高芳瑜, 刘煊, 田娣, 陈溪渊, 田雪艳, 焦子铭, 侯朋邑, 王发选. lncRNA MRAK050699对二氧化硅粉尘诱导大鼠肺泡Ⅱ型上皮细胞上皮-间质转换的影响[J]. 环境与职业医学, 2021, 38(3): 231-237. DOI: 10.13213/j.cnki.jeom.2021.20477
引用本文: 高芳瑜, 刘煊, 田娣, 陈溪渊, 田雪艳, 焦子铭, 侯朋邑, 王发选. lncRNA MRAK050699对二氧化硅粉尘诱导大鼠肺泡Ⅱ型上皮细胞上皮-间质转换的影响[J]. 环境与职业医学, 2021, 38(3): 231-237. DOI: 10.13213/j.cnki.jeom.2021.20477
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

lncRNA MRAK050699对二氧化硅粉尘诱导大鼠肺泡Ⅱ型上皮细胞上皮-间质转换的影响

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

  • 摘要: 背景

    矽肺发病机制未明且没有特效治疗方法,长链非编码RNA(lncRNA)在矽肺纤维化进程中或可发挥作用。

    目的

    探讨lncRNA MRAK050699对SiO2粉尘诱导大鼠肺泡Ⅱ型上皮细胞(RLE-6TN细胞)发生上皮-间质转换(EMT)过程的影响。

    方法

    建立大鼠肺泡巨噬细胞(NR8383细胞)与RLE-6TN细胞共培养体外矽肺模型,分为正常组、模型组、敲减对照组、敲减组。采用CCK8法检测NR8383细胞在10、20、40、80、160、320μg·cm-2 SiO2粉尘刺激下的活力,酶联免疫吸附试验(ELISA)法检测在0、20、40、80μg·cm-2粉尘刺激下的转化生长因子-β(TGF-β)含量,实时荧光定量PCR(RT-qPCR)法检测MRAK050699敲减效率。取四个6孔板,在前两个6孔板中正常培养RLE-6TN细胞24 h,后两个6孔板分别培养转染阴性对照病毒的RLE-6TN细胞24 h,和转染敲减MRAK050699病毒的RLE-6TN细胞24 h;同时在另四个6孔板中,插入Transwell小室,正常培养NR8383细胞24 h。NR8383细胞与RLE-6TN细胞铺板比例为1∶2,之后将Transwell小室连同NR8383细胞一同转入RLE-6TN细胞的6孔板中,向后三组共培养体系的Transwell小室中加入40 μg·cm-2 SiO2粉尘悬液150 μL后,四组同时继续培养24 h,取Transwell小室下室的RLE-6TN细胞置于显微镜下观察,使用Western blotting及RT-qPCR法检测E-cadherinN-cadherinα-SMA的mRNA和蛋白表达水平。

    结果

    CCK8、ELISA法结果显示SiO2剂量在40 μg·cm-2时,细胞活力下降较为明显且可保证后续实验的有效进行,上清液中TGF-β表达水平是对照组的2.94倍,可用于后续实验;敲减组MRAK050699的表达水平相比敲减对照组明显下调,敲减效率约为50%(P < 0.05)。显微镜下观察共培养后RLE-6TN细胞形态,正常组细胞呈现典型的上皮样特征,为多边形、卵圆形且紧密连接。模型组细胞呈现间质样细胞形态,表现为长梭形、纺锤形,并且细胞间隙变宽。敲减对照组与模型组类似,呈现间质细胞形态。而敲减组的细胞基本处于紧密连接状态。各组细胞中E-cadherinN-cadherinα-SMA mRNA和蛋白表达水平差异均有统计学意义(F=28.11、35.72、15.26,P < 0.05;F=328.78、51.84、13.39,P < 0.05)。与正常组相比,模型组E-cadherin mRNA和蛋白表达水平分别下降32%、67%,N-cadherin mRNA和蛋白表达水平分别上升至2.28、1.64倍,α-SMA mRNA和蛋白表达水平分别上升至2.74、1.66倍(均P < 0.05);敲减组相较于敲减对照组,E-cadherin mRNA和蛋白表达水平上升,而N-cadherinα-SMA的mRNA和蛋白表达水平下降(P < 0.05);模型组与敲减对照组的各基因mRNA和蛋白表达水平差异无统计学意义(P>0.05)。

    结论

    MRAK050699在矽肺纤维化中发挥着重要的作用,可能参与了SiO2诱导的肺泡Ⅱ型上皮细胞EMT过程。

     

    Abstract: 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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