Effect of Baricitinib on the epithelial-mesenchymal transition of alveolar epithelial cells induced by IL-6.

OBJECTIVE: Interstitial lung disease (ILD) is one of the common complications of Connective tissue disease (CTD). Epithelial-mesenchymal transition (EMT) is one of the main pathological mechanisms of ILD. IL-6 may induce ILD through the JAK/STAT pathway. Therefore, exploring the mechanism of IL-6 on the EMT of alveolar epithelial cells and inhibition JAK/STAT pathway with Baricitinib on the EMT of alveolar epithelial cells is helpful in revealing the pathogenesis of CTD-ILD and guiding treatment. METHODS: Electrochemiluminescence was applied to detect the changes in serum IL-6 levels before and after treatment in 37 patients with anti-synthetase syndrome-associated ILD; A549 cells (a human AEC cell line) were incubated with IL-6, Baricitinib, or both IL-6 and Baricitinib, and changes in EMT-related markers levels were measured using real-time PCR, western blotting and fluorescence microscopy. The related proteins in the JAK/STAT signaling pathways were examined by western blot. The level of Connective tissue growth factor (CTGF) and Hydroxyproline (Hyp) in cell supernatants was measured by ELISA. RESULTS: Serum IL-6 level in patients with anti-synthetase syndrome-associated ILD was significantly higher than that in health (6.78(4.19, 16.14)pg/ml vs. 2.10(1.43, 5.18)pg/ml, p < 0.01). The level of IL-6 in the improvement group of ASS-ILD was considerably decreased than that before treatment(before(7.48(4.54, 22.76) pg/mL vs. 5.00(3.46, 11.32)pg/mL, p < 0.01), p < 0.01), and the level of IL-6 in the progressive group of ASS-ILD was significantly higher than that before treatment(before(7.49(6.77, 35.80) pg/mL vs. 30.02(8.01, 82.98) pg/mL, p < 0.05). IL-6 increased the expression of epithelial phenotypic marker E-cadherin and inhibited mesenchymal phenotypic markers, including vimentin and N-cadherin in A549 cells. Moreover, IL-6-induced EMT was attenuated by Baricitinib. Furthermore, we found that IL-6 activated the phosphorylation of JAK1/2, STAT3, and Baricitinib, partially inhibiting these changes in this process. Baricitinib reduced the secretion of CTGF and Hyp in A549 cells. CONCLUSION: The significant higher level of IL-6 in patients with anti-synthase syndrome-associated ILD may be related to disease activity and recurrence. Our results suggest that Baricitinib attenuates epithelial-mesenchymal transition in alveolar epithelial cells in the presence of IL-6 through the JAK/STAT signaling pathway.

LncRNA HOXC-AS3 promotes non-small-cell lung cancer growth and metastasis through upregulation of YBX1.

NSCLC is common and is the primary cause of cancer-related deaths due to a lack of early diagnosis and its propensity for metastasis. The pathogenesis of NSCLC is still unclear. Here, we explored the molecular mechanisms underlying NSCLC development, focusing on the HOXC-AS3/YBX1/HOXC8 axis. Human NSCLC specimens and cell lines were used. qRT-PCR and western blotting were utilised to examine the levels of HOXC-AS3/YBX1/HOXC8. CCK-8, colony formation, scratch wound healing and Transwell assays were performed to evaluate cancer cell proliferation, migration and invasion. A nude mouse xenograft model was used to examine tumour growth and metastasis in vivo. RNA pull-down, chromatin immunoprecipitation, coimmunoprecipitation and dual-luciferase assays were applied to validate the interactions of HOXC-AS3/YBX1, MDM2/YBX1 and the YBX1/HOXC8 promoter. The levels of HOXC-AS3 and HOXC8 were increased in human NSCLC specimens and cells. Knockdown of HOXC-AS3 suppressed NSCLC cell proliferation, migration and invasion, as well as tumour growth and metastasis in vivo. HOXC-AS3 directly bound to YBX1 to suppress its ubiquitination mediated by MDM2. YBX1 bound to the HOXC8 promoter and enhanced its transcription. Knockdown of HOXC8 inhibited the effects of HOXC-AS3 overexpression on NSCLC. HOXC-AS3 promotes NSCLC growth and metastasis by stabilising YBX1 and thus increasing HOXC8 transcription. Our study indicates that the HOXC-AS3/YBX1/HOXC8 axis could serve as a biomarker for NSCLC diagnosis or as a target for therapy development.

YBX1 regulated by Runx3-miR-148a-3p axis facilitates non-small-cell lung cancer progression.

BACKGROUND: Y-box binding protein 1 (YBX1) is a common oncogene in non-small-cell lung cancer (NSCLC), which is regulated by microRNAs (miRNAs) and transcription factors. This research aims to explore the function of YBX1, miR-148a-3p and Runt-related transcription factor 3 (Runx3) in NSCLC development, and analyze their interactions. METHODS: YBX1, miR-148a-3p and Runx3 levels were detected using quantitative reverse transcription polymerase chain reaction(RT-PCR), Western blotting or immunohistochemical staining. The functions of YBX1, miR-148a-3p and Runx3 were assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, wound healing, transwell, flow cytometry, xenograft model and Western blotting analyses. The binding correlation was validated through dual-luciferase reporter analysis and chromatin immunoprecipitation (ChIP). RESULTS: YBX1 expression was upregulated, and miR-148a-3p and Runx3 levels were reduced in NSCLC samples and cell lines. YBX1 silence restrained NSCLC cell proliferation, migration, invasion and tumor growth, and enhanced apoptosis. YBX1 was targeted via miR-148a-3p. MiR-148a-3p knockdown promoted cell proliferation, migration, invasion and tumor growth, and repressed apoptosis, and these effects were abolished by YBX1 silence. Runx3 upregulation restrained cell proliferation, migration, invasion and tumor growth, and facilitated apoptosis. Runx3 bound with miR-148a-3p promotor to regulate miR-148a-3p expression. Runx3 silence modulated YBX1 expression though miR-148a-3p to promote NSCLC progression by increasing Cyclin D1, Cyclin B1, Slug-1, MMP-2 and MMP-9 levels. CONCLUSION: Runx3-miR-148a-3p axis targeted YBX1 to modulate NSCLC progression.