南方医科大学学报

南方医科大学学报 ›› 2023, Vol. 43 ›› Issue (7): 1081-1092.doi: 10.12122/j.issn.1673-4254.2023.07.04

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miR-30e-5p过表达促进结直肠癌细胞的增殖和迁移：基于下调PTEN激活CXCL12轴

魏 可，石纪雯，肖雨寒，王文锐，杨清玲，陈昌杰   

  1. 蚌埠医学院癌症转化医学安徽省重点实验室，肿瘤基础研究与临床检验诊断重点实验室，生物技术教研室，生物化学与分子生物学教研室，安徽 蚌埠 233000
  • 出版日期:2023-07-20 发布日期:2023-07-20

MiR-30e-5p overexpression promotes proliferation and migration of colorectal cancer cells by activating the CXCL12 axis via downregulating PTEN

WEI Ke, SHI Jiwen, XIAO Yuhan, WANG Wenrui, YANG Qingling, CHEN Changjie   

  1. Anhui Provincial Key Laboratory of Cancer Translational Medicine, Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis, Department of Biotechnology, Department of Biochemistry and Molecular Biology, Bengbu Medical College, Bengbu 233000, China
  • Online:2023-07-20 Published:2023-07-20

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摘要： 目的 探讨miR-30e-5p通过PTEN/CXCL12轴对结直肠癌生物学活性的影响及机制。方法 实验设置空白对照、阴性对照、miR-30e-5p mimics组、miR-30e-5p inhibitor组及共转染miR-30e-5p mimics+LV-PTEN（PTEN过表达）组或miR-30e-5pinhibitor+si-PTEN（PTEN干扰）组。生物信息学分析miR-30e-5p在结直肠癌组织和正常组织中的表达差异；qRT-PCR检测miR-30e-5p在肠上皮细胞和结直肠癌细胞中的差异表达；生物信息学和双荧光素酶实验预测并验证miR-30e-5p与PTEN的靶向关系；平板克隆、CCK-8增殖、细胞周期、细胞凋亡、划痕愈合和Transwell实验检测癌细胞的生物学活性；Western blotting检测癌细胞PTEN、CXCL12表达情况；小鼠体内实验检测miR-30e-5p（miR-NC、miR-30e-5p inhibitor组）对结直肠癌发生发展的影响。结果 生信分析结直肠癌组织miR-30e-5p较癌旁组织表达升高（P<0.01）；在癌细胞中miR-30e-5p较肠上皮细胞表达升高（P<0.01）；双荧光素酶实验证实miR-30e-5p与PTEN存在靶向关系（P<0.05）；相对于Control组，miR-30e-5p mimics能增强癌细胞增殖、转移能力并抑制凋亡（P<0.05），共转染miR-30e-5p mimics+LV-PTEN能够逆转miR-30e-5p mimics的作用（P<0.05）；miR-30e-5p mimics能抑制PTEN表达，增强CXCL12表达（P<0.01）；然而miR-30e-5p inhibitor与mimics效果相反，将细胞阻滞在G0/G1期，共转染miR-30e-5p inhibitor+si-PTEN能逆转miR-30e-5p inhibitor的作用（P<0.05）；体内实验发现相对于miR-NC组，miR-30e-5p inhibitor可抑制肿瘤发生发展。结论 miR-30e-5p过表达通过下调PTEN激活CXCL12轴，从而促进结直肠癌细胞的增殖和迁移。

关键词: 结直肠癌；miR-30e-5p；PTEN；CXCL12

Abstract: Objective To investigate the regulatory effects of miR-30e-5p on biological behaviors of colorectal cancer cells and the role of PTEN/CXCL12 axis in mediating these effects. Methods Bioinformatic analysis was performed to explore the differential expression of miR-30e-5p between colorectal cancer tissues and normal tissues. RT-qPCR was used to detect the differential expression of miR-30e-5p in intestinal epithelial cells and colorectal cancer cells. Bioinformatics and dual luciferase assay were used to predict and validate the targeting relationship between miR-30e-5p and PTEN. Human and murine colorectal cancer cell lines were transfected with miR-30e-5p mimics, miR-30e-5p inhibitor, miR-30e-5p mimics+LV-PTEN, or miR-30e-5p inhibitor + si-PTEN. The changes in biological behaviors of the cells were detected using plate clone formation assay, CCK-8 assay, flow cytometry, scratch healing and Transwell assays. PTEN and CXCL12 expressions in the cancer cells were detected by Western blotting. The effects of miR-30e-5p inhibitor on colorectal carcinogenesis and development were observed in nude mice. Results Bioinformatic analysis showed that miR-30e-5p expression was significantly elevated in colorectal cancer tissues compared with the adjacent tissue (P<0.01). Higher miR-30e-5p expression was detected in colorectal cancer cell lines than in intestinal epithelial cells (P<0.01). Dual luciferase assay confirmed the targeting relationship between miR-30e-5p and PTEN (P<0.05). Transfection with miR-30e-5p mimics significantly enhanced proliferation and metastasis and inhibited apoptosis of the colorectal cancer cells (P<0.05), and co-transfection with LV-PTEN obviously reversed these changes (P<0.05). MiR-30e-5p mimics significantly inhibited PTEN expression and enhanced CXCL12 expression in the cancer cells (P<0.01), and miR-30e-5p inhibitor produced the opposite effect. Transfection with miR-30e-5p inhibitor caused cell cycle arrest in the cancer cells, which was reversed by co-transfection with si-PTEN (P<0.05). In the in vivo experiments, the colorectal cancer cells transfected with miR-30e-5p inhibitor showed significantly lowered tumorigenesis. Conclusion Overexpression of miR-30e-5p promotes the malignant behaviors of colorectal cancer cells by downregulating PTEN to activate the CXCL12 axis.

Key words: colorectal cancer; miR-30e-5p; PTEN; CXCL12