Polymorphism Effect Of Pre-miR-146a Promoted Cell Proliferation Of Cervical Cancer Cells Mainly By Targeting IRAK1 And TRAF6

Background and ObjectivesMiRNAs are class of small non-coding endogenous RNAs of about 22 nucleotides that bind to the 3'untranslated region(3'UTR)of their target mRNAs as gene regulators.MiRNAs regulate gene expression by translational repression or mRNA degradation,and influence multiple cellular activities including proliferation,apoptosis and tumor occurrence.Single nucleotide polymorphisms(SNPs)is the change,insertion or deletion of nucleotide.Epidemiological studies suggest that genetic variations in the host may also contribute to the pathogenesis of cervical cancer.MiR-146a precursor SNP(rs2910164)involves a G>C nucleotide substitution that causes change from a G:U pair to a C:U mismatch.Higher expression of miR-146a was found in many solid cancer types,such as papillary thyroid carcinoma,breast cancer,and lung cancer.Cervical cancer is the third most common female cancer,the incidence rate of cervical cancer was second in female malignant tumors which lower than the breast cancer,with an estimated over 500,000 new cases and 274,000 death in global per year.High risk type HPV infect is a necessary factor in the occurrence of cervical intraepithelial neoplasia and cervical cancer.However,the HPV infection can be cleared within 1 or 2 years in most individuals,HPV persists in the epithelium of infected individuals can progress to cancer.Women who carrying HPV-16 and-18 subtypes are tend to developing into cervical cancer.A number of studies have indicated that the abnormal expression of miRNA is closely related to the occurrence of cervical cancer.MiR-34a and miR-21 are high expression in cervical cancer,while miR-182,miR-125b,miR-133a and let-7a are low expression in cervical cancer.In the present study,to further study the latent function of polymorphism,we investigated the impact of the SNP on the expression of miR-146a in cervical cancer cells and the immortalized non-tumorigenic cells.Soon afterwards,we sought to determine the association of miR-146a and IRAK1,TRAF6 in cervical cancer cells.Our study showed that overexpression of miR-146a promotes proliferation of cervical cancer cells via decreasing IRAK1 and TRAF6,thus providing new insights into the understanding of cervical cancer.Materials and Methods(1)The immortalized non-tumorigenic cells Endocervical Endl/E6E7(CRL-2615)was cultured in Keratinocyte-Serum Free medium with 0.1 ng/ml human recombinant EGF,0.05 mg/ml bovine pituitary extract,and additional calcium chloride 44.1 mg/L(final concentration 0.4 mM).Both HeLa and C-33 A cells were cultured in DMEM supplemented with 10%fetal bovine serum(FBS).QPCR was used to check the miR-146a,IRAK1 and TRAF6 on mRNA level.HeLa cells have been reported to contain human papilloma virus 18(HPV-18)sequences,while C-3 3 A cells and CRL-2615 were negative.(2)CRL-2615,HeLa and C-33 A cells were transfected with expression plasmid vector DNA containing either the G or C allele of miR-146a rs2910164(Pre-miR-146a-G or Pre-miR-146a-C),and using Lipofectamine 2000 according to the manufacturer's instructions.The pcDNA3.1(+)vector without an insert was used as a negative control.24 hours after the cell was up to 70%?60%for transfection,miR-146a expression was verified by QPCR.(3)Cervical cancer cells were transfected with miR-146a overexpression mimics and negative control(NC)using lipofectamine 2000 according to the manufacturer's recommendations.cell proliferation were performed using a CCK-8.stained with 50mg/mL of propidium iodide(PI)for DNA content analysis by flow cytometry on a FACS Calibur system,Results were expressed as a percentage of cells in each cell-cycle phase.Western Blot were used to check the CyclinD1 protein level.(4)Cervical cancer cells were transfected with miR-146a overexpression mimics and negative control(NC),Cells were lifted off the plates,washed twice with PBS and determined with an Annexin V-FITC/propidium iodide(PI)kit at room temperature for 15 min in the dark,and then analyzed by BD Biosciences FACS Calibur Flow Cytometry according to the manufacturer's protocol.Used flow cytometry to examine the apoptosis rate of cells in each group.Additionally,the expression of cleaved-caspase3 protein which related to cell apoptosis were tested using Western Blot.(5)The potential targets of miR-146a were predicted and analyzed using 3 computer-aided algorithms,including TargetScan,miRanda and PicTar,As a result,IRAK1 and TRAF6 were predicted by these software packages.Cervical cancer cells were transfected with miR-146a overexpression mimics and negative control(NC),QPCR and Western Blot were used to check the expression of IRAK1 and TRAF6 on mRNA and protein level.(6)Statistical Analysis:SPSS 20.0 was used for statistical analysis.The results in this study are represented as means ± SD from three separate experiments or more.Statistical analysis was performed by ANOVA for experiments involving more than two groups.The two-tailed Student's t-test was performed for comparison with two groups.and the p<0.05 was considered significant,*,P<0.05;**,P<0.01.Results(1)Compared with the immortalized non-tumorigenic cell line Endocervical End1/E6E7(CRL-2615)cell line,the expression of miR-146a were 44.3,11.5 fold of that in negative control(NC)group in HeLa and C-33 A cells,respectively(P<0.05).both the IRAK1 and TRAF6 mRNA expression were elevated significantly(P<0.05).(2)After transfected with expression plasmid vector DNA containing either the G or C allele of miR-146a rs2910164(Pre-miR-146a-G or Pre-miR-146a-C),the expression of miR-146a were 333.0,177.6 fold than in negative control(NC)group in CRL-2615,respectively(P<0.05).the expression of miR-146a were 517.0,639.8 fold than in negative control(NC)group in HeLa,respectively(P<0.05).the expression of miR-146a were 307.5,491.4 fold than in negative control(NC)group in C-33 A,respectively(P<0.05).(3)After transfected with miR-146a mimics,the expression of miR-146a were 562.6,599.0 fold in HeLa and C-33 A cells than in CRL-2615,respectively(P<0.05).The results of CCK-8 assay displayed that miR-146a significantly promoted cell growth in HeLa and C-33 A cells(P<0.05).The cell-cycle distribution of HeLa and C-33 A cells showed that cell counts in the G1-phase were significantly decreased in response to miR-146a mimic transfected in HeLa(P=0.048)and C-33 A(P=0.022)cells,compared with NC,whereas the cell population in S-phase was moderately increased(P = 0.018 in HeLa cells and P = 0.046 in C-33 A cells)(P<0.05).CyclinDl in HeLa and C-33 A cells was upregulated(P<0.05).(4)The percentage of early apoptotic cells measured by flow cytometry and found to be no significantly decreased in miR-146a mimic transfected cervical cancer cells(P>0.05).Western blot results revealed that cleaved caspase-3 protein levels were also no decreased in HeLa and C-33 A cells with miR-146a overexpression(P>0.05).(5)We transfected HeLa and C-33 A cells with miR-146a mimics and examined IRAK1 and TRAF6 expression levels,ectopic expression of miR-146a led to a decrease in IRAK1 and TRAF6 mRNA(P<0.05)and protein levels(P<0.05).Conclusion(1)MiR-146a was high expression Cervical cancer cells,the expression of miR-146a was higher in HeLa than C-33 A cells.The expression of miR-146a may be related to the occurrence of cervical cancer caused by HPV infection.(2)MiR-146a precursor SNP(rs2910164)affect the expression of miR-146a,and influence target gene IRAKI and TRAF6 by miR-146a.(3)MiR-146a in cervical cancer cells promotes cell proliferation effects on inhibiting IRAK1 and TRAF6,but have no effect on the apoptosis.(4)MiR-146a may play an important role in the pathogenesis of cervical cancer by promoting the proliferation of cervical cancer.The relationship between miR-146a and HPV infection may provide a new perspective for the prevention and treatment of cervical cancer.