Regulation Of IC-N-miR-20 On The Malignant Behavior Of Human Cervical Cancer Cells

【Objective】Cervical cancer(CC)is one of the most common malignant tumor in women.It mainly occurs in developing countries with high mortality.Studies have shown that the occurrence and development of CC was mainly associated with human papillomavirus(HPV)infection.At the same time,a large number of studies have proved that the persistence of nonresolving inflammation plays an important role in the initiation,maintenance and promotion of tumorigenesis.Nonresolving inflammation can not only promote tumorigenesis,but also participate in tumor progression,escape,angiogenesis and metastasis.However,the molecular mechanism of inflammation causing malignant tumor is still unclear.It is well-known that micro RNAs(mi RNAs)mainly regulate the expression of gene and affect disease progression at the post-transcriptional level.In order to explore the relationship among mi RNA,inflammation and CC,He La cells treated with tumor necrosis factor alpha(TNF-α)and 5-Aza-2’-deoxycytidine(5-Aza-Cd R)were analyzed by deep sequencing.Among these differentially expressed mi RNAs,we chose a new mi RNA,which expressed significantly increased and was named as IC-N-mi R-20,for subsequent research.The purpose of this study is to explore the effect of IC-N-mi R-20 on malignant behaviors of cervical cancer and its detailed mechanism,so as to provide a new molecular target for early detection,diagnosis and treatment of cervical cancer.【Method】First,we selected 10 differentially expressed mi RNAs according to the results of deep sequencing,and verified the expression of these mi RNAs in He La cells treated with TNF-α and 5-Aza-Cd R by RT-q PCR.Then,IC-N-mi R-20 biosynthesis was further confirmed through knocking down the expression of Dicer and Drosha in He La cells.Next,colony formation assay,MTT assay,transwell migration/invasion assay and Western blot were performed to investigate the effect of IC-N-mi R-20 on malignant behaviors of cervical cancer cells.Furthermore,Target Scan Human 5.2 Custom was used to predict the target gene of IC-N-mi R-20.The EGFP fluorescent reporter experiment was used to verify the relationship between IC-N-mi R-20 and target gene,meanwhile,the regulatory effect of IC-N-mi R-20 on the target gene was confirmed by RT-q PCR and Western blot at the m RNA and protein level.Finally,the function of target gene was verified by cell function experiments.The rescue experiment was applied to evaluate whether IC-N-mi R-20 could directly regulate the expression of target gene to affect the malignant behaviors of cervical cancer cells.【Results】RT-qPCR results showed that the expression of IC-N-miR-20 was significantly increased with the treatment of TNF-α and 5-Aza-Cd R,and its formation was dependent on the classical mi RNA biosynthesis pathway.Cell functional experiments showed that over-expression of IC-N-mi R-20 could promote colony formation ability and migration/invasion ability of He La and Si Ha cells,and Western blot assays demonstrated that IC-N-mi R-20 could down-regulate the expression of E-cadherin and up-regulate the expression of vimentin,thus promoting the EMT process,which suggested that IC-N-mi R-20 may function as an oncogene in cervical cancer.Bioinformatics predicted that IC-N-mi R-20 had a potential binding site in SOCS6 3’ untranslated region.The EGFP fluorescent reporter experiment confirmed that IC-N-mi R-20 could directly target SOCS6.At the same time,RT-q PCR and Western blot assays showed that IC-N-mi R-20 could negatively regulate the expression of SOCS6 at m RNA and protein level.SOCS6 had an inhibitory effect on the colony formation ability,migration/invasion ability and EMT process of cervical cancer cells,which was contrary to the effect of IC-N-mi R-20.Finally,the rescue experiment between IC-N-mi R-20 and SOCS6 further verified that IC-N-mi R-20 promoted malignant behaviors of cervical cancer cells by negatively regulating the expression of SOCS6.【Conclusions】This study confirmed that IC-N-mi R-20 promoted the colony formation,migration and invasion of cervical cancer cells by negatively regulating SOCS6 expression,thus promoting the EMT progress.Therefore,our findings may provide a new molecular basis for the diagnosis and treatment of cervical cancer.