X Shi1,2, K Wei1,2, Y Wu1, W Wang3, Q Yang4, C Chen4. 1. Anhui Provincial Key Laboratory of Cancer Translational Medicine, Bengbu Medical College, Bengbu 233000, China. 2. Key Laboratory of Cancer Research and Clinical Laboratory Diagnosis, Bengbu Medical College, Bengbu 233000, China. 3. Department of Biotechnology, Bengbu Medical College, Bengbu 233000, China. 4. Department of Biochemistry and Molecular Biology, Bengbu Medical College, Bengbu 233000, China.
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.
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.
Authors: M Katie Conley-LaComb; Allen Saliganan; Pridvi Kandagatla; Yong Q Chen; Michael L Cher; Sreenivasa R Chinni Journal: Mol Cancer Date: 2013-07-31 Impact factor: 27.401