The Effects Of TMPO-AS1 On Thyroid Cancer Through Regulating TMPO By Sponging Mir-498

Thyroid cancer is the most general endocrine malignancy,and its occurrence rate has been increasing rapidly worldwide in past decades.Although the 5-year overall survival rate of PTC patients is about 95%,tumors can metastasize to distant organs and lymph nodes,leading to poor prognosis and high recurrence rate in some patients.Therefore,the identification of new treatment strategies to reduce the incidence of lung metastasis will achieve long-term remission of thyroid cancer patients,and the study of its potential molecular mechanisms is of great significance to improve the diagnosis and prognosis of PTC patients.Long non-coding RNAs(lncRNAs)are composed of a group of more than 200 nucleotide ncRNAs.Numerous studies have shown that lncRNAs are involved in human pathology by regulating downstream target effectors.In relation to clinical practice,lncRNAs can act as potential markers or therapeutic targets in different types of human cancers.An increasing number of studies have shown that lncRNAs play an important role in regulating cell biological functions such as proliferation,apoptosis,migration,invasion,drug resistance and immune response.Many studies have observed dysregulation of lncRNA expression in various types of cancer including thyroid cancer,but their role and molecular mechanism in thyroid cancer are still unclear.TMPO antisense RNA 1(TMPO-AS1)is an antisense lncRNA located in chromosome 12p23.1 region,which is transcribed by the antisense of the 5’ end of the coding gene thymosin(TMPO).TMPO-AS1 has been identified as an oncogene involved in cell proliferation in a variety of cancers.In recent years,many studies have shown that TMPO-AS1,as an oncogene,is significantly upregulated in a variety of cancers and is associated with poor prognosis and tumor progression.Thymopoietin(TMPO),also known as laminar associated polypeptide 2(LAP2),has six selectively splicing isomers with diverse functions.TMPO can interact with lamellar and BAF to regulate the dynamics of nuclear structure and cell cycle.The study has found that TMPO plays an important role in cancer biology.According to bioinformatics prediction,TMPO-AS1 has a binding sequence with miR-498,and TMPO is the target gene of miR-498,so it can be speculated that TMPO-AS1 plays an important role in the occurrence and development of cancer by adsorbing miR-498 and regulating TMPO.In order to explore the expression and correlation of TMPO-AS 1 and TMPO in thyroid cancer,this study first analyzed the expression of TMPO-AS 1 and TMPO in thyroid cancer tissues from the TCGA database and their correlation by qRT-PCR and Pearson correlation coefficient.Then,the expression of TMPO-AS1 and TMPO in thyroid cancer tissues was determined by qRT-PCR and their correlation was analyzed.Then,the position relationship of TMPO-AS1 and TMPO genes was analyzed by UCSC database,and the relationship between TMPO-AS1 and TMPO was verified by double luciferase and immunoprecipitation assays.In order to study the effects of interference with TMPO-AS 1 on the biological behavior of thyroid cancer cells and the growth of transplanted tumor,the expression of TMPO-AS1 in thyroid cancer cell lines was detected by qRT-PCR,and then the expression positions of TMPO-AS1 in TPC-1 and IHH-4 cells were confirmed.EDU,clony formation,flow cytometiy,TUNEL and Transwell assays were used to detect the effects of interference with TMPO-AS 1 on the proliferation,clony formation,apoptosis,invasion and migration of TPC-1 and IHH-4 cells.In addition,a nude mouse model was established to investigate the effect of interference with TMPO-AS1 on the growth of thyroid carcinoma xenograft.In order to explore the effect of TMPO-AS1 adsorption of miR-498 on the function of thyroid cancer cells through regulation of TMPO,the relationship between TMPO-AS1,TMPO and miR-498 was detected by immunoprecipitation and dual luciferase assay.In addition,the effects of overexpression of miR-498 on proliferation,clony formation,apoptosis,invasion and migration of TPC-1 and IHH-4 cells were investigated.In addition,the effects of interference with TMPO on proliferation,clony formation,apoptosis,invasion and migration of TPC-1 and IHH-4 cells were analyzed.And the effects of TMPO-AS1+TMPO overexpression experiment on the proliferation,clony formation,apoptosis,invasion and migration of TPC-1 cells.This research was mainly divided into three parts:Part Ⅰ:The expression and correlation analysis of TMPO-AS1 and TMPO in thyroid cancer;Part Ⅱ:Effects of interfering TMPO-AS1 on the biological behavior of thyroid cancer cells and the growth of xenografts;Part Ⅲ:The effects of TMPO-AS1 on thyroid cancer cell function through regulating TMPO by sponging miR-498.The main content:Part Ⅰ:The expression and correlation analysis of TMPO-AS1 and TMPO in thyroid cancerMethods1.The expression and correlation of TMPO-AS1 and TMPO in TCGA database were analyzed by qRT-PCR and Pearson correlation coefficient.2.Expression and correlation analysis of TMPO-AS1 and TMPO in thyroid cancer tissues was detected by qRT-PCR.3.UCSC database was used to analyze the positional relationship between TMPO-AS1 and TMPO genes.Results1.TCGA database analyses showed that TMPO-AS1 and TMPO were markedly increased in 118 thyroid cancer tissues compared with 339 paracancerous tissues(*P<0.05),and the expression levels of TMPO-AS1 and TMPO were positively correlated,R=0.23,P=2.5e-07.2.UCSC database analysis found that TMPO was a gene near TMPO-AS 1 in the genome.3.qRT-PCR detection showed that TMPO-AS1 and TMPO were up-regulated in thyroid cancer tissues compared with adjacent normal tissues(**P<0.01),and the expression levels of TMPO-AS1 and TMPO were positively correlated,R=0.625,P<0.001.ConclusionThe expression of TMPO-AS1 and TMPO was significantly up-regulated in thyroid cancer,and the expression of the two was positively correlated,and the distance between TMPO-AS1 and TMPO in the genome was close.Part Ⅱ:Effects of interfering TMPO-AS1 on the biological behavior of thyroid cancer cells and the growth of xenograftsMethods1.The expression of TMPO-AS 1 in thyroid cancer cells was detected by qRT-PCR.2.Cytoplasmic separation and fluorescence in situ hybridization(FISH)were used to detect the location of TMPO-AS1 expression in thyroid carcinoma.3.shRNA sequence was designed to construct the interfering TMPO-AS1 lentivirus vector.After packaging,TPC-1 and IHH-4 cells were infected to obtain the interfering TMPO-AS 1 stable cell lines for subsequent functional studies.4.EdU was used to detect the effect of interference with TMPO-AS 1 on cell proliferation.5.Clony formation assay was performed to determin the impact of interference with TMPO-AS1 on cell clone formation.6.The influence of interference with TMPO-AS1 on apoptosis was tested by TUNEL assay.7.Flow cytometry assay was carried out to detect the impact of interference TMPO-AS1 on cell apoptosis.8.Trans well assay was performed to determin the effect of interference with TMPO-AS1 on cell invasion.9.Transwell assay was implemented to detect the effect of interference with TMPO-AS1 on cell migration.10.The thyroid cancer xenograft of nude mice was constructed to analyze the effect of interference with TMPO-AS1 on the growth of thyroid cancer xenograft.Results1.Compared with the control group,the expression levels of TMPO-AS1 in thyroid cancer cell lines TPC-1,IHH-4,A-PTC and CUTC5 were significantly increased(**P<0.01),and the highest expression was found in TPC-1 and IHH-4 cells.TMPO-AS1 was mainly localized in the cytoplasm of TPC-1 and IHH-4 cells.2.Interference of TMPO-AS1 could inhibit the proliferation,clony formation,invasion and migration of TPC-1 and IHH-4 cells as well as the growth of thyroid cancer xenograft(**P<0.01),and promote cell apoptosis(**P<0.01).ConclusionTMPO-AS1 was mainly expressed in the cytoplasm of thyroid cancer cells,and its expression level was significantly up-regulated.Interference of MPPO-AS1 could inhibit the proliferation,clonogenesis,invasion and migration of TPC-1 and IHH-4 cells,as well as the growth of thyroid cancer transplanted tumor,and promote cell apoptosis.Part Ⅲ:The effect of TMPO-AS1 on thyroid cancer cell function through regulating TMPO by sponging miR-498Methods1.The relationship between TMPO-AS1,TMPO and miR-498 was detected by immunoprecipitation and dual luciferase assays.2.The effects of overexpression of miR-498 and interference of TMPO on proliferation,clony formation,apoptosis,invasion and migration of TPC-1 and IHH-4 cells were detected by EDU,clony formation,TUNEL,flow cytometry and Transwell assays.3.EDU,clony formation,TUNEL,flow cytometry and Transwell assays were used to detect the effects of interference with TMPO-AS1 and/or TMPO overexpression response assay on the proliferation,clone formation,apoptosis,invasion and migration of TPC-1 cells.Results1.miR-498 had binding sequences in TMPO-AS1,while TMPO is the target gene of miR-498.Overexpression of miR-498 could inhibit the expression of TMPO(**P<0.01),restrain the proliferation,invasion and migration of thyroid cancer cells(**P<0.01),and accelerate cell apoptosis(**P<0.01).2.Interference of TMPO could inhibit the proliferation,clony formation,invasion and migration of thyroid cancer cells(**P<0.01),and facilitate cell apoptosis(**P<0.01).3.Interference with TMPO-AS 1 expression could suppress the proliferation,invasion and migration of thyroid cancer cells(**P<0.01),and expedite cell apoptosis(**P<0.01),while overexpression of TMPO could reverse this suppression and promotion effects(**P<0.01).ConclusionBy positively regulating TMPO,TMPO-AS1 could promote the proliferation,clony formation,invasion and migration of thyroid cancer cells,and inhibit cell apoptosis.General conclusionIn thyroid cancer,TMPO-AS1 could regulate the expression of TMPO by sponging miR-498,thereby promoting the proliferation,clony formation,invasion and migration of cancer cells,as well as the growth of transplanted tumors,and inhibiting cell apoptosis,thus promoting cancer.