Regulation Of MicroRNA-155 On HTERT In Gastric Cancer Cell

Gastric cancer is one of the most common malignant tumors in China.It is a major disease to endanger the health of residents.At present,the medical science lack of ideal therapeutic method for gastric cancer.Therefore,the research on the pathogenesis of gastric cancer has profound theoretical value and clinical significance.Activation of telomerase is one of the important factors in tumorigenesis.Telomerase highly expressed in more than 85%~90% tumor cells,which makes the tumor cells immortalized.hTERT is the rate limiting subunit of telomerase,which directly affects the activity of telomerase.Therefore,the activation of telomerase induced by hTERT is an important factor for malignant transformation and unlimited proliferation of tumor cells.Previous studies on the regulation of hTERT are mostly focused on the transcriptional level,whereas the studies on the post-transcriptional level is less.MicroRNA(miRNAs)is a class of small endogenous non-coding RNA,which is involved in the regulation of gene post-transcriptional level.MiRNA plays an important role in many pathological and physiological processes,including the development and metastasis of tumor.Studies have shown that some miRNA can directly or indirectly regulate the expression level of hTERT,thereby affecting the process of tumor disease.However,there are few reports about the regulation mechanism of microRNA 155(miR-155)on hTERT.Therefore,this study used bioinformatics prediction software TargetScan and Miranda to predict whether SP1 and E2F2 mRNA 3'-UTR is the target of human miR-155.According to the SP1 and E2F2 mRNA3'-UTR design of two complementary oligonucleotide chains,including the target sequence(SP1,E2F2 wild type sequence)and mutation sequence(SP1-mu,E2F2-mu)to construct reporter gene vector pMIR-SP1,pMIR-E2F2,pMIR-SP1-mu and pMIR-E2F2-mu.Cultured HEK-293 cells,the wild-type and mutant pMIR-Luc plasmids of SP1 and E2F2 were co-transfected into HEK-293 cells by liposome method for 24 h.The cells were divided into SP1 control group,SP1 target sequence group,SP1 mutation sequence group,E2F2 control group,E2F2 target sequence group,E2F2 mutation sequence group,treated with 0.2?g recombinant plasmid and 0.01?g control plasmid and 0.375?l oligonucleotide,cultured for 24 h.Dual-luciferase reporter gene system was used to detect the double luciferase activity,and the firefly luciferase activity value(F)/ ranilla luciferase activity value(R)ratio was used to evaluate the combined effect of miR-155 and SP1,E2F2 mRNA 3'-UTR.Cultured SGC-7901 cells,the miRNA control?miR-155 mimics?miR-155 inhibitors were co-transfected into SGC-7901 cells by liposome method for 24 h.Real-time PCR and Western blot were used to verify the effect of miRNA-155 on the expression of target gene and hTERT in gastric cancer cells.The study found that online prediction results showed that SP1 mRNA3'-UTR and E2F2 mRNA 3'-UTR had a sequence of complete complementary pairing with miR-155,and the two genes were predicted to be target genes for miRNA-155.Reporter gene recombinant plasmid of SP1 and E2F2 were successfully constructed.The SP1 target sequence group F/R was lower than that of the SP1 control group and the SP1 mutant sequence group(P < 0.05),the E2F2 target sequence group F/R was lower than that of the E2F2 control group and the E2F2 mutant sequence group(P< 0.05).The RT-PCR and Western blot proved that miR-155 combined with SP1 mRNA 3'-UTR to inhibit the expression of SP1(P<0.05),thereby inhibiting the expression of hTERT(P<0.05).MiR-155 played a regulatory role in the mechanism of gastric cancer cells in hTERT pathway.In this study,we successfully predicted and verified some of the target genes of miR-155 in the regulation of hTERT in gastric cancer cells.Theseresults suggest that SP1 plays an important role in the activation of telomerase and tumorigenesis,and is involved in the regulation of hTERT expression in gastric cancer cells by miR-155.As a new negative feedback regulation factor,miR-155 constituted a new gene regulatory network with target genes,which is involved in the regulation of hTERT expression in gastric cancer cells.MiR-155 provides a new direction for the study of the pathogenesis of gastric cancer.