MicroRNA-141 protects PC12 cells against hypoxia/reoxygenation-induced injury via regulating Keap1-Nrf2 signaling pathway.

To understand the role of microRNA-141 (miR-141) in hypoxia/reoxygenation (H/R)-induced PC12 cell injury via modulation of Keap1/Nrf2 signaling pathway. PC12 cells were divided into Control, H/R, H/R + miR-141 mimics, H/R + NC, H/R + miR-141 inhibitor, H/R + siKeap1 and H/R + miR-141 inhibitors+siKeap1 groups. The expression of miR-141 and Keap1/Nrf2 pathway was measured by qRT-PCR and western blotting, cell viability evaluated by MTT assay while cell apoptosis tested by flow cytometry. Besides, MDA (malondialdehyde), SOD (Super Oxide Dismutase) and LDH (lactate dehydrogenase) levels were determined. DCFH-DA and JC-1 staining were used to measure ROS and mitochondrial membrane potential (MMP) respectively. Compared with Controls, PC12 cells induced by H/R exhibited decreased cell viability and increased cell apoptosis rate, with elevated MDA, LDH and ROS and reduced SOD levels; and meanwhile, MMP and miR-141 expression were declined, whereas cytoplasmic Nrf2 levels were enhanced with the downregulated nuclear Nrf2 level (all P < 0.05). However, these cells treated with miR-141 mimics and siKeap1 showed obvious improvement in H/R-induced cell injury, while miR-141 inhibitors presented significantly aggravated cell injury (both P < 0.05). Besides, siKeap1 can reverse the effect of miRNA-141 inhibitors on aggravating H/R-induced PC12 cell injury. miR-141-mediated Keap1/Nrf2 signaling pathway to promote cell viability, inhibit cell apoptosis and reduce oxidative stress of PC12 cells, thereby alleviating H/R-induced cell injury.

Protective Role of SOCS3 Modified Bone Marrow Mesenchymal Stem Cells in Hypoxia-Induced Injury of PC12 Cells.

We attempted to explore the possible effects of SOCS3 (suppressor of cytokine signaling 3)-modified bone marrow mesenchymal stem cells (BMSCs) on the hypoxic injury of rat adrenal gland pheochromocytoma (PC-12) cells. PC12 cells were cultured with EGFP (enhanced green fluorescent protein)-BMSCs and SOCS3-BMSCs respectively under hypoxia in vitro and classified into control, hypoxia, EGFP-BMSCs, and SOCS3-BMSC groups. CCK-8, Hoechst 33258 staining, and Annexin V-FITC/PI staining were assessed to measure the viability and apoptosis of hypoxia-induced PC12 cells. The JAK/STAT3 pathway- and apoptosis-related proteins were identified by Western blot. Finally, rat models of permanent middle cerebral artery occlusion (pMCAO) were established to verify the potential influences of SOCS3-BMSCs in vivo. SOCS3-modified BMSCs can stably express SOCS3 protein. EGFP-BMSCs, especially SOCS3-BMSCs, can improve cell viability and SOD content, and reduce cell apoptosis, LDH viability, and MDA content in hypoxia-induced PC12 cells (all P < 0.05). Besides, EGFP-BMSCs and SOCS3-BMSCs decreased cleaved caspase-3 level and increased Bcl-2/Bax of hypoxia-induced PC12 cells, while SOCS3-BMSCs could also elevate SOCS3 protein and reduce p-STAT3 protein level in hypoxia-induced PC12 cells (all P < 0.05). In vivo experiments confirmed that EGFP-BMSCs, particularly SOCS3-BMSCs, could ameliorate infarct size and inhibit neuronal apoptosis of different degrees in pMACO rats (all P < 0.05). SOCS3-modified BMSCs can alleviate oxidative stress, improve cell viability, and reduce neuronal apoptosis by downregulation of JAK/STAT3 pathway, thereby exerting the neuroprotective role in ischemic brain injury.

MiR-203a-3p regulates the biological behaviors of ovarian cancer cells through mediating the Akt/GSK-3ß/Snail signaling pathway by targeting ATM.

OBJECTIVE: To investigate whether miR-203a-3p can regulate the biological behaviors of ovarian cancer cells by targeting ATM to affect the Akt/GSK-3ß/Snail signaling pathway. METHODS: The expression levels of miR-203a-3p and ATM were detected by qRT-PCR, immunohistochemical staining and Western blotting in ovarian cancer tissues and adjacent normal tissues obtained from 152 subjects. A dual-luciferase reporter gene assay was performed to verify the relationship between miR-203a-3p and ATM. Human ovarian cancer cell lines (A2780 and SKOV3) were used to generate the Blank, miR-NC, miR-203a-3p mimic, Control siRNA, ATM siRNA, and miR-203a-3p inhibitor + ATM siRNA groups. The biological behaviors of ovarian cancer cells were evaluated by CCK-8, wound healing, and Transwell invasion assays, annexin V-FITC/PI staining and flow cytometry. The levels of Akt/GSK-3ß/Snail pathway-related proteins were assessed by Western blotting. RESULTS: Ovarian cancer tissues showed lower miR-203a-3p levels and higher ATM levels than adjacent normal tissues, both of which were associated with the FIGO stage, grade and prognosis of ovarian cancer. As confirmed by a dual-luciferase reporter gene assay, miR-203a-3p could target ATM. Furthermore, the miR-203a-3p mimic had multiple effects, including the inhibition of the proliferation, invasion and migration of A2780 and SKOV3 cells, the promotion of cell apoptosis, the arrest of the cell cycle at the G1 phase, and the blockage of the Akt/GSK-3ß/Snail signaling pathway. ATM siRNA had similar effects on the biological behaviors of ovarian cancer cells, and these effects could be reversed by a miR-203a-3p inhibitor. CONCLUSION: miR-203a-3p was capable of hindering proliferation, migration, and invasion and facilitating the apoptosis of ovarian cancer cells through its modulation of the Akt/GSK-3ß/Snail signaling pathway by targeting ATM, and therefore it could serve as a potential therapeutic option for ovarian cancer.