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.
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-5p inhibitor+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。
Keywords: CXCL12; PTEN; colorectal cancer; miR-30e-5p.