作者：凌旭坤，陈超，钟晓华，胡琛

单位：惠州市中心人民医院 胃肠外科，广东 惠州 516001

Authors: Ling Xukun，Chen Chao，Zhong Xiaohua，Hu Chen

Unit: Gastrointestinal Surgery Department, Huizhou Municipal Central Hospital, Huizhou, 516000, Guangdong, China

摘要：

目的 研究沉默circSATB2对结直肠癌细胞Caco2迁移、侵袭和凋亡的影响及其可能的机制。方法 qRT-PCR方法测定circSATB2在结直肠癌细胞SW480、HR-8348、Caco2和正常肠黏膜上皮细胞中的表达差异。结直肠癌细胞Caco2分成对照组、sh-NC(shRNA对照)组、sh-circSATB2(circSATB2 shRNA)组、sh-circSATB2+anti-miR-NC (circSATB2 shRNA、抑制剂对照)组、sh-circSATB2+anti-miR-150-5p(circSATB2 shRNA、miR-150-5p 抑制剂)组,CCK-8 实验检测细胞增殖变化,流式细胞术检测细胞凋亡,Transwell小室检测细胞迁移和侵袭,蛋白质印迹法检测C-胱天蛋白酶-3(C-caspase-3)、C-caspase-9、基质金属蛋白酶(matrix metalloproteinase,MMP)-2、MMP-9 表达。荧光素酶报告系统鉴定circSATB2和miR-150-5p的靶向关系。结果 结直肠癌细胞SW480、HR-8348、Caco2中circSATB2水平高于正常肠黏膜上皮细胞(P<0.05)。结直肠癌细胞Caco2中circSATB2水平高于结直肠癌细胞SW480、HR-8348(P<0.05)。与对照组、sh-NC组比较,sh-circSATB2组结直肠癌细胞存活率降低,细胞迁移数目减少,细胞侵袭数目也减少,细胞凋亡率升高,细胞中C-caspase-3、C-caspase-9 表达增多,MMP-2、MMP-9 表达减少(P<0.05)。与 sh-circSATB2+anti-miR-NC 组比较,sh-circSATB2+anti-miR-150-5p组结直肠癌细胞中miR-150-5p水平降低,细胞存活率升高,细胞凋亡率降低,细胞迁移和侵袭数目增加,细胞中C-caspase-3、C-caspase-9表达减少,MMP-2、MMP-9表达增多。沉默circSATB2靶向促进结直肠癌细胞Caco2中miR-150-5p表达。结论 沉默circSATB2可能通过靶向促进miR-150-5p表达来发挥其抑制结直肠癌细胞Caco2迁移、侵袭,诱导细胞凋亡的作用。

关键词：结直肠癌；circSATB2；凋亡；迁移；miR-150-5p

Abstract：

Objective To explore the effect and mechanism of silencing circSATB2 on the migration, invasion and apoptosis of colorectal cancer cells Caco2. Methods The qRT -PCR method was used to determine the expression differences of circSATB2 in colorectal cancer cells SW480, HR-8348, Caco2 and normal intestinal mucosal epithelial cells. Colorectal cancer cells Caco2 were divided into Control, sh -NC (shRNA control), sh-circSATB2 (circSATB2 shRNA), sh-circSATB2+anti-miR-NC (circSATB2 shRNA, inhibitor control), sh-circSATB2+anti-miR-150-5p group (circSATB2 shRNA, miR-150-5p inhibitor). CCK-8 experiment was used to detect cell proliferation, flow cytometry was used to detect cell apoptosis. We used Transwell chamber to detect cell migration and invasion, Western blot to detect C-caspase-3, C-caspase-9, MMP-2, MMP-9 expression. The luciferase reporter system identified the targeting relationship between circSATB2 and miR-150-5p. Results The level of circSATB2 in colorectal cancer cells SW480, HR-8348, and Caco2 were higher than that in normal intestinal mucosal epithelial cells (P<0.05). The level of circSATB2 in colorectal cancer cells Caco2 was higher than that in colorectal cancer cells SW480 and HR-8348 (P<0.05). Compared with control and sh-NC groups, the survival rate of colorectal cancer cells in the sh-circSATB2 group was reduced , the number of cell migration decreased , the number o f cell invasions also decreased , the rate of cell apoptosis increased , and C -caspase -3 and C-caspase -9 in the cells increased , MMP- 2 , MMP-9 expression decreased ( P< 0 . 05 ) . Silencing circSATB2 induced colorectal cancer cell proliferation, migration, invasion inhibition and apoptosis promotion. Compared with the sh -circSATB2 +anti -miR -NC group, in the sh -circSATB2 +anti -miR -150 -5 p group, the level of miR-150-5p in colorectal cancer cells decreased, the cell survival rate increased, and the cell apoptosis rate decreased, the number of cell migration and invasion increased, the expression of C-caspase-3 and C-caspase-9 in the cells decreased, and the expression of MMP-2 and MMP-9 increased. Silencing circSATB2 targeting promotes the expression of miR-150-5p in colorectal cancer cells Caco2. Conclusion Silencing circSATB2 may inhibit the migration,invasion and induces cell apoptosis of colorectal cancer cells Caco2 by promoting the expression of miR-150-5p.

Key Words: Colorectal cancer；CircSATB2；Apoptosis；Migration; miR-150-5p