The Roel And Mechanism Of Long Non-Coding RNA MIAT In Papillary Thyroid Carcinoma

Background and ObjectiveBackground:Thyroid cancer is the most common endocrine malignancy,and papillary thyroid cancer is the most common pathological type.At present,no breakthrough has been made in the study of the molecular mechanism of papillary thyroid carcinoma.MIAT is a common long-chain non-coding RNA,which is highly expressed in many malignant tumors,such as lung cancer,liver cancer,prostate cancer,breast cancer and so on.It has been proved that MIAT plays an important role in the regulation of various tumors.As non-small cell lung cancer,inhibiting the expression of MIAT can reduce the invasive ability of the cancer cells,and the expression level of MIAT is positively correlated with the size of tumors.p21 is a most common cancer suppressor gene,which can inhibit cell proliferation by reducing the replication of the damaged DNA through cell cycle arrest.But recent studies have found that the expression of P21 is very low in normal thyroid tissues,but increased in thyroid malignant tumors.The expression of P21 in thyroid cancer tissues is related to the size of primary tumors and invasion of tumors,and higher in larger tumors or papillary thyroid carcinomas with invasion of capsules.GSK-3β/β-catenin signaling pathway is a key part of Wnt signaling pathway.Abnormal activation of GSK-3β/β-catenin signaling pathway may promote the development of many diseases in such ways:leading to abnormal differentiation,proliferation or apoptosis of cells.At present,GSK-3β/β-catenin signaling pathway has become a hot research topic in the medical field,and a potential target for the treatment of many malignant tumors.In view of the above research background,we analyzed the expression difference of MIAT in papillary thyroid carcinoma and normal thyroid tissue from GEO public database,and concluded that that MIAT was expressed in papillary thyroid carcrnoma tissue.Objective:To investigate the regulation of MIAT on biological behavior of papillary thyroid carcinoma by transfection of MIAT or silencing MIAT in papillary thyroid carcinoma cells.It is speculated that MIAT may play a role in regulating p21 gene.Finally,the coordination and regulation of GSK-3 β/β-catenin signaling pathway in papillary thyroid carcinoma are studied.Our objective is to investigate the regulation of long non-coding RNA MIAT by p21 gene and GSK-3 β/β-catenin signal pathway to promote the study and analysis of the development mechanism of papillary thyroid carcinoma.Chapter I:expression and clinical significance of MIAT in papillary thyroid carcinomaObjective:To investigate the expression of MIAT in papillary thyroid cancer and its correlation with clinical.features.Materials and Methods:1.78 cases of papillary thyroid carcinoma and adjacent tissues were collected and randomly divided into three groups:screening group(3 cases,microarray technology to detect the differentiated expression of LncRNAs,screening differentiated expression genes),verification group(15 cases,real-time PCR to verify the differentiated expression of LncRNAs,in papillary thyroid cancer and adjacent tissues)and clinical correlation study group(detection of MIAT expression)The correlation between the difference and clinicopathological parameters in patients with papillary thyroid cancer.2.Real time PCR was used to detect the expression of MIAT in KTC-1,B-CPAP,TPC-1 and NPA87 cells.Results:1.In papillary thyroid carcinoma,53 lncrnas were up-regulated and 109 lncrnas were down regulated.MALAT1,H19,PVT1,MIAT and UCA1 were up-regulated in papillary thyroid carcinoma,and the most significant increase was in MIAT.2.The expression of MIAT was correlated with tumor grade and lymph node metastasis,but not with gender,age and distant metastasis..3.The expression of MIAT in KTC-1,B-CPAP,TPC-1 and NPA87 cells was higher than that in normal thyroid cells.The expression of MIAT in TPC-1 cells increased most significantly.Conclusions:1.MIAT is highly expressed in papillary thyroid carcinoma.and papillary thyroid carcinoma cell line.2.The expression level of MIAT is related to TNM grade and lymph node metastasis of papillary thyroid carcinoma.Chapter Ⅱ:the biological role of MIAT in papillary thyroid carcinoma cell lineObjective:MIAT overexpression vector and MIAT siRNA were constructed and transfected into TPC-1 cells.Detect the proliferation,apoptosis,invasion and energy metabolism of TPC-1 cells.Materials and Methods:1.Construct the over expression vector pcdna3.1-MIAT and siRNA of MIAT.PcDNA3.1 empty vector and meaningless siRNA were used as control.They were transfected into TPC-1 cells.2.CCK-8 assay was used to detect the cell proliferation.3.Transwell method was used to measure the invasion and migration ability.4.Cell apoptosis was detected by flow cytometry.5.Mitochondrial transmembrane potential detection and analysis of the changes of mitochondrial transmembrane potential in each group.6.Transmission electron microscope was used to detect the morphological changes of mitochondria in cell nucleus.Results:1.Over expression of MIAT decreased the apoptosis rate,but silencing MIAT increased it.2.Overexpression of MIAT can accelerate the growth of cells.After silencing MIAT,the growth trend of cells gradually slowed down.3.The overexpression of MIAT can promote the invasion ability of TPC-1 cells,while silencing MIAT can reduce the invasion ability of TPC-1 cells.4.Overexpression of MIAT can increase mitochondrial transmembrane potential and ATP synthesis in TPC-1 cells.The silencing of MIAT resulted in the decrease of mitochondrial transmembrane potential and ATP synthesis.5.After the over expression of MIAT,many vacuoles were observed in the cells,accompanied by lipid precipitation and lysosome to a certain extent;while the mitochondria and Golgi apparatus were swollen,and the pathological degree of the cells decreased significantly after the silencing of MIAT,and the volume of mitochondria gradually recovered.Conclusions:1.MIAT could improve the proliferation,invasion and inhibit the apoptosis of TPC-1 cells;2.MIAT could enhance the mitochondrial membrane potential and ATP synthesis ability of TPC-1 cells,making the cells in a high activity state.Chapter Ⅲ:The molecular mechanism of MIAT in the regulation of p21Objective:The role of MIAT and p21 in papillary thyroid carcinoma was studied in vitro and in vivo.On the basis of constructing cell and animal models with MIAT overexpression,p21 inhibitor was added to detect the biological function of cells or the growth status of transplanted tumor,and the expression of signal pathway protein GSK-3β/β-catenin was further detected to clarify the mechanism of MIAT..Materials and Methods:1)In vitro experiments1.The overexpression vector pcdna3.1-MIAT was constructed and transfected into TPC-1 cells.2.CCK-8 assay was used to detect the cell proliferation.3.Transwell method was used to measure the invasion and migration ability.4.Cell apoptosis was detected by flow cytometry.5.Mitochondrial transmembrane potential detection and analysis of the changes of mitochondrial transmembrane potential in each group.6.Transmission electron microscope was used to detect the morphological changes of mitochondria in cell nucleus.7.The transcriptional promoter activity of GSK-3β/β-catenin was detected by dual luciferase reporter system.8.The protein expression levels of p21,GSK-3β and β-catenin were detected by Western blot.(2)In vivo experiments1.The xenograft tumor model was established in nude mice.Pcdna3.1-MIAT was injected into the tumor and p21 inhibitor was injected into the tail vein.2.After 6 weeks of culture,the nude mice were killed and the tumor volume was measured.3.The protein expression levels of p21,GSK-3β and β-catenin were detected by Western blot.Results:1.Over expression of MIAT promotes the expression of p21 gene and protein.2.The ability of MIAT to inhibit apoptosis was weakened after inhibiting the expression of p21.3.The growth promoting effect of MIAT on TPC-1 cells can be weakened by p21 inhibitor.4.After p21 was inhibited,the ability of MIAT to promote the invasion of TPC-1 cells decreased.5.After p21 was inhibited,the effect of MIAT on mitochondrial transmembrane potential was weakened.6.After p21 was inhibited,the effect of MIAT on the change of intracellular structure was weakened.7.After over expression of MIAT,the activities of GSK-3β and β-catenin promoter were increased.When p21 inhibitor was added,the activities of GSK-3βand β-catenin were decreased.8.After over expression of MIAT,the expression of GSK-3β and β-catenin increased.When p21 inhibitor was added,the expression of GSK-3β and β-catenin decreased correspondingly.9.The tumor volume of nude mice with MIAT overexpression wa2019s significantly increased compared with the control group.The tumor volume of nude mice treated with p21 inhibitor decreased significantly.10.The expression of p21 protein in tumor tissue of nude mice with MIAT overexpression group was significantly higher than that of control group.11.The expression levels of GSK-3 β and β-catenin protein in tumor tissue of nude mice in MIAT overexpression group were significantly higher than those in control group.After p21 inhibitor treatment,the expression levels of GSK-3β andβ-catenin protein in tumor tissue of nude mice were significantly decreased.Conclusions:1.MIAT affects the biological function of papillary thyroid carcinoma by regulating p21.2.The downstream signaling pathway of MIAT may be GSK-3β/β-catenin signaling pathway.