Overexpression of miR-431-5p impairs mitochondrial function and induces apoptosis in gastric cancer cells via the Bax/Bcl-2/caspase3 pathway / 南方医科大学学报

OBJECTIVE@#To investigate the expression of microRNA miR-431-5p in gastric cancer (GC) tissues and its effects on apoptosis and mitochondrial function in GC cells.@*METHODS@#The expression level of miR-431-5p in 50 clinical samples of GC tissues and paired adjacent tissues was detected using real-time fluorescence quantitative PCR, and its correlation with the clinicopathological features of the patients was analyzed. A cultured human GC cell line (MKN-45 cells) were transfected with a miR-431-5p mimic or a negative control sequence, and the cell proliferation, apoptosis, mitochondrial number, mitochondrial potential, mitochondrial permeability transition pore (mPTP), reactive oxygen species (ROS) production and adenosine triphosphate (ATP) content were detected using CCK-8 assay, flow cytometry, fluorescent probe label, or ATP detection kit. The changes in the expression levels of the apoptotic proteins in the cells were detected with Western blotting.@*RESULTS@#The expression level of miR-431-5p was significantly lower in GC tissues than in the adjacent tissues (P < 0.001) and was significantly correlated with tumor differentiation (P=0.0227), T stage (P=0.0184), N stage (P=0.0005), TNM stage (P=0.0414) and vascular invasion (P=0.0107). In MKN-45 cells, overexpression of miR-431-5p obviously inhibited cell proliferation and induced cell apoptosis, causing also mitochondrial function impairment as shown by reduced mitochondrial number, lowered mitochondrial potential, increased mPTP opening, increased ROS production and reduced ATP content. Overexpression of miR-431-5p significantly downregulated the expression of Bcl-2 and increased the expressions of pro-apoptotic proteins p53, Bcl-2 and cleaved caspase-3 protein.@*CONCLUSION@#The expression of miR-431-5p is down-regulated in GC, which results in mitochondrial function impairment and promotes cell apoptosis by activating the Bax/Bcl-2/caspase3 signaling pathway, suggesting the potential role of miR-431-5p in targeted therapy for GC.

Asymmetric Opening of Mitochondrial Permeability Transition Pore in Mouse Brain Hemispheres: A Link to the Mitochondrial Calcium Uniporter Complex

Abstract The prolonged entry of large amounts of calcium into the mitochondria through the mitochondrial calcium uniporter complex (MCUC) may cause the permeability transition pore (mPTP) to open, which contributes to the pathogenesis of several diseases. Tissue-specific differences in mPTP opening due to variable expression of MCUC components may contribute to disease outcomes. We designed this study to determine differential mPTP opening in mitochondria isolated from different regions of mouse brain and kidney and to compare it with the expression of MCUC components. mPTP opening was measured using mitochondria isolated from the left/right brain hemispheres (LH/RH, respectively) and from kidney cortex/medulla, while the expression level of MCUC components was assessed from total cellular RNA. Interestingly, LH mitochondria showed less calcium-induced mPTP opening as compared to RH mitochondria at two different calcium concentrations. Conversely, mPTP opening was similar in the renal cortex and renal medulla mitochondria. However, the kidney mitochondria demonstrated bigger and faster mPTP opening as compared to the brain mitochondria. Furthermore, asymmetric mPTP opening in the LH and RH mitochondria was not associated with the expression of MCUC components. In brief, this study demonstrates thus far unreported asymmetric mPTP opening in mouse brain hemispheres that is not associated with the mRNA levels of MCUC components.

Study on the mechanism of Wuzi-Yanzong-Wan-medicated serum interfering with the mitochondrial permeability transition pore in the GC-2 cell induced by atractyloside / 中国天然药物

Wuzi-Yanzong-Wan (WZYZW) is a classic prescription for male infertility. Our previous investigation has demonstrated that it can inhibit sperm apoptosis via affecting mitochondria, but the underlying mechanisms are unclear. The purpose of the present study was to explore the actions of WZYZW on mitochondrial permeability transition pore (mPTP) in mouse spermatocyte cell line (GC-2 cells) opened by atractyloside (ATR). At first, WZYZW-medicated serum was prepared from rats following oral administration of WZYZW for 7 days. GC-2 cells were divided into control group, model group, positive group, as well as 5%, 10%, 15% WZYZW-medicated serum group. Cyclosporine A (CsA) was used as a positive control. 50 μmol·L-1 ATR was added after drugs incubation. Cell viability was assessed using CCK-8. Apoptosis was detected using flow cytometry and TUNEL method. The opening of mPTP and mitochondrial membrane potential (MMP) were detected by Calcein AM and JC-1 fluorescent probe respectively. The mRNA and protein levels of voltage-dependent anion channel 1 (VDAC1), cyclophilin D (CypD), adenine nucleotide translocator (ANT), cytochrome C (Cyt C), caspase 3, 9 were detected by RT-PCR (real time quantity PCR) and Western blotting respectively. The results demonstrated that mPTP of GC-2 cells was opened after 24 hours of ATR treatment, resulting in decreased MMP and increased apoptosis. Pre-protection with WZYZ-medicated serum and CsA inhibited the opening of mPTP of GC-2 cells induced by ATR associated with increased MMP and decreased apoptosis. Moreover, the results of RT-qPCR and WB suggested that WZYZW-medicated serum could significantly reduce the mRNA and protein levels of VDAC1 and CypD, Caspase-3, 9 and CytC, as well as a increased ratio of Bcl/Bax. However, ANT was not significantly affected. Therefore, these findings indicated that WZYZW inhibited mitochondrial mediated apoptosis by attenuating the opening of mPTP in GC-2 cells. WZYZW-medicated serum inhibited the expressions of VDAC1 and CypD and increased the expression of Bcl-2, which affected the opening of mPTP and exerted protective and anti-apoptotic effects on GC-2 cell induced by ATR.