| Objectives:Screening differentially expressed genes between thyroid papillary carcinoma(PTC)and thyroid follicular carcinoma(FTC)based on bioinformatics to explore the potential causes of high metastatic rate of thyroid follicular carcinoma,combined with survival analysis to determine the prognostic impact of differential genes on follicular thyroid carcinoma Further screening,through tissue verification and cell experiments to verify its biological function in FTC cells.Combined with KEGG enrichment analysis to explore the main differential signaling pathways leading to the malignant biological behavior of FTC,and verify their function in FTC,to further clarify the regulatory relationship between differentially expressed genes that affect survival and this signaling pathway.Methods and materials:1.Database retrievalComprehensively screen the GEO database,read the relevant datasets in detail,and process the datasets that meet the inclusion requirements in the next step.Using the GEO2R function,the data of the selected datasets were analyzed,and the differential genes of the three datasets were extracted.cBioPortal(http://www.cbioportal.org/)is an open platform to explore multidimensional cancer genome data based on TCGA database,including 516 samples of survival information of FTC patients.The overall survival rate(OS)was adjusted according to sex,age and tumor stage.Calculate and display the log rank P value.According to the survival rate of further screening to determine the prognosis of FTC has a clear role in the differential gene to carry out a part of the test expression and functional verification.2.Clinical tissue validationAccording to the inclusion criteria,FTC and PTC pathological sections were obtained from the pathology department of Qilu Hospital,Shandong University.This study was reviewed by the ethics committee of Qilu Hospital,Shandong University,and all patients had informed consent.The expression of DUSP5 in FTC and PTC was confirmed by immunohistochemistry.3.To investigate the migration and proliferation of FTC 133 mediated by DUSP5 in vitroHuman thyroid follicular carcinoma cell line(FTC-133)was divided into the following four groups:A:DUSP5 overexpression plasmid transfection,B:empty plasmid control transfection,C:DUSP5 small interference transfection.D:FTC-133 was transfected empty small interference transfection.western blotting and qRT-PCR were used to detect the expression of DUSP5 afer transfected with four groups,and the transfection effect of DUSP5 was confirmed.EdU assay was used to detect the ability of proliferation,Transwell assay was used to detect the ability of migration and invasion,and western blotting was used to detect the expression of EMT related proteins at the same time.4.KEGG enrichment analysis identified the key pathway of FTC and PTC differential genes and conducted in vitro verificationFurther KEGG enrichment analysis of differentially expressed genes in the three datasets found that the MAPK signaling pathway was significantly enriched in the three datasets,suggesting that the MAPK signaling pathway plays an important role in mediating the difference in biological behavior between FTC and PTC.We used Edu and Transwell inhibitors of the three sub-pathways in the MAPK signaling pathway to verify the role of the three sub-pathways(ERK MAPK,JNK,p38 MAPK)in FTC cells.Further,western blotting was used to detect the activation of the three sub-pathways of MAPK signaling pathway after intervention of DUSP5 expression.Subsequently,Edu and Transwell identified the effects of DUSP5 and its activated MAPK sub-pathway on cell proliferation,migration and invasion.5.Statistical analysisStatistical analysis:the data was showed by mean ąSE,and statistical analysis software was used SPSS 22.0.The data between the different groups were compared by the method of non paired t-test.If P<0.05,it is considered statistically significant(bilateral),if P<0.01,it is considered statistically significant(bilateral).Results:1.Comprehensive analysis results of the database:Three datasets of GSE27155,GSE53157 and GSE29315 met the inclusion criteria,including 26 FTC samples and 67 PTC samples.After differential gene analysis by GEO2R tool and summary by Venn(Figure 1.2),26 genes(see Table 1)were found to be differentially expressed in these three databases,and the screening process is shown in Figure 1.Two of the 26 differentially expressed genes were up-regulated in FTC and 24 were down regulated(Figure 1.3).Based on the survival analysis of 26 common differential genes with the original data of thyroid cancer patients in cBioPartol database,we found that DUSP5 of these 26 common differential genes had a significant impact on the survival rate of thyroid cancer(Figure 1.4).2.To verify the expression of dusp5 in pathological tissues:Immunohistochemistry and RT-PCR results showed that DUSP5 expression was rare in the tissues of FTC group,while there was more DUSP5 expression in PTC group(Figure 2.2,2.3)(P<0.05).The same results were obtained in tissue microarray(Figure 2.1)(P<0.05).3.Transfection DUSP5:Transfection DUSP5 overexpression plasmid,small interference and corresponding blank control in FTC-133 cell line.western blottingting,RT-PCR to assess the transfection effect,the results showed that the expression of DUSP5 was increased in the overexpression DUSP5 group with protein level and mRNA level,compared with the empty plasmid control group(P<0.05).The expression of DUSP5 was decreased in small interference group compared with the control group(Figure 3.1)(P<0.05).4.Edu assay was used to detect the cell proliferation,migration and invasion ability:Compared with the empty plasmid group,the ability of proliferation,migration and invasion in the overexpression DUSP5 group was decreased(P<0.05);compared with the control group,the cell proliferation,migration and invasion ability was increased in the DUSP5 downregulation group(Figure 4.1-4.3)(P<0.05).5.Cell migration and invasion related protein expression:western blotting verified the expression of cell migration protein.The results showed that the expression of MMP9 and Vimentin were decreased in the DUSP5 upregulation group compared with the control group,however,E-cadherin was increased;compared with the control group,the expression of MMP9 and Vimentin in the group of siDUSP5 increased,the expression of E-cadherin was decreased.(Figure 5.1)(P<0.05).6.KEGG enrichment analysis and screening of key signal pathways:The KEGG enrichment analysis of differentially expressed genes in the three datasets of GSE27155,GSE53157 and GSE29315,respectively,found that the MAPK signaling pathway was significantly enriched in the three datasets(Figure 6.1-6.3).7.The effect of MAPK signaling pathway on FTC:Inhibitors inhibit the three sub-pathways of the MAPK signaling pathway(ERK MAPK,JNK,p38 MAPK)(Figure 7.1).EdU verified cell proliferation and Transwell verified cell migration and invasion.The results showed that inhibition of ERK MAPK and JNK signaling pathways can significantly inhibit the proliferation,migration and invasion of FTC-133 cells,while p38 MAPK signaling pathway Wu influences(Figure 7.2-7.4).8.Verify the regulation of DUSP5 on the MAPK signaling pathway:Overexpression plasmid and small interference transfection interfered with the expression of DUSP5.western blotting was used to detect the activation of ERK MAPK,JNK,and p38 MAPK signaling pathways(Figure 8.1).The results showed that down-regulation of DUSP5 could activate ERK MAPK and JNK signaling pathways.9.DUSP5 affects FTC proliferation,migration and invasion through the ERK MAPK/JNK signaling pathway:Based on the down-regulation of DUSP5 expression,inhibitors of ERK MAPK and JNK signaling pathways were given.In the "rescue" experiment,EdU and Transwell found that inhibition of ERK MAPK and JNK signaling pathways can reduce cell proliferation,migration,and invasion caused by DUSP5 down-regulation(Figure 9.1-9.3).Conclusion:1.DUSP5 is closely related to the survival period of FTC patients,which suggests that dusp5 may be a new prognostic marker of FTC.2.In vitro experiments show that overexpression of DUSP5 in FTC-133 can inhibit cell migration,proliferation and invasion,while inhibition of the expression of DUSP5 can promote cell migration and proliferation.And invasion play an important role in FTC cells.3.ERK MAPK and JNK signaling pathway play an important role in FTC.4.DUSP5 down-regulation promotes the proliferation,migration and invasion of FTC cells by activating ERK MAPK and JNK signaling pathways. |
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