MiRNA-146b-5p And Autophagy Of Papillary Thyroid Cancer

BackgroundMicro RNAs represent a class of non-coding RNAs acting as posttranscriptional gene expression regulators by inhibiting translation or destabilizing m RNAs,highly conserved in structure,concluding 19-25 nucleotides.The binding area of m RNAs distribute in 5'-UTR,coding zone,promoter besides 3'-UTR,which means one single m RNA regulates hundreds of genes to participate in the process of cell proliferation,differentiation,aging and apoptosis.Thyroid gland carcinoma is the most frequent endocrine system malignancy and top the cancer rank developing fastest.Papillary thyroid carcinoma(PTC)is the most frequent thyroid carcinomas(more than 90%)with a lower risk and survive well after standard surgical treatment and postoperative therapy.Autopagy represents a active process,in which cell digests itself to acquire necessary material in a state of stress or hunger,presenting some double membrane structure containing large number of proteins(long live proteins)and organelles in the cytoplasm digested to be necessary material in order to satisfy the metabolic needs,complete organelle transformation and renewal and maintain internal stability.Autophgy relates to many diseases including tumer effected in two opposite ways by autophagy in the process of tumer development.In the previous study,we have selected differently expressed Mi RNAs between PTC tissues in different stages and paracancer tissues with high-throughput micro RNA microarrays and figure out that expression of mi R-146b-5p is higher in the early and middle-late stage of PTC tissues increasing with the tumer stages indicating that mi R-146b-5p may play a key role in the occurrence,development and metastasis of PTC.The following study have confirmed mi R-146b-5p promote theproliferation,migration and invasion of PTC.ObjectOn the basis of previous studies about the relation of mi R-146b-5p and the biological behavior of TPC-1,furtherly,the study aims at exploring the relation of mi R-146b-5p and the autophagy of TPC-1 which lay a theoretical foundation to study the relation of autophagy and the biological behavior of TPC-1 and whether mi R-146b-5p affect the biological behavior of TPC-1 through the path of autophagy.Method 1.Construction of stable tranfected TPC-1 cell monoclone with over expressed and depleted mi R-146b-5p.(1)Construction of overexpression of mi R-146b-5p lentivirus plasmid(2)The overexpressed mi R-146b-5p lentivirus plasmid was transfected into TPC-1 cells,to acquire stable overexpressed mi R-146b-5p cell monoclonal.(3)The previous study has constructed depleted mi R-146b-5p TPC-1 2.To explore the connection between mi R-146b-5p and TPC-1 autophagy(1)Groups:micro-up group;TPC-1 group;micro-down group.(2)Observe the autophagosome of the individual groups in transmission electron mircroscope(3)Compare the difference of LC3 point-like aggregation of the individual groups with Immunofluorescence(4)Compare the difference of LC3?/?of the individual groups with western blot(5)Compare the difference of p62/SQSTM1 of the individual groups with western blotResult(1)Transmission electron microscopy shows autophagy is mostly frequently observed in the micro RNA-down group which is higher than the micro RNA-normal group and micro RNA-up.group is the least.(p<0.05)(2)WB test shows differences of LC3-II/I in the three groups:micro RNA-down>micro RNA-normal> micro RNA-up.(3)WB test shows differences of p62/SQSTM1 : micro RNA-down<micro RNA-normal<micro RNA-up.(4)Immunofluorescence test shows the different levels of the membrane LC3? cluster: micro RNA-down>micro RNA-normal> micro RNA-up.ConclusionThe above results suggest that micro RNA-146b-5p can inhibit the autophagy in TPC-1 cells,but by what path and with what target genes it produces this effect and how it can be linked to other biological properties of TPC-1 by this action need further study.