| Background and objective:Thyroid cancer(TC)is a common endocrine malignant tumor,which mostly occurs in women and is one of the main causes of death.Papillary thyroid carcinoma(PTC)is a dominant histological type TC,accounts for about 80%of all TC cases.Although great progress has been made in the treatment of PTC,the 5-year survival rate of PTC is usually more than 95%,its prognosis is still very poor,especially the distant metastasis of PTC can promote its development into a more aggressive cancer,reducing the survival rate to about 50%,which has become a major threat to PTC patients,so it is very important to further study the potential mechanism of PTC pathogenesis and metastasis.Long-chain non-coding RNA(lncRNA)plays an important role in the development and process of human diseases,especially cancer.It is abnormally expressed in a variety of cancers and is closely related to the changes of cancer biological behavior.It is involved in cancer invasion,metastasis,autophagy,differentiation and other biological processes.In PTC,lncRNA also plays the role of"oncogene" or "tumor suppressor gene" and participates in its development.Papillary thyroid cancer susceptibility candidate gene 3(PTCSC3)is one of the first identified lncRNA in thyroid gland,which is located near the single nucleotide polymorphism(SNP)rs944289 associated with PTC susceptibility.PTCSC3 gene is significantly suppressed in PTC tumor tissues.The risk allele of SNP rs944289 suppresses PTCSC3 by destroying the transcription factor binding site in the PTCSC3 promoter.The data of microarray expression assay showed that PTCSC3 changed the expression of many genes which are important for the development and progress of PTC.MicroRNA(miRNA)is a highly conserved group of non-coding RNA,with a length of 18-22 nucleotides.By binding to the 3’ untranslated regions(UTR)of the target gene,they play a role in RNA silencing and post-transcriptional regulation of gene expression,resulting in target gene degradation or transcriptional inhibition.miRNA regulates various cellular processes,including proliferation,apoptosis,differentiation and metastasis.It is reported that the imbalance of miRNA is related to a variety of human malignant tumors.miRNA can act as an "oncogene" or "tumor suppressor gene".The expression of miR-574-5p is up-regulated in many diseases.Previous studies have shown that miR-574-5p is abnormally expressed in TC by targeting PTCSC3,but it is not clear whether it plays an important role in PTC.Wnt signal pathway can participate in physiological processes such as embryonic development,cellular immunity and maintenance of homeostasis by regulating β-catenin protein.Wnt/β-catenin signal is a classic pathway in tumorigenesis,which can regulate downstream target genes such as c-myc.LncRNA can participate in the changes of tumor biological behavior by regulating Wnt/βcatenin signal.Existing studies have found that Wnt/β-catenin are also important in the progression of PTC.Some studies have found that the tumor growth of PTC is induced by the activation of Wnt/β-catenin.However,it is not clear whether Wnt/β-catenin mediate the proliferation and migration of PTC cells through PTCSC3 and miR-574-5p.Distant metastasis of PTC is a major threat to patients with PTC.Therefore,it is very important to study the potential pathogenic mechanism of the treatment of PTC.This study mainly discusses the role and mechanism of PTCSC3,miR-574-5p and Wnt/β-catenin signaling pathways in the pathogenesis of PTC,which provides a theoretical basis for revealing the occurrence and development,metastasis mechanism and finding a more effective treatment of PTC.In this study,we.first detected the levels of PTCSC3 and miR-574-5p in PTC patients and their adjacent normal tissues,analyzed the correlation between PTCSC3 and miR-574-5p in PTC tissues,and detected the expression of PTCSC3,SCAI,miR574-5p and β-catenin in TPC-1 cells.Secondly,the cell line overexpressing PTCSC3 was constructed,and the changes of cell viability,invasion and migration ability after overexpression of PTCSC3 were investigated,and the expression changes of miR547-5p,SCAI,E-cadherin and β-catenin protein were detected.The interaction between PTCSC3,miR-574-5p,SCAI and β-catenin was studied to determine the relationship between PTCSC3,miR-574-5p and SCAI protein and Wnt/β-catenin pathway and their effects on cell proliferation and migration.Finally,in vivo experiments were carried out to explore the effects of PTCSC3 overexpression on the growth of xenografts and the expression of miR-574-5p,SCAI,β-catenin,c-myc and E-cadherin in xenografts using TPC-1 cells overexpressed by PTCSC3.This paper is divided into three parts:Part one:the expression and significance of PTCSC3 and miR-574-5p in PTC tissues and cells.Part two:the effect of PTCSC3 and miR-574-5p on the biological behavior of PTC cells and its molecular mechanism.Part Ⅲ:the inhibitory effect of overexpression of PTCSC3 on the growth of transplanted tumor in mice.Main research content:Part 1:The expression and significance of PTCSC3 and miR-574-5p in PTC tissues and cellsObjectiveTo study the expression of PTCSC3 and miR-574-5p in PTC tissues and cell lines,and to further understand the clinical significance of PTCSC3 and miR-574-5p.Methods1.qRT-PCR to detect the mRNA expression of PTCSC3 and miR-574-5p in PTC and its adjacent normal tissues.2.qRT-PCR detection of mRNA expression of PTCSC3 and miR-574-5p in papillary thyroid carcinoma cell line TPC-1 and thyroid epithelial cell line Nthy-ori31.3.Western blot detection of SCAI and β-catenin protein expression in PTC and its adjacent normal tissues.4.The TOP/FOP ratio of TPC-1 cells and Nthy-ori3-cells was measured by double luciferase reporter gene.Results1.The expression of PTCSC3 mRNA in PTC tissue decreased significantly,while the expression of miR-574-5p mRNA increased significantly.2.The expression of PTCSC3 mRNA in TPC-1 cells decreased significantly,while the expression of miR-574-5p mRNA increased significantly.3.The expression of SCAI protein decreased while the expression of β-catenin protein increased in PTC tissue.4.The ratio of TOP/FOP in TPC-1 cells was significantly higher than that in Nthy-ori3-1 cells.Part 2:The effect of PTCSC3 and miR-574-5p on the biological behavior of PTC cells and its molecular mechanism.ObjectiveTo study the effects of PTCSC3 and miR-574-5p on the viability,migration and invasion of PTC cell line TPC-1 cells,as well as the expression of migration and invasion-related proteins,and to explore the molecular mechanism of the effects of PTCSC3 and miR-574-5p on the occurrence and development of PTC.Methods1.Transfection or cotransfection of pcDNA3.1-PTCSC3,miR-574-5p inhibitor,miR-574-5p mimic,si-SCAI,pcDNA-SCAI,si-β-catenin,empty vector,miRNC into TPC-1 cells.2.qRT-PCR detection of PTCSC3,miR-574-5p and SCAI mRNA expression in TPC-1 cells after transfection or cotransfection.3.Western blot detection of SCAI,E-cadherin and β-catenin protein expression in TPC-1 cells after transfection or cotransfection.4.Transwell assay to detect the metastatic ability of TPC-1 cells after transfection or cotransfection.5.Cell scratch assay to detect the invasive ability of TPC-1 cells after transfection or cotransfection.6.Detection of TOP/FOP ratio of TPC-1 cells after transfection or cotransfection with double luciferase reporter gene.7.The viability of TPC-1 cells after transfection or cotransfection was detected by MTT assay.8.The combination of PTCSC3 and miR-574-5p was detected by RNA pull-down test.Results1.Overexpression of PTCSC3 significantly decreased the viability,migration and invasion of TPC-1 cells.2.The expression of miR-547-5p was significantly decreased and the expression of SCAI was significantly increased in TPC-1 cells overexpressing PTCSC3.3.The expression of SCAI and E-cadherin protein was significantly increased and the expression of β-catenin protein was significantly decreased in TPC-1 cells overexpressing PTCSC3.4.Overexpression of miR-574-5p inhibited the protein expression of SCAI,while down-regulation of miR-574-5p promoted the protein expression of SACI.5.Inhibition of miR-574-5p significantly inhibited the ratio of TOP/FOP in TPC1 cells,which was reversed by down-regulation of SCAI.Enhanced miR-574-5p significantly increased the ratio of TOP/FOP and β-catenin protein expression in TPC-1 cells,while overexpression of SCAI significantly eliminated this effect.6.Inhibition of miR-574-5p in TPC-1 cells significantly increased the expression of SCAI protein and decreased the expression of β-catenin protein.After interfering with SCAI,the inhibition of miR-574-5p was significantly eliminated.7.Up-regulation of SCAI significantly decreased the ratio of TOP/FOP,downregulation of SCAI significantly increased the ratio of TOP/FOP,and the expression of β-catenin decreased gradually with the increase of SCAI.8.RNA pull-down experiments confirmed that miR-574-5p could be enriched by PTCSC3,which indicated that PTCSC3 could bind to miR-574-5p.9.TPC-1 cells significantly inhibit the growth,migration and invasion of TPC-1 cells after interfering with β-catenin;overexpression of PTCSC3 inhibits the growth,migration and invasion of TPC-1 cells and enhances the effect of miR-574-5p on overexpression of PTCSC3,but cannot reverse the effect of interfering β-catenin;interfering with β-catenin can eliminate the effects of PTCSC3 and miR-574-5p.10.Interference with β-catenin did not affect the expression of SCAI protein,overexpression of PTCSC3 promoted the expression of SCAI,interference with miR574-5p could eliminate the effect of PTCSC3;interference with β-catenin decreased the expression of E-cadherin,overexpression of PTCSC3 could inhibit the expression of β-catenin and E-cadherin,which could be enhanced and reversed by miR-574-5p;interference β-catenin eliminated overexpression of PTCSC3 and enhanced the effect of miR-574-5p.Part 3:The inhibitory effect of overexpression of PTCSC3 on the growth of transplanted tumor in mice.ObjectiveTo study whether PTCSC3 can inhibit the growth of tumor in mice and its effect on the expression of miR-574-5p,SCAI,β-catenin,c-myc and E-cadherin in mice.Methods1.Transfection of pcDNA-PTCSC3 into TPC-1 cells.2.The mouse xenograft tumor model was constructed by transfected cells,and the tumor volume was measured.3.qRT-PCR to detect the expression of miR-574-5p and SCAI mRNA;4.Western blot was used to detect the expression of SCAI,β-catenin,c-myc and Ecadherin protein.Results1.Overexpression of PTCSC3 significantly inhibited tumor volume and tumor weight.2.Overexpression of PTCSC3 significantly inhibited miR-574-5p and significantly increased mRNA level of SCAI.3.Overexpression of PTCSC3 increased the expression of E-cadherin and decreased the expression of β-catenin and its downstream molecule c-myc protein.Conclusion:1.The expression of PTCSC3 and β-catenin in PTC increased,while the expression of miR-574-5p and SCAI decreased.2.The combination of PTCSC3 with SCAI and β-catenin plays a key role in cell viability;3.miR-574-5p regulates Wnt/β-catenin pathway through SCAI;4.PTCSC3/miR-574-5p mediate the proliferation and invasion of PTC cells by regulating the Wnt/β-catenin pathway through SCAI;5.Overexpression of PTCSC3 can inhibit tumor growth in vivo. |
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