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Mesenchymal cell proliferation is critical for the growth of the palate shelf. All-trans retinoic acid (atRA), as well as pathways associated with TGF-β/Smad signaling, play crucial roles in the proliferation of mouse embryonic palate mesenchymal (MEPM) cells. We have found that MEPM-cell proliferation was regulated by atRA and exogenous TGF-β3 could significantly antagonize the atRA-mediated suppression of MEPM cell proliferation, which is closely associated with the regulation of TGF-β/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been reported to activate TGF-β/Smad signaling, thereby regulating cellular proliferation, differentiation, and related processes. Here, we found that Meg3 expression increased significantly in atRA-treated MEPM cells while TGF-β3 treatment markedly inhibited Meg3 expression and antagonized the effect of atRA on Meg3. Moreover, Smad2 was found to interact directly with Meg3, and atRA treatment significantly enriched Meg3 in Smad2-immunoprecipitated samples. After Meg3 deletion, the effects of atRA on the proliferation of MEPM cells and TGF-β3-dependent protein expression were lost. Hence, we speculate that Meg3 has a role in the RA-induced suppression of MEPM cell proliferation by targeting Smad2 and thereby mediating TGF-β/Smad signaling inhibition., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Palatal mesenchymal cell proliferation is essential to the process of palatogenesis, and the proliferation of mouse embryonic palate mesenchymal (MEPM) cells is impacted by both all-trans retinoic acid (atRA) and the TGF-β/Smad signaling pathway. The long non-coding RNA (lncRNA) MEG3 has been shown to activate TGF-β/Smad signaling and to thereby regulate cell proliferation, differentiation, and related processes. Herein, we found that atRA treatment (100 mg/kg) promoted Meg3 upregulation in MEPM cells, and that such upregulation was linked to the suppression of MEPM cell proliferation in the context of secondary palate fusion on gestational day (GD) 13 and 14. Moreover, the demethylation of specific CpG sites within the lncRNA Meg3 promoter was detected in atRA-treated MEPM cells, likely explaining the observed upregulation of this lncRNA. Smad signaling was also suppressed by atRA treatment in these cells, and RNA immunoprecipitation analyses revealed that Smad2 can directly interact with Meg3 in MEPM cells following atRA treatment. Therefore, we propose a model wherein Meg3 is involved in the suppression of MEPM cell proliferation, functioning at least in part via interacting with the Smad2 protein and thereby suppressing Smad signaling in the context of atRA-induced cleft palate., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2021. Published by Elsevier B.V.)

Gemcitabine is the first-line chemotherapy drug for cholangiocarcinoma (CCA), but acquired resistance has been frequently observed in CCA patients. To search for potential long noncoding RNAs (lncRNAs) involved in gemcitabine resistance, two gemcitabine resistant CCA cell lines were established and dysregulated lncRNAs were identified by lncRNA microarray. Long intergenic non-protein coding RNA 665 (LINC00665) were found to rank the top 10 upregulated lncRNAs in our study, and high LINC00665 expression was closely associated with poor prognosis and chemoresistance of CCA patients. Silencing LINC00665 in gemcitabine resistant CCA cells impaired gemcitabine tolerance, while enforced LINC00665 expression increased gemcitabine resistance of sensitive CCA cells. The gemcitabine resistant CCA cells showed increased EMT and stemness properties, and silencing LINC00665 suppressed sphere formation, migration, invasion and expression of EMT and stemness markers. In addition, Wnt/β-Catenin signaling was activated in gemcitabine resistant CCA cells, but LINC00665 knockdown suppressed Wnt/β-Catenin activation. B-cell CLL/lymphoma 9-like (BCL9L), the nucleus transcriptional regulators of Wnt/β-Catenin signaling, plays a key role in the nucleus translocation of β-Catenin and promotes β-Catenin-dependent transcription. In our study, we found that LINC00665 regulated BCL9L expression by acting as a molecular sponge for miR-424-5p. Moreover, silencing BCL9L or miR-424-5p overexpression suppressed gemcitabine resistance, EMT, stemness and Wnt/β-Catenin activation in resistant CCA cells. In conclusion, our results disclosed the important role of LINC00665 in gemcitabine resistance of CCA cells, and provided a new biomarker or therapeutic target for CCA treament.

Background: Cholangiocarcinoma (CCA) is a malignant tumor in the world. LncRNA HOX transcript antisense intergenic RNA (HOTAIR) was identified as a crucial regulator in various cancers including CCA. This study aimed to unravel the functions of HOTAIR and its biological mechanism in CCA, hinting for the new therapeutic targets in CCA., Methods: The levels of HOTAIR, miR-204-5p and HMGB1 in CCA tissues and cell lines (HuB28 and HuCCT1) were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot was conducted to detect the protein levels of LC3-I, LC3-II, Beclin-1 and HMGB1. The relationships among HOTAIR, miR-204-5p and HMGB1 were examined by dual-luciferase reporter assay, RNA immunoprecipitation (RIP) assay and RNA pull down assay. Cell proliferation ability and apoptosis rate were assessed by CCK8 assay and flow cytometry, respectively. in vivo experiment was conducted to examine the bio-functions of HOTAIR in nude mice., Results: HOTAIR and HMGB1 were over-expressed, while miR-204-5p was lowly expressed in CCA tissues and cells. The dual-luciferase reporter assay indicated that miR-204-5p was a target of HOTAIR, and HMGB1 was a target of miR-204-5p. The restoration experiments showed that HOTAIR repressed cell apoptosis, autophagy and promoted cell proliferation via miR-204-5p/HMGB1 axis. Additionally, HOTAIR silencing retarded the xenograft tumor growth by up-regulation of miR-204-5p and down-regulation of HMGB1., Conclusion: These data unraveled that lncRNA HOTAIR regulated HMGB1 to suppress cell apoptosis, autophagy and induce cell proliferation by sponging miR-204-5p in CCA. Thus, this new regulatory pathway may provide new therapeutic targets for CCA., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)