Study On The Role And Mechanism Of Brca1 In Drug Resistance Of Colon Cancer Cells

Colon cancer is one of the common malignancies in the digestive system.Recent studies have found that the incidence and mortality of colon cancer in the world have risen,threatening people's health and longevity.These treatments have also achieved significant clinical results,but in recent years chemotherapy sensitivity was significantly reduced due to the emergence of tumor resistance,which leads to an increase in the recurrence rate and mortality of patients.In order to overcome the tolerance of tumor cells to chemotherapy,the researchers have studied the possible mechanism and found some genes related to tumor cell tolerance,which provides a certain directionfor overcoming the tolerance of cancer.This study aims to find the possible drug-resistance related genes of human colon cancer and to provide theory basis and direction for the reversing of drug-resistanceof colon cancer in the clinical area.We used HCT-8 cells,which is commonly used to establish the drug-resistance model of colon cancer,to conduct this study.The ultrastructure and growth characteristics of HCT-8 cell lines resembles malignant epithelial cells.Tumors from HCT-8 cells inoculated in mice are similar to the original tumor tissue of patients.Vincristine(VCR)is likely to induce drug-resistance when HCT-8 cells are cultured in vitro.For this reason,we used VCR of different concentrations to induce HCT-8 cells and to investigate its drug-resistance mechanism.VCR is one of the most commonly used chemotherapeutic drugsintreatingcolon cancer.It is cell cycle-specific,whichsuppresses the assemblyof microtubule and the progress of the metaphase of mitosis by binding with microtubulin.VCR has been widely used in the treatment of leukemia,lung cancer and other malignant tumors.Considering that many genes participate in the adjustment of drug-resistance of tumor cells,we have consulted literature about drug-resistance and colon cancer.Then we selected three genes that are possibly related to the drug-resistance of colon cancer,which are TGF-?1(transforming growth factor-?1,TGF-?1),SIKE1(suppressor of Ikappa B kinase-epsilon)and BRCA-1(breast cancer susceptibility gene 1),to investigate whether they participate in the drug-resistance of HCT-8 cells and the mechanism of drug-resistance.We have found that BRCA1 is related to the drug-resistance of colon cancer by screening and have discussed its drug-resistance mechanism.This paper includes the following three parts: Part one: Screening of colon cancer cell resistance-related genes;Part II: Investigation of the relationship between BRCA1 and colon cancer cell resistance,proliferation and apoptosis;Part III: Investigation of drug resistance mechanism of BRCA1 in colon cancer cells.Part One: Screening of resistance-related genes in colon cancer cell Methods:1.HCT-8 colon cancer cells resistant to vincristine were constructed.2.MTT assay was used to detect the IC50 of HCT-8(sensitive cells)and HCT-8 / V(vincristine-resistant HCT-8)cells.3.The expression of LAP,SIKE1 and BRCA1 m RNA in HCT-8 and HCT-8 / V cells were detected by real-time quantitative RT-PCR.4.The expression of SIKE1 and BRCA1 protein in HCT-8 and HCT-8 / V cells was detected by Western blotting.5.The expression of TGF-?1 in HCT-8 and HCT-8 / V cells was detected by ELISA.6.The expression of TGF-?1 and BRCA1 in the cells was inhibited by si RNA,and the changes of IC50 were detected by MTT assay.Results:1.IC50 results showed that HCT-8 / V had a significant tolerance to vincristine,the IC50 value of HCT-8 / V(151.3±4.2)was about 18 times that of the HCT-8(8.2±1.3).2.RT-PCR results showed that the expression of LAP and SIKE1 m RNA in HCT-8 / V cells did not change significantly compared with HCT-8 cells,and the expression of BRCA1 m RNA was significantly increased(In HCT-8/V cells,the expression level was 3.05 times higher than that in HCT-8 cells)(P<0.05).3.The results of Western Blotting showed that the level of SIKE1 protein in HCT-8 / V cells did not change significantly compared with HCT-8 cells,and the expression level of BRCA1 protein was significantly increased in HCT-8 / V cells(0.83 ±0.010)compared with HCT-8 cells(0.41±0.05)(P<0.05).4.The results of ELISA showed that the expression of TGF-?1 was significantly increased in HCT-8 / V cells(3323.615 ±118.991ng/ml)compared with HCT-8 cells(1607ng/ml±67.473)(P<0.05).5.Transfection of TGF-?1 si RNA did not cause significant changes in IC50 in HCT-8 cells,whereas transfection with BRCA1 si RNA significantly reduced HCT-8 / V IC50 levels,HCT-8/V NC si RNA Treated team :1.055 ng/m L,HCT-8/V BRCA1 si RNA Treate team: 0.178 ng/m L(P<0.05).Conclusion:BRCA1 gene may be involved in colon cancer cell HCT-8 tolerance to vincristine.Part two: Investigation of the relationship between BRCA1 and colon cancer cell resistance,proliferation and apoptosisMethods:1.The expression of BRCA1 protein in tumor tissue from patient sensitive to and nonsensitive to vincristine therapy was detected by immunohistochemistry.2.The expression of BRCA1 in HCT-8 / V cells inhibited by si RNA,and the cell viability was detected by MTT assay and CCK-8 method.Flow cytometry was used to detect the apoptosis rate of HCT-8 / V cells induced by BRCA1.The changes of apoptosis-related proteins were detected by Western blotting,and the mitochondrial membrane potential was detected by Rhodamine 123.3.The mice were transplanted into BABL / c nude mice by stable screening of the control group and the HCT-8 / V cells of BRCA1 si RNA plasmid to establish the mouse model of transplanted tumor.Western blotting was used to detect the apoptosis-related protein in tumor tissue.Results:1.There was a significant difference in the expression of BRCA1 protein in the clinical tissues tolerated to the vincristine.Positive rate for sensitive patients was 16.7%(5/30),with nonsensitive 36.7%(11/30))(P<0.05).2.After the expression of BRCA1 was inhibited by si RNA in HCT-8/V cells,the results of MTT showed that The survival rate in cells transfeted with BRCA1 si RNA(71%)was significantly decreased compared with NC si RNA(89%)(P<0.01).the results of CCK-8 showed that The survival rate in cells transfeted with BRCA1 si RNA(53%)was significantly decreased compared with NC si RNA(92%))(P<0.01),consistent with the results of MTT ? BRCA1 was inhibited by si RNA could significantly reduce the survival rate of cells,the difference was statistically significant.The results of flow cytometry showed that the apoptosis rate of BRCA1 si RNA(0.079±0.009)was significantly increased compared with NC si RNA(0.042±0.007)(P<0.05),and the expression of cleaved caspase-3 in BRCA1 si RNA cells(0.63±0.05)was significantly increased compared with NC si RNA cells(0.86±0.09)(P<0.05)the expression of cleaved cleaved PARP(0.71±0.10)was significantly increased compared with NC si RNA cells(0.92±0.05)(P<0.05).and cleaved PARP in BRCA1 si RNA cells(0.58±0.015)was significantly decrease compared with NC si RNA cells(0.91±0.04).The mitochondrial membrane potential was reduced after transfection of BRCA1 si RNA plasmid(P<0.05).3.The expression of BRCA1 in HCT-8 / V cells could significantly decrease the growth rate of tumor cells,Mice tumor weight of BRCA1 si RNA was 0.213±0.022 g,mice tumor weight of NC si RNA was 0.522±0.043 g,P<0.05.the ratio of Bcl-2 / Bax significantly decrease in BRCA1 si RNA(1.20)than in NC si RNA(2.54)(P<0.05).Conclusion:Inhibition of BRCA1 expression in drug-resistant cells HCT-8 / V can inhibit tumor growth and induce tumor apoptosis.Inhibition of BRCA1 expression in vivo can effectively inhibit the growth of transplanted tumor in nude mice and promote tumor cell apoptosis.Part three: Investigation of drug resistance mechanism of BRCA1 in colon cancer cells Methods:1.The expression of P-gp protein in HCT-8 cells and HCT-8 / V cells and the expression of P-gp protein inhibited by BRCA1 si RNA was detected by western blotting.2.Western blotting was used to detect the expression of P-gp in BRCA1 inhibition transplanted tumor mice model.The fluorescence intensity of Rhodamine 123 was detected by flow cytometry Flow cytometry was used to detect the intracellular accumulation by Rhodamine 123.3.The fluorescence intensity of Rhodamine 123 was detected by flow cytometry Flow cytometry was used to detect the intracellular accumulation by Rhodamine 123.Rhodamine 123 has important value in the functional detection of P-gp.It can indirectly reflect the function of P-gp by the accumulation of intracellular Rhodamine 123.Result:1.The expression of P-gp protein in HCT-8 / V cells(0.89±0.07)was significantly higher than that in HCT-8 cells(0.35±0.04).The expression of P-gp protein in HCT-8/V BRCA1 si RNA(0.41±0.01)was significantly decreased than that in HCT-8 / V cells.Inhibition of BRCA1 expression can significantly reduce the expression of P-gp protein(P<0.01).2.Compared with the control group(1.35+0.23),the transfection of BRCA1 plasmid could significantly decrease the expression level of P-gp in tumor tissue(0.78+0.19)(P<0.05)3.The fluorescence intensity of HCT-8/V BRCA1_si RNA group was(38.68±4.75)×104,The fluorescence intensity of HCT-8/V NC si RNA group was(5.78±0.34)×104.Compared with HCT-8/V BRCA1 si RNA group and HCT-8/V NC si RNA group,the fluorescence intensity increased(P<0.01),inhibition of BRCA1 can significantly increase the intensity of intracellular Rhodamine 123,suggesting that weakened the function of P-gp,indicating that BRCA1 could increase the P-gp expression,increased discharge of Rhodamine 123(P<0.01).Conclusion:P-gp protein is involved in BRCA1-mediated colon cancer cell resistance.