微小RNA-545-3p对结直肠癌细胞进展的影响研究

Effect of miR-545-3p on the progression of colorectal cancer cells

目的 研究微小RNA-545-3p在结直肠癌(CRC)进展中的作用和调控机制。方法 正常培养的人结直肠黏膜上皮细胞系NCM460以及人CRC细胞系(HCT-15、HCT-116和HCT-8)分为NCM460组、HCT-15组、HCT-116组和HCT-8组。将miR-545-3p模拟物、模拟物阴性对照、miR-545-3p抑制剂、抑制剂阴性对照和血管内皮生长因子A(VEGFA)过表达载体、miR-545-3p模拟物和VEGFA过表达载体分别转染至HCT-116细胞,分别为miR-545-3p mimic组、mimic-NC组、miR-545-3p inhibitor组、inhibitor-NC组、mimic-NC+VEGFA组、miR-545-3p mimic+VEGFA组。以实时定量逆转录聚合酶链反应(qRT-PCR)法检测CRC患者癌组织和细胞系中miR-545-3p和VEGFA的表达水平。以细胞计数法-8(CCK-8)检测细胞增殖能力;以Transwell实验检测细胞迁移和侵袭能力;以原位末端标记(TUNEL)法检测细胞的凋亡;以蛋白质印迹(Western blot)法检测B淋巴细胞瘤-2(Bcl-2)、Bcl-2相关X蛋白(Bax)和VEGFA蛋白的表达;以双荧光素酶实验检测miR-545-3p和VEGFA之间的靶向关系。结果 mimic-NC组、miR-545-3p mimic组、mimic-NC+VEGFA组、miR-545-3p mimic+VEGFA组在48 h时的光密度值分别为1.12±0.06,0.58±0.04,1.26±0.08,0.61±0.09,细胞迁移数分别为96.24±7.20,53.32±8.60,134.24±9.97,57.34±10.62,细胞侵袭数分别为85.24±6.71,47.64±8.59,121.35±11.28,63.21±7.42。以上指标,miR-545-3p mimic组与mimic-NC组相比差异均有统计学意义(均P<0.05),miR-545-3p mimic+VEGFA组与mimic-NC+VEGFA组相比,差异均有统计学意义(均P<0.05)。结论 miR-545-3p可通过调控VEGFA的表达进而调控结直肠癌的进展。

Objective To explore the role and regulatory mechanism of miR-545-3p in the progression of colorectal cancer (CRC).MethodsThe normal cultured human colorectal epithelial cell lines NCM460 and human CRC cell lines (HCT-15,HCT-116 and HCT-8) were divided into NCM460,HCT-15,HCT-116 and HCT-8 groups.miR-545-3p mimics,mimics negative control,miR-545-3p inhibitors,inhibitor negative control and vascular endothelial growth factor A(VEGFA) overexpression vector,miR-545-3p mimics and VEGFA overexpression vector were transfected into HCT-116 cells.The cells were divided into miR-545-3p mimic group,mimic-NC group,miR-545-3p inhibitor group,mimic-NC+VEGFA group and miR-545-3p mimic+VEGFA group according to different transfected substances.Real-time quantitative polymerase chain reaction (qRT-PCR) was used to detect the expression levels of miR-545-3p and VEGFA in cancer tissues and cell lines of CRC patients.CCK-8 experiment was used to detect cell proliferation ability;Transwell experiment was used to detect cell migration and invasion ability;in situ end labeling method (TUNEL) was used to detect cell apoptosis;Western blot method was used to detect the expression of B lymphoma-2 (Bcl-2),Bcl-2 related X protein (Bax) and VEGFA protein;dual luciferase assay was used to detect the targeting relationship between miR-545-3p and VEGFA.Results The optical density values of mimic-NC group,miR-545-3p mimic group,mimic-NC+VEGFA group and miR-545-3p mimic+VEGFA group at 48 h were 1.12±0.06,0.58±0.04,1.26±0.08 and 0.61±0.09,cell migration numbers were 96.24±7.20,53.32±8.60,134.24±9.97 and57.34±10.62,cell invasion numbers were 85.24±6.71,47.64±8.59,121.35±11.28 and 63.21±7.42.The above indicators were significantly different between miR-545-3p mimic group and mimic-NC group (all P<0.05),and between miR-545-3p mimic+VEGFA group and mimic-NC+VEGFA group (all P<0.05).Conclusion MiR-545-3p can regulate the progression of colorectal cancer by regulating the expression of VEGFA.