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Zhenjun Xu,1,* Kai Zhong,2,* Guanjun Guo,1,* Can Xu,1 Zhizhao Song,1 Dongjin Wang,1 Jun Pan1 1Department of Thoracic and Cardiovascular Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, 210008, People’s Republic of China; 2Nanjing Drum Tower Hospital Clinical College of Nanjing Medical University, Nanjing, 210008, People’s Republic of China*These authors contributed equally to this workCorrespondence: Jun Pan; Zhenjun XuDepartment of Thoracic and Cardiovascular Surgery, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, 321 Zhongshan Road, Nanjing, 210008, People’s Republic of ChinaTel +86 25 68182222-60721Fax +86 25 83105117Email pj791028@163.com; xzj881225@163.comBackground: Aortic dissection (AD) is a threatening and catastrophic vascular disease with high mortality rate and limited therapeutic strategies. There is emerging evidence showing that circular RNAs play crucial role in regulating various cardiovascular diseases. However, the biological functions and molecular mechanisms of circRNAs in AD still remains elusive. The purpose of this study was to illustrate the potential functional roles and mechanisms of hsa_circ_TGFBR2 in vitro and in vivo.Methods: The vascular smooth muscle cells (VSMCs) and AD-VSMCs were isolated from normal aorta and AD tissues. The expression of circ_TGFBR2, miR-29a and KLF4 were detected by realtime polymerase chain reaction (RT-PCR) and fluorescence in situ hybridization (FISH). Cell proliferation was assessed by CCK-8 assay, colony formation and EDU assay. Cell migration was evaluated through transwell assay. Dual-luciferase reporter assay and RNA pulldown were performed to identify the interaction between circ_TGFBR2 and miR-29a or between miR-29a and KLF4. The wild-type sequence of circ_TGFBR2 or KLF4 were cloned into the luciferase reporter plasmid, and the activity was measured using dual-luciferase reporter assay system. And for RNA pulldown, the relative RNA enrichment of circ_TGFBR2 and miR-29a were confirmed using RT-PCR. Western Blot measured the expression of phenotype switch-related proteins. AD rat model induced by β-aminopropionitrile monofumarate (BAPN) was used to verify the role and mechanism of circ_TGFBR2.Results: Circ_TGFBR2 inhibited cell proliferation and migration of AD-VSMCs cells. Overexpression of circ_TGFBR2 promoted the expression of contractile markers (α-SMA, SM22α) and inhibited the expression of synthetic markers (MGP, OPN) in AD-VSMCs cells. Circ_TGFBR2 served as a sponge for miR-29a targeting KLF4. MiR-29a mimics rescued biological roles induced by circ_TGFBR2 overexpression. The in vivo experiments revealed that overexpression of TGFBR2 suppressed the progression of AD and increased the expression of contractile markers while inhibited the expression of synthetic markers.Conclusion: Our study revealed that circ_TGFBR2 regulated VSMCs phenotype switch and suppressed the progression of AD.Keywords: aortic dissection, circ_TGFBR2, miR-29a, KLF4, vascular smooth muscle cells

Wei Yuan Hao, Li Wen Guo, Jun Luo, Guo Liang Shao, Jia Ping Zheng Cancer Hospital Affiliated to University of Chinese Academy of Sciences, Hangzhou, Zhejiang 310022, People’s Republic of ChinaCorrespondence: Jia Ping Zheng Email yuzhengdijiao5369@163.comBackground: As a highly malignant tumor, cholangiocarcinoma poses a serious threat to human life and health, so exploring the mechanisms of its development and progression at a molecular level is of great significance to the diagnosis and treatment of the disease.Objective: This study was aimed at investigating the effects and related mechanisms of LncRNA TUG1 on cholangiocarcinoma cells.Methods: Cholangiocarcinoma tissues and adjacent tissues (n=82 each), human cholangiocarcinoma cell lines (RBE, QBC939, HuH28), and a human normal biliary epithelial cell line (HIBE) were collected. miR-29a-mimics, miR-29a-inhibitor, miR-NC, si-TUG1, pcDNA3.1 TUG1, and NC were transfected into the cholangiocarcinoma cells. qRT-PCR was performed to detect TUG1 and miR-29a expression in the cholangiocarcinoma tissues and cells. Western blotting (WB) was conducted to detect the expression of Bax, Caspase-3, and Bcl-2 in the cells. CCK-8 assay, Transwell, and flow cytometry were carried out to detect cell proliferation, invasion, and apoptosis. Dual luciferase reporter gene assay (DLRGA) was performed to confirm the correlation of TUG1 with miR-29a.Results: TUG1 was highly expressed while miR-29a was poorly expressed in cholangiocarcinoma cells. TUG1 expression was negatively correlated with miR-29a expression, and TUG1 had a relatively high diagnostic value for cholangiocarcinoma. Cell experiments showed that inhibiting TUG1 expression or up-regulating miR-29a expression could inhibit cholangiocarcinoma cells from proliferation and invasion, and promote their apoptosis, while up-regulating TUG1 or inhibiting miR-29a could promote the proliferation and invasion but inhibit the apoptosis. Rescue experiment showed that overexpressing miR-29a could reverse the effects of high TUG1 expression on cholangiocarcinoma cells. DLRGA confirmed that there was a regulatory relationship between TUG1 and miR-29a.Conclusion: TUG1 is highly expressed in cholangiocarcinoma tissues. It can promote the growth and metastasis of cholangiocarcinoma cells by inhibiting miR-29a, so it may be a new target for diagnosing and treating cholangiocarcinoma.Keywords: LncRNA TUG1, miR-29a, cholangiocarcinoma, proliferation, invasion, apoptosis

Xianhua Huang*, Zhixi Chen*, Weimei Shi, Rui Zhang, Linfu Li, Hai Liu, Longhuo WuCollege of Pharmacy, Gannan Medical University, Ganzhou, People’s Republic of China*These authors contributed equally to this work Background: miR-29a, a downstream factor of Wnt/β-catenin signaling, promotes the activity of the Wnt/β-catenin signaling in a positive feedback loop. Our previous work showed that 5,7,3ʹ,4ʹ-tetramethoxyflavone (TMF), a major constituent from Murraya exotica L., exhibited chondroprotective activity by inhibiting the activity of Wnt/β-catenin signaling.Purpose: To investigate whether TMF showed the inhibitory effects on miR-29a/β-catenin signaling by up regulation of Foxo3a expression.Methods: Rat knee OA models were duplicated by using Hulth’s method. TMF (5 μg/mL and 20 μg/mL) was used for administration to cultured cells, which were isolated from the rat cartilages. Analysis of chondrocytes apoptosis, gene expression, and protein expression were conducted. In addition, miR-29a mimics and pcDNA3.1(+)-Foxo3a vector were used for transfection, luciferase reporter assay for detecting the activity of Wnt/β-catenin signaling, and co-immunoprecipitation for determining proteins interaction.Results: TMF down regulated miR-29a/β-catenin signaling activity and cleaved caspase-3 expression and up regulated Foxo3a expression in OA rat cartilages. In vitro, miR-29a mimics down regulated the expression of Foxo3a and up regulated the activity of Wnt/β-catenin signaling and cleaved caspase-3 expression. TMF ameliorated miR-29a/β-catenin-induced chondrocytes apoptosis by up regulation of Foxo3a expression.Conclusion: TMF exhibited chondroprotective activity by up regulating Foxo3a expression and subsequently inhibiting miR-29a/Wnt/β-catenin signaling activity. Keywords: osteoarthritis, chondrocytes apoptosis, miR-29a, Wnt/β-catenin, Foxo3a, TMF