Mir-181b Targets Adenylyl Cyclase 9(AC9) And Participates In The Positive Feedback Regulated By NF-κB In Cervical Carcinoma Cells

[Aims]MicroRNAs(miRNAs)are a class of small non-coding RNAs that post-transcriptionally regulate gene expression and involved in many physiological and pathological progresses.Recent evidence indicates that special miRNAs may function as oncogenes or tumor suppressors and play a critical role in cancer initiation and progression by negatively regulating their target genes.Using MTT assay,we found that microRNA-181b(miR-181b)promotes the growth of cervical carcinoma cells.In this study,we focused on the effects of miR-181b on the phenotypes of cervical carcinoma cells as well as the identification and effects on the phenotypes of cervical carcinoma cells of the direct target genes of miR-181b,and research the relationship between miR-181b and NF-κB,in order to illuminate the molecular mechanisms of miR-181b in the initiation and progression of human cervical carcinoma.[Methods]We detected the changes of cell viability and growth effeted by miR-181b with MTT assay and colony formation assay.Subsequently,we used bioinformation and identified the candidate target genes for miR-181b.The reliability of the direct target genes was confirmed by fluorescent reporter experiment.Furthermore,the mRNA levels and protein levels of target genes in miR-181b-enhanced cervical carcinoma cells were detected with real-time PCR and Western blot,in order to confirm the regulating role of miR-181b in target gene expression.Then,the function of target gene was inhibited in human cervical carcinoma cell line and the changes of cell phenotypes were detected with MTT assay and colony formation assay.We used TUNEL assay to search the effects of target gene to apoptosis.Finally,we assumed AC9-cAMP axis participated in the poaitive loop between NF-κB and miR-181b and study the changes of effectors in the loop and cell phenotypes by inhibiting NF-κB activation.[Results]Experimental results showed that after overespression of miR-181b,the cervical carcinoma cell proliferation activity and the colony formation activity were both improved.Subsequently,we identified tumor related gene Adenylyl cyclase(AC9)as one of the candidate target genes for miR-181b.The AC9 mRNA 3’-untranslated region(3’UTR)contains the potential binding site of miR-181b.The fluorescent reporter experiment also confirmed that miR-181b can directly bind to the AC9 mRNA 3’UTR and negatively regulate the gene expression.After overexpression of miR-181b in cervical carcinoma cells,mRNA level and protein level of AC9 were both descended.What’s more,AC9 promoted apoptosis of cervical carcinoma cells.We also found that after knockdown of AC9,the cell proliferation activity and the colony formation activity were both improved.Finally,after inhibiting NF-κB activation,the downstream effectors and miR-181b were downregulated,AC9 upregulated,and cell viability declined,which were similar as overexpression of AC9.[Conclusions]We provide evidence that miR-181b restricts cAMP production by targeting AC9 and that the pathway participates in the positive feedback loop between miR-181b and NF-κB.This new mechanism of the regulation of miR-181b expression should aid in the identification of potential targets for therapy.