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Effect of Lentivirus Expressing MRPL18 shRNA on Mitochondrial Energy Metabolism of MIN6 Cells
Shi Mengru, Chen Lin, Zhou Linshuang, Yang Yu, Lü Jianxin, Li Wei*
School of Laboratory Medicine and Life Science, Wenzhou Medical University, Zhejiang Provincial Key Laboratory of Medical Genetics, Wenzhou 325035, China
Abstract: The effects of lentivirus expressing mitochondrial ribosomal protein L18 (MRPL18) shRNA on mitochondrial energy metabolism of MIN6 cells were observed in this study. MRPL18 gene low-expressing MIN6 cell models were established using lentivirus expressing MRPL18 shRNA. The expressions of MRPL18 mRNA and protein in MIN6 cells were detected by Real-time PCR and Western blot. Flow cytometric analysis was used to detect the reactive oxygen species (ROS) in MIN6 cells. The Seahorse XF96 Extracellular Flux Analyser was used to evaluate cell mitochondrial function. The contents of ATP， ADP and AMP in cells were detected by high-performance liquid chromatography (HPLC). Compared with control group， the mRNA and protein expressions of MRPL18 decreased
significantly in MRPL18-shRNA group (P<0.05， P<0.01)， and there was no significant difference between control
group and MRPL18-NC group. After MRPL18 interference， the cellular ROS increased significantly (P<0.05)， while the basal respiratory capacity， maximal respiratory capacity， pare respiratory capacity and proton leak， ATP content and energy charge (EC) in cells decreased significantly (P<0.05， P<0.01) compared with control group. Meanwhile， the content of ADP and AMP increased significantly (P<0.05， P<0.01). The results proved that there were intracellular metabolism disorders after down-regulation of MRPL18. MRPL18 might play an important role in energy metabolism that maintain the mitochondrial function. MIN6 cell models with mitochondrial dysfunction caused by down-regulation of MRPL18 gene can be used to clarify the mechanism of mitochondrial diabetes mellitus.
significantly in MRPL18-shRNA group (P<0.05， P<0.01)， and there was no significant difference between control
group and MRPL18-NC group. After MRPL18 interference， the cellular ROS increased significantly (P<0.05)， while the basal respiratory capacity， maximal respiratory capacity， pare respiratory capacity and proton leak， ATP content and energy charge (EC) in cells decreased significantly (P<0.05， P<0.01) compared with control group. Meanwhile， the content of ADP and AMP increased significantly (P<0.05， P<0.01). The results proved that there were intracellular metabolism disorders after down-regulation of MRPL18. MRPL18 might play an important role in energy metabolism that maintain the mitochondrial function. MIN6 cell models with mitochondrial dysfunction caused by down-regulation of MRPL18 gene can be used to clarify the mechanism of mitochondrial diabetes mellitus.
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