Let-7f-5p Induces Chemotherapeutic Resistance By Promoting Expression Of Anti-apoptotic Protein And Inhibiting Pro-apoptotic Protein In Colorectal Carcinoma

Backgroud and Purposes:Colorectal cancer(CRC)has been one of the most common malignancies threatening human life and health.It ranks second place in male tumors and third place in female tumors respectively.The main treatments include surgical excision,radiotherapy,chemotherapy and targeted therapy.But in clinic half of the patients with colorectal canceroccuredneoplasm metastasis just before radical operation.However,the form of chemotherapy resistance leads to the majority of the patients react poorly to chemotherapy.MicroRNA(miRNA)has a wide participation in the process of various life actions,also has a close relation to drug resistant mechanism of malignant tumor.Meanwhile,its abnormal expression involves progression and metastasis of different cancers.Besides,up-regulation or down-regulation the expression of some specific miRNA influences response of chemotherapeutics directly.New research evidences also indicate that miRNA is the key regulator of chemotherapy resistance of colorectal cancer.Otherstudieshaveshown that Let-7 family plays a role as tumor inhibitor by regulating various kinds of carcinogenic signal transductions.Therefore,how to prevent and control chemotherapy resistance in treatment is an urgent clinical problem to solve.This study aims to explore the effect and molecular mechanism of miRNAlet-7f-5p to CRC chemotherapy resistance.Methods:1.miRNA sequence database of colorectal cancer genome maps was analyzed and the relationship between let-7f-5p and chemotherapy resistance of CRC was observed.2.HCT116 cells were transfected with let-7f-5p simulant and SW480 CRC cells were transfected with let-7f-5p stimulant inhibitor to analyze expression of let-7f-5p and comparation with control group by real time PCR,as well transcriptionallevel was standardized by U6 formula;the effects of let-7f-5p on expression of apoptins Bcl-2 and Bcl-x L were tested by Western-blot;the effects of let-7f-5p on activity of caspase-3/-9 were tested by detection kit;further confirmed the function of let-7f-5p in CRC chemotherapy resistance when treated with first-line drug 5-fluorouracil(5-FU).3.The effect of let-7f-5p on the mitochondrial potential of CRC cells was examined that let-7f-5p promotes the chemoresistance of CRC cells to 5-FU via.4.TargetScan and miRanda algorithm was utilized to predict potential targets which may regulate target genes;the effects of overexpression and silence of let-7f-5p on the mRNA express level of TP53,TP53INP1,TP53INP2 and caspase-3 were analyzed by real-time PCR;let-7f-5p was used to overexpress and silent pmirGLO-3'UTR reporter gene of TP53?TP53INP1?TP53INP2 and Caspase-3 in HCT116 cells and SW480 cells.Then tested it with luciferase to further confirm whether TP53?TP53INP1?TP53INP2 and Caspase-3 were target genes or not.Results:1.It was found that the expression of let-7f-5p increased in chemotherapy resistant CRC tumor cells by analyzing TCGA database and let-7f-5p may increase chemical resistance of CRC.2.let-7f-5p improves chemotherapy resistance of CRC cells by increasing expression of Bcl-2 and Bcl-xLwhich are anti-apoptotic proteins.2.1 Overexpression of let-7f-5p decreases the apoptotic rate and increases mitochondrial potential in CRC cells.2.2 let-7f-5p up-regulation may increase expression of Bcl-2 and Bcl-xL;on the contrary,let-7f-5p down-regulation may decrease its expression.2.3 The activity of let-7f-5 up-regulate caspase-3/-9 decreased,however,silent let-7f-5 may increase activity of caspase-3/-9.It shows that let-7f-5p improves chemical resistance of CRC cells by inhibiting activity of caspase-3/-9.3.It was found that TP53,TP53INP1,TP53INP2,caspase-3 as well as other pro-apoptotic proteins may be the potential targets of let-7f-5p by using TargetScan and miRanda.The analysis of real-time PCR shows that overexpression of let-7f-5p decreases mRNA expression of TP53,TP53INP1,TP53INP2 and Caspase-3.On the contrary,silent let-7f-5p increases their expression which indicates that let-7f-5p negatively regulates mRNA expression of TP53,TP53INP1,TP53INP2 and Caspase-3.Besides,results of luciferase test show that decreases let-7f-5p overexpression and silents it may increase activity of reporter genes which driven by their 3'UTRs.These results indicates that let-7f-5p targets TP53?TP53INP1?TP53INP2 and Caspase-3 directly and further improve chemotherapy resistance of CRC.Conclusions:In current studies,our research results indicate that the expression of let-7f-5p in chemotherapy resistant CRC tissues increases when compared with chemotherapy sensitive tissues.Besides,let-7f-5p up-regulation can increase the expression of Bcl-2 and Bcl-x L,meanwhile,decreased the activity of caspase-3 or-9 in CRC cells.On the contrary,let-7f-5p down-regulation has opposite effects.More importantly,our discovery shows that let-7f-5p improves chemotherapy resistance by inhibiting TP53,TP53INP1,TP53INP2,caspase-3 as well as other pro-apoptotic proteins directly.This study reveals new mechanism of let-7f-5p increases chemotherapy resistance of CRC cells.Therefore,our results indicate that the overexpression of let-7f-5p is significant for chemotherapy resistance of CRC.