NEAT1₂ Functions As A Competing Endogenous RNA To Regulate ATAD2 Expression By Sponging MicroRNA-106b-5p In Papillary Thyroid Cancer

Background:Thyroid cancer is the most common type of endocrine malignancy.The incidence of thyroid cancer has been increasing rapidly around the world,constituting5 % of all female malignancies.Papillary thyroid cancer?PTC?is the most common histological subtype of thyroid cancer,constituting 85–90 % of all thyroid malignancies.However,some patients do not respond to RAI therapy or progress to metastatic disease.In these cases,prognosis is poor and the 10-year survival rate drops to 40%.Long non-coding RNAs?lnc RNAs?are RNAs consisting of more than 200 nucleotides and do not encode proteins.Recent studies indicated that lnc RNAs are involved in various biological processes and diseases in humans.NEAT1 was first found in the rat brain injected with Japanese encephalitis virus and rabies virus.NEAT1 is essential for the formation and maintaining the stability of paraspeckles.Recent studies have suggested that NEAT1 contributes to tumorigenesis in various cancers,such as hepatocellular cancer,breast cancer,non-small cell lung cancer,prostate cancer,colon cancer.Our previous lnc RNA expression profile microarray study in PTC showed that NEAT1₂expression was significantly upregulated in PTC compared with that in non-cancerous tissues.The fold change was 4.65 in our genome-wide analysis.However,the expression,clinicopathological significance,and function of NEAT1₂ in PTC tissues and cell lines require further investigation on a large scale.The ATAD2 gene is located in the human chromosome 8q24.13.Its encoded protein contains 2 AAA?ATPase associated with diverse celluar activities?regions and a brominated domain?Bromodomain?.The AAA region is used as a molecular chaperone to perform the function of assembly,operation and decomposition of protein complexes.Bromine domain is involved in gene transcription regulation,cell cycle regulation and signal transduction.It is reported that ATAD2 is highly expressed in hepatocellular carcinoma,prostate cancer,lung cancer and ovarian cancer.The high expression of ATAD2 is positively correlated with tumor stage,poor histological grade,lymph node metastasis and poor prognosis.However,the expression and function of ATAD2 have not been reported in PTC.The purpose of this study is to explore the expression of NEAT1₂ in the PTC tissues and adjacent non-cancerous tissues and the functions in the PTC cell line.To demonstrate that NEAT1₂ can play a role in promoting PTC by regulating the expression of ATAD2.In addition,we want to explain the specific regulatory mechanism between NEAT1₂ and ATAD2.Methods: 1.We collected 87 pairs of PTC tissues and adjacent non-cancerous tissues treated by thyroid surgery in the First Affiliated Hospital of China Medical University between October 2014 and August 2015.The expression of NEAT1₂ was detected in 87 pairs of PTC tissues and adjacent non-cancerous tissues by q RT-PCR.The relationship between the expression of NEAT1₂ and clinicopathological features in PTC tissues was analyzed by chi-square test.The expression of NEAT1₂ was downregulated by transfection of si-NEAT1₂,and the interference efficiency was detected by q RT-PCR.CCK8 was used to detect NEAT1₂ on the growth of PTC cells,Transwell was used to detect NEAT1₂ on PTC cell migration and invasion effect,wound healing assay was used to detect the effect of NEAT1₂ on the migration of PTC cells,flow cytometry was used to detect the effect of NEAT1₂ on the apoptosis of PTC cells,western blot was used to detect NEAT1₂ on the apoptosis and invasion related proteins.3.QRT-PCR and western blot were used to detect the expression of ATAD2 after NEAT1₂downregulation.4.The expression of ATAD2 in 87 PTC tissues and adjacent non-cancerous tissues was detected by q RT-PCR,and the relationship between the expression of ATAD2 and clinicopathological features was analyzed by chi-square test.The expression of ATAD2 was downregulated by transfection of si-ATAD2,and the interference efficiency was detected by q RT-PCR.CCK8 was used to detect the effect of ATAD2 on the growth of PTC cells.Transwell was used to detect the effect of ATAD2 on migration and invasion of PTC cells.The effect of ATAD2 on PTC migration was detected by Wound healing assay,and the effect of ATAD2 on apoptosis of PTC cells was detected by flow cytometry.5.After the overexpression of ATAD2,western blot,CCK8,transwell,wound healing assay and flow cytometry were used to detect the effects of ATAD2 protein expression,cell growth,migration and invasion ability and apoptosis in si-NC group,si-NEAT1₂ group and si-NEAT1₂+ATAD2 group.6.The "seed-pairing" and Targetscan were used to predict the mi RNAs both binding to NEAT1₂ and 3 'UTR of ATAD2.The expression of potential binding mi RNAs in group si-NC and si-NEAT1₂ group was detected by q RT-PCR.The PTC cells were transfected with mi R-106a-5p mimic,mi R-106b-5p mimic and mi R-17-5p mimic to overexpress the mi R-106a-5p,mi R-106b-5p and mi R-17-5p.Western blot was used to detect the protein expression in different groups.7.The expression level of mi R-106b-5p in 87 pairs of PTC and adjacent non-cancerous tissue was detected by q RT-PCR.Pearson correlation was used to analyze the correlation between the expression of NEAT1₂ and mi R-106b-5p in 87 PTC tissues.8.Luciferase assays were used to prove whether mi R-106b-5p can directly bind to NEAT1₂ and the 3 'UTR of ATAD2,and whether NEAT1₂ can regulate ATAD2 expression through competitive binding to mi R-106b-5p.Results: 1.The expression of NEAT1₂ in PTC tissues was significantly higher than that in matched adjacent non-cancerous tissues,and the expression of NEAT1₂ in PTC was correlated with the tumor size.2.Knockout NEAT1₂ in PTC cells could inhibit cell growth,induce apoptosis,and down regulate the expression of apoptosis related protein Bcl-2 and Bcl-xl.Knockout NEAT1₂ in PTC cells could inhibit cell migration and invasion and change the expression of EMT-related proteins and MMP proteins associated with invasion.3.The knockout of NEAT1₂ in PTC cells could significantly reduce the m RNA and protein expression of ATAD2.4.The expression of ATAD2 in PTC tissues was significantly higher than that of adjacent non-cancerous tissues,and the expression in PTC tissues was associated to the tumor size.Knockdown of ATAD2 could significantly inhibit cell growth,induce cell apoptosis,and inhibit cell migration and invasion in PTC cells.5.Overexpression of ATAD2 could partially restore the inhibitory effect of si-NEAT1₂ on the growth,migration and invasion and promotion of apoptosis in PTC cells.6.Through the use of "seed paird" and targetscan prediction,a total of 28 mi RNAs was initially predicted directly binding to NEAT1₂ and ATAD2.Further q RT-PCR result showed that mi R-106a-5p,mi R-106b-5p and mi R-17-5p may be mi RNAs directly binding to NEAT1₂.Overexpression of mi R-106a-5p,mi R-106b-5p and mi R-17-5p in PTC cells indicated that the expression of ATAD2 protein significantly downregulated in mi R-106b-5p overexpression group,but there was no significant change in ATAD2 protein expression after overexpression of mi R-106a-5p and mi R-17-5p.7.Mi R-106b-5p was low expressed in 87 PTC tissues than that in adjacent non-cancerous tissues.The expression of mi R-106b-5p was negatively correlated with NEAT1₂ in PTC tissues.8.The luciferase assays showed that mi R-106b-5p could directly bind to NEAT1₂ and ATAD2 3 'UTR.NEAT1₂ can regulate the expression of ATAD2 through a competitive combination of mi R-106b-5p.Conclusion: 1.The expression of NEAT1₂ in PTC is significantly up-regulated and is associated with tumor size.Down regulation of the expression of NEAT1₂ can significantly inhibit the growth of PTC cells,induce apoptosis,inhibit invasion and metastasis.2.ATAD2 is highly expressed in PTC and is associated with tumor size.ATAD2 can play a role in promoting cancer in PTC.3.NEAT1₂ can play a role in promoting the growth,anti apoptosis,promoting migration and invasion of PTC cells through functioning as a competing endogenous RNA to regulate ATAD2 expression by sponging mi R-106b-5p.