Research Of The MiR-130a-TNF-a-NF-?B Pathway In Cervical Cancer Cell

Objective:NF-?B as a member of the transcription factors family,is the most important intracellular nuclear transcription factor,in a variety of signal transduction and regulation mediated by cell stimulation,NF-?B play a central role,it involved in a large number of genes expression and regulation,it is an important indicator of cell activation.Studies have shown that NF-?B exhibit different ability in different cells in the regulation of cell proliferation,and it is able to induce abnormal expression of multiple miRNA.MicroRNAs(miRNAs)are a class of single-stranded non-coding RNAs with a size of 21-24 nucleotides encoded by an endogenous gene,they involved in the regulation of gene expression at the post-transcriptional level in plants and animals,and the adjusting role involves the growth and development,cell differentiation,apoptosis,fat metabolism,and many other life processes.Numerous studies show that in tumor cells,there are big differences in the expression of miRNAs compare with normal tissue,this prompts us miRNAs inevitable play acertain role in the development of tumor.This study we mainly study how does NF-?B affect cell growth and verify whether have microRNAs downstream is involved in the regulation,researching the deeper mechanism of the regulatory pathways.Methods:We adopted MTT and colony formation assay to detect the growth activity and multiplication capacity in HeLa and C33A cells.After confirmed that NF-?B is associated with cervical cancer cell growth,we used real-time quantitative PCR to detect miR-130a,one microRNA may be affected by the change in the expression level of NF-KB.Next,we try to study miR-130a in the proliferation of cervical cancer cells and we still using the MTT assay and colony formation assay to detect.Afterwards,we use bioinformatics methods predicted its possible targets and chose the gene closely related to tumor growth used for the fluorescent reporter vector experiment to determine the target function.Followed by real-time quantitative PCR and Western blot experiments,we detect the mRNA and protein level of target gene in HeLa and C33A cells with different miR-130a level,which further prove the regulation of miR-130a.Then we constructed a target gene overexpression plasmid without the 3'UTR,transfected the plasmid into cells,through the change in the cells phenotype,llustrate the miR-130a regulatory mechanism of cell growth.Finally,we over expressed the NF-?B plasmid,by Western blot,detected the target gene expression.And then,stimulate cells with purification target gene encoding protein,detecting its stimulation of NF-?B,checking whether a feedback regulation is exists.Results:MTT and colony formation assay described NF-?B increase cell growth ability,Real-time quantitative PCR showed an increase in miR-130a follow the overexpression of NF-?B.Cell growth experiment show that overexpression of miR-130a promote the growth and proliferation of cervical cancer cell and knock down miR-130a get the opposite results.After gene screening,we select TNF-alpha as the candidate target and its 3 'non-translated region contains a potential miR-130a binding sites.Fluorescent reporter vector experiments confirmed that miR-130a can inhibit the expression of the reporter gene with a TNF-alpha binding site.Real-time quantitative PCR and Western blot results also showed that miR-130a can suppress TNF-alpha mRNA and protein levels.The gene function studies showed that overexpression of TNF-alpha can reduce the raising of cell viability and proliferative capacity caused by miR-130a.Western blot analysis showed that overexpression of NF-?B reduced the TNF-alpha level,and immunofluorescence results suggest that TNF-alpha can make more NF-?B into the nucleus so increased the activation of NF-?B.Conclusion:NF-?B can promote the growth of cervical cancer cells,and the mechanism may like NF-?B go into the nucleus cause the improvement of miR-130a thereby inhibiting the TNF-alpha expression,but the TNF-alpha reduction caused NF-?B weakening the ability to enter the nucleus,all these form a negative feedback regulation mechanism.