MicroRNA-372Affects Cervical Carcinoma Cell Growth By Targeting Multiple Cell Cycle Related Genes

[Aims] MicroRNAs (miRNAs) are a wide class of small, noncoding RNAs that negatively regulate gene expression at the post-transcriptional level and involve in many physiological and pathological processes. Recent evidence indicates that miRNAs play a critical role in cancer initiation and progression. In the previous study, we showed that HepG2cell cycle can be arrested and cell growth was inhibited when AFP was silenced. The high expression of microRNA-372(miR-372) may contribute to AFP-mediated cell cycle regulation. This paper is to further study the expression level of miR-372in cervical carcinoma and the effects of miR-372on cervix cancer cell growth and cell cycle. To explore which genes participate in miR-372-mediated tumor suppression, we identified its direct target genes. This study enriches our understanding of the regulation mechanisms of miR-372on cancer cell, providing more molecular mechanisms and a new therapy choice for cancer.[Methods] Quantitative real time PCR was performed to detect the expression level of miR-372in eight pairs of cervical carcinoma tissue samples and matched normal cervical tissue samples. We overexpessed and/or blocked miR-372in human cervix cancer cell HeLa, then used quantitative real time PCR to ascertain the changes of miR-372. We observed cell growth and cell cycle process using growth curve and FACS, respectively. According to bioinformatics analysis, cDNA array and the known gene function, we screened the candidate target genes of miR-372. The mRNA and protein expression of the target genes was detected by RT-PCR and Western blot. Furthermore, fluorescent reporter assay was used to confirm that miR-372regulate the expression of the target genes by binding to their3’untranslated region (3’UTR).[Results] miR-372is down-expressed in cervical carcinoma tissue, compared to normal cervical tissue. The over-expressed of miR-372inhibited HeLa cell growth, while miR-372blocking increased cell growth speed. FACS showed that miR-372overexpression caused cell cycle arrest that more cells accumulate in S and fail to entry into G2stage. Conversely, blocking miR-372made S into G2phase. CDK2, cyclin A1and PPP6C were screened to be target genes, whose3’UTR contained the potential binding sites of miR-372. High level of miR-372deregulated the mRNA and protein expression of the target genes, while miR-372blocking upregulated the mRNA and protein expression of the target genes. The fluorescent reporter assay confirmed that miR-372can directly bind to the specific site of target genes3’UTR and negatively regulate their expression.[Conclusions] Our results indicate that in HeLa, miR-372can directly target cell cycle regulators-CDK2, cyclin A1and PPP6C, then regulate the cell cycle through altering the expression of these target genes. We fully confirmed that miR-372induced an accumulation of S phase and inhibited the process of S to G2phase, which is the key factor of miR-372inhibiting the growth speed of HeLa. In additon, miR-372is down-expressed in cervical carcinoma tissue. Then, we support that miR-372plays a role as a tumor suppresor in cervical carcinoma. Thus, gene regulation function of miR-372enriched the content of miRNA and tumor, which provided a new clue to the research of cancer initiation, progression, diagnosis and therapy.