| Background and purpose:Papillary thyroid carcinoma(PTC)is the most common endocrine malignancy.The incidence of PTC has been increasing rapidly all around the world in recent years,PTC is relatively inert,most of which can be cured if it is detected early and treated regularly.However,in clinical work,it was noted that some of the small cancers were associated with a wide range of metastasis and poor prognosis.Therefore,how to achieve accurate early diagnosis,to find the risk factors predicting for tumor invasion,metastasis,recurrence,to find a more effective treatment procedure,has become an urgent problem to be solved.The purpose of this study includes two aspects:Firstly,Through analyzing BRAFV600 E mutations and miRNA363 expression,it is aimed to clear whether the BRAFV600 E mutation is associated with high risk tumor behavior and whether double molecular detection can be used as an index for early diagnosis of papillary thyroid microcarcinoma;Secondly,Our goal is to study the effect of miRNA363 on TPC-1 cell proliferation,differentiation,metastasis using papillary thyroid carcinoma cell line(TPC-1 cells),to look for potential new targets for the treatment of PTC.Part one : Expression of miRNA363 and BRAF in papillary thyroid microcarcinoma PurposeOur goal was to analyze the association between clinicopathologic characteristics and BRAF mutation in papillary thyroid microcarcinoma,analyze the association between of BRAF mutation and miRNA363 expression in papillary thyroid carcinoma and adjacent normal tissue and benign nodular goiter,analyze the value of miRNA363 and BRAFV600 E in the early-diagnosis of papillary thyroid microcarcinoma.MethodParaffin specimens from 62 patients with papillary thyroid microcarcinoma who underwent surgery from January 2016 to February 2017 were enrolled,All patients were operated by the same surgeon.Information including sex,age,family history,multifocal,capsule invasion,lymph node metastasis,and TNM were collected;PCR was used to detect the mutation of BRAFV600 E.Fresh specimens were collected from another 49 patients,including 26 cases of papillary thyroid microcarcinoma,23 cases of benign thyroid tumors or the adjacent normal tissues,Information of their sex,age,family history,multifocal,capsule invasion,lymph node metastasis,and TNM;miRNA363 expression and BRAFV600 E mutation was evaluated by PCR.ResultThere were 13 males and 49 females with an average age of 45.8 years,30 patients had lymph node metastasis;A single lesion occurred in 37 patients,and 25 cases had multifocal thyroid disease.There were 15 patients having capsular invasion,13 cases having family history,BRAFV600 E mutation was noted in 44 cases,with a rate of 71%.Statistical analysis showed that there were no differences in sex,family history,multifocal,capsule invasion,lymph node metastasis,and TNM in patients with BRAF600 E mutation and without mutation except age.A total of 49 fresh specimens including 26 papillary thyroid carcinoma specimens and 23 thyroid benign tumors and normal tissue specimens were collected for detecting miRNA363 and BRAF mutations.The level of miRNA363 expression was markedly decreased in all papillary thyroid microcarcinoma,but was not significantly related to BRAF mutant.The accuracy of the two molecular assays was further analyzed.BRAFV600 E was 87.8%,and miRNA363 was 100%.Summary: 1.The rate of BRAFV600 E mutation is higher in papillary thyroid microcarcinoma,but was not significantly related to common risk factors except age.2.Low expression of miRNA363 was common in papillary thyroid carcinoma,not correlated with the mutation of BRAF.The level of miRNA363 expression has high accuracy.If it combined with BRAFV600 E mutation detection,the early diagnosis rate of papillary thyroid microcarcinoma can be improved.Part two: Mechanism of miRNA363 in the progression of thyroid papillary carcinoma PurposeOur goal was to study the effect of miRNA363 on the proliferation and differentiation and metastasis of thyroid papillary carcinoma cells(TPC-1),and to explore the mechanism of the influence of the cell.For providing a theoretical basis for miRNA363 as a target gene for the treatment of papillary thyroid carcinoma.Method1.Culturing TPC-1 cells in good condition,up regulation of miRNA363 expression by lentivirus which was observed by fluorescence microscope under fluorescent microscope.2.Real time quantitative PCR(RT-PCR)was used to detect the expression of miRNA363 in TPC-1-miRNA363 cell line,blank plasmid control cell line(TPC-1-LV)and blank cell line(TPC-1-NC).3.Cell viability was detected by MTT assay to verify the effect of target gene on cell proliferation.4.Effect of migration through the detection of containing target gene to the cell culture medium containing serum in the Transwell cell to verify the gene transfer ability of cells.5.The migration ability of tumor cells in experimental group and control group was observed by scratch test.6.The ability of clone formation of the cells in the cell culture plate after infection was compared with that of the cells in the experimental group and the control group.7.Using the bioinformatics,prediction and screening of CACNA1 C of Calcium Sinaling Pathyway and DUSP10 of MAPK is the target gene of miRNA363,construct luciferase plasmid miRNA363 over expression plasmid,CACNA1 C 3-UTR,DUSP10 3-UTR,CACNA1 C 3-UTR-Mu,DUSP10 3-UTR-Mu,after the transfection,after processing,determination and calculate the relative luciferase activity groups.8.Western-Blot method was used to detect the expression of CACNA1 C and DUSP10 protein in experimental group and control group.Result1.TPC-1 cells were cultured by 1640+10%FBS medium,According to the experimental method,the empty plasmid and miRNA363 plasmid vector were transfected successfully.The cells grew well.2.The results of qPCR showed that the expression of miRNA363 was significantly higher in the experimental group.3.TPC-1 cells infected by lentivirus were cultured for 5 days.After MTT treatment for a period of 4 hours,the absorption rate of 490 nm in each group changed with time were recorded.Compared with TPC-1-NC and TPC-1-LV control group,The proliferation rate of TPC-1-miRNA363 group was was significantly inhibited,analyzed by T-test,P<0.05.The contrast between the two control groups,P > 0.05,no significant difference in cell value.4.Transwell experiments showed that the cell counts in the 200 X microscope,The average TPC-1-NC and TPC-1-LV control group were 193 and 185,respectively,The count of the TPC-1-miRNA363 group was 81,and the number of cells in the experimental group was decreased.5.Scratch test,the infected cells were cultured,according to the experimental method of scratch,Compared with control group,The TPC-1-miRNA363 group decreased migration ability.6.Clone formation assay,The number of clone formation of TPC-1-NC group and TPC-1-LV group was 202 and 198,respectively,and the number of TPC-1-miRNA363 group was 188,The number of TPC-1-miRNA363 group was reduced.7.Luciferase assay showed that miRNA363 specifically cut the expression of CACNA1 C and DUSP10 genes.8.The results of Western-Blot showed that miRNA363 could specifically degrade the expression of CACNA1 C and DUSP10 protein after miRNA363 overexpression in TPC-1.Summary1.miRNA363 is a tumor suppressor gene in TPC-1,which can inhibit the growth of TPC-1,It has a certain influence on the metastasis of tumor cells.2.By the specific targeting of the 3′terminal untranslated region of the CACNA1 C gene in the Ca signaling pathway and the DUSP10 gene in the MAPK signaling pathway,miRNA363 leads to the degradation of mRNA,which leads to the failure of protein translation.3.As a tumor suppressor gene in TPC-1,miRNA363 may be used as a target gene for the treatment of papillary thyroid carcinoma in future.Conclusion:1.In papillary thyroid microcarcinoma,BRAFV600 E mutation rate is higher,but there is no significant difference between the patients with high risk factors and those with low risk factors except age.Low expression of miRNA363 is common in papillary thyroid carcinoma,not correlated with the mutation of BRAF.The level of miRNA363 expression has high accuracy.If it combined with BRAFV600 E mutation detection,the early diagnosis rate of papillary thyroid microcarcinoma can be improved.2.miRNA363 as a tumor suppressor gene,by the specific targeting of the 3 terminal untranslated region of the CACNA1 C gene in the Ca signaling pathway and the DUSP10 gene in the MAPK signaling pathway,leads to the degradation of mRNA,the failure of protein translation and the growth inhibition of TPC-1,which may be used as a papillary thyroid carcinoma targeted gene. |
PDF Full Text Request