MiR-1975 Suppresses The Growth Of Cervical Carcinoma Hela Cells

[Background and Aims]MicroRNAs(miRNAs)are a class of small non-coding RNAs that include 18-26 nucleotides,which post-transcriptionally regulate gene expression.MiRNAs are involved in many physiological and pathological progress.Recent evidence indicates that many miRNAs function as oncogenes or tumor suppressors and play an important role in cancer initiation and progression by regulating their target genes negatively.Using Real-time PCR,we found that microRNA-1975 were significantly up-regulated in human cervical carcinoma HeLa cells which were transfected with P53.In this study,we focused on the effects of miR-1975 on the phenotypes of cervical carcinoma HeLa cells as well as the identification of their direct target genes,in order to illuminate the molecular mechanisms of miR-1975 in the initiation and progression of human cervical carcinoma.[Methods]In order to verify the result of gene chip,we detected the expression of miR-1975 in HeLa cells which were transfected with P53 by real-time PCR.The changes of cell phenotypes were detected with MTT assay,colony formation assay and cell growth curve with the up-regulation of miR-1975 in HeLa cell.Subsequently,we combined bioinformatics and BLAST analysis and identified the candidate target genes for miR-1975.Then fluorescent reporter assay was performed to confirm the reliability of the direct target genes.Furthermore,the mRNA levels and protein levels of target genes in miR-1975 overexpressed cervical carcinoma cells were detected with Real-time PCR and Western blot,in order to confirm the regulative role of miR-1975 on WZ expression.Finally,the target gene was knocked down using RNA interference and changes of cell phenotypes were detected both with MTT assay,colony formation assay and cell growth curve..[Results]Real-time PCR results showed that the expression of miR-1975 was elevated when the P53 expression level was up-regualted.We report that after overexpression of miR-1975,the cell viability,the colony formation activity and cell growth activity of HeLa cells were both inhibited.Subsequently,we identified oncogene Homo sapiens hypothetical protein LOC100292005(LOC100292005)which short for WZ as congenerous candidate target genes for miR-1975.The WZ mRNA CDS(coding sequence)contains the potential binding site both of miR-1975.The fluorescent reporter assay also confirmed that miR-1975 can directly bind to the specific site of WZ mRNA CDS and negatively regulate the gene expression.When miR-1975 was over-expressed in HeLa cells,mRNA level and protein level of WZ were both depressed.We also discovered that when the target gene was knocked down,the cell viability,colony formation activity and cell growth activity of cervical carcinoma cells were both inhibited apparently,consistent with the results of the overexpression of miR-1975.[Conclusions]Our results show that in HeLa cells,miR-1975 functions as a tumor suppressor and inhibits cell proliferation.The over-expression of miR-1975 lead to an abnormally low expression level of oncogene WZ and an decreased ablility of cell proliferation in HeLa cells.The elucidation of the mechanisms of miR-1975 in cervical carcinoma helps us to further understand the mechanism of cervical cancer initiation and progression,and pave a way for clinical diagnosis and therapy of cervical carcinoma.