Studies On Mechanism Of RPA1 Regulating The Sensitivity Of Colorectal Cancer To Chemotherapy With 5 Fluorouracil

BackgroundThe digestive system is the place where human malignant tumors occur frequently.Esophageal cancer,gastric cancer,liver cancer and colorectal cancer are the top ten malignant tumors in morbidity and mortality.Gastric and hepatocellular carcinoma highly occurred in China.In recent years,with the development of social economy,viral hepatitis effectively controlled,hygienic conditions improved and dietary structure changed,both hepatocellular and gastric carcinoma have already declined.However,the incidence and mortality of colorectal cancer rise with the high protein and fat consumption,and the patients,characteristics appear to younger age.Despite advances in surgical techniques and chemotherapy and targeted therapy,the mortality of colorectal cancer has decreased,but the prognosis of advanced colorectal cancer,especially with liver and lung metastasis,is still poor.Reproduction protein A(RPA)is a single-stranded DNA(ssDNA)binding protein in eukaryotic cells.It plays an important role in DNA metabolism,including DNA replication,recombination and repair of various DNA damage.At the same time,RPA also participates in regulating cell apoptosis and gene expression.Studies have shown that it is highly expressed in tumor tissues and is involved in tumor development.Recent years,with the deepening of the research on the etiology and mechanism of cancer,especially on oncogenes,tumor suppressor genes and related signal transduction pathways,molecular targeted therapy has opened up a new way for the specific treatment of cancer,but the etiology of the occurrence and development of colorectal cancer has been improved.And the mechanism has not been fully elucidated so far,and the current treatment effect is not satisfactory.Therefore,exploring new molecular regulatory mechanisms for the occurrence and development of colorectal cancer can provide new ideas and new therapeutic targets for the diagnosis and treatment of colorectal cancer.The purpose of this study is to explore the role of RPA1 in the development of digestive tract malignant tumors and how RPA1 affects the sensitivity of tumor cells to 5-FU through clinicopathological and in vitro cell experiments.ObjectiveThe tissue experiment was to investigate the expression of replication protein A1(RPA1)in colorectal carcinoma and adjacent tissues,and preliminary explore its clinical significance.The cell experiments were aimed to investigate the expression of RPA1 in digestive system malignant tumor cell lines(HT-29,SGC-7901 and SMMC-7721).After silencing RPA1,the biological behavior of these cells and their sensitivity to 5-FU were observed.And the signal pathway and mechanism of silencing RPA1 regulating the sensitivity of HT-29 cells to 5-FU were preliminarily explored.MethodsThe expression of RPA1 protein was examined in 30 paired colorectal carcinoma(CRC)and adjacent tissues by immunohistochemistry(IHC).The expression of RPA1 mRNA in these tissues was detected by reverse transcription polymerase chain reaction(RT-PCR).Meanwhile,the clinical data of these patients were collected and analyzed.In this study,the three cell lines were divided into seven groups:control group,solvent group,5-Fu group(5-Fu),lentiviral no-load group(NV),lentiviral no-load +5-Fu group(NV + Fu),RPA1 silencing lentiviral transfection group(RPA1 KD)and RPA1 silencing lentiviral transfection + 5-Fu group(RPA1 KD + Fu).The expression of RPA1 was silenced by infecting cells with knocked down RPA1 lentiviral,and 5-FU was added to interfere with cell proliferation,and we detected the biological indicators of each group in vitro as follows.RPA1 mRNA expression was detected by qRT-PCR,RPA1 protein expression was detected by Western-Blot(WB),and cloning formation assay was used to detect cell proliferation,flow cytometry was used to detect cell cycle and apoptosis,and WB was used to detect the expression of caspase-3.Moreover,CRC cell line HT-29 was divided into five groups:control group,5-FU group,silent RPA1 group(RPA1 KD),lentiviral no-load group(NV)and silent RPA1+5-FU group(RPA1 KD+Fu).The expression of RPA1 was silenced by infecting cells with knocked down RPA1 lentiviral,and 5-FU was added to interfere with cell proliferation.The differences of ERK1/2 and p-ERK1/2 proteins,JNK1/2 and p-JNK1/2 proteins,P38 and p-P38 proteins in each group were detected by WB.Intervention experiments were carried out with ERK inhibitors and caspase-3 inhibitors immediately,and the changes of ERK1/2 and p-ERK1/2 proteins expression were detected by WB.ResultsThe tissue experiment resultsIHC showed that RPA1 protein was expressed in both CRC tissues and adjacent tissues,but the expression in the former was significantly higher than that in the latter(P<0.05).The results of RT-PCR detection was highly consistent with the results of IHC examination.The labeling index(LIs)of RPA1 protein was 63.8%and 17.7%in CRC and adjacent tissues respectively,which was significantly higher in the former(P<0.05).By statified analysis,the LIs of well-differentiated and moderately-differentiated patients were found to have no obvious regularity,but the LIs of poorly differentiated,undifferentiated and mucinous adenocarcinoma patients were all more than 50%.The LIs levels had no correlation with tumor size,lymph node metastasis and clinical stage.The cell experiment resultsSuccessfully,the silencing cell lines were established by using lentivirus vector system in HT-29,SGC-7901 and SMMC-7721.WB and qRT-PCR detection showed that there was no significant difference in the expression of RPA1 protein and RPA1 mRNA between the control group and the solvent group(P>0.05);compared with the solvent group,the expression of RPA1 were inhibited both in 5-Fu group and in RPA1 KD group;while the lowest expression occurred in RPA1 KD +Fu group,indicating the most obvious inhibition.The difference was statistically significant(P<0.05).The clone formation assay showed that no significant difference was observed between the control group and the solvent group(P>0.05).Compared with solvent group,the clone formation rate of 5-Fu group in HT-29,SGC-7901 and SMMC-7721 cells was markedly inhibited(P<0.05).The clone formation rate in RPA1 KD groups was lower than that in NV group(P<0.05).Compared with NV+Fu group,the RPA1 KD +Fu group more significantly repressed the clone formation rate(P<0.05).Flow cytometry to measure cell cycle showed that there was no markedly difference on the percentage of G1 phase between control and solvent group in HT-29,SGC-7901 and SMMC-7721 cells(P>0.05).However,the addition of 5-Fu obviously increased the percentage of G1 phase.Compare witht NV group,the supplement of 5-Fu or the RPA1 KD group enhanced the percentage of G1 phase.And in the RPA1 KD+Fu group,the percentage of G1 phase showed the highest level(P<0.05).The apoptosis results showed that no significant difference on the apoptotic rate was observed in Control and Solvent groups in HT-29 cells(P>0.05).However,compared with Solvent group,the application of 5-Fu improved the apoptotic rate.What's more,compared with NV group,the RPA1 KD group also increased the apoptotic rate,and the application of 5-Fu into NV group significantly enhanced the apoptotic rate.Furthermore,the combination of RPA1 knockdown and 5-Fu displayed the more significant level of apoptotic rate.Similar results were observed in SGC-7901 and SMMC-7721 cells.In the control,solvent and NV group in HT-29 cells,SGC-7901 and SMMC-7221,WB assay showed that the levels of Caspase-3 were very low and there was no significant differences among them(P>0.05).The expression of Caspase-3 in the 5-Fu group was higher than that in the solvent group.Compared with the NV group,both the RPA1 KD group and the NV+Fu group enhanced the expression of Caspase 3.What's more,the level of Caspase 3 in RPA1 KD+ Fu group displayed the highest.The preliminary mechanism experiment resultsHT-29 cells transfected with knocked down RPA1 lentivirus and interfered with 5-FU,we obtained the results of WB examination as follows.?The sequence of ERK1/2 protein expression from high to low was RPA1 KD+FU group,RPA1 KD group,NV group,control group and 5-FU group.The order of p-ERK1/2 protein expression was 5-FU group,control group,NV group,RPA1 KD group and RPA1 KD+FU group.There was statistically significant between each group(P<0.05).The order of JNK1 protein expression was 5-FU group,RPA1 KD+FU group,RPA1 KD group,control group and NV group,The sequence of JNK2 protein expression was 5-FU group,NV group,RPA1 KD+FU group,RPA1 KD group and control group,and the p-JNKl/2 expression in turn was 5-FU group,RPA1 KD+FU group,NV group,control group and RPA1 KD group.The order of P38 expression was RPA1 KD group,control group,RPA1 KD+FU group,NV group and 5-FU group,then the p-P38 expression in turn was 5-FU group,NV group,control group,RPA1 KD+FU group and RPA1 KD group.?In ERK inhibitors intervention experiment,the ERK1/2 protein expression increased as following:5-FU group,control group,NV group,RPA1 KD group and RPA1 KD+FU group.The expression of p-ERKl and p-ERK2 decreased slightly in RPA1 KD group,but slinghly increased in RPA1 KD+FU group,decreased significantly in NV and control group,decreased mostly 5-Fu group.?After interveneing with caspase-3 inhibitor,the expression of ERK1/2 decreased in RPA1 KD and RPA1 KD+FU group,but increased in control,NV and 5-FU group.The expression of p-ERKl/2 increased mostly in RPA1 KD+FU group,secondly in RPA1 KD group,but the changes in 5-FU,control and NV group were relatively slight.ConclusionThe expressions of RPA1 protein and RPA1 mRNA in CRC tissues were higher than those in adjacent tissues,and the LIs was higher in poorly differentiated tissues.High expression of RPA1 may be associated with the occurrence and development of colorectal cancer.RPA1 was highly expressed in gastrointestinal cancer cells.Only 5-Fu or the knockdown of RPA1 suppressed cell clone formation,induced cell cycle arrest at the G1 phase and promoted cell apoptosis by regulating the protein level of Caspase 3.And the combination of the application of 5-Fu and RPA1 silencing significantly enhanced the above effects.Therefore,it is suggested that RPA1 serves as an oncogene during colorectal cancers progression,and silencing RPA1 can enhance the sensitivity of colorectal cancer cells to 5-Fu.The mechanism may through the ERK/caspase-3 pathway.Silencing RPA1 can induce the decrease of ERK phosphorylation and the increase of Caspase-3 expression,thus inducing cell apoptosis.