| Background and purposePapillary thyroid carcinoma(PTC)accounts for about 85%of thyroid cancer.It can occur at any age.It is a common and low grade thyroid carcinoma.In recent years,the incidence of thyroid papillary carcinoma is increasing year by year due to the influence of environment,heredity,hormone and so on.MiRNA is a kind of non RNA encoding widely exists in plants,mainly involved in the regulation of gene transcription level,through its mRNA 3 'UTR,complementary degradation of target mRNA or inhibiting the translation,widely involved in various physiological processes,including cell growth,development,proliferation,differentiation and apoptosis.In the study of thyroid cancer,a large number of miRNA have been excavated.Meanwhile,studies have shown that miR-146b,miR-221,miR-222 and miR-135b are all specifically expressed in papillary thyroid carcinoma.It is confirmed that miRNA-637 is expressed in the process of acute cerebral ischemia,and it also inhibits the growth,migration and invasion of glioma cells.However,whether miRNA-637 is associated with invasion and metastasis of papillary thyroid carcinoma is not clear about the mechanism by which it promotes the development of papillary thyroid carcinoma.The potential target genes in this study through the application of miRNA target gene prediction software Target Scan,miRanda miR-637 and miRWalk analysis,found that miR-637 can target AKT3 3 'CCCCCAG sequence of UTR,and the dual luciferase reporter assay system to verify the miR-637 with specific binding to PI3K-AKT3-mTOR signaling pathway and affect the papillary thyroid carcinoma cells the invasion and metastasis.Therefore,we used RT-PCR to detect the expression of miRNA-637 in papillary thyroid carcinoma.Western blot was used to detect the expression of AKT3 protein in PTC,and its correlation with clinical pathology was analyzed.Then the lentivirus mediated transfection of papillary thyroid carcinoma cells,the expression of miRNA-637 in cells,the biological behavior of thyroid papillary carcinoma cells before and after transfection analysis,between the application of molecular biology techniques to verify miRNA-637 and AKT3 targeted regulation,through the knockout of AKT3 gene using RNA interference technology to observe the effect of invasion the migration ability of TPC-1 cells.This study will clarify the mechanism of miR-637 regulating the invasion and metastasis of thyroid cancer cells,and obtain important microRNA to regulate the invasion and metastasis of thyroid cancer cells,so as to provide experimental evidence for targeted therapy of thyroid cancer.Materials and methods1,randomly selected 56 cases of PTC tumor tissues and cancer are more than 2cm non epithelial carcinoma(non-carcerous epithelium,NCE),the expression of RT-PCR miRNA-637 was detected in PTC tumor tissues and adjacent tissues.The expression of AKT3 protein was detected in Western blot PTC in tumor tissue and tumor adjacent tissues the differential expression analysis of miRNA-637 and AKT3 in PTC tumor tissues and adjacent tissues,the expression of miRNA-637 and the relationship between AKT3 level and clinical pathological features,analysis of the relationship between miRNA-637 expression and AKT3 expression;2,The human miR-637 lentivirus overexpression vector was constructed and the TPC-1 cell strain of thyroid papillary carcinoma cell line was established stable over expression of miR-637.3,cell proliferation,flow cytometry,cell cycle and apoptosis,scratch test,invasion and migration test,and plate cloning were used to detect the biological behavior of TPC-1 cells before and after transfection by CCK-8.4,the application of Western blot and PCR were detected by overexpression of miR-637 transfection group,empty vector group and control group in PI3K-AKT3-mTOR cell signaling pathway key protein AKT3 expression;through the application of RNA interference AKT3 gene knockout effect by scratch test and Transwell chamber invasion assay.After silencing AKT3 on TPC-1 cells migration and invasion.5.Data analysis is carried out by SPSS17.0 software.The ratio of two samples is compared with four grid data.A single factor analysis of variance was used for the chi square test.The quantitative data of two samples were t.The difference of P<0.05 was statistically significant.Result1,MiR-637 is low in thyroid papillary carcinoma tissue,and its expression is closely related to TNM stage,pathological grading and lymph node metastasis.The expression of miR-637 is negatively correlated with AKT3 expression in papillary thyroid carcinoma.2,Through the restriction endonuclease identification and DNA sequencing analysis,the plasmid of GV369-miR-637 lentivirus expression vector was successfully constructed.GV369-miR-637-TPC-1 and GV369-NC-TPC-1 cells emit green fluorescence under the fluorescence microscope,and the transfection efficiency is high.3,the CCK-8 results showed that the inhibition of TPC-1 cells stably overexpressing miRNA-637 after proliferation;cell cycle analysis showed that over expression of miRNA-637 TPC-1 cells in G1 phase increased by flow cytometry,S phase decreased;the detection of cell apoptosis after transfection showed that over expression of miRNA-637 promotes cell apoptosis;scratch test the experimental results show that the stable cell migration and migration capacity of over expression of miRNA-637 in TPC-1 cells decreased significantly.4,we detected the transfection and expression of PI3K-AKT3-mTOR signal pathway group and the control group and the control group of AKT3 protein in the cells,the results showed that the expression of AKT3 protein in the transfection group decreased,indicating that miR-637 in papillary thyroid carcinoma cell had inhibited the expression of PI3K-AKT3-mTOR signaling pathway.After knocking the AKT3 gene of TPC-1 cells,the invasiveness and migration ability of TPC-1 cells was found to be decreased by scratch test and Transwell cell invasion test.conclusion1,MiR-637 may play an important role in the occurrence and development of papillary thyroid carcinoma.There is a negative correlation between the expression of miRNA-637 and AKT3 protein in papillary thyroid carcinoma specimens,suggesting that both can participate in the development of thyroid cancer.2,the lentivirus can infect the TPC-1 cell line and can be stably expressed in the TPC-1 cells.3,TPC-1 cells overexpressing miR-637 through lentivirus,TPC-1 cells transfected after transfection.The inhibition of physical behavior is further confirmed that miRNA-637 has the role of tumor suppressor gene.4,AKT3 is the target gene of miR-637,and miR-637 regulates AKT3 at the level of mRNA and protein.The expression of TPC-1 decreased after silencing AKT3 cell invasion and migration,the PI3K-AKT3-mTOR pathway mediated the effects of MiRNA-637 on migration and invasion of thyroid papillary carcinoma cells,and provide a theoretical basis for gene targeting therapy to reduce the invasion and metastasis of papillary thyroid carcinoma. |
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