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• Research Paper Volume 12, Issue 18 pp 18436-18452

  Nudt21-mediated alternative polyadenylation of HMGA2 3′-UTR impairs stemness of human tendon stem cell

  Relevance score: 7.65606

  Yangbai Sun, Hua Chen, Hui Ye, Wenqing Liang, Kun-kuan Lam, Biao Cheng, Yong Lu, Chaoyin Jiang

  Keywords: HMGA2, tendon stem cells, stemness, pluripotency, Nudt21

  Published in Aging on September 26, 2020

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  Tendon-derived stem cells (TSCs) play a primary role in tendon physiology, pathology, as well as tendon repair and regeneration after injury. TSCs are often exposed to mechanical loading-related cellular stresses such as oxidative stress, resulting in loss of stemness and multipotent differentiation potential. Cytoprotective autophagy has previously been identified as an important mechanism to protect human TSCs (hTSCs) from oxidative stress induced impairments. In this study, we found that high-mobility AT-hook 2 (HMGA2) overexpression protects hTSCs against H₂O₂-induced loss of stemness through autophagy activation. Evidentially, H₂O₂ treatment increases the expression of Nudt21, a protein critical to polyadenylation site selection in alternative polyadenylation (APA) of mRNA transcripts. This leads to increased cleavage and polyadenylation of HMGA2 3′-UTR at the distal site, resulting in increased HMGA2 silencing by the microRNA let-7 and reduced HMGA2 expression. In conclusion, Nudt21-regulated APA of HMGA2 3′-UTR and subsequent HMGA2 downregulation mediates oxidative stress induced hTSC impairments.

  

  HMGA2 overexpression protects hTSCs from H₂O₂-induced loss of self-renewal capacity and pluripotency. hTSCs transfected with HMGA2 overexpression lentivirus (HMGA2 OE) or the negative control (nc) were subjected to H₂O₂ treatment at indicated concentrations for 24 h. Non-transfected cells (mock) were included for comparison. (A, B) The HMGA2 mRNA (a) and protein (b) levels were determined by qRT-PCR and western blot analysis, respectively. (C) Clonogenicity was evaluated by the colony formation assay. (D) The quantitative expression of stemness markers Nanog, Nucleostemin, Oct-4, and SSEA-4 of treated hTSCs were measured by qRT-PCR. (E) The multilineage differentiation capability was assessed as described in materials and methods. Scale bar = 50 μm. (F) The expression of markers for lineage-specific differentiation (PPARγ for adipogenesis, Runx-2 for osteogenesis and Sox-9 for chondrogenesis) at day 0 and day 14 of differentiation was evaluated by qRT-PCR. The data shown are from three replicates and are presented as mean ± SD. *p < 0.05, **p < 0.01.

  
  
  

  

  HMGA2 overexpression protects hTSCs from H₂O₂-induced cell death, cell senescence and decreased S-phase cell number. hTSCs transfected with lenti-HMGA2 (HMGA2 OE) or the negative control (nc) were subjected to H₂O₂ treatment at indicated concentrations. Non-transfected cells (mock) were included for comparison. (A) Cell viability was determined by the CCK-8 assay after 24 h of H₂O₂ treatment. (B) Cell senescence was assessed by β-gal staining after 72 h of H₂O₂ treatment. (C) Cell cycle analysis was performed by flow cytometry after 24 h of H₂O₂ treatment. The data shown are from three replicates and are presented as mean ± SD. *p < 0.05, **p < 0.01.

  
  
  

  

  HMGA2 overexpression activates autophagy in H₂O₂-treated hTSCs. (A) hTSCs were incubated with rapamycin and 3-MA for 24 h, alone or in combination as indicated, and subsequently exposed to 0.5 mM H₂O₂ for 24 h. hTSCs were transfected with lenti-HMGA2 (HMGA2 OE) or the negative control (nc) in the presence or absence of 3-MA for 24 h, and subsequently exposed to 0.5 mM H₂O₂ for 24 h. Normal cells (mock) were included for comparison. The protein levels of the autophagic markers LC3-I, LC3-II, and Beclin-1 were determined by western blot analysis. (B) GFP-LC3-transfected hTSCs were subjected to treatments as described in a. GFP-LC3-labeled vacuoles (puncta) were detected by fluorescence imaging at 60× magnification. The data shown are from three replicates and are presented as mean ± SD. *p < 0.05, **p < 0.01.

  
  
  

  

  Autophagy blockage abolishes the protective effects of HMGA2 overexpression against H₂O₂-induced loss of potential regenerative power and pluripotency. hTSCs transfected with lenti-HMGA2 (HMGA2 OE) or the empty virus (nc) were incubated with 3-MA and rapamycin for 24 h, alone or in combination as indicated, and subsequently exposed to 0.5 mM H₂O₂ for 24 h. Untransfected cells (mock) were included for comparison. (A) Clonogenicity was assessed by the colony formation assay. (B) The quantitative expression of stemness markers Nanog, Nucleostemin, Oct-4, and SSEA-4 were measured by qRT-PCR. (C–E) The multilineage differentiation capability and the expression of markers for lineage-specific differentiation (PPARγ for adipogenesis, Runx-2 for osteogenesis and Sox-9 for chondrogenesis) was evaluated by qRT-PCR. Scale bar = 50 μm. The data shown are from three replicates and are indicated as mean ± SD. *p < 0.05, **p < 0.01.

  
  
  

  

  Autophagy blockage abolishes the protective effects of HMGA2 overexpression against H₂O₂-induced cell death, cell senescence and decreased S-phase cell number. hTSCs transfected with lenti-HMGA2 (HMGA2 OE) or the empty virus (nc) were incubated with 3-MA and rapamycin for 24 h, alone or in combination as indicated, and subsequently exposed to 0.5 mM H₂O₂ treatment. Untransfected cells (mock) were included for comparison. (A) Cell viability was determined by the CCK-8 assay after 24 h of H₂O₂ treatment. (B) Cell senescence was assessed by β-gal staining after 72 h of H₂O₂ treatment. (C) Cell cycle analysis was performed by flow cytometry after 24 h of H₂O₂ treatment. The data shown are from three replicates and are indicated as mean ± SD. *p < 0.05, **p < 0.01.

  
  
  

  

  Nudt21 is induced by H₂O₂ and down-regulates HMGA2 in hTSCs. (A) hTSCs were subjected to H₂O₂ treatment at indicated concentrations for 24 h. The protein levels of Nudt21 were determined by western blot analysis. n = 3; *p < 0.05, **p < 0.01 vs. 0 mM H₂O₂. (B) hTSCs transfected with shNudt21-1, shNudt21-2 or a scrambled control shRNA (nc) lentivirus were cultured in the presence or absence of 0.5 mM H₂O₂ for 24 h. Nudt21 and HMGA2 protein levels were determined by western blot analysis. Untransfected cells (mock) were included for comparison. (C–E) hTSCs transfected with a scrambled control shRNA (nc), shNudt21-2 and shHMGA2, alone or in combination as indicated were cultured in the presence or absence of 0.5 mM H₂O₂ for 24 h. (C) Clonogenicity was evaluated by the colony formation assay. (D) Cell viability was determined by the CCK-8 assay. (E) Cell senescence was assessed by β-gal staining 72 h after H₂O₂ treatment. The data shown are from three replicates and are indicated as mean ± SD. *p < 0.05, **p < 0.01.

  
  
  

  

  Nudt21 upregulates let-7-mediated HMGA2 silencing through APA. (A, B) hTSCs transfected with shNudt21-1, shNudt21-2 or a scrambled control shRNA (nc) lentivirus were cultured in the presence or absence of 0.5 mM H₂O₂ for 24 h. (A) The short and long HMGA2 3′-UTR transcripts were detected by RT-PCR. The long transcript-to-total transcript ratios are shown. (B) The short (Proximal) and long (Distal) HMGA2 3′-UTR transcripts were detected by 3′-RACE. (C) hTSCs were transfected with 100 ng psiCHECK2-HMGA2 along with shNudt21-1, shNudt21-2 or a scrambled control shRNA (control) and 0, 10 or 20 μg let-7 as indicated in the presence or absence of 0.5 mM H₂O₂. The luciferase activity was determined 48 h after transfection. (D) hTSCs were transfected with 100 ng psiCHECK2-HMGA2 or psiCHECK2-HMGA2 MUT along with 0, 10 or 20 μg let-7 as indicated in the presence or absence of 0.5 mM H₂O₂. The luciferase activity was determined 48 h after transfection. The data shown are from three replicates and are indicated as mean ± SD. *p < 0.05, **p < 0.01.

  
  
  

• Research Paper Volume 11, Issue 24 pp 11937-11954

  Hsa_circ_0006948 enhances cancer progression and epithelial-mesenchymal transition through the miR-490-3p/HMGA2 axis in esophageal squamous cell carcinoma

  Relevance score: 7.2834682

  Zihao Pan, Jiatong Lin, Duoguang Wu, Xiaotian He, Wenjian Wang, Xueting Hu, Lei Zhang, Minghui Wang

  Keywords: circRNA, esophageal squamous cell carcinoma, EMT, hsa_circ_0006948, HMGA2

  Published in Aging on December 26, 2019

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  Increasing studies have indicated that circular RNAs (circRNAs) are important in cancer progression. However, few circRNAs associated with epithelial-mesenchymal transition (EMT) have been elucidated in esophageal squamous cell carcinoma (ESCC). In this study, we aimed to identify whether hsa_circ_0006948 promotes ESCC cell EMT and explore its biological mechanisms. We first screened circRNA expression profiles using a circRNA microarray, and found that the expression of a novel circRNA, hsa_circ_0006948, is increased in 153 ESCC tissues and cell lines compared with noncancerous tissues and cell lines. Additionally, high hsa_circ_0006948 levels were positively associated with lymphatic metastasis and poor prognosis. Functionally, the assays indicated that cell proliferation, migration and invasion were promoted by hsa_circ_0006948 both in vitro and in vivo. Furthermore, we analyzed the relationship between hsa_circ_0006948 and miR-490-3p through bioinformatics, luciferase reporter assays, RNA immunoprecipitation and qRT-PCR. We found that hsa_circ_0006948 could bind directly to miR-490-3p which targets the 3’UTR of the oncogene HMGA2 to induce EMT. In conclusion, hsa_circ_0006948 was overexpressed in ESCC tissues and promoted cancer progression, and it could induce EMT by enhancing HMGA2 by sponging miR-490-3p, suggesting that hsa_circ_0006948 could be a biomarker for ESCC.

  

  The identification and characteristics of hsa_circ_0006948 in ESCC cells. (A) Heat map showing the differential expression and hierarchical clustering of circRNAs between ESCC and adjacent normal tissues. (B) Volcano plot, x-axis: log2 (fold change); y-axis: -log10 (P-value). The vertical lines correspond to 2.0-fold up and down, and the horizontal line represents a P-value of 0.05. The red points in the plot represent differentially expressed circRNAs with statistical significance. (C) The relative hsa_circ_0006948 was significantly high in ESCC cells. (D) q RT-PCR analyses of expression of hsa_circ_0006948, linFNDC3B and GAPDH in various ESCC cell lines. Y-axis is the raw CT value. (E) Above: Divergent primers detected circular RNAs in cDNA but not gDNA. Below: Three exons form hsa_circ_0006948 by back splicing from chromosomal region and Sanger sequencing of hsa_circ_0006948 showed the back-splice junction (∇). (F) Fluorescence in situ hybridization assay was conducted to determine the subcellular localization of hsa_circ_0006948.

  
  
  

  

  The hsa_circ_0006948 was up-regulated in ESCC tissues and correlates with poor patients prognosis. (A) hsa_circ_0006948 expression was assessed in cancer tissues and normal tissues. (B) The relative hsa_circ_0006948 expression was assessed in 153 ESCC patients. Blue: low level: Red: high level. (C) Present the ROC curve analysis of hsa_circ_0006948 for the diagnosis of ESCC. (D) The expression of hsa_circ_0006948 was significantly higher in patients with lymph nodes at N1-N3 stage than in those with lymph nodes at NO stage. (E) Kaplan-Meier’s analyses of correlations between the hsa_circ_0006948 expression levels and OS (overall survival) of 153 ESCC patients. (F) Present the ROC curve analysis of hsa_circ_0006948 for the prognosis of ESCC.

  
  
  

  

  The function of hsa_circ_0006948 in ESCC cells. (A) Expression of hsa_circ_0006948 and FNDC3B mRNA in TE-1 cells transfected with siRNAs. and (B) KYSE30 cells overexpressing hsa_circ_0006948. (C and D) The effect of hsa_circ_0006948 on cell proliferation in vitro using colony formation assay and CCK8 assay after knocking down hsa_circ_0006948 in TE-1. (E) Cell migration and invasion abilities were assessed by transwell assay after knocking down hsa_circ_0006948 in TE-1 cells. (F and G) The effect of hsa_circ_0006948 on cell proliferation in vitro using colony formation assay and CCK8 assay after overexpressing hsa_circ_0006948 in KYSE30 cells. (H) Cell migration and invasion abilities were assessed by transwell assay after overexpressing hsa_circ_0006948 in KYSE30 cells. (I) Western blot analysis comparing downregulated and upregulated-hsa_circ_0006948 ESCC cells with control cells were shown for vimentin, E-cadherin and N-cadherin* P<0.05,**P<0.01, ***P<0.001.

  
  
  

  

  Hsa_circ_0006948 could serve as a miR-490-3p sponge in ESCC cells. (A) Top five miRNA targets are displayed. (B) A schematic drawing showing the putative binding sites of the miR-490-3p associated with hsa_circ_0006948. (C) Luciferase reporter assay for the luciferase activity of Luc-circ_0006948 WT or Luc-circ_0006948 mutant in cells cotransfected with miRNA mimics. Data are the mean±SD of three experiments. (D) Hsa_ciic_0006948 expression was negatively correlated with miR-490-3p expression in 153 patients with ESCC. (E and F) RNA immunoprecipitation (RIP) assays were performed using an anti-AGO2 antibody with the transfection of miR-490-3p mimics (miR-490-3p) or miR-NC in TE-1 and KYSE30 cells to detect hsa_circ_0006948 expression according to qRT-PCR. ***P<0.001.

  
  
  

  

  The function of miR-490-3p in ESCC cells. (A) and (B) The effect of miR-490-3p on cell proliferation in vitro using CCK8 assay and colony formation assay after overexpressing miR-490-3p in TE-1. (C) Cell migration and invasion abilities were assessed by transwell assay after overexpressing miR-490-3p in TE-1 cells. (D and E) The effect of miR-490-3p on cell proliferation in vitro using CCK8 assay and colony formation assay after knocking down miR-490-3p in KYSE30 cells. (F) Cell migration and invasion abilities were assessed by transwell assay after knocking down miR-490-3p in KYSE30 cells. (G) Luciferase reporter assay for the luciferase activity of HMGA2-3 ′UTR WT or HMGA2-3 ’UTR mutant in cells cotransfected with miRNA mimics. (H) The invasion ability was evaluated by transwell Matrigel invasion assays (I) Knockdown of HMGA2 inhibits EMT (J) Western blot analysis comparing upregulated and downregulated- miR-490-3p ESCC cells with control cells were shown for vimentin, E-cadherin, N-cadherin and HMGA2. (K) The expression of HMGA2 and EMT markers was detected by Western blot after transfection with mimics or HMGA2 overexpression plasmids.* P<0.05,**P<0.01, **P<0.001.

  
  
  

  

  Overexpression of hsa_circ_0006948 reverses the suppressive roles of miR-490-3p in ESCC. The migration (A) and invasion (B) abilities inhibited by miR-490-3p were reversed after co-transfection with hsa_circ_0006948 using transwell assay. (C) The expression of levels of vimentin, N-cadherin and HMGA2 were inhibited by down-regulated hsa_circ_0006948, but this effect was reversed by miR-490-3p inhibitor presented in Western blot analysis. (D) The expression of levels of vimentin, N-cadherin and HMGA2 were enhanced by up-regulated hsa_circ_0006948, but this effect was reversed by miR-490-3p mimics presented in Western blot analysis. * P<0.05, **P<0.01, ***P<0.001.

  
  
  

  

  Knockdown of HMGA2 abolishes the oncogenic effect induced by hsa_circ_0006948 in ESCC. (A and B) The cell proliferation was measured by CCK8 assays. (C) The invasion ability was evaluated by transwell Matrigel invasion assays. (D) The upregulation of vimentin, N-cadherin. HMGA2 and the downregulation of E-cadherin in TE-1 and KYSE30 cells transfected with hsa_ciic_0006948 overexpression plasmid were abolished by knockdown of HMGA2 as detected by Western blot analysis. (E) A mechanistic model: hsa_circ_0006948 functions as a miR-490-3p sponge and regulates HMGA2 through inhibiting miR-490-3p activity in ESCC cells' EMT. *P<0.05, **P<0.01, ***P<0.001.

  
  
  

  

  The images of tumor-bearing nude mice from three treatment groups (n = 5 for each group) on the 30^th day. (A) Tumor volumes were monitored with a caliper during the time course of 30 days, and tumor weights were also measured at the end of this study.(B) IHC analysis of E-cadherin, N-cadherin, vimentin and HMGA2. **P<0.01 vs control group, ## P<0.01 vs si-hsa_circ_0006948 group.

  
  
  

• Research Paper pp undefined-undefined

  LncRNA HAGLROS contribute to papillary thyroid cancer progression by modulating miR-206/HMGA2 expression

  Relevance score: 7.6818542

  Zhaohui Zeng, Liping Chen, Shengtao Tang, Haiwen Hou, Yuan Liu, Juhui Li

  Keywords: papillary thyroid cancer, HAGLROS, miR-206, HMGA2

  Published in Aging on Invalid Date

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  Objective: Papillary thyroid cancer (PTC) is one of the most serious diseases of the endocrine system. In view of the limited therapeutic effects of current medical methods, this study starts from the molecular level and looks for potential treatments. The interaction between HAGLROS/miR-206/HMGA2 was studied using multi-omics methods, which provided new ideas and methods for future treatments.

  Method: Microarray analysis and R language were used for differential analysis to screening experimental targets of lncRNA, miRNA, and mRNA. qRT-PCR was used to detect RNA expression in tissues and cells. Double luciferase reporter assays analyzed and validated binding relationships between different RNAs. Colony formation, flow cytometry, and transwell assays were used to measure the effect of them on cell proliferation, apoptosis, and migration.

  Result: Microarray analysis identified lncRNAs, miRNAs, and mRNAs differentially expressed in PTC and normal cells, and selected lncRNA HAGLROS, miR-206, and mRNA HMGA2 as study subjects. LncRNA HAGLROS and mRNA HMGA2 were highly expressed in PTC cells while miR-206 was lowly expressed in PTC cells. LncRNA HAGLROS/HMGA2 can inhibit apoptosis of PTC cells, promote proliferation and migration, and miR-206 promotes the above process. HAGLROS and HMGA2 were negatively correlated with miR-206. shHAGLROS promoted miR-206 expression, inhibited HMGA2 expression and repressed PTC tumor growth in mice.

  Conclusions: HAGLROS promotes the growth of PTC by competitively binding to miR-206 to promote HMGA2 expression.