miR-222 通过靶向 c-kit 调节慢传输型便秘大鼠分离的 Cajal 间质细胞的细胞生长、凋亡和自噬。
miR-222 regulates cell growth, apoptosis, and autophagy of interstitial cells of Cajal isolated from slow transit constipation rats by targeting c-kit.
机构信息
Nanjing University of Chinese Medicine, Nanjing, 210029, Jiangsu Province, China.
Department of Rehabilitation, Linyi People's Hospital, Linyi, 276003, Shandong Province, China.
出版信息
Indian J Gastroenterol. 2021 Apr;40(2):198-208. doi: 10.1007/s12664-020-01143-7. Epub 2021 Apr 1.
BACKGROUND
Excessive autophagy and apoptosis of the interstitial cells of Cajal (ICC) have been identified in gastrointestinal (GI) motility disorders including slow transit constipation (STC). MicroRNA 222 (miR-222) has been shown to affect GI motility. This study aimed to explore whether miR-222 influences apoptosis and excessive autophagy of isolated ICC.
METHODS
miR-222, c-kit, and stem cell factor (SCF) were evaluated in colon tissues in STC rats compared with normal control by qRT-PCR and western blot analysis. The condition of autophagy of colon tissue was observed by transmission electron microscope. ICC were isolated from the colon of STC rats. Cell Counting Kit-8 (CCK-8) assay and wound healing assay were carried out to examine the cell viability and migration rate. Cell apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) and Annexin V-Flourescein Isothiocyanate/Propidine Iodide (FITC/PI) apoptosis detection kit. Western blot analysis was performed to detect the c-kit and SCF expression; apoptosis-related proteins Bcl-2, Bax, caspase-3, and pro-caspase-3; and autophagy-related proteins LC3B and Beclin-1. The connection between miR-222 and c-kit was detected by bioinformatics and luciferase activity analysis.
RESULTS
miR-222 expression was significantly higher, whereas c-kit and SCF expressions were markedly lower in STC rats' colon tissue compared with normal control. Meanwhile, STC rats exhibited excessive autophagy in colon tissue than normal control. Inhibition of miR-222 expression promoted cell proliferation as well as migration and inhibited autophagy, whereas upregulation of miR-222 had the opposite effect. In addition, miR-222 upregulation induced apoptosis and excessive autophagy compared with normal controls (NC). Western blot analysis showed that miR-222 overexpression caused decreased c-kit and SCF protein levels compared with NC. Bioinformatics and luciferase activity analysis revealed that miR-222 could be a predictive regulator of c-kit.
CONCLUSION
miR-222 induces apoptosis and excessive autophagy of ICC and may serve as potential biomarker for ICC loss in STC.
背景
在包括慢传输型便秘（STC）在内的胃肠动力障碍中，已经发现 Cajal 间质细胞（ICC）的过度自噬和凋亡。microRNA 222（miR-222）已被证明会影响胃肠动力。本研究旨在探讨 miR-222 是否会影响分离的 ICC 的凋亡和过度自噬。
方法
通过 qRT-PCR 和 Western blot 分析比较 STC 大鼠与正常对照组结肠组织中 miR-222、c-kit 和干细胞因子（SCF）的表达。通过透射电子显微镜观察结肠组织的自噬情况。从 STC 大鼠的结肠中分离 ICC。通过细胞计数试剂盒-8（CCK-8）测定和划痕愈合试验检测细胞活力和迁移率。通过末端脱氧核苷酸转移酶 dUTP 缺口末端标记（TUNEL）和 Annexin V-Flourescein Isothiocyanate/Propidine Iodide（FITC/PI）凋亡检测试剂盒检测细胞凋亡。Western blot 分析检测 c-kit 和 SCF 表达；凋亡相关蛋白 Bcl-2、Bax、caspase-3 和 pro-caspase-3；以及自噬相关蛋白 LC3B 和 Beclin-1。通过生物信息学和荧光素酶活性分析检测 miR-222 与 c-kit 的联系。
结果
与正常对照组相比，STC 大鼠结肠组织中 miR-222 表达明显升高，而 c-kit 和 SCF 表达明显降低。同时，STC 大鼠结肠组织自噬过度。抑制 miR-222 表达促进细胞增殖和迁移，抑制自噬，而上调 miR-222 则产生相反的效果。此外，与正常对照组（NC）相比，miR-222 上调诱导细胞凋亡和过度自噬。Western blot 分析显示，与 NC 相比，miR-222 过表达导致 c-kit 和 SCF 蛋白水平降低。生物信息学和荧光素酶活性分析表明，miR-222 可能是 c-kit 的预测调节因子。
结论
miR-222 诱导 ICC 凋亡和过度自噬，可能作为 STC 中 ICC 缺失的潜在生物标志物。
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