Study Of Effects Of MiRNA-146b-5p On The Invasion And Migration Of Papillary Thyroid Carcinoma Cells Mediated By Wnt/?-catenin Signal Pathway

BackgroundPapillary thyroid carcinoma(PTC)is the most commonendocrine tumor,and the morbidity of the disease accounted for 3% of malignant tumors.In recent years,the global reports about the morbidity of PTCskeep increasing and a latest research indicates the five-year survival rate of PTCs in China only reaches 67.5%,of which the regional invasion and metastasis are principal.Micro RNA(miRNA),non-coding single strand RNA,containing 21~25nucleotides,is a characteristicendogenous RNA of eucaryon.MiRNAsidentify and adjust the transcription of mRNA,managing the representative of related genes,boosting the degradation and inhibitingthe translation process,so as to manage and control the growth,apoptosis proliferation,and differentiation process of cells and tissues.Plenty of studies indicate that the abnormal expression of miRNAs which locate in the tumor associated part of the chromosome firmly relate to the occurrence,development,invasion and metastasis of tumers.Our research group have tested the differential expression profile of PTC tissues and normal thyroid tissues with high flux of Micro RNA microarray biochip technology in the earlier research and verified the differently expressed miRNAs,selecting some miRNAs related to PTC,in which the miRNA-146b-5p upregulates most distinctly.In following research,we found the expression of miRNA-146b-5p increases with the growing stages of PTC which indicates the key role of miRNA-146b-5p in the occurrence and development of PTC.It is neither clear that whether miRNA-146b-5p relates to the invasion and metastasis of PTC nor how miRNA-146b-5p promotes the evelopment of PTC.And a further research about the influence and mechanism of miRNA-146b-5p to the biological behavior of PTC will be of great importance.Purpose Study the relationship between miRNA-146b-5p and the ccurrence and development of PTC and investigate the mechanism of the progress.Methods1.Build overexpression lentivirus plasmid and transfect it into human PTC cell TPC-1 to acquire stable transfected monoclonal cells,testing the expression of miRNA-146b-5p of cells in transfected group.Observe the metastasis of cells in transfected group,empty carrier group and control group with wound healing test and the invasion of cells in these three groups withtranswellchamber invasion assay.2.Use biology information technology to predict the miRNA-146b-5p target genes,confirm the target genes use dual-luciferase reporter gene assay;test the expression of essential proteins and mRNAs such as ?-catenin,Axin and APC in Wnt/?-catenin signal pathway in the transfected group,empty carrier group and control group with Western blot and RT-PCR;block the Wnt/?-catenin signal pathway in transfected group with NF2.Observe the metastasis of cells in the blocked group and control group with wound healing test and the invasion of cells in both groups with transwell chamber invasion assay.3.Test the expression of epithelial-mesenchymal transition(EMT)related proteins and mRNAs E-cadherin?N-cadheri and Vimetin in transfected group,empty carrier group and control group with Western blot and RT-PCR;test the expression of EMT signal pathway related proteins and mRNAs E-cadherin?N-cadheri and Vimetinin the blocked group and control group with Western blot and RT-PCR.Result1.The result of PT-PCR indicates a higher expression of mRNA of mic RNA-146b-5p in the carrier group than control group.The result ofwound healing test indicates a larger number of metastasis cells in the transfected group than empty carrier group and control group with time of 48h(p<0.05);the result oftranswell chamber invasion assay indicates a larger number of penetrating cells in the transfected group than empty carrier group and control group with timeof 48h(p<0.05).2.Biology information technology predict that NF2 may be the target gene of miRNA-146b-5p;Dual-luciferase report display that NF2 is the target gene of miRNA-146b-5p;The result of Western blot and RT-PCR indicates a higher expression of ?-catenin,lower expression of Axin and APC(p<0.05)in the empty carrier group than control group;the result of wound healing test indicates less metastasis cells in the in the blocked group than control group(p<0.05)in the time of 24 h,48h;the result of transwell chamber invasion assay indicates less penetrating cells in the blocked group than control group(p<0.05)in the time of 24 h,48h.3.The result of Western blot and RT-PCR indicates a lower expression of E-cadherin,a higher expression of N-cadherin and Vimetin(p<0.05)in the transfected group than empty carrier group and control group;a higher expression of E-cadherin,lower expression of N-cadherin and Vimetin(p<0.05)in the blocked group than control group.Conlusion1.The miRNA-146-b-5 p through regulating target genes NF2 activation of Wnt/beta-catenin signaling pathway,promote the TPC-1 cell migration.2.miRNA-146b-5p promotes TPC-1 cell invasion and migration by epithelial mesenchymal transition.3.Enhanced expression of NF2 gene in TPC-1 cell can inhibit the Wnt/?-catenin signaling pathway,then EMT progression reverse.