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中国人民解放军总医院老年心血管病研究所
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中华老年多器官疾病杂志编辑委员会
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创刊人 王士雯
总编辑 范利
副总编辑 陈韵岱
执行主编 叶大训
编辑部主任 王雪萍
ISSN 1671-5403
CN 11-4786
创刊时间 2002年
出版周期 月刊
邮发代号 82-408
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王争,裴小华,赵卫红.miR-200c改善转化生长因子-β1诱导的肾小管上皮细胞纤维化的机制[J].中华老年多器官疾病杂志,2020,19(8):612~616
miR-200c改善转化生长因子-β1诱导的肾小管上皮细胞纤维化的机制
Mechanism of miR-200c in alleviating fibrosis in renal tubular epithelial cells induced by transforming growth factor-β1
投稿时间:2019-08-01  
DOI:10.11915/j.issn.1671-5403.2020.08.143
中文关键词:  miR-200c;自噬;转化生长因子-β1;纤维化
英文关键词:miR-200c; autophagy; transforming growth factor-β1; fibrosis This work was supported by National Natural Science Foundation of China
基金项目:国家自然科学基金(H0511-81670677);江苏省医学重点学科项目(ZDXKA2016003);江苏省医学重点人才项目(ZDRCA2016021);江苏省干部保健课题(BJ17018,BJ16016)
作者单位E-mail
王争 南京医科大学第一附属医院老年肾科,江苏省老年医学重点实验室,南京 210029 zhaoweihongny@njmu.edu.cnmechanism 
裴小华 南京医科大学第一附属医院老年肾科,江苏省老年医学重点实验室,南京 210029 zhaoweihongny@njmu.edu.cnmechanism 
赵卫红 南京医科大学第一附属医院老年肾科,江苏省老年医学重点实验室,南京 210029 zhaoweihongny@njmu.edu.cnmechanism 
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中文摘要:
      目的 探讨miR-200c是否通过调节自噬缓解转化生长因子-β1(TGF-β1)诱导的肾小管上皮细胞纤维化。方法 不同浓度(0、5、10、15、20、30ng/ml)TGF-β1刺激HK2细胞48h后倒置显微镜下观察细胞形态,CCK-8检测细胞增殖情况。选择致HK2细胞增殖最快的TGF-β1浓度(c)进行转染实验:细胞转染40nmol/L无意义序列RNA(NC组);细胞转染40nmol/L无意义序列RNA后c浓度TGF-β1处理细胞48h(NC+T组);细胞转染40nmol/L miR-200c mimics(M组);细胞转染40nmol/L miR-200c mimics后c浓度TGF-β1处理细胞48h(M+T组)。qRT-PCR检测miR-200c转染效率。Western blotting检测4组细胞Smad通路蛋白Smad2和Smad3、α-平滑肌肌动蛋白(α-SMA)、钙黏蛋白(E-cad)表达量,以及自噬相关蛋白LC3(包括LC3Ⅱ/LC3Ⅰ比值)和P62。结果 10ng/ml的TGF-β1刺激HK2细胞48h后增殖最快,镜下细胞形态由正常的卵圆形铺路石状变为长梭形,纤维化形态最明显。qRT-PCR显示M组miR-200c表达量是NC组(212.42±12.25)倍(t=24.41,P=0.000),说明转染有效。Western blotting结果显示,与NC组比较,NC+T组加入10ng/ml TGF-β1的HK2细胞Smad2、Smad3和α-SMA表达量增加,E-cad蛋白表达量降低(P<0.05)。与NC+T组比较,M+T组Smad2、Smad3和α-SMA表达量减少,E-cad表达量增加(P<0.05),提示miR-200c表达增加抑制了纤维化。进一步发现,M组较NC组的LC3-Ⅱ/LC3-Ⅰ表达水平增加,P62表达水平降低,提示自噬活性增加;M+T组较NC+T组LC3-Ⅱ/LC3-Ⅰ表达水平增加,P62表达水平降低,提示miR-200c表达升高缓解了纤维化程度。结论 miR-200c可能通过激活细胞自噬缓解TGF-β1诱导的肾小管上皮细胞纤维化。
英文摘要:
      Objective To investigate whether miR-200c can alleviate fibrosis in renal tubular epithelial cells induced by transforming growth factor-β1 (TGF-β1-induced) via the regulation of autophagy. Methods HK2 cells were stimulated with TGF-β1 at different concentrations (0,5, 10,15, 20,30ng/ml) for 48h, the cell morphology was observed under an inverted microscope, and the cell proliferation was detected by CCK-8. The concentration (c) of TGF-β1, which induced the most rapid proliferation of HK2 cells, was selected for transfection. The cells were transfected with 40nmol/L nonsense RNA (NC group), being treated with 40nmol/L nonsense RNA (NC+T group) for 48 h, with 40 nmol/L miR-200c mimics (M group), and with 40nmol/L miR-200c mimics (M+T group). qRT-PCR was used to detect the transfection efficiency of mir-200c. Western blotting was used to detect the expression of Smad pathway protein Smad2 and Smad3, α-smooth muscle actin(α-SMA), E-cadherin (E-cad), autophagy-related protein LC3 and p62. Results HK2 cells stimulated by TGF-β1 at 10ng/ml proliferated the fastest 48h later. Microscopic morphology of HK2 cells changed from normal oval paving stone to long fusiform with the most obvious fibrosis. qRT-PCR showed that the expression of miR-200c in group M was(212.42±12.25) times higher than that in group NC (t=24.41, P=0.000), indicating that the transfection was effective. Western blotting showed that expression of Smad2, Smad3 and α-SMA in HK2 cells in NC+T group increased and E-cad protein expression decreased compared with NC group (P<0.05). Compared with NC+T group, the expression of Smad2, Smad3 and α-SMA in M+T group decreased, and the expression of E-cad increased (P<0.05), suggesting that the increase of miR-200c expression inhibited fibrosis. It was further found that the expression of LC3-Ⅱ/LC3-Ⅰin M group was higher than that in NC group, and the expression of p62 was lower, suggesting increased autophagy; the expression of LC3-Ⅱ/LC3-Ⅰ in M+T group was higher than that in NC+T group, and the expression of p62 was lower, suggesting that the increased expression of miR-200c alleviated fibrosis. Conclusion miR-200c may alleviate TGF-β1-induced renal tubular epithelial fibrosis by activating autophagy.
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