Association Between MicroRNA Gene Polymorphism And Cervical Cancer In PI3K/AKT Signaling Pathway

Background:Cervical cancer is the second-largest female malignancy after breast cancer which seriously threatens the health of women.According to the estimation of the World Health Organization,there are about 490,000 new cases each year in the world and the mortality rate is about 47%.In China,there are approximately 131.000 new cases of cervical cancer each year,accounting for 28.8%of all new cases in the world.Recent statistics show that the age of incidence and death appears to be younger.The occurrence and development of cervical cancer can be divided into two stages;cervical intraepithelial neoplasia(CIN)and cervical cancer(CC).High-risk human papilomavirus(HPV)infection is one of the necessary conditions for the development of cervical cancer.In addition,host genetic factors also play an important role in this process.At the same time,intracellular signaling pathways are involved in the regulation of the development and progression of cervical cancer,such as PI3K,MAPK and P53.PI3K signaling pathway is mainly involved in cell proliferation,differentiation,apoptosis and other processes.Studies have found that microRNA is a very important regulatory molecule in the PI3K signaling pathway,and abnormal expression of miRNAs that regulate the PI3K signaling pathway is found in cervical cancer cells.MicroRNAs are small,non-encoding,single-stranded RNA molecules,about 20-24 nucleotides in length,that bind to a target gene by the way of base complementation,causing degradation or translational inhibition of target gene mRNA.It has been found that mutations in pri-or pre-sequences in miRNA genes may affect miRNA cleavage.processing and maturation processes and thus affect the expression of mature miRNAs.Variations in mature miRNA sequences may affect miRNAs regulation on target genes.In summary,mutations in the miRNA gene of the PI3K signaling pathway may affect the regulation of PI3K signaling pathway by affecting the expression of mature miRNAs or affecting the recognition of target genes by miRNAs and have a correlation with the occurrence and development of cervical cancer.Objective:To search for the mutation sites(Single nucleotide polymorphisms,SNPs)in the miRNA gene of PI3K/AKT pathway to analyze the the relationship between SNPs of PI3K/AKT pathway and cervical cancer as well as its possible mechanism of occurrence and development of cervical cancer.At last we may provide new ideas for the study of the pathogenesis of cervical cancer and provide new targets for the study of cervical cancer diagnosis and treatment.Method:Thirty-two miRNAs related to the PI3K/AKT signaling pathway were selected in this study,and SNPs in these miRNA gene sequences were searched by sequence analysis.TaqMan Assay genotyping and Massarray genotyping were used to genotype SNPs in the miRNA gene of 416 healthy controls,179 patients with cervical intraepithe-lial neoplasia and 245 patients with cervical cancer.The distribution characteristics of SNPs in three groups were obtained.The distribution of alleles and genotypes of these SNPs in the three groups was analyzed to find SNPs that were related to the development of cervical cancer.SNPstatus software was used to analyze genotypes that correlated with the development of cervical cancer.RNAfold software was used to analyze the effect of SNPs on miRNA secondary structure.The miRNA target genes were analyzed by many databases(miRNAanda,Microcode,Targetscan,miRNATarget2,microT,etc.).Finally,the above results were combined to analyze the association of SNPs in the miRNA gene of PI3K/AKT signaling pathway with the occurrence and development of cervical cancer and its possible mechanisms.Results:1.miRNAs of the PI3K/AKT signaling pathway and their target genes:By searching the miRPath database and reading articles we obtained 32 miRNAs of PI3K/AKT signaling pathway.Through target gene prediction and reports we have confirmed miRNA target genes in the PI3K pathway.These miRNAs and target genes were p21(miRNA-125a),p27Kipl(miRNA-200b),AKT(miRNA-7-1,miRNA-7-2,miRNA-7-3,miRNA-155,miRNA-125a,miRNA-124,let-7a2,miRNA-214,miRNA-424,miRNA-133a2,miRNA-145,miRNA-200b,miRNA-92a and miRNA-31),PI3K(miRNA-126 and mi-RNA-10b),mTOR(miRNA-127,mi-RNA-100,miRNA-218-2,miRNA-218-1,miRNA-101-1 and miRNA-101-2),PDK(mi-RNA-23b and miRNA-155),PTEN(miRNA-149,miRNA-21,miRNA-196a2,miRNA-3529,miRNA-499 and miRNA-106b),BAD(miRNA-106b),CDK(miRNA-9-1,miRNA-9-2,let-7a2 and miRNA-205).2.Search for polymorphic sites in miRNA gene:Through analysis of sequencing results,we found 18 polymorphic sites in 14 miRNA genes:rs353292(pri-miRNA-145),rs353293(pri-miRNA-145),rs74693964(pri-miRNA-145),rs1143770(pri-let-7a2),rs629367(pri-let-7a2),rs4078756(miRNA-10b promoter region),rs767649(pri-miRNA-155),rs999885(miRNA-106b promoter region),rs4636297(pri-miRNA-126),rs531564(pri-miRNA-124),rs8111742(pri-miRNA-125a),rs6062251(pri-miRNA-133a),rs2292832(pre-miRNA-149),rs9660710(pri-miRNA-200b),rs11614913(miRNA-196a-3p),rs11134527(pri-mi RNA-218),rs543412(pri-miRNA-100),rs1834306(pri-miRNA-100).3.Association analysis results:?The allele frequency of rs4636297 in miRNA-126 between control group and CIN group as well as between control group and CC group are statistically significant(P=0.005 and P=0.036).The frequency of allele C in control group is higher than in CIN groups(OR=0.507;95%CI:0.372-0.691)and in CC group(OR=0.637;95%CI:0.475-0.853).The allele C may be a protective factor for the development of cervical cancer.The genotype frequency of rs4636297 between control group and CIN group is statistically significant(P=0.004).However there are no statistically significant between control group and CC group as well as CIN group and CC group(P>0.05).The results of genetic model analysis showed that the C/C genotype of rs4636297 may reduce the risk of CIN(P<0.002;OR=0.60,95%CI:0.41-0.88)using codominant model in a comparison of the control group and CIN group.The 2C/C-C/T genotype of rs4636297 may decrease the risk of cervical cancer in the comparison of control group and CC group(P=0.038,OR=0.62;95%CI:0.45-0.84).?The frequency of allele C of rs2292832 in miRNA-149 among control group,CIN group and CC group were not statistically significant(P>0.05)after Bonferroni correction.The frequency of the genotype frequency between control group and CIN group was statistically significant(P=0.036),but the genotype frequency of rs2292832 were not statistically significant in a comparison of control CIN group with CC group(P>0.05).The results of genetic model analysis showed that the T/T-C/C genotype of rs2292832 may reduce the risk of CIN(P=0.001,OR=0.51;95%CI:0.34-0.75).?The frequency alleles and genotypes in the remaining SNPs(rs531564,rs8111742,rs6062251,rs74693964,rs353292,rs353293,rs9660710,rs629367,rs1143770,rs4078756,rs999885,rs11134527,rs543412,rs767649,rs11614913 and rs 1834306)were not statistically significant after Bonferroni correction(P>0.05)among this three groups.4.Prediction of the secondary structure of miRNA sequences:?The predicted secondary structure of miRNA-126 showed that the secondary structure of miRNA-126 carrying allele C would be more stable than the secondary of miRNA-126 carrying allele T.?The predicted secondary structure of miRNA-149 showed that the secondary structure of miRNA-149 carrying allele T would be more stable than the secondary of miRNA-149 carrying allele C.The prediction of miRNA secondary structure is consisted with the results of the association study.Conclusion:The results of this study show that the allele C and genotype C/C of rs4636297 in miRNA-126 gene may be the protective factors of the occurrence of cervical cancer in Yunnan Han population.Similarly,the genotype T/T-C/C of rs2292832 may be the protective factors of the occurrence of CIN in Yunnan Han population.Through the analysis of the miRNA secondary structure,it was found that the rs4636297 and rs2292832 may affect the stability of the pre-miRNA-126 and the pre-miRNA-149 secondary structure,respectively.Therefore these two SNPs may affects the expression of mature miRNA by affecting the cleavage,processing and mature process of miRNA-126 and miRNA-149 and may further affect the expression of the target genes PI3K and PTEN,ultimately affect the PI3K/AKT pathway which plays an important role in the development of cervical cancer.