| Part I Expression and function of LncRNAEGOT in thyroid carcinomaObjective: This part mainly studies the expression of LncRNAEGOT in thyroid carcinoma and its role in the phenotype of proliferation and invasion of thyroid carcinoma.Methods: Papillary thyroid carcinoma tissues were collected from affiliated tumor hospital from January to June 2015,including paired cancer tissues,adjacent normal tissues and extraglandular invasive tissues,and detected by ce RNA microarray.The high expression of lnc RNA,in invasive thyroid carcinoma was screened by bioinformatics analysis,and the corresponding genes were knocked down and tested in vitro after being verified by Q-PCR.Results: 1.ce RNA chip not only compared cancer and normal tissue,but also compared invasive thyroid carcinoma and cancer tissue,invasive thyroid cancer tissue and normal tissue.Then three sets of data were selected and intersected.A total of 15 LncRNA were upregulated and 39 were down-regulated.2.Total RNA was extracted from thyroid cancer tumor tissue and was used to Q-PCR test,paired normal tissue and invasive thyroid carcinoma tissue,and the results showed that the expression of LncRNAEGOT in cancer tissue was higher than that in normal tissue and invasive carcinoma tissue compared with non-invasive carcinoma tissue,and the difference was statistically significant.3.Using RNA probe to detect FISH,it was found that LncRNAEGOT was localized in the nucleus and cytoplasm,and the cytoplasmic separation experiment also proved that both the cytoplasm and nucleus of LncRNAEGOT were present.4.Colony formation,CCK8,Transwell and cell scratch experiments were carried out after siRNA knockdown LncRNAEGOT was transfected with thyroid cancer cell line.It was found that the cell proliferation and invasion decreased after knockout LncRNAEGOT.Conclusion: 1.Compared with normal tissues,the expression of LncRNAEGOT in invasive thyroid carcinoma is higher than that in non-invasive thyroid carcinoma,which indicates that LncRNA.EGOT plays a certain role in the occurrence and invasion of thyroid carcinoma.2.The expression of LncRNAEGOT in a large number of clinical samples is similar to that obtained by gene chip sequencing.3.LncRNAEGOT exists not only in the nucleus but also in the cytoplasm.4.LncRNAEGOT can promote the proliferation and invasion of thyroid cancer cells.Part II LncRNAEGOT binding to splicing factor QKIObjective: This part mainly studies the binding of LncRNA EGOT to QKI protein in thyroid carcinoma.Methods: First of all,the human proteome chip hybridization technique of Hu Prot was used to hybridize with LncRNAEGOT,and the best protein that might bind to LncRNAEGOT was screened,and then the protein bound to LncRNA was confirmed by RNAPulldown experiment.Finally,we tested again whether the protein could bind to LncRNA by RIPsequence.Results: After hybridization between human proteome chip and LncRNAEGOT,it was found that four proteins were most likely to bind to LncRNAEGOT,namely,QKI,RPUSD4,NFE2L2 and MEX3 B.2.RNAPulldown assay showed that the protein QKI could bind to the sense chain of LncRNAEGOT,but not to its antisense chain,while the protein RPUSD2 could bind to both the sense chain and the antisense chain of LncRNA,and the other two proteins had no obvious binding to the antisense chain of LncRNA.3.RIP-PCR confirmed that QKI can be enriched to LncRNAEGOT,RIP-sequence data analysis results show that QKI can bind to more m RNA,including LncRNAEGOT.Conclusion: 1.LncRNAEGOT can bind to four proteins: QKI,RPUSD4,NFE2L2 and MEX3 B.2.The binding of LncRNAEGOT to QKI is specific,but to RPUSD4 is nonspecific,and it cannot bind to two proteins,NFE2L2 and MEX3 B.Both 3.RIP-PCR and RIP-sequence can prove that QKI can bind to LncRNA.Part III Downregulation of QKI expression in thyroid carcinoma leads to enhancement of tumor proliferation and migration phenotypeObjective: This part mainly studies the regulatory relationship between LncRNAEGOT and QKI,the expression of QKI in thyroid and its role in the phenotype of proliferation and migration of thyroid carcinoma.Methods: through the analysis of TCGA database,the expression of QKI in thyroid carcinoma and the relationship between its expression and clinical stage,pathological classification,invasion and other phenotypes were confirmed.After knocking down QKI in thyroid carcinoma,transcriptome sequencing was performed,and the main function of QKI in thyroid carcinoma was confirmed by bioinformatics analysis,function and pathway enrichment analysis,and the role of QKI in thyroid cancer was confirmed by in vitro function test.Results: 1.The analysis of QKI expression in TCGA database showed that the expression of QKI in thyroid carcinoma was significantly down-regulated compared with normal tissue,especially in invasive carcinoma,and QKI was significantly correlated with pathological tumor size,pathological stage and invasiveness,that is,the low expression of QKI predicted larger tumor and more severe stage.Through immunohistochemical analysis,we found that the expression of QKI protein was down-regulated in thyroid carcinoma.2.The QKI studied in this paper is QKI-5,.Immunofluorescence detection showed that QKI-5 was located in the nucleus.3.After the TPC-1 cell line was transfected with siRNA,81 differential genes that splicing were affected by QKI were screened,including 9 down-regulated and 72 up-regulated.Enrichment analysis showed that they were the same as RIP-sequence and many pathways in TCGA data analysis,such as cell adhesion,TNF signal pathway,actin cytoskeleton and so on.4.After knocking down LncRNA and transfecting lentivirus overexpressed LncRNAEGOT in thyroid carcinoma,QKIm RNA and protein were detected and negative regulation of QKI by LncRNA was found.Conclusion: 1.The expression of QKI is down-regulated in thyroid carcinoma,which indicates larger tumor,more severe clinical stage and invasiveness.2.QKI-5 localizes in nucleus.3.Down-regulation of QKI can promote the proliferation,invasion and migration of thyroid carcinoma.4.LncRNAEGOT negatively regulates QKI.Part IV Shear factor QKI regulates ESYT2 variable splicing to inhibit thyroid proliferation and migrationObjective: This part mainly studies which genes QKI as a splicing factor can affect the variable splicing of which genes and which genes play an inhibitory role in thyroid cancer.Methods: The transcriptional group sequence after QKI knockout was analyzed by variable cutting analysis,and the variable cutting events were screened.The existence of variable cutting events was verified by PCR.Minigene test confirmed that QKI affected the variable cutting of ESYT2.The thyroid function was detected by transfection of siRNA knockdown ESYT2 long transcripts.Results: 1.QKI affected 535 significant events in thyroid cancer,including skipped exon(SE).Through PCR,we verify that ESYT2,PLEKHM2 and DEPDC are consistent with the data analysis.2.Minigene experiment showed that the splicing of LncRNAEGOT,exon14 was decreased and ESYT2_L was up-regulated,while the exon14 splicing of ESYT2 was increased and ESYT2_L was down-regulated after overexpression of QKI.3.The ability of proliferation,migration and invasion of thyroid carcinoma decreased after ESYT2_L knockdown.Conclusion: 1.As a splicing factor,QKI can affect the variable splicing of multiple genes in thyroid carcinoma.2.Minigene experiments show that QKI can affect the variable shear of ESYT2.3.The ability of proliferation,migration and invasion of thyroid cancer cells decreased after knocking down ESYT2 L. |
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