GAO Fangyu, LIU Xuan, TIAN Di, CHEN Xiyuan, TIAN Xueyan, JIAO Ziming, HOU Pengyi, WANG Faxuan. Effect of lncRNA MRAK050699 on epithelial-mesenchymal transition in rat type Ⅱ alveolar epithelial cells induced by SiO2 dust[J]. Journal of Environmental and Occupational Medicine, 2021, 38(3): 231-237. DOI: 10.13213/j.cnki.jeom.2021.20477
Citation: GAO Fangyu, LIU Xuan, TIAN Di, CHEN Xiyuan, TIAN Xueyan, JIAO Ziming, HOU Pengyi, WANG Faxuan. Effect of lncRNA MRAK050699 on epithelial-mesenchymal transition in rat type Ⅱ alveolar epithelial cells induced by SiO2 dust[J]. Journal of Environmental and Occupational Medicine, 2021, 38(3): 231-237. DOI: 10.13213/j.cnki.jeom.2021.20477

Effect of lncRNA MRAK050699 on epithelial-mesenchymal transition in rat type Ⅱ alveolar epithelial cells induced by SiO2 dust

  • Background The pathogenesis of silicosis is unknown and there is no specific treatment for the disease. Long-chain non-coding RNA (lncRNA) may play a role in the process of silicosis fibrosis.
    Objective This experiment aims to investigate the effect of lncRNA MRAK050699 on epithelial-mesenchymal transition (EMT) in rat type Ⅱ alveolar epithelial cells induced by SiO2 dust.
    Methods The rat alveolar macrophages (NR8383 cells) and RLE-6TN cells were co-cultured in vitro and divided into a normal group, a model group, a knockdown control group, and a knockdown group. The viabilities of NR8383 cells stimulated by 10, 20, 40, 80, 160, and 320 μg·cm-2 SiO2 dust were detected by CCK8 method, the levels of transforming growth factor-β (TGF-β) stimulated by 0, 20, 40, and 80 μg·cm-2 SiO2 dust were detected by ELISA, and the knockdown efficiency was tested by real-time quantitative PCR (RT-qPCR). RLE-6TN cells in two 6-well plates, RLE-6TN cells transfected with knockdown control virus in a plate, and RLE-6TN cells transfected with knockdown virus in a plate were cultured for 24 h. At the same time, the other four 6-well plates were inserted into the Transwell chamber, and NR8383 cells were cultured normally for 24 h. The ratio of NR8383 cells to RLE-6TN cells was 1:2. Then the Transwell chamber together with NR8383 cells were transferred into the 6-well plates of RLE-6TN cells, and 40 μg·cm-2 SiO2 dust suspension was added to the Transwell chamber of the latter three groups of co-culture system. After further 24 h, the RLE-6TN cells in the lower compartment of the Transwell chamber were observed under microscope. The mRNA and protein expression levels of E-cadherin, N-cadherin, and α-SMA were detected by Western blotting and RT-qPCR respectively.
    Results CCK8 and ELISA results showed that the viability of NR8383 cells decreased significantly, and the survival rate was considered eligible when SiO2 was 40 μg·cm-2, as the level of TGF-β in supernatant was high enough for subsequent experiments; the knockdown efficiency of the knockdown group was about 50%, and the difference was statistically significant (P < 0.05) comparing to the knockdown control group. After co-culture, the morphology of RLE-6TN cells was observed under microscope: The normal cells showed typical epithelioid characteristics of polygonal and oval appearance and tight junction; the cells in the model group showed interstitial-like cell morphology, including long fusiform and spindle shape, and the intercellular space became wider; the knockdown control group was similar to the model group, showing the morphology of interstitial cells; however, in the knockdown group, the cells were basically in a state of tight junction. The mRNA and protein expression levels of E-cadherin, N-cadherin, and α-SMA in each group were significantly different (F=28.11, 35.72, 15.26, P < 0.05; F=328.78, 51.84, 13.39, P < 0.05). Compared with the normal group, the mRNA and protein expression levels of E-cadherin in the model group decreased by 32% and 67%, while the levels of N-cadherin increased by 228% and 164%, and those of α-SMA increased by 274% and 166% (P < 0.05). Compared with the knockdown control group, the mRNA and protein expression levels of E-cadherin in the knockdown group increased, while the levels of N-cadherin and α-SMA decreased (P < 0.05). There were no significant differences in mRNA and protein expressions of selected genes between the model group and the knockdown control group (P>0.05).
    Conclusion MRAK050699 plays an important role in silicotic fibrosis and may be involved in the EMT of type Ⅱ alveolar epithelial cells induced by SiO2.
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