Study On The Mechanism Of MiR-126 Inhibiting Cervical Cancer And The Function Of MiR-126 Gene Mutation

Background:Cervical cancer is a common gynecological malignant tumor and ranks the fourth in the incidence and mortality of female malignant tumors all the year round in the world,posing a serious threat to women’s normal life and health.Cervical cancer is a multi-factory-induced disease,in which the persistent infection of High risk Human papillomavirus(HR-HPV)plays an important role.In addition,individual factors(living habits and host genetic factors,etc.)also play important roles in the occurrence of cervical cancer.The study found that the expression of a large number of microRNAs(miRNAs)in cervical cancer tissues was dysregulated.MiR-126(miR-126-3p and miR126-5p)expressed low in a variety of tumors,including cervical cancer,and may be involved in the development of cervical cancer.We found that Single nucleotide polymorphisms(SNPs)in miRNA genes may be associated with the risk of tumorigenesis by affecting the expression of mature miRNAs.Our previous association study between the SNPs locus in miRNA genes and cervical cancer showed that the SNP locus rs4636297 in miR-126 gene was associated with the risk of cervical cancer in the Han population in Yunnan.Based on the above data,this study intends to study the role of miR-126 in cervical cancer and its related mechanisms,as well as the function of SNP locus in miR-126 genes.Objective:Current studies only focus on the relationship between miR-126-5p or miR126-3p and cervical cancer.In this study,miR-126 eukaryotic expression vector will be used to simultaneously express miR-126-3p and miR-126-5p in cervical cancer cells.The role of miR-126 in cervical cancer and its mechanism would be systematically studied.Furthermore,based on the results of previous genetic studies,the influence of miR-126 gene mutation on the expression of mature miR-126 in cervical cancer cells was analyzed to reveal the possible mechanism of rs4636297 in the risk of cervical cancer.Methods:1.The expression of miR-126 in cervical cancer tissues was analyzed by qPCR.2.The role of miR-126 in cervical cancer:(1)The transient expression vector pcDNA3.1-miR-126 and the stable expression vector plvshRNA-EGFP-miR-126 were constructed,and the negative control expression vector of miRNA was constructed at the same time;(2)Through overexpression of miR-126 in cervical cancer HeLa cells,the effects of miR-126 on proliferation,migration,invasion and other characteristics of HeLa cells were studied.3.The mechanism of miR-126 in cervical cancer HeLa cells:(1)The possible target genes of miR-126-3p and miR-126-5p in tumor-related signaling pathways were predicted by database;(2)The effects of overexpression of miR-126-3p or miR-126-5p on the expression level of target genes in the tumor signaling pathway in HeLa cells were analyzed by qPCR and Western blot;(3)The interaction between miR126-3p or miR-126-5p and target genes was verified by the dual luciferase reporter gene detection system.4.To analyze the mechanism of rs4636297 and the risk of cervical cancer:(1)To construct transient and stable expression vectors carrying different alleles(A or G)of rs4636297,and to construct negative control expression vectors of miRNA;(2)The relationship between the expression vectors of different alleles in transient expression and stable expression system and the expression of mature miR-126 was analyzed,and the mechanism of rs4636297 and cervical cancer susceptibility was preliminarily analyzed.Results:1.The effect of miR-126(miR-126-3p and miR-126-5p)on tumor suppressor gene in cervical cancer was studied.(1)The relative expression level of mature miR-126(miR-126-3p and miR-126-5p)in cervical cancer tissues was lower than that in adjacent tissues and normal tissues,and the difference was statistically significant(P<0.05).(2)Overexpression of miR-126 in cervical cancer HeLa cells can inhibit the proliferation,migration and invasion of HeLa cells.2.In cervical cancer HeLa cells,miR-126 plays a role of tumor suppressor gene by targeting genes in the PI3K signaling pathway:(1)The target genes of miR-126-3p and miR-126-5p in tumor-related signaling pathway were predicted by miRNA-mRNA interaction database respectively,and the results showed that both miR-126-3p and miR-126-5p could be targeted to molecules in the PI3K signaling pathway;(2)After transfection of miR-126-5p and miR-126-3p analogs into HeLa cells,miR-126-3p target gene CRK and PIK3R2 were down-regulated in both mRNA and protein levels,while the expression of miR-126-5p target gene PIK3CA was also inhibited.(3)The results of the dual luciferase reporter gene detection system showed that miR-126-3p could be targeted to PIK3R2 and CRK in HeLa cells,while miR-126-5p could be targeted to PIK3CA in HeLa cells.3.There was no association between different alleles of rs4636297 and the expression level of mature miR-126:(1)(1)There was no significant difference in the relative expression level of mature miR126 in HeLa cells carrying different alleles of rs4636297,the SNP locus of miR-126,which was transient expression and stable overexpression of miR-126(P>0.05).(2)The results suggested that rs4636297 was not associated with cervical cancer susceptibility by affecting the expression of mature miR-126,but may be associated with cervical cancer susceptibility through other mechanisms.Conclusions:In summary,miR-126 can play a role of tumor suppressor gene by targeting CRK,PIK3R2 and PIK3CA in the PI3K signaling pathway in cervical cancer HeLa cells.Moreover,rs4636297,the SNP locus in miR-126 gene,was not associated with the risk of cervical cancer by affecting the expression of mature miR-126.