cleavage.
  • the sgRNA we selected: 5’-GCATTTTTCTTAAGCGTCGA-3’.
  • Target sequence(PAM) GCATTTTTCTTAAGCGTCGA(TGG);
  • Restriction enzyme digestion Use Bbs I restriction enzyme to digest pSpCas9(BB)-2A-puro(PX459)V2.0 plasmid, perform agarose electrophoresis and gel recovery and purification.
  • the restriction conditions are as follows:
  • connection Use T4 ligase to connect PX459 and gRNA, then agarose electrophoresis identification and gel recovery and purification, denoted as PX459/hKRAS gRNA.
  • the connection conditions are as follows:
  • A. Gene retrieval First use genomic DNA extraction kit to extract LoVo genomic DNA, and then amplify about 3000bp DNA sequence including sgRNA and exon2 by high-fidelity PCR method, run electrophoresis for identification and gel recovery and purification.
  • the PCR reaction system and conditions are as follows:
  • T vector the DNA purified in the previous step, after the A reaction, is connected to the pGM-T vector, transferred to competent bacteria for amplification, and a single clone is selected for sequencing verification. Verify that the sequence is correct and record it as pGM-T/KRAS-homology.
  • the reaction conditions for adding A and ligation are as follows:
  • the PCR reaction system of PrimeSTAR Max Premix is the same as above, and the reaction conditions are changed to 15s for the extension time. After the two PCR products were recovered and purified by gel, they were connected to the pGM-T vector using seamless cloning, and then gel recovery, purification, transformation, amplification and single clone sequencing were performed.
  • the seamless cloning reaction system and reaction conditions are as follows:
  • the pDONR 221 connection reaction system and conditions are as follows:
  • KRAS interference lentivirus (sh-KRAS) was constructed and packaged by Shanghai Jikai Gene Company. A total of 2 targets were designed. See Table 7 for details.
  • results show that compared with KRAS wild-type, patients with KRAS mutant colorectal cancer using metformin have longer overall survival time and progression-free survival time, and the same results have been verified in tumor tissue sections and cell viability experiments. .
  • PDX model Use the PDX model: take clinical KRAS wild-type and mutant colorectal cancer patient tumor tissues, digest the tumor cells, identify the KRAS mutation, and plant the tumor cells in the axillary area of nude mice.
  • the size of the tumor site is about 1cm 3 when the tumor is transplanted.
  • the tumor tissue is taken out and trimmed into 2 ⁇ 2 ⁇ 2mm tissue blocks for passage.
  • the passaging operation is the same as above;
  • mice 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;
  • mice were sacrificed 30 days later, and the tumor tissues were taken for photographing, measurement, embedding and sectioning.
  • metformin (equivalent to 1000 mg for humans) was dissolved in water, and the KRAS wild-type and mutant tumor animals were drunk with metformin, the tumor size was measured, and the tumor tissue was sacrificed 30 days later, and the tumor tissue was weighed.
  • metformin can significantly inhibit the size and weight of KRAS mutant tumors. 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 for KRAS mutant patients (Figure 7).
  • Example 3 The effect of metformin on inhibiting the proliferation of KRAS mutant colorectal cancer cells
  • Annexin V/PI double staining was used to detect the effect of metformin on the apoptosis of colorectal cancer cells.
  • Apoptosis kit (A211-02) was purchased from KGI; Cell cycle PI single staining detection kit (558662) was purchased from BD.
  • Annexin V/PI double staining was used to detect cell apoptosis.
  • the results showed that 2.5mM, 5mM, 10mM metformin did not promote the apoptosis of KRAS wild-type colorectal cancer cell SW48 and KRAS mutant colorectal cancer cell LoVo.
  • Edu was used to detect the proportion of proliferating cells, plate clone formation experiment, and PI/Rnase single staining to detect cell cycle distribution, to clarify the effect of metformin on the proliferation of colorectal cancer cells.
  • EdU-treated cells Plant the cells in a petri dish (sterilized coverslips have been placed), adhere to the wall for 12 hours, starve overnight, dosing for the corresponding time, and add EdU (final concentration 10 ⁇ M) in the last 2-6h (EdU treatment time depends on cell growth rate).
  • Cell inoculation aspirate and discard the culture medium, collect the cells, suspend the cells in the treatment condition culture medium, count 3 times to take the average value, and adjust the cell suspension concentration to 1 ⁇ 10 3 /ml.
  • Each well of the 6-well culture plate was added with 2.5ml of the treatment condition culture medium, and each well was added with 0.5ml of cell suspension (ie 500 cells per well), the final volume was 3ml.
  • Cell culture Cells are cultured under standard conditions for 2 to 3 weeks, the formation of clones is observed, and the conditioned medium is changed about every 3 days.
  • Clone count Place the culture plate in the gel imaging system, under visible light conditions, count the number of clones with the software that comes with the machine, scan and save the image.
  • Clone formation rate (%) number of clones/number of inoculated cells ⁇ 100%. Each group of cell samples were seeded in 3 replicate wells, and the experiment was repeated 3 times independently.
  • Metformin reduces the proportion of EdU-positive proliferative cells in KRAS mutant colorectal cancer cell LoVo, inhibits the LoVo cloning ability, but has no obvious effect on KRAS wild-type colorectal cancer cell SW48 ( Figure 8A-B).
  • Lentiviral shRNA was used to construct a KRAS knockdown LoVo cell line, and the results showed that interference with the expression of KRAS (G13D) can down-regulate the inhibitory effect of metformin on the viability of LoVo cells, and down-regulate the inhibitory effect of metformin on the transformation of LoVo cells from the G1 phase to the S phase ( Figure 8D)
  • the KRAS (G13D) mutation constructed using the CRISPR/Cas9 system enhances the anti-tumor cell proliferation effect of metformin, and the cell cycle (Figure 8E).
  • the cells are seeded in a petri dish, cultured to 60% confluence, and treated with medicine for 24h.
  • KRAS mutant colorectal cancer cell LoVo was treated with metformin for 24 hours, the phosphorylation levels of ERK, RB, AMPK, AKT, mTOR and 4E-BP1 were inhibited, but there was no significant change in KRAS wild-type colorectal cancer cell SW48 ( Figure 9A -C). It shows that metformin can inhibit the proliferation of KRAS mutant colorectal cancer cells by simultaneously inhibiting ERK and AKT signaling pathways.
  • metformin promotes G1 arrest of KRAS mutant colorectal cancer cells, inhibits tumor cell proliferation, but does not induce apoptosis; metformin can simultaneously inhibit ERK and AKT, and down-regulate phosphorylation of RB and 4E-BP1 to inhibit KRAS Mutant colorectal cancer cells.
  • Example 4 KRAS mutation enhances the sensitivity of colorectal cancer cells to metformin
  • the KRAS (G13D) mutant cells constructed by the CRISPR/Cas9 system and the KRAS stable knockdown LoVo cell line verified that the concentration of metformin increased in KRAS mutant cells.
  • HCT-116 and LoVo were treated with 5 ⁇ M and 10 ⁇ M lansoprazole at the same time, and the cells were treated with metformin for 48h, and CCK8 was used to detect cell viability.
  • the KRAS (G13D) mutant cells constructed by the CRISPR/Cas9 system and the KRAS stable knockdown LoVo cell line were treated with metformin, cell lysates were collected at different time points, and the intracellular metformin concentration was detected by mass spectrometry.
  • the PDX model uses mass spectrometry to detect the concentration of metformin in KRAS wild-type and mutant tumor tissues.
  • RNA level of metformin channel protein in the cells By analyzing the TCGA-COAD database, detecting the RNA level of metformin channel protein in the cells, screening KRAS mutant colorectal cancer cell lines HCT-116 and LoVo, and KRAS wild-type colorectal cancer cell lines SW48 and CaCO2 differential expression.
  • RNA extraction kit (CW0581) of Kangwei Century Biotechnology Co., Ltd.
  • RNA concentration with Nanodrop UV spectrophotometer. Reverse transcription of 500ng NA into cDNA, as follows:
  • the instrument is a lightcycler fluorescent quantitative PCR instrument or BIO-RAD's CFX 96 fluorescent quantitative PCR instrument.
  • the reagent is Takara Green I dye.
  • the primers were designed and synthesized using PubMed's primer-blast program and synthesized by Life Technology. The primer sequences are as follows:
  • the fluorescence quantitative PCR reaction procedure is as follows:
  • PCR After PCR, refer to the melting curve to determine the specificity of the primer, and according to the Cp value obtained from the reaction, correct it with the standard curve of different genes and the internal reference, and set the control group as 1, analyze and combine the pictures.
  • the differentially expressed MATE1 was screened out, and then verified on clinical specimens by immunohistochemistry, and verified by intracellular gene overexpression or knockdown experiments.
  • MATE1 and transcription factor Sp1 were detected by Western blot, and the results showed that the expression of MATE1 in colorectal cancer cells was not related to the expression of Sp1.
  • CDX cell-derived xenograft model
  • metformin treatment in the SW48 xenograft model had no significant anti-tumor effect compared with the control, but after knocking down MATE1, metformin treatment significantly inhibited the growth of SW48+sh-MATE1 tumors.
  • metformin treatment significantly inhibited the growth of SW48+sh-MATE1 tumors.
  • metformin was ineffective in KRAS G13D SW48 xenografts ( Figure 12G-L).
  • the CpG island of the MATE1 promoter is amplified.
  • the sequence and amplification primer labels are as shown in Figure 14 (the box is the primer sequence, and the underline is the CpG site).
  • the PCR reaction system and conditions are as follows:
  • genomic DNA can be broken into fragments of about 400bp.
  • RT-qPCR primers are as follows, and the RT-qPCR reaction system and conditions are the same as described above. Use unenriched DNA as a correction.
  • KRAS mutant colorectal cancer cells HCT-116 and LoVo had higher methylation levels of the MATE1 promoter ( Figure 13B) .
  • KRAS mutation regulates the expression of MATE1 by regulating the methylation level of MATE1 promoter
  • Lentiviral shRNA was used to construct a KRAS knockdown LoVo cell line, and a KRAS (G13D) mutant SW48 cell line constructed by the CRISPR/Cas9 system. It was verified that the KRAS mutation regulates the expression of MATE1 by regulating the methylation level of the MATE1 promoter. . Detect the methylation level of MATE1 in PDX KRAS mutant and wild-type tumor tissues.
  • the methylation level detection method is the same as above.
  • the KRAS(G13D) mutant SW48 cell line constructed with the CRISPR/Cas9 system showed increased methylation levels of the MATE1 promoter (Figure 13C); the LoVo cell line with KRAS knockdown constructed using lentiviral shRNA, the MATE1 promoter The methylation level was reduced ( Figure 13D-E); the methylation level of MATE1 in PDX KRAS mutant tumor tissue was significantly higher than that of the wild type ( Figure 13F).
  • RNA levels screen differentially expressed methyltransferases and demethylases, and construct KRAS knockdown LoVo cell lines in clinical samples and lentiviral shRNA, and KRAS (G13D) mutations constructed by CRISPR/Cas9 system Type SW48 cell line for verification.
  • RNA detection method is the same as above, the primers are as follows
  • methylase inhibitor azacitidine in LoVo cells and KRAS (G13D) mutant SW48 cell line can up-regulate the expression of KRAS mutant colorectal cancer cells MATE1, thereby inhibiting the anti-tumor proliferation effect of metformin ( Figure 16E) -F).
  • Interfering with TET1/2 in the KRAS knockdown LoVo cell line will re-regulate the expression of MATE1 and promote the effect of metformin on inhibiting tumor cell proliferation ( Figure 16G).
  • the 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.
  • the genotype of KRAS exon 2 can be detected, and metformin can be selectively used to treat colorectal cancer.

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Abstract

本发明公开了一种确定结直肠癌治疗方案的标志物，所述标志物为KRAS基因和/或蛋白，本发明公开了KRAS突变型结直肠癌患者使用二甲双胍治疗，二甲双胍排出通道MATE1表达下调是KRAS突变型结直肠癌细胞对二甲双胍敏感的关键机制，通过使MATE1转录水平降低增加肿瘤细胞内二甲双胍的浓度，从而增强二甲双胍抑制肿瘤细胞增殖的作用。

Description

二甲双胍在治疗KRAS突变型结直肠癌中的应用 技术领域
本发明涉及结直肠癌的治疗技术领域，更具体地，涉及二甲双胍在治疗KRAS突变型结直肠癌中的应用。
背景技术
结直肠癌(colorectal cancer,CRC)是最常见的恶性肿瘤之一。目前以奥沙利铂或伊立替康为主的化疗，并联合抗表皮生长因子受体(epidermal growth factor receptor,EGFR)单抗，能使结直肠癌患者的中位总体生存时间提高到2年以上。但是，在中国约1/4的患者在诊断时候已发生肿瘤转移，化疗效果欠佳。此外，结直肠癌是一种基因异质性疾病，APC,KRAS,TP53,BRAF,PIK3CA等基因的改变(突变或缺失)以及微卫星不稳定(microsatellite instability,MSI)和染色体不稳定(chromosomal instability,CIN)等表观遗传改变在肠息肉到癌变的过程、肿瘤转移过程和化疗药物抵抗中起到了重要作用。其中，中国结直肠癌患者的KRAS基因突变概率高达30％～50％，大量临床研究表明此类患者未能从抗EGFR靶向治疗中获益，从而导致我国结直肠癌肿瘤相关性死亡率呈现快速的上升趋势。
因此，研发针对结直肠癌患者个体表观遗传改变的治疗方案和药物，由传统无差别杀伤细胞的化疗药物向多靶点靶向药物治疗的过度，是肿瘤精准治疗的关键。
目前针对存在KRAS突变型CRC的治疗策略有抑制KRAS激活或抑制KRAS下游促增殖信号通路MEK/ERK的活化，但均以二期临床试验失败而告终。前者因为法尼基化转移酶抑制剂不能完全抑制KRAS活化，后者可能与反馈激活PI3K/AKT信号通路有关。
二甲双胍是目前治疗2型糖尿病的一线药物，能够有效的降低和维持患者的血糖水平和胰岛素水平，改善胰岛素抵抗。近年来有较多的回顾性研究显示二甲双胍对结直肠癌有一定的预防和治疗作用，其机制主要包括对肿瘤细胞的直接作用：抑制MEK-ERK、PI3K-AKT以及mTOR信号通路激活的作用；以及对肿瘤细胞的间接作用：如降低和维持血糖、胰岛素水平，抑制炎症反应，提高CD8+ T细胞的比例，从而改善肿瘤的细胞免疫功能等。但同时也有部分研究报道二甲双胍不能改善结直肠癌患者的总体生存时间和无进展生存时间。
上述研究提示二甲双胍对结直肠癌的治疗效果可能存在型别和个体差异，目前尚未明确二甲双胍治疗直肠癌的有效治疗型别和个体，也并未并阐明其机制。
发明内容
本发明的目的是为了克服现有技术的不足，提供二甲双胍在治疗KRAS突变型结直肠癌中的应用。
本发明的第一个目的是提供一种确定结直肠癌治疗方案的标志物。
本发明的第二个目的是提供KRAS基因和/或蛋白作为确定结直肠癌治疗方案的标志物的应用。
本发明的第三个目的是提供KRAS基因突变的检测试剂在制备确定结直肠癌治疗方案的试剂盒中的应用。
本发明的第四个目的是提供KRAS蛋白的检测试剂在制备确定结直肠癌治疗方案的试剂盒中的应用。
本发明的第五个目的是提供一种确定结直肠癌治疗方案的试剂盒。
本发明的第六个目的是提供二甲双胍在治疗结直肠癌或制备治疗结直肠癌药物中的应用。
本发明的第七个目的是提供MEK信号通路和AKT信号通路的组合作为结直肠癌治疗靶点的应用。
本发明的第八个目的是提供MEK信号通路的抑制剂和AKT信号通路的抑制剂联合使用治疗KRAS突变型结直肠癌。
本发明的第九个目的是提供一种治疗KRAS突变型结直肠癌的药物组合物。
为了实现上述目的，本发明是通过以下技术方案予以实现的：
1.采用分层Cox比例风险模型进行了回顾性研究，首次明确KRAS突变型结直肠癌患者使用二甲双胍获益更显著。为临床上选择性使用二甲双胍治疗结直肠癌提供循证医学证据。
2.在细胞实验上构建了KRAS(G13D)点突变模型和KRAS敲低模型，从正反向验证KRAS突变型结直肠癌细胞对二甲双胍的抗肿瘤作用敏感。阐明二甲双胍同时抑制ERK/cyclin D1/RB和AKT/mTOR/4E-BP1两条通路抑制KRAS突变型结直肠癌细胞增殖。为临床上联合使用MEK和AKT抑制剂增强治疗 KRAS突变型结直肠癌细胞的效果提供了佐证，同时也提供了二甲双胍作为可选药物的实验依据。
3.首次明确二甲双胍排出通道MATE1表达下调，是KRAS突变型结直肠癌细胞对二甲双胍敏感的关键机制。阐明突变型KRAS蛋白，通过上调甲基转移酶DNMT1和下调去甲基化酶TET1/2，促进MATE1启动子CpG岛甲基化，使MATE1转录水平降低。
4.临床标本和细胞实验证据提示，临床上可通过检测KRAS基因型，选择性使用二甲双胍治疗结直肠癌。
因此本发明要求保护以下内容：
一种确定结直肠癌治疗方案的标志物，所述标志物为KRAS基因和/或蛋白，KRAS突变型结直肠癌选用二甲双胍治疗结直肠癌。
KRAS基因和/或蛋白作为确定结直肠癌治疗方案的标志物的应用。
具体的，KRAS突变型结直肠癌选用二甲双胍治疗结直肠癌，这里所述KRAS突变型结直肠为KRAS基因突变后持续激活高表达
KRAS基因突变的检测试剂在制备确定结直肠癌治疗方案的试剂盒中的应用，也属于本发明的保护范围。
KRAS蛋白的检测试剂在制备确定结直肠癌治疗方案的试剂盒中的应用，也属于本发明的保护范围。
一种确定结直肠癌治疗方案的试剂盒，所述试剂盒包括KRAS突变型结直肠癌检测试剂。
二甲双胍在治疗结直肠癌或制备治疗结直肠癌药物中的应用，所述结直肠癌为KRAS突变型结直肠癌。
以上所述KRAS突变型结直肠癌并不限制KRAS突变的类型，目前实验数据支持codon12.13常见的突变都可以。
由于MEK和AKT是KRAS下游重要的调控细胞增殖的信号通路，单用MEK抑制剂或AKT抑制剂临床II期实验失败，二甲双胍同时抑制MEK和AKT信号通路，因此可有效抑制KRAS突变型结直肠癌细胞增殖。
因此本发明进一步要求保护以下内容：
MEK信号通路，和AKT信号通路的组合作为结直肠癌治疗靶点的应用；
MEK信号通路的抑制剂和AKT信号通路的抑制剂联合使用在制备治疗 KRAS突变型结直肠癌；
一种治疗KRAS突变型结直肠癌的药物组合物，其特征在于，包括MEK信号通路的抑制剂，以及AKT信号通路的抑制剂。
优选地，以上所述治疗结直肠癌为促进结直肠癌细胞G1停滞期、抑制肠癌细胞增殖、抑制肿瘤增大、抑制肿瘤增重、延长患者总生存时间和/或延长化疗的无进展时间。
更优选地，所述延长化疗的无进展时间为延长一线化疗的无进展时间。
以上所述抑制剂为任意能够降低相应蛋白、基因或信号通路的物质。
与现有技术相比，本发明具有如下有益效果：
本发明首次明确KRAS突变型结直肠癌患者使用二甲双胍获益更显著，进一步明确二甲双胍排出通道MATE1表达下调，是KRAS突变型结直肠癌细胞对二甲双胍敏感的关键机制，通过使MATE1转录水平降低增加肿瘤细胞内二甲双胍的浓度，从而增强二甲双胍抑制肿瘤细胞增殖的作用。本发明对于结直肠癌的治疗提供了一个新的思路和方法，意义深远，值得大力推广。
附图说明
图1为临床患者纳入分组示意图。
图2为使用二甲双胍改善合并糖尿病的结直肠癌患者总体生存时间。
图3为二甲双胍提高KRAS突变型结直肠癌患者的总体生存时间和一线化疗期间的无进展生存时间。
图4为SW48KRAS(G13D)细胞株细胞内基因修饰模式图。
图5为KRAS exon2 GGC>GAC突变型为阳性克隆序列比对图。
图6为二甲双胍抑制KRAS突变型结直肠癌细胞生长。
图7为二甲双胍抑制KRAS突变型PDX肿瘤生长。
图8为二甲双胍促进KRAS突变型结直肠癌细胞G1期停滞抑制肿瘤细胞的增殖。
图9为二甲双胍促进KRAS突变型结直肠癌细胞G1期停滞抑制肿瘤细胞增殖的机制。
图10为KRAS突变结肠癌细胞对二甲双胍敏感性高于KRAS野生型结肠癌细胞。
图11为二甲双胍浓度在KRAS突变型结直肠癌细胞和PDX肿瘤组织内积聚。
图12为KRAS突变下调MATE1从而提高了二甲双胍在细胞内的浓度，增强二甲双胍抗结直肠癌细胞增殖的效果。
图13为KRAS突变调控MATE1甲基化进而下调MATE1表达。
图14为基因组DNA样本经过重亚硫酸盐修饰后对MATE1启动子CpG岛扩增序列及扩增引物。
图15为DNMT/TET在SW48KRAS(G13D)细胞和敲低KRAS的Lovo细胞中的表达
图16为KRAS突变通过调控DMNT1/TET下调MATE1表达。
具体实施方式
下面结合说明书附图和具体实施例对本发明做出进一步地详细阐述，所述实施例只用于解释本发明，并非用于限定本发明的范围。下述实施例中所使用的试验方法如无特殊说明，均为常规方法；所使用的材料、试剂等，如无特殊说明，为可从商业途径得到的试剂和材料。
一、实验材料
新鲜肿瘤组织
经中山大学肿瘤防治中心确诊并收治的结直肠癌患者，手术切除原位肿瘤后，取约5×5×5mm的癌组织，。病例的收集严格遵守中山大学肿瘤防治中心的操作流程，经医院批准并征得患者本人及其家属同意。
实验动物
4～6周龄雄性裸鼠(BALB/c nude mice)，体重14～18g，购自北京维通利华实验动物技术有限公司，生产许可证号：SCXK(京)2016-0011，饲养于中山大学(实验动物中心北校园)无特定病原体(Specific pathogen free，SPF)的环境中，实验单位使用许可证号：SYXK(粤)2017-0081。检疫合格后做实验。在饲养动物过程中，保证实验动物五项基本福利，实验过程遵循Replacement，Reduction和Refinement的3R原则。
细胞株
SW48购自深圳华拓生物科技有限公司，CaCO2、HCT-116和LoVo由中山大学附属第六医院胃肠病研究所惠赠，SW480和SW620细胞为实验室保存细胞株。这6株结直肠癌细胞株的STR鉴定均由广州赛库生物科技有限公司完成，100％与ATCC提供的信息匹配，无其他细胞污染和STR改变。上述细胞的KRAS 基因型查询自ATCC官网，并通过PCR测序验证。
二、实验方法
1、拍片与肿瘤病理分级和增殖细胞定量
1)使用全自动数字玻片扫描系统(Axio Scan Z1)对全组织样本进行拍摄(10×和20×)；
2)导出图像后由病理科医生对肿瘤进行病理分级，分为高分化、中分化、低分化和未分化；
3)Ki67染色的肿瘤组织图像使用ImageJ的IHC Profiler插件对Ki67(+)的细胞核进行定量分析，记为增殖期细胞；
2、细胞活性检测(CCK-8法)
将细胞以5000个细胞/孔(48孔板，每孔体积200μl)的浓度种板。按实验需求用不同方式处理细胞，一定时间后加入10μl的CCK8溶液。培养箱中孵育1h，吸取200μl上清至96孔酶标板中，酶标仪测定OD 450。
实施例1二甲双胍对转移性结直肠癌患者预后的影响
一、合并2型糖尿病的转移性结直肠癌患者分组及组间临床特征
1、实验样本
从中山大学肿瘤防治中心2004～2016年收治的4751名转移性结直肠癌(metastatic colorectal cancer,mCRC)患者中，纳入在确诊前即患有2型糖尿病(type 2 diabetes,T2DM)的患者282名，分为服用二甲双胍组(metformin use，n＝109)、胰岛素或胰岛素促泌剂(insulin or insulin-releasing，n＝141)服用其他降糖药组(other anti-diabetic drugs use，n＝22)以及未治疗组(without anti-diabetic treatments,n＝32)。患者纳入分组见图1。
2、实验方法
收集患者的一般临床特征，如性别、年龄、体重指数(body mass index,BMI)；以及已有研究报道影响mCRC预后的临床特征(如肿瘤原发部位、病理分级、转移部位、一线化疗方案以及KRAS基因型)用以排除混杂因素，进而明确服用二甲双胍对合并2型糖尿病的结直肠癌患者的总体生存时间(overall survival,OS)和一线化疗的无进展生存时间(progression free survival,PFS)的影响。患者基本临床特征信息见表1。
表1合并2型糖尿病的结直肠癌患者基本临床信息：
Figure PCTCN2020099974-appb-000001
统计个体临床特征在二甲双胍组和非二甲双胍组中的分布是否存在差异。连续变量(年龄、BMI)采用单因素方法分析，分类变量(性别、肿瘤原发部位、病理分级、KRAS基因型、转移部位、一线化疗方案，以及年龄分组、BMI分组)采用卡方检验。
3、实验结果
对该180例患者进行统计分析显示，性别、年龄、BMI、肿瘤原发部位、病理分级、转移部位、KRAS基因型在二甲双胍组和非二甲双胍组组之间无统计学 差异(P＞0.05)，见表2。
表2 Kras基因型明确的180例合并2型糖尿病的转移性结直肠癌患者临床特征在二甲双胍组和其他降糖药物组中的分布情况
Figure PCTCN2020099974-appb-000002
二、二甲双胍提高合并2型糖尿病的转移性结直肠癌患者的总体生存时间和一线化疗的无进展生存时间
使用Kaplan-Meier生存曲线分析，明确与服用其他降糖药物组比较，二甲双胍是否改善该中心合并2型糖尿病的转移性结直肠癌患者的预后。
1、实验方法
使用GraphPad Prism 7作Kaplan-Meier生存曲线，并进行Log-rank(Mantel-Cox)统计分析；另外使用校正的风险比(stratified HR)统计不同降糖药与未降糖治疗组相比，对OS和PFS的影响。
2、实验结果
如-图2所示，与未合并糖尿病组相比，合并糖尿病未治疗组中位生存时间减少11.2个月(P＝0.007)，二甲双胍组的中位生存时间延长11.3个月(P＝0.022)，其他降糖药无明显改善。表明在中山大学肿瘤防治中心2004～2016年确诊的282例合并2型糖尿病的转移性结直肠癌患者中，二甲双胍可以显著延长总体生存时间。另外使用校正的风险比统计显示(见表3)，降糖治疗改善预后(HR＝0.547，95％CI：0.327-0.913)；单纯使用或联用二甲双胍均能提高mCRC患者的预后，而其他降糖药的作用无统计学差异，因此说明除了降低血糖外，二甲双胍对mCRC预后的改善作用还可能与其他因素有关。
表3不同降糖药与未降糖治疗组相比对OS和PFS的影响。
Figure PCTCN2020099974-appb-000003
三、二甲双胍对患者死亡或肿瘤进展的效应符合等比例风险假设
1、实验方法
进行等比例风险假设(proportional hazard assumption)，即假设使用二甲双胍对患者死亡或者肿瘤进展的效应不能随时间的变化而改变，应该是一个固定的值。我们使用Kolmogorov-Smirnov test和Cramer von Mises test进行假设检验。
2、实验结果
表4的结果表明二甲双胍对患者死亡或者肿瘤进展的效应符合等比例风险假设(P＞0.05)。
表4二甲双胍对患者死亡或肿瘤进展的效应的等比例风险假设
Figure PCTCN2020099974-appb-000004
四、KRAS突变型转移性结直肠癌患者使用二甲双胍获益更显著
采用分层Cox比例风险模型对上述收集的临床特征进行分层，探索影响二甲双胍有效性的个体因素。
1、实验方法
经过等比例风险假设后，我们进一步假设性别、年龄、BMI、肿瘤原发部位、病理分级、转移部位以及KRAS基因型，可能作为混杂因素对二甲双胍的抗肿瘤作用造成影响。因此，我们对上述临床特征进行分层回归分析(hierarchical regression analysis)，从而确定与二甲双胍起交互作用的个体差异。
2、实验结果
如表5所示，将上述临床特征作为混杂因素纳入比例风险回归模型的方程后，我们可以发现二甲双胍与其他降糖药物的使用相比，降低死亡的风险比(hazard ratio，HR)为0.746，95％可信区间(confidence interval，CI)为0.496-1.121，区间跨越1，没有统计学差异(P＞0.05)；降低一线化疗期间肿瘤进展的HR为0.737，95％CI为0.501-1.086，也没有统计学差异(P＞0.05)，说明使用二甲双胍与其他降糖药物相比，是否能使转移性结直肠癌患者获益存在个体差异。
进一步对上述临床特征进行分层回归分析，结果显示，对于KRAS突变型的转移性结直肠癌患者，使用二甲双胍比其他降糖药物更能降低死亡的风险(HR＝0.272，95％CI为0.120-0.617)，同时也能降低一线化疗期间肿瘤进展的风险(HR＝0.405，95％CI为0.212-0.774)。此外，使用基于R语言的EmpowerStats软件进行交互作用检验，我们发现KRAS突变能显著增强二甲双胍降低死亡风险 的作用(P interaction<0.001)以及降低一线化疗期间肿瘤进展风险的作用(P interaction ＝0.02)。此外，采用Kaplan-Meier生存曲线分别对KRAS野生型和KRAS突变型的mCRC患者进行分析，二甲双胍显著延长KRAS突变型的mCRC患者的总体生存时间(P<0.001)和一线化疗的无进展生存时间(P<0.01)(图3A-B)，而在KRAS野生型的mCRC患者中无效(图3C-D)。
表5使用二甲双胍与总体生存时间和无进展生存时间的比例风险回归模型及其与各临床特征交互作用的分析：
Figure PCTCN2020099974-appb-000005
Figure PCTCN2020099974-appb-000006
五、二甲双胍抑制KRAS突变型结直肠癌细胞增殖
从结直肠癌组织切片的免疫组化和细胞学实验上验证该临床特征对二甲双胍抗肿瘤治疗的作用。
1、实验方法
(1)为了进一步明确二甲双胍对KRAS突变型结直肠癌细胞的作用，收集了28例确诊为转移性结直肠癌并行肿瘤切除术前已服用二甲双胍患者的病理切片(包括原发灶和转移灶)，提取石蜡组织的DNA进行KRAS基因型鉴定。
通过苏木素-伊红染色(hematoxylin-eosin stain,H&E)判断肿瘤细胞位置(核深染、异质，腺上皮结构破坏)，并用Ki67染色标记增殖期的细胞。
①苏木素-伊红染色
1)将固定好的肿瘤组织，制作成石蜡切片；
2)用二甲苯使切片脱蜡；
3)切片放入甲醇中固定2min；
4)苏木素染色3min；
5)分色液：70％酒精+冰醋酸10ml(数分钟，至颜色适当即可)
6)流水返蓝5min；
7)伊红染色1min；
8)75％，80％，90％，95％无水乙醇各30sec；
9)100％酒精2-3缸，每缸30sec；
10)100％二甲苯3缸，第一缸10min，第二、三缸各5分钟。
11)中性树胶封片。
②Ki67染色
1)烤片：65℃，2h；
2)二甲苯脱蜡：30min×3次，室温；
3)无水乙醇：10min×1次，室温；
4)水化：100％，95％，90％，80％，70％乙醇各5min；
5)dH 2O洗：5min×1次；
6)PBST洗：5min×3次；
7)抗原修复：10mmol/L柠檬酸盐缓冲液(PH 6.0)1L置高压锅煮沸后，将切片架置入其中全部浸没，盖紧锅盖，等出气后控制时间2min，将缓冲液自然冷却至室温后取出切片；
8)dH 2O洗：2min×2次；
9)PBST洗：5min×3次；
10)阻断：使用免疫组化笔画圈，加3％H2O2，室温30min；
11)PBST洗：5min×3次；
12)封闭：山羊血清，室温1h，然后PBST洗5min×3次；
13)一抗孵育：抗体稀释液1:400稀释Ki67，覆盖组织，切片放于湿盒内防止干片，4℃过夜；
14)PBST洗：5min×3次；
15)二抗：免疫组化鼠兔通用型检测试剂盒A液，室温孵育30min；
16)PBST洗：5min×3次；
17)DAB显色：免疫组化鼠兔通用型检测试剂盒B液以1:50比例稀释C液，自来水中止反应后继续水洗30min；
18)苏木素复染：1min，盐酸乙醇(1:1000)分化10s，去除非特异着色；自来水中止反应后继续水洗20min；
19)脱水：70％、80％、90％、95％、100％乙醇各5min；
20)二甲苯脱水透明：5min×2次，中性树胶封片。
(2)在体外使用梯度浓度的二甲双胍处理KRAS野生型结直肠癌细胞系SW48和CaCO2，以及KRAS G13D突变型结直肠癌细胞系HCT-116和LoVo，和KRAS G12V突变型结直肠癌细胞系SW480和SW620。另外，使用梯度浓度的二甲双胍处理转染KRAS G12V，KRAS G13D和KRAS G12D质粒的KRAS野生型结直肠癌细胞系SW48。
(3)在体外使用梯度浓度的二甲双胍处理CRISPR/Cas9系统构建的KRAS(G13D)突变型SW48细胞株，正向验证二甲双胍抑制KRAS突变结直肠癌细胞活性。
①KRAS G13D点突变CRISPR/Cas9质粒构建
1)sgRNA(sequence of guide RNA)设计：Zhang Lab,MIT提供的网站http://crispr.mit.edu上对人12号染色体上KRAS基因的2号外显子起始密码ATG 前250bp的碱基进行设计。取评分>85分的脱靶效率较低的sgRNA，使用CRISPR RGEN tool Cas-OFFinder网站http://www.rgenome.net/cas-offinder/，设置错配碱基数≤2，不使用出现错配的sgRNA，避免出现非特异性的切割。我们选取的sgRNA：5’-GCATTTTTCTTAAGCGTCGA-3’。
2)gRNA合成与重组载体连接：
A.化学方法合成gRNA oligo(PAGE纯化)，下划线为BbsⅠ酶切连接位点，反向序列的3’-端不是胞嘧啶需要加一个胞嘧啶碱基，如下：
Target sequence(PAM):GCATTTTTCTTAAGCGTCGA(TGG)；
Forward: CACCGCATTTTTCTTAAGCGTCGA；
Reverse: AAACTCGACGCTTAAGAAAAATG C；
B.每OD的oligo使用ddH 2O(10μL/1nmol的比例)溶解，终浓度为100μM。按以下反应将oligo合成双链核苷酸：
Figure PCTCN2020099974-appb-000007
C.酶切：使用Bbs Ⅰ限制性内切酶，酶切pSpCas9(BB)-2A-puro(PX459)V2.0质粒，进行琼脂糖电泳和胶回收纯化。酶切条件如下：
Figure PCTCN2020099974-appb-000008
D.连接：使用T4连接酶连接PX459和gRNA，然后琼脂糖电泳鉴定和胶回收纯化，记作PX459/hKRAS gRNA。连接条件如下：
Figure PCTCN2020099974-appb-000009
Figure PCTCN2020099974-appb-000010
3)KRAS G13D点突变供体质粒的构建：
A.基因调取：首先使用基因组DNA提取试剂盒提取LoVo的基因组DNA，然后通过高保真PCR的方法扩增出包含sgRNA和exon2在内的约3000bp的DNA序列，跑电泳鉴定并胶回收纯化。PCR反应体系和条件如下：
Figure PCTCN2020099974-appb-000011
B.构建T载体：上一步纯化的DNA，进行加A反应后，连接到pGM-T载体上，转入感受态菌进行扩增，挑单克隆进行测序验证。验证序列正确，记为pGM-T/KRAS-homology。加A和连接反应条件如下：
Figure PCTCN2020099974-appb-000012
C.对sgRNA的PAM进行TGG>TGA点突变：使用高保真PCR的方法扩增sgRNA PAM TGG前的序列，引物为hKRAS-Left arm-F和hKRAS-TGG mut-Left arm-R，上游引入了15bp与pGM-T 3’断端同源的序列，下游引物5’端引入了点突变序列CCA>TCA；另外一个DNA片段的引物为hKRAS-TGG mut-Right arm-F和hKRAS-Right arm-R，上游引物5’端引入与前面一个DNA片 段3’端15bp的同源序列(包括ACC>ACT)，3’端引入了与pGM-T 5’断端同源的序列。PrimeSTAR Max Premix的PCR反应体系同上，反应条件将延伸时间改为15s。2个PCR产物经胶回收纯化后，与pGM-T载体使用无缝克隆进行连接，然后进行胶回收纯化、转化、扩增和挑单克隆测序。无缝克隆反应体系及反应条件如下：
Figure PCTCN2020099974-appb-000013
D.将用于同源重组的序列连接到供体载体pDONR 221上：通过引物设计，分别在5’端和3’端引入attB1，通过BP反应将片段连接到pDONR 221上，形成Entry Clone，记为pDONR 221/hKRAS-homology质粒。引物设计见表6。
表6所用引物自命名及相应序列：
Figure PCTCN2020099974-appb-000014
Figure PCTCN2020099974-appb-000015
pDONR 221连接反应体系及条件如下：
Figure PCTCN2020099974-appb-000016
②构建SW48KRAS(G13D)细胞株
1)将SW48传代至3cm 3皿中，待细胞融合度达到80％-90％的时候，提前2h更换新鲜培养液。分别取1.5μg PX459/hKRAS gRNA和1.5μg pDONR 221/hKRAS-homology质粒，加入150μL opti-MEM中，并加入9μL P3000；另外取3.5μL Lipo3000加入150μL opti-MEM中，室温孵育5min。然后将质粒预混液加入Lipo3000预混液中，室温孵育15min后，逐滴加入SW48细胞培养液中。
2)转染12h后，更换新鲜培养液，并加入1μM的Scr7抑制NHEJ反应，提高同源重组的效率(文献报道称可提高4-5倍)。转染24h后，加入1μM嘌呤霉素，筛选转染阳性的细胞。
3)转染72h后，更换新鲜培养液，彻底去除嘌呤霉素。如果细胞融合度为70％-90％，活力较好，则进行传代。
4)传代后取1×10 6个细胞提取基因组DNA，使用hKRAS-Homology-F和hKRAS-Homology-R进行PCR扩增，步骤及反应条件同上。连接仅pGM-T并转化入DH5α进行扩增后，涂板挑取约30个克隆进行测序。如果测序结果存在sgRNA PAM TGG>TGA以及KRAS exon2 GGC>GAC突变，则表示同源重组成功。细胞内基因修饰模式图见图4。
5)确保SW48混合细胞株细胞活力较好的情况下，消化和重悬细胞，计数200个细胞将其浓度调整为1个/200μL，种到96孔板中，每孔200μl培养液约1个细胞。
6)隔日观察，注意培养箱湿度，防止96孔板内培养液蒸发。待单个细胞长成克隆后，挑20-50个克隆进行传代。
7)单克隆细胞株经过扩增后，操作同上，每个细胞克隆取一个细菌克隆进行测序。存在KRAS exon2 GGC>GAC突变型为阳性克隆。保种，记为SW48KRAS(G13D)细胞株。阳性序列比对如图5。
(4)在体外使用梯度浓度的二甲双胍处理慢病毒shRNA构建KRAS敲低的LoVo细胞株，反向验证二甲双胍抑制KRAS突变结直肠癌细胞活性。
①构建KRAS稳定敲低的LoVo细胞株
1)KRAS干扰慢病毒(sh-KRAS)由上海吉凯基因公司构建和包装，共设计2个靶点，详见表7。
表7 sh-KRAS靶点序列信息：
Figure PCTCN2020099974-appb-000017
2)将LoVo传代至6孔板中(约1×10 5/孔)，当贴壁后，细胞融合度不超过50％时，提前2h更换新鲜培养液，并加入慢病毒感染增强剂10μg/mL polybrene。LoVo的病毒感染复数(Multiplicity of infection,MOI)约为20，根据公式：每孔加病毒量(μl)＝MOI×细胞数/滴度(TU/ml)×1000加入慢病毒。16～18h换液，常规加入1μM嘌呤霉素进行KRAS干扰RNA稳定表达的细胞株的筛选。
2、实验结果
免疫组化结果显示，共有KRAS野生型mCRC 18例，KRAS突变型mCRC 10例。如图3C所示，KRAS突变型结直肠癌组织的Ki67(+)细胞的比例明显低于KRAS野生型，具有显著的统计学差异(P<0.01)。
细胞实验结果表明，二甲双胍对KRAS G13D，G12D和G12V突变型结直肠癌细胞有抑制细胞活性的作用，而对KRAS野生型结肠癌细胞没有作用(图6)。
综上所述，结果显示相较于KRAS野生型，使用二甲双胍的KRAS突变型结直肠癌患者总体生存时间和无进展生存时间更长，并且同样的结果在肿瘤组织切片和细胞活性实验上得到验证。
实施例2在肿瘤动物模型上验证二甲双胍对KRAS突变型肿瘤的治疗作用
一、PDX模型的构建
使用PDX模型：取临床KRAS野生型和突变结直肠癌病人肿瘤组织，消化法培养肿瘤细胞，鉴定KRAS突变之后将肿瘤细胞种植在裸鼠腋窝区。
1、实验方法
1)病人肿瘤标本的收集：手术切除后的标本，取边缘未坏死的癌组织，浸泡于5％胎牛血清、含1×青霉素及链霉素双抗的PRMI1640培养液中，4℃运输；
2)于超净工作台内将肿瘤组织切成约2×2×2mm大小的小块，用上述培养液清洗3遍，将淤血清除；
3)使用4.8％水合氯醛对4-6周龄的BALB/C裸鼠进行麻醉，麻醉后于腋下皮肤切开一个约3mm小口，使用镊子进行钝性分离，将修剪后的肿瘤组织埋皮下，6-0缝线缝合切口，使用双抗防止伤口感染；
4)取50mg肿瘤组织提基因组DNA进行KRAS基因型鉴定，剩余组织-80℃保存备用；
5)约4～6周后移植肿瘤部位隆起小结约1cm 3大小，则将肿瘤组织取出，修剪成2×2×2mm大小的组织块进行传代。传代操作与上面相同；
6)约2～3代后，将裸鼠分为4组：KRAS野生型肿瘤对照组、KRAS野生型肿瘤二甲双胍组、KRAS突变型肿瘤对照组、KRAS突变型肿瘤二甲双胍组，各10只；
7)当移植肿瘤长至100-200mm 3后，连续28天每日早上9点进行灌胃给药。对照组给予生理盐水灌胃，二甲双胍组给予二甲双胍溶液灌胃(每日100mg/kg裸鼠体重，生理盐水溶解)。期间每日测量移植肿瘤的大小，volume(mm 3)＝[length×width 2]/2；
8)30日后处死裸鼠，取肿瘤组织拍照、测量、包埋和切片。
二、二甲双胍对KRAS突变型肿瘤的治疗作用
1、实验方法
将二甲双胍200mg/kg(相当于人1000mg)溶于水，分别给KRAS野生型和突变肿瘤动物二甲双胍饮用，测量肿瘤大小，30天后处死取肿瘤组织，称重。
2、实验结果
相对于KRAS野生型肿瘤模型，二甲双胍能够明显抑制KRAS突变型肿瘤的大小和重量。二甲双胍对于KRAS突变型结直肠癌具有较好的治疗效果，提示临床KRAS突变患者可选择使用二甲双胍，为KRAS突变型患者药物开发提供依据(图7)。
实施例3二甲双胍抑制KRAS突变结直肠癌细胞增殖的作用
一、二甲双胍对结直肠癌细胞凋亡的作用
在KRAS野生型细胞SW48和KRAS(G13D)突变型细胞LoVo上，使用Annexin V/PI双染检测二甲双胍对结直肠癌细胞凋亡的作用。
1、实验方法
1)将细胞种在6孔板中，贴壁12h后，饥饿过夜，加药处理24h，使用不含EDTA的胰蛋白酶消化细胞，PBS洗涤一次，不需要固定。
2)加入300μl Binding Buffer重悬细胞，加入3μl Annexin V-FITC和3μl Propidium Iodide(PI)，混匀，避光室温孵育30min，上机进行流式细胞仪测定。
3)流式检测抗体：凋亡试剂盒(A211-02)购自凯基公司；细胞周期PI单染检测试剂盒(558662)购自BD公司。
2、实验结果
使用Annexin V/PI双染检测细胞凋亡，结果显示2.5mM，5mM，10mM的二甲双胍均不促进KRAS野生型结直肠癌细胞SW48以及KRAS突变型结直肠癌细胞LoVo的细胞凋亡。
二、二甲双胍对结直肠癌细胞增殖的作用
使用Edu检测增殖细胞比例、平板克隆形成实验以及PI/Rnase单染检测细胞周期分布情况，明确二甲双胍对结直肠癌细胞增殖的作用。
1、实验方法
(1)增殖检测(Edu法)
1)EdU处理细胞：将细胞种在培养皿中(已放置灭菌盖玻片)，贴壁12h后，饥饿过夜，加药处理相应时间，并在最后2-6h加入EdU(终浓度10μM)(EdU处理时间依据细胞生长速率而定)。
2)固定：加入4％多聚甲醛1mL/孔，室温固定15分钟。
3)洗片：用3％BSA(用PBS溶解)1mL/孔，洗2次。
4)破膜：0.5％TritonX-100，1mL/孔，室温20分钟。
5)洗片：同步骤3。
6)加ClickiT反应混合物：50μL/张，避光，室温30分钟。
7)洗片：用3％BSA(用PBS溶解)1mL/孔，洗2次，再用PBS洗一次。
8)染核：50μL DAPI(1：3000，PBS配置)避光染色10分钟。
9)洗片：PBS洗2次。
10)避光晾干，加抗淬灭剂，用指甲油封片。
11)全自动正置荧光显微镜镜下观察分析结果，并拍照记录(100×，200×，400×，保存原图)。
(2)平板克隆形成
1)细胞接种：吸弃培养液，收集细胞，将细胞悬浮于处理条件培养液中，计数3次取均值，调整细胞悬液浓度为1×10 3/ml。6孔培养板每孔分别加入2.5ml处理条件培养液，每孔加入0.5ml细胞悬液(即每孔500个细胞)，终体积3ml。接种时注意多次十字方向摇晃培养板，使细胞尽量均匀分布。
2)细胞培养：细胞在标准条件下培养2～3周，观察克隆形成情况，约每3天更换条件培养基。
3)克隆染色：当细胞形成肉眼可见的克隆(每个孔克隆细胞数在50～150个左右)时终止培养。弃培养基，PBS小心浸洗2次，每孔分别加4％多聚甲醛1.5ml室温固定15min；弃固定液，流水缓慢冲洗干净，每孔加入1.5ml的结晶紫使用液，室温静置染色30min，以流水缓慢洗净染色液，通风橱中干燥。
4)克隆计数：将培养板置于凝胶成像系统，在可见光条件下，以随机所带软件计算克隆数目，扫描并保存图像。克隆形成率(％)＝克隆数目/接种细胞数×100％。每组细胞样本接种3个复孔，独立重复3次实验。
(3)细胞周期检测
1)将细胞种在6孔板中，贴壁12h后，饥饿过夜，加药处理24h，消化细胞并用PBS洗3遍，离心弃上清，逐滴加入-20℃预冷的70％乙醇，涡旋混匀细胞，-20℃固定过夜。
2)2000rpm水平离心10min，弃上清，并用PBS洗2-3遍去除残余乙醇，每遍均离心5min。
3)细胞用500μL PI/RNase染液重悬，避光孵育15min，2h内上流式细胞仪检测，激发波长488nm。
4)使用FlowJo 7.6软件进行作图分析。
2、实验结果
二甲双胍减少KRAS突变型结直肠癌细胞LoVo中EdU阳性的增殖期细胞的比例，抑制LoVo克隆形成能力，而对KRAS野生型结直肠癌细胞SW48无明显作用(图8A-B)。
细胞周期的流式结果显示，二甲双胍浓度依赖性地增加KRAS突变型结直肠癌细胞LoVo G1期细胞的比例，降低S期细胞的比例，但是对KRAS野生型结直肠癌细胞SW48无明显作用(图8C)。
使用慢病毒shRNA构建KRAS蛋白敲低的LoVo细胞株，结果显示干扰KRAS(G13D)的表达能够下调二甲双胍对LoVo细胞活力的抑制作用、下调二甲双胍对LoVo细胞G1期向S期转化的抑制作用(图8D)，使用CRISPR/Cas9系统构建的KRAS(G13D)突变增强二甲双胍抗肿瘤细胞增殖的作用，细胞周期(图8E)。
三、二甲双胍抑制KRAS突变结直肠癌细胞增殖的机制
使用Western blot检测二甲双胍对增殖信号通路MEK/ERK/cyclin D1/RB和PI3K/AKT/mTOR/4E-BP1相关分子的改变，阐明二甲双胍抑制KRAS突变影响结直肠癌细胞增殖的机制。
1、实验方法
①体外细胞实验
1)将细胞接种在培养皿中，培养至60％融合度，加药处理24h。
2)收蛋白，PBS清洗3次后加入100μl 1×SDS缓冲液(100mmol/l Tris-Cl pH6.8，2％SDS，10％甘油)，此时细胞裂解液粘稠。
3)用细胞刮刀将细胞裂解液收集于0.5ml离心管中，100℃煮30min。
4)BCA试剂盒法(BIO-RAD公司)对细胞总蛋白提取物进行蛋白定量。
5)按9μl变性蛋白质样品：1μl变性缓冲液(β-巯基乙醇，0.4％溴酚兰)的比例处理样品，100℃煮沸30min，备用。
6)取已灌制好的SDS-PAGE凝胶(10％分离胶和5％浓缩胶)，加入样品40ug/孔，80V电泳30min，120V电泳90min。
7)以300mA恒流转膜180min，将凝胶上的蛋白转移至PVDF膜。
8)将膜用7％脱脂牛奶封闭90min，加入相应一抗4℃振摇过夜。
9)次日用TBST洗膜3次，每次10min，后加入相应二抗4℃孵育4h。
10)TBST洗膜3次后，加入ECL曝光。
②取PDX动物肿瘤组织，匀浆提取蛋白，进行WB。
2、实验结果
KRAS突变型结直肠癌细胞LoVo经过二甲双胍处理24h后，ERK、RB、AMPK、AKT、mTOR和4E-BP1的磷酸化水平受到抑制，在KRAS野生型结直肠癌细胞SW48则无明显改变(图9A-C)。说明二甲双胍可以通过同时抑制ERK和AKT信号通路从而抑制KRAS突变型结直肠癌细胞增殖。
使用CRISPR/Cas9系统构建的KRAS(G13D)突变型SW48细胞株，cyclinD1/RB和AKT/mTOR/4E-BP1信号通路的改变(图9D)与KRAS突变结直肠癌细胞LoVo一致。使用慢病毒shRNA构建KRAS蛋白敲低的LoVo细胞株，结果显示干扰KRAS(G13D)的表达能够下调二甲双胍抑制LoVo RB蛋白和4E-BP1磷酸化的作用(图9E)。
PDX肿瘤组织中，二甲双胍抑制KRAS突变型结直肠癌组织中ERK和AKT信号通路(图9F)。
综上所述，二甲双胍促进KRAS突变型结直肠癌细胞G1期停滞，抑制肿瘤细胞的增殖，而非诱导细胞凋亡；二甲双胍可以同时抑制ERK和AKT，下调RB和4E-BP1的磷酸化抑制KRAS突变型结直肠癌细胞。
首次验证了二甲双胍对KRAS突变型结直肠癌细胞的作用及机制，同时也为联合抑制ERK/cyclin D1/RB和AKT/mTOR/4E-BP1两条通路增强治疗KRAS突变型结直肠癌细胞的效果提供了佐证。
实施例4 KRAS突变增强结直肠癌细胞对二甲双胍敏感性
一、二甲双胍抑制结直肠癌细胞增殖的作用与细胞内浓度相关
使用兰索拉唑抑制细胞对二甲双胍的吸收，通过CCK8检测细胞活性，明确二甲双胍抑制结直肠癌细胞增殖的作用与细胞内浓度相关。
使用CRISPR/Cas9系统构建的KRAS(G13D)突变细胞和KRAS稳定敲低的LoVo细胞株验证二甲双胍在KRAS突变型细胞中浓度增加。
1、实验方法
使用5μM和10μM的兰索拉唑(lansoprazole)同时处理HCT-116和LoVo，同时使用二甲双胍处理细胞48h，CCK8检测细胞活性。
使用二甲双胍处理CRISPR/Cas9系统构建的KRAS(G13D)突变细胞和KRAS稳定敲低的LoVo细胞株，收集不同时间点的细胞裂解液，质谱检测细胞内二甲双胍浓度。
PDX模型采用质谱检测KRAS野生型和突变型肿瘤组织中二甲双胍浓度。
2、实验结果
CCK8结果显示二甲双胍对KRAS野生型结直肠癌细胞SW48的IC50为90.83mM(95％可信区间为68.60-127.60)，CaCO2为88.12mM(95％可信区间为74.71-106.92)，而对于KRAS突变型结直肠癌细胞HCT-116的IC50为23.71mM (95％可信区间为17.22-33.54)，LoVo为8.18mM(95％可信区间为6.52-10.13)(图10)。使用兰索拉唑抑制细胞对二甲双胍的吸收后，HCT-116和LoVo的IC 50增加了1.2～2.5倍以上(图11A-B)。
质谱结果显示CRISPR/Cas9系统构建的KRAS(G13D)突变细胞相比较对照细胞，细胞内二甲双胍浓度明显增加(图11C)；而KRAS稳定敲低的LoVo细胞株细胞内二甲双胍浓度相对于LoVo明显下降(图11D)。
质谱结果显示二甲双胍治疗KRAS突变型肿瘤组织中二甲双胍浓度比KRAS野生型肿瘤组织中升高(图11F)。
二、细胞中二甲双胍通道蛋白的RNA水平
通过分析TCGA-COAD数据库，检测细胞中二甲双胍通道蛋白的RNA水平，筛选KRAS突变型结直肠癌细胞系HCT-116和LoVo，以及KRAS野生型结直肠癌细胞SW48和CaCO2之间的差异表达。
1、实验方法
(1)实时荧光定量PCR(real-time quantitative PCR,RT-qPCR)
1)RNA的提取
具体步骤参见康为世纪生物科技有限公司的RNA提取试剂盒(CW0581)说明书。
2)逆转录
用Nanodrop紫外分光光度计测量RNA浓度。将500ng NA逆转录为cDNA，如下：
Figure PCTCN2020099974-appb-000018
3)RT-qPCR
仪器为lightcycler荧光定量PCR仪或BIO-RAD的CFX 96荧光定量PCR仪。试剂为Takara公司
Figure PCTCN2020099974-appb-000019
Green I染料。引物利用PubMed的primer-blast程序设计合成，由Life technology公司合成，引物序列如下表：
Figure PCTCN2020099974-appb-000020
Figure PCTCN2020099974-appb-000021
荧光定量PCR反应程序如下：
1)三步法(效率高，但特异性较差)：
①预变性：95℃ 30s，1个循环；
②PCR反应：95℃变性5s，55℃退火30s，72℃延伸45s，35个循环；
③融解曲线分析：95℃ 0s，65℃ 15s，95℃ 0s，1个循环。
2)二步法(特异性高，但效率较低)：
①预变性：95℃ 30s，1个循环；
②PCR反应：95℃变性5s，60℃退火加延伸30s，40个循环；
③融解曲线分析：95℃ 0s，65℃ 20s，95℃ 0s，1个循环。
(引自
Figure PCTCN2020099974-appb-000022
Premix Ex TaqⅡ试剂说明书)。
PCR结束后，参考溶解曲线，判断引物特异性，并根据反应得到的Cp值，以不同基因的标准曲线和内参进行校正，并设对照组为1，分析并组图。
2、实验结果
TCGA-COAD数据库中分析和细胞系二甲双胍通道的RNA水平筛选发现，与KRAS野生型结直肠癌细胞相比，KRAS突变型结直肠癌细胞(HCT-116和LoVo)MATE1(SLC47A1)表达降低(图12A)。KRAS(G13D)突变型SW48细胞株MATE1(SLC47A1)表达降低(图12B)，sh-KRAS-LoVo MATE1(SLC47A1)表达增加(图12C)。
三、MATE1的表达水平
筛选出差异表达的MATE1，然后通过免疫组化在临床标本上进行验证，通过细胞内基因过表达或敲低实验进行验证。
1、实验方法
免疫组化(p-RB标记)同实施例1。
2、实验结果
临床标本免疫组化结果显示，与KRAS野生型结直肠癌相比，KRAS突变型结直肠癌MATE1蛋白水平降低；而且在服用二甲双胍的转移性结直肠癌患者的临床标本中，MATE1的表达与细胞增殖指标p-RB表达成正比(图12D)。
在LoVo上过表达MATE1进行正向实验或在SW48上干扰MATE1进行反向实验，证明MATE1表达下调，促进二甲双胍抑制结直肠癌细胞增殖的作用(图12E-F)。
通过Western blot检测MATE1与转录因子Sp1的表达，结果显示结直肠癌细胞MATE1的表达与Sp1的表达不相关。
四、体内验证低表达MATE1在二甲双胍抑制肿瘤效果中的作用
通过细胞源性异种移植模型(CDX)实验验证MATE1在二甲双胍抑制肿瘤效果中的作用。
1、实验方法
在细胞源性异种移植模型(CDX)实验中，我们首先利用CRISPR/Cas9建立了KRAS G13DSW48细胞株；在SW48中通过shRNA慢病毒转导敲除MATE1构建sh-MATE1-SW48细胞株；KRAS G13DSW48细胞中慢病毒感染过表达MATE1。1×10 6个细胞悬浮在基底膜基质(100μl高浓度基底膜基质和100μl PBS))皮下注入BALB/c裸小鼠。将CDX老鼠随机分为二甲双胍治疗组和对照组。将二甲双胍200mg/kg(相当于人1000mg)溶于水，分别给四组动物饮用，测量肿瘤大小，30天后处死取肿瘤组织，称重。
2、实验结果
结果表明，SW48异种移植模型中二甲双胍治疗与对照相比无明显抗肿瘤作用，但是敲低MATE1后，二甲双胍治疗显著抑制SW48+sh-MATE1肿瘤生长。相反，和KRAS G13DSW48肿瘤相比，过表达MATE1后，二甲双胍在KRAS G13DSW48异种移植体中无效(图12G-L)。
五、MATE1基因启动子CpG岛甲基化水平的差异
通过TCGA-COAD数据库中mRNA-seq和MethyArray 450K的数据进行分析和相关统计，提示在结直肠癌细胞中MATE1的表达与MATE1启动子甲基化呈负相关关系(图13A)。
使用重亚硫酸盐测序的方法，检测KRAS突变型结直肠癌细胞系HCT-116和LoVo，以及KRAS野生型结直肠癌细胞SW48和CaCO2之间，MATE1基因 启动子CpG岛甲基化水平的差异。
1、实验方法
(1)DNA重亚硫酸盐测序(bisulfite sequencing PCR,BSP)
1)DNA重亚硫酸氢钠修饰：
A.提取基因组DNA，然后按以下反应体系和反应条件进行5mC>U的重亚硫酸氢钠反应：
Figure PCTCN2020099974-appb-000023
B.重亚硫酸盐处理后的DNA纯化，见DNA重亚硫酸盐转化试剂盒(DP215)产品说明书。
2)PCR扩增和T载体连接：
A.基因组DNA样本经过重亚硫酸盐修饰后，对MATE1启动子CpG岛进行扩增，序列及扩增引物标记如下图14(带框为引物序列，下划线为CpG位点)。
PCR反应体系及条件如下：
Figure PCTCN2020099974-appb-000024
B.胶回收纯化，然后进行平末端PCR产物加A反应，连接到T载体pGM-T中，转化入感受态菌并进行扩增，挑取5个单克隆测序，具体步骤同实施例3。
3)分析甲基化位点及甲基化水平：测序结果直接使用网站http://quma.cdb.riken.jp/QUantification tool for Methylation Analysis(QUMA)进行分析，选择点图，每个圆点代表1个CpG位点，白色为非甲基化，黑色为甲基化，共34个CpG位点。
(2)羟甲基化DNA定量
1)羟甲基化DNA免疫共沉淀(Hydroxymethylated DNA immunoprecipitation,hMeDIP)：
A.提取基因组DNA，使用Sonics超声破碎仪25％的能量，按10s/pulse×4pulses，每次间隔40s的频率进行超声破碎，可将基因组DNA断裂为约400bp的片段。
B.使用Abcam公司的hMeDIP ChIP kit富集含有5hmC的DNA片段。具体步骤参考Abcam公司hMeDIP ChIP Kit(ab117134)说明书。
2)RT-qPCR进行半定量分析：RT-qPCR引物如下，RT-qPCR反应体系和条件同上所述。使用未富集的DNA作为校正。
Figure PCTCN2020099974-appb-000025
2、实验结果
通过重亚硫酸盐测序PCR，发现与KRAS野生型结直肠癌细胞SW48和CaCO2相比，KRAS突变型结直肠癌细胞HCT-116和LoVo中MATE1启动子的甲基化水平更高(图13B)。
六、KRAS突变通过调控MATE1启动子甲基化水平，从而调控MATE1表达
使用慢病毒shRNA构建KRAS敲低的LoVo细胞株，以及CRISPR/Cas9系统构建的KRAS(G13D)突变型SW48细胞株，正反向验证KRAS突变通过调控MATE1启动子甲基化水平，从而调控MATE1表达。检测PDX KRAS突变型和 野生型肿瘤组织中MATE1的甲基化水平。
1、实验方法
甲基化水平检测方法同上。
2、实验结果
使用CRISPR/Cas9系统构建的KRAS(G13D)突变型SW48细胞株，MATE1启动子的甲基化水平升高(图13C)；使用慢病毒shRNA构建KRAS敲低的LoVo细胞株，MATE1启动子的甲基化水平降低(图13D-E)；PDX KRAS突变型肿瘤组织中MATE1的甲基化水平较野生型明显升高(图13F)。
七、甲基转移酶和去甲基化酶的差异表达和验证
检测细胞系RNA水平，筛选差异表达的甲基转移酶和去甲基化酶，并在临床样本和慢病毒shRNA构建KRAS敲低的LoVo细胞株，以及CRISPR/Cas9系统构建的KRAS(G13D)突变型SW48细胞株中进行验证。
1、实验方法
RNA检测方法同上，引物如下
Figure PCTCN2020099974-appb-000026
2、实验结果
检测细胞系甲基转移酶和去甲基化酶的差异表达，结果提示甲基转移酶DNMT1、DNMT3A在KRAS突变型结直肠癌细胞中上调，去甲基化酶TET1/2在KRAS突变型结直肠癌细胞中下调(图15A)。在使用慢病毒shRNA构建KRAS敲低的LoVo细胞株中，TET1/2上调(图15B)。
同时，使用组织化学染色验证了59例结肠癌样本中DNMT1与TET1的表达，并使用ImageJ软件计算核染色强阳性的细胞数比例。结果显示KRAS突变 型结肠癌组织中DNMT1蛋白水平较KRAS野生型高(P＝0.0003)，而TET1的表达在两组间没有显著差异(P＝0.2989)。Spearman秩相关分析的结果显示，DNMT1强阳性细胞的比例与MATE1的表达存在显著的负相关关系(r＝-0.66P<0.0001,n＝59)(图16A)。在KRAS突变型PDX肿瘤组织中DNMT1表达增加，TET1表达减少(图16B)
CRISPR/Cas9系统构建的KRAS(G13D)突变型SW48细胞株上，结果显示DNMT1表达增加，TET1/2表达减少(图16C)，在慢病毒shRNA构建KRAS敲低的LoVo细胞株中，DNMT1表达减少，TET1/2表达增加(图16D)。
在LoVo细胞中和KRAS(G13D)突变型SW48细胞株使用甲基化酶抑制剂阿扎胞苷，能够上调KRAS突变型结直肠癌细胞MATE1的表达，从而抑制二甲双胍抗肿瘤增殖的作用(图16E-F)。在KRAS敲低的LoVo细胞株中干扰TET1/2会重新下调MATE1的表达，促进二甲双胍抑制肿瘤细胞增殖的作用(图16G)。
综上所述，KRAS突变下调MATE1从而提高了二甲双胍在细胞内的浓度，增强二甲双胍抑制结直肠癌细胞增殖的效果。临床上可通过检测KRAS 2号外显子基因型，选择性使用二甲双胍治疗结直肠癌。

Claims (9)

  1. 一种确定结直肠癌治疗方案的标志物，其特征在于，所述标志物为KRAS基因和/或蛋白。
  2. KRAS基因和/或蛋白作为确定结直肠癌治疗方案的标志物的应用。
  3. KRAS基因突变的检测试剂在制备确定结直肠癌治疗方案的试剂盒中的应用。
  4. KRAS蛋白的检测试剂在制备确定结直肠癌治疗方案的试剂盒中的应用。
  5. 一种确定结直肠癌治疗方案的试剂盒，其特征在于，所述试剂盒包括KRAS突变型检测试剂，KRAS突变型结直肠癌选用二甲双胍治疗结直肠癌。
  6. 二甲双胍在治疗结直肠癌或制备治疗结直肠癌药物中的应用，其特征在于，所述结直肠癌为KRAS突变型结直肠癌。
  7. MEK信号通路和AKT信号通路的组合作为结直肠癌治疗靶点的应用。
  8. MEK信号通路的抑制剂和AKT信号通路的抑制剂联合使用治疗KRAS突变型结直肠癌。
  9. 一种治疗KRAS突变型结直肠癌的药物组合物，其特征在于，包括MEK信号通路的抑制剂，以及AKT信号通路的抑制剂。
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