| Thyroid carcinoma has become the most common endocrine malignancy with a rapid growth of incidence in recent 20 years.Papillary thyroid carcinoma(PTC)is the most common pathological type of thyroid carcinoma.Although patients usually have better prognoses,but a few PTC are very aggressive,appeared on the tumor invasion and lymph node metastasis in early stage,prone to repeated recurrence and metastasis after operation.It need more effective molecular targets to help further improve the therapeutic effect of this part of thyroid carcinoma.We used transcriptome sequencing technologies and bioinformatic analysis to find key genes and the gene signal transduction pathways related to generation and development of human papillary thyroid carcinoma.Then we chose differentially expressed gene PIK3R1 through transcriptome sequencing technologies and explored the expression and significance of phosphatidylinositol3-kinase regulatory subunit alpha(PI3Kp85α)in PTC.Next,we studied on the molecular mechanisms involved in the pathogenesis of PTC through in vitro experimental study about the contribution of PI3Kp85α on TPC-1 cells.Part Ⅰ The study of differentially expressed genes on papillary thyroid carcinoma by transcriptome sequencingObjective: To systematically screen key genes and the gene signal transduction pathways related to generation and development of human papillary thyroid carcinoma using transcriptome sequencing technologies and bioinformatic analysis,provide a theoretical basis and genomic strategy for molecular mechanism,early diagnosis,targeting therapy and prognosis prediction for human papillary thyroid carcinoma.Methods: Total RNA of a frozen high-risk papillary thyroid carcinoma tissue and the low-risk papillary thyroid carcinoma tissue were extracted and the qualities of RNA samples were detected.The integrity and purification of the total RNA isolated were tested using formaldehyde-agarose gel electrophoresis and UV spectrophotometer before c DNA libraries were constructed.The Illumina Hiseq 4000 sequencing platform was used for transcriptome sequencing.Then,the original data was sorted,filtered and quality evaluated.Bioinformatic analysis were used to analyze the transcriptome sequencing results(including GO/KEGG enrichment analysis of different genes and cluster analysis)to identify key genes associated with generation and development of papillary thyroid carcinoma.Results:1)In this study,1892 differentially expressed genes were identified through transcriptome sequencing technology,of which 1402 genes were up-regulated and 489 genes were down-regulated.2)GO pathway analysis showed that differentially expressed genes were enriched in signal transduction,small molecule metabolic process,cell adhesion,regulation of transcription and extracellular matrix organization in biological process,plasma membrane,integral component of membrane,cytoplasm,extracellular exosome and nucleus in cellular component,protein binding,metal in binding,ATP bindingand calcium ion binding in molecular function.3)KEGG pathway analysis showed that differentially expressed genes were related to pathways in cancer,focal adhesion,cytokine-cytokine receptor,ECM-receptor interaction.4)We selected candidate differentially expressed genes that involved in the carcinogenesis of carcinoma and could serve as potential biomarkers of prognosis,such as ECM1,CCND2,PARP4,TNFRSF1 B,PLK3,PROX1,LAMC2,SMAD9,CENPJ,STARD13,PI K3R1 and MMP9.Conclusions: There were specific differentially expressed genes with more up-regulated genes than down-regulated ones on papillary thyroid carcinoma through transcriptome sequencing.The enrichment analysis of GO and KEGG showed that those genes were involved in particular functions and pathways which indicated particular effect on the tumorigenesis of papillary thyroid carcinoma.There are some differential genes were found in this study to be correlated with the tumorigenesis of cancer which have not been reported to involve in previous literature.Part Ⅱ The expression and significance of PI3Kp85αon papillary thyroid carcinomaObjective: To explore the expression and significance of phosphatidylinositol3-kinase regulatory subunit alpha(PI3Kp85α)in papillary thyroid carcinoma.Methods: PI3Kp85α expressions were detected in papillary thyroid carcinoma tissues(n=116)and para-carcinoma thyroid normal tissues(NT,n=116)by immunohistochemistry,western blot and enzyme-linked immunosorbent assay(ELISA).The relationships between PI3Kp85α expressions and clinical pathological features in PTC were analyzed.Diagnostic values of PI3Kp85αin PTC were evaluated by receiver operating characteristic(ROC)curve.Results: PI3Kp85α expression levels in PTC tissues were statistically different from those in NT tissues(P<0.05).PI3Kp85α expression was associated with lymph node metastasis and TNM stage(P<0.05).The diagnostic sensitivity and specificity of PI3Kp85α were 92.2 and 91.1%,respectively,with a cutoff value of 2.100 and an area under curve(AUC)of 0.966.Conclusions: PI3Kp85α may be related to the tumorigenesis and progression of PTC.Moreover,PI3Kp85α may serve as potential diagnostic markers to the biological behavior of PTC.Part Ⅲ The Effects of PI3Kp85α on biological behavior of papillary thyroid carcinomaObjective: Providing a theoretical basis for further study on the molecular mechanisms involved in the pathogenesis of PTC through in vitro experimental study about the contribution of PI3Kp85α on TPC-1 cells.Methods: TPC-1 cells stably down-expressed PI3Kp85α were established by si RNA.the fluorescence microscope was used to observe the infection effect.We Detected the level of PI3Kp85α in si RNA,CONTROL and NC groups with real-time PCR(RT-PCR)and western blot and observed the cellularability of proliferation in vitro TPC-1 cells line by MTT and colony forming assay.We detected the changes of apoptosis rate of si RNA,CONTROL and NC groups by Annexin V-FITC/PI flowcytometry.Through observing the healing velocity of the artificial scratch on monolayer cell to indirectly and transwell cell migration assay evaluated the cell migration ability.Transwell assay with spread matrige glue was used to observe the changes of invasion ability of TPC-1 cells.P-Akt,p-m TOR,p-S6K1 and Bad expression were detected by RT-PCR and Western blot.Results:1.We infected TPC-1 cells with PI3Kp85α-si RNA successfully.Under the fluorescence microscope,we saw more than 80% of the cells expressing green fluorescent.The results of RT-PCR and western blot showed that PI3Kp85α in si RNA group was lower than that of CONTROL and NC groups,the difference was statistically significant(P<0.05),CONTROL and NC groups had no difference(P>0.05).2.Clone formation results showed that the average number of clones of TPC-1 and number of clones in si RNA group was obviously less than that of CONTROL and NC groups(P<0.05).Si RNA significantly inhibited the proliferation of TPC-1 cells through MTT assay.Compared with the three groups,the cell proliferation was significantly inhibited in si RNA group.IC50 value of si RNA group was significantly lower than that of CONTROL and NC groups same time(P<0.05).3.The apoptosis rate of NC,CONTROL and si RNA groups was respectively 2.15±0.53%,2.61±0.36% and 19.28±0.79% detected by Annexin V-FITC/PI flowcytometry.Compared with the other groups,the apoptosis rate in si RNA group was the highest(P<0.01),and the apoptosis rate of CONTROL and NC groups had no difference(P>0.05).4.The migration ability of the cells in si RNA group decreased significantly.According to the monolayer cell scratch test,the distance of scratch reduced greatly in CONTROL and NC groups,while in si RNA group,the distance of scratch was wider than that in the other two groups(P<0.01).The results of transwell migration assay showed that compared with the CONTROL and NC groups,the cell number of polycarbonate membrane under-chambers surface in si RNA group decreased significantly(P<0.01).5.PI3Kp85α had obvious effect on cell invasion of TPC-1.The results of transwell invasion assay showed that compared with the CONTROL and NC groups,the cell number of polycarbonate membrane under-chambers surface in si RNA group decreased significantly(P<0.01).6.Three groups of cells were detected in the expression of p-Akt,p-m TOR,p-S6K1 m RNA and Bad m RNA.In si RNA group,Bad was significantly higher than that of the CONTROL and NC groups,and p-Akt,p-m TOR,p-S6K1 were lower than that of the CONTROL and NC groups(all P<0.01).In si RNA group,Bad was obviously enhanced and p-Akt,p-m TOR,p-S6K1 were reduced compared with the CONTROL and NCgroups(all P<0.01).Conclusions: PI3Kp85α can promote the proliferation,migration and invasion of TPC-1 cells,inhibit cell apoptosis.PI3Kp85α may promote the proliferation,migration and invasionof TPC-1 cells,inhibit cell apoptosis through PI3K/Akt/m TOR/S6K1 and PI3K/Akt/Bad signaling pathway. |
