Shi X, Wei K, Wu Y, Wang W, Yang Q, Chen C
Anhui Provincial Key Laboratory of Cancer Translational Medicine, Bengbu Medical College, Bengbu 233000, China.
Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis, Bengbu Medical College, Bengbu 233000, China.
Nan Fang Yi Ke Da Xue Xue Bao. 2022 Aug 20;42(8):1191-1197. doi: 10.12122/j.issn.1673-4254.2022.08.11.
To investigate whether miR-372-5p regulates PI3K/AKT/CXCL12 signaling pathway by targeting PTEN to promote metastasis of colorectal cancer cells.
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.
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 ( < 0.01). Double luciferase assay confirmed that PTEN was a potential target gene of miR-372-5p ( < 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 ( < 0.05); si-PTEN obviously neutralized the effect of miR-372-5p inhibitor ( < 0.01). PI3K inhibitor significantly decreased CXCL12 expression and inhibited the cell migration ( < 0.05), and this effect was mitigated by miR-372-5p mimics ( < 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 ( < 0.01).
MiR-372-5p activates PI3K/AKT signaling pathway by targeting PTEN and up-regulates CXCL12 expression to promoting metastasis of colorectal cancer cells.
探讨miR-372-5p是否通过靶向PTEN调控PI3K/AKT/CXCL12信号通路来促进大肠癌细胞转移。
采用RT-qRCR检测miR-372-5p在结直肠癌组织及癌旁组织、结直肠癌细胞及正常肠上皮细胞中的差异表达。进行生物信息学分析和双荧光素酶报告基因检测以验证miR-372-5p与PTEN的靶向关系。采用蛋白质免疫印迹法评估单独转染miR-372-5p抑制剂和miR-372-5p模拟物、miR-372-5p抑制剂与si-PTEN共转染、miR-372-5p模拟物与PI3K抑制剂共转染对PTEN和CXCL12表达及PI3K/AKT信号通路激活的影响；采用Transwell实验和划痕实验检测转染细胞、miR-372-5p模拟物与si-CXCL12共转染细胞以及用miR-372-5p模拟物转染的HCT116细胞的条件培养基处理的细胞迁移能力的变化。
miR-372-5p在结直肠癌组织中的表达明显高于癌旁组织，在HCT116和SW620细胞中的表达高于NCM460细胞（<0.01）。双荧光素酶报告基因检测证实PTEN是miR-372-5p的潜在靶基因（<0.05）。用miR-372-5p模拟物转染HCT116细胞明显降低PTEN蛋白表达，增加CXCL12表达和AKT的磷酸化水平，并降低细胞迁移能力，而转染miR-372-5p抑制剂则产生相反的效果（<0.05）；si-PTEN明显中和了miR-372-5p抑制剂的作用（<0.01）。PI3K抑制剂明显降低CXCL12表达并抑制细胞迁移（<0.
