南方医科大学学报

南方医科大学学报 ›› 2022, Vol. 42 ›› Issue (8): 1191-1197.doi: 10.12122/j.issn.1673-4254.2022.08.11

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miR-372-5p促进结直肠癌细胞的转移：通过靶向PTEN调控PI3K/AKT/CXCL12信号通路

时秀茹，魏 可，吴雨伦，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:2022-08-20 发布日期:2022-09-05

MiR-372-5p regulates PI3K/AKT/CXCL12 signaling pathway by targeting PTEN to promote colorectal cancer cell metastasis

SHI Xiuru, WEI Ke, WU Yulun, 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:2022-08-20 Published:2022-09-05

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摘要： 目的 探讨miR-372-5p通过靶向PTEN调控PI3K/AKT/CXCL12信号通路对结直肠癌细胞迁移能力的影响及机制。 方法 qRT-RCR检测miR-372-5p在结直肠癌和癌旁组织及结直肠癌细胞和正常肠上皮细胞中的表达；生物信息学和双荧光素酶实验验证miR-372-5p与PTEN的靶向关系；Western blotting检测转染inhibitor-NC/mimics-NC（miR-372-5p抑制剂/类似物的阴性对照）、miR-inhibitor（miR-372-5p抑制剂）、miR-mimics（miR-372-5p类似物）以及共转染miR-inhibitor+si-PTEN（PTEN干扰）、miR-mimics+PI3K抑制剂对PTEN、CXCL12表达和PI3K/AKT信号通路激活水平的影响；Transwell实验、划痕实验检测以上各组、共转染miR-mimics+si-CXCL12（CXCL12干扰）以及转染mimics-NC、miR-mimics后的HCT116条件培养基对细胞迁移能力的影响。结果 结直肠癌组织中miR-372-5p较癌旁组织表达升高，相对于NCM460，HCT116较SW620中miR-372-5p表达上调更显著（P<0.01）；双荧光素酶实验证实PTEN是miR-372-5p的潜在靶基因（P<0.05）。相较于NC组，miR-mimics可降低PTEN蛋白表达水平，增加CXCL12蛋白表达水平和AKT磷酸化水平，提高HCT116迁移能力；而miR-inhibitor则导致相反的结果（P<0.05），si-PTEN可中和miR-inhibitor的作用（P<0.01）；PI3K抑制剂可降低CXCL12的蛋白表达水平，并抑制HCT116迁移能力（P<0.05），而miR-mimics可中和这一作用（P<0.01）；miR-mimics转染HCT116的条件培养基可提高NCM460的迁移能力（P<0.01），联合si-CXCL12可逆转miR-mimics对HCT116转移的促进作用（P<0.01）。结论 miR-372-5p通过靶向PTEN激活PI3K/AKT信号通路，上调CXCL12的表达，从而促进结直肠癌细胞的迁移能力。

关键词: 结直肠癌；转移；miR-372-5p；CXCL12

Abstract: Objective To investigate whether miR-372-5p regulates PI3K/AKT/CXCL12 signaling pathway by targeting PTEN to promote metastasis of colorectal cancer cells. Methods We detected the differential expression of miR-372-5p using RT-qRCR in colorectal cancer and adjacent tissues, colorectal cancer cells and normal intestinal epithelial cells. Bioinformatic analysis and double luciferase assay were performed for verification of the targeting relationship between miR-372-5p and PTEN. Western blotting was used to assess the effects of transfection with miR-372-5p inhibitor and miR-372-5p mimics alone, co-transfection with miR-372-5p inhibitor and si-PTEN, and co-transfection with miR-372-5p mimics and PI3K inhibitor on the expressions of PTEN and CXCL12 and the activation of PI3K/AKT signal pathway; Transwell assay and scratch assay were used to examine the changes in the migration ability of the transfected cells, the cells co-transfected with miR-372-5p mimics and si-CXCL12, and the cells treated with conditioned medium from HCT116 cells transfected with miR-372-5p mimics. Results The expression of miR-372-5p was significantly higher in colorectal cancer tissues than in adjacent tissues, and higher in HCT116 and SW620 cells than in NCM460 cells (P<0.01). Double luciferase assay confirmed that PTEN was a potential target gene of miR-372-5p (P<0.05). Transfection of HCT116 cells with miR-372-5p mimics obviously decreased PTEN protein expression, increase CXCL12 expression and the phosphorylation level of AKT, and lowered the cell migration ability, while transfection with miR-372-5p inhibitor produced the opposite effects (P<0.05); si-PTEN obviously neutralized the effect of miR-372-5p inhibitor (P<0.01). PI3K inhibitor significantly decreased CXCL12 expression and inhibited the cell migration (P<0.05), and this effect was mitigated by miR-372-5p mimics (P<0.01). Treatment with the conditioned medium from HCT116 cells transfected with miR-372-5p mimics significantly enhanced the migration ability of NCM460 cells, and this effect was suppressed by transfection with si-CXCL12 (P<0.01). Conclusion MiR-372-5p activates PI3K/AKT signaling pathway by targeting PTEN and up-regulates CXCL12 expression to promoting metastasis of colorectal cancer cells.

Key words: colorectal cancer; metastasis; miR-372-5p; CXCL12