摘要
目的miR-637靶向SCUBE3调控结肠癌的作用仍未完全清晰,文章旨在探讨miR-637在结肠癌的表达及其是否靶向SCUBE3影响结肠癌的生长。方法选取20份结肠癌组织和癌旁组织样本及结肠癌细胞系HCT116细胞为研究对象,细胞转染miR-637-mimic、miR-637-inhibitor或/和pcDNA3.1-SCUBE3。采用实时荧光定量PCR(qRT-PCR)或免疫组织化学法检测组织miR-637或信号肽-CUB-EGF样结构域蛋白3(SCUBE3)表达;qRT-PCR和CCK-8检测miR-637表达及其对细胞增殖的影响;流式细胞凋亡和蛋白质印迹法(Western blot)分析miR-637对细胞凋亡的影响;荧光素酶实验、qRT-PCR和Western blot检测miR-637与SCUBE3的靶向关系;Western blot检测miR-637对TGFβ/Smad信号通路的调节作用。结果与癌旁组织相比,结肠癌组织miR-637的表达水平(0.61±0.03)显著下调,SCUBE3的表达(1.81±0.09)显著上调(P<0.01);与阴性对照组相比,miR-637 mimic转染后HCT-116细胞中的miR-637表达(1.53±0.08)显著增强(P<0.05),细胞增殖被显著抑制(P<0.01),细胞凋亡显著增加(P<0.01),且Bax蛋白表达显著升高,Bcl-2、p-smad2、p-smad3、TGF-βR2蛋白表达显著降低(P<0.01);SCUBE3是miR-637的靶基因;与miR-637 mimic组相比,pcDNA3.1-SCUBE3明显抑制了miR-637 mimic的促凋亡作用(P<0.01),miR-637 mimic+pcDNA3.1-SCUBE3组Bax蛋白表达显著降低,Bcl-2、p-smad2、p-smad3、TGF-βR2蛋白表达显著升高(P<0.01)。结论miR-637对结肠癌细胞具有抑制增殖及促凋亡作用,并可能通过靶向SCUBE3和抑制TGF-β/Smad信号通路来抑制结肠癌。
Objective It is still not fully explored for role of miR-637 targeting SCUBE3 to regulate colon cancer(CC) This paper aims to investigate the expression of miR-637 in colon cancer and whether it targets SCUBE3 to affect the growth of CC. Methods Twenty pairs of CC and adjacent normal tissues and CC cell line HCT116 cells were selected for the study, cells were transfected with miR-637-mimic, miR-637-inhibitor or/and pcDNA3.1-SCUBE3. The levels of miR-637 and the signal peptide CUB EGF-like domain-containing protein 3(SCUBE3) were quantified using real-time quantitative PCR or immunohistochemistry assays. The expression of miR-637 mimic and its effect on cell proliferation were analyzed by qRT-PCR and CCK-8 assays. Then, the effect of miR-637 on cell apoptosis was analyzed using flow cytometry and western blot. Dual-luciferase reporter assay, qRT-PCR and Western blot were performed to explore the target relationship between miR-637 and SCUBE3. Western blot was conducted to explore the regulatory effect of miR-637 on the TGF-β/Smad signaling pathway. Results Compared with adjacent normal tissues, the expression of miR-637(0.61±0.03) were significantly down-regulated and the expression of SCUBE3(1.81±0.09) was significantly up-regulated in CC tissues(P<0.01);compared with the negative control group, miR-637 expression(1.53±0.08) was significantly enhanced(P<0.05), cell proliferation was significantly inhibited(P<0.01), apoptosis was significantly increased(P<0.01), and Bax protein expression was significantly increased and Bcl-2, p-smad2, p-smad3, and TGF-β R2 protein expression was significantly decreased(P<0.01) in HCT-116 cells after miR-637 mimic transfection. SCUBE3 was identified as the target gene of miR-637. Compared with the miR-637 mimic group, pcDNA3.1-SCUBE3 significantly inhibited the pro-apoptotic effect of miR-637 mimic(P<0.01), and the miR-637 mimic+ pcDNA3.1-SCUBE3 group showed Bax protein expression were significantly decreased and Bcl-2, p-smad2, p-smad3, and TGF-β R2 protein expression were significantly increased(P<0.01). Conclusion miR-637 has inhibitory proliferative and pro-apoptotic effects on CC cells and it may inhibit CC by targeting SCUBE3 and suppressing the TGF-β/Smad signaling pathway.
作者
黄洋峰
赵海明
罗玉明
汪英
唐鹏
HUANG Yang-feng;ZHAO Hai-ming;LUO Yu-ming;WANG Ying;TANG Peng(Department of Public Health,International College,Krirk University,Bangkok 10220,Thailand;Department of Gastroenterology,Sichuan Academy of Medical Sciences&Sichuan Provincial People′s Hospital,Chengdu 610101,Sichuan,China;Department of Oncology,Eastern Hospital,Sichuan Academy of Medical Sciences&Sichuan Provincial People′s Hospital,Chengdu 610101,Sichuan,China)
出处
《医学研究生学报》
CAS
北大核心
2022年第9期930-937,共8页
Journal of Medical Postgraduates
作者简介
黄洋峰,医学硕士研究生,现在四川省医学科学院/四川省人民医院中医科工作;通信作者:唐鹏,E⁃mail:tp3531@163.com。
