Prohibitin Promote Gastrointestinal Stromal Tumor Cell GIST-T1 Proliferation Through Mitochondria-dependent Pathway

Gastrointestinal stromal tumors(GISTs) were the most common mesenchymal tumors of the gastrointestinal tract.The long-term outcome for GIST patients with different risk stratification showed significant differences.In the early proteomics study of the risk stratification of gastrointestinal stromal tumor GISTs(high risk and low risk)and positive immune phenotype KIT GISTs of organization,We found that prohibitin was up-regulated in the high risk gastrointestinal stromal tumor.In this section,to explore the PHB function in GIST-T1 cells and find new target of gastrointestinal stromal tumor,we knocked down the PHB expression by lentivirus-mediated RNA interference and measured the cell proliferation in vitro and vivo.In the first part of our study four shRNA-PHB sequences were designed and constructed shRNA-PHB lentiviral vector for virus packaging.After GIST-T1 infected with packaging virus,we used puromycin to screen positive cell lines.The knockdown effect was detected by real-time PCR and Western blot.Cell immunofluorescence and mitochondrial separation were used to identify where the PHB located.Results showed that shPHB-1?shPHB-4 could effectively knock down PHB,which was verified repeat and repeat.Cell immunofluorescence and mitochondrial separation verified that PHB was mainly located in mitochondria,only tiny amounts of PHB in the nucleus.In the second part,to study PHB proliferation function in gastrointestinal stromal tumor GIST-T1,deficiency PHB cell model in vitro and in vivo were established.In vitro,the change of cell proliferation was observed with CCK8 colorimeter.Colony formation assay and soft-agar colony formation were used to measure the colony formation ability.In vivo,the tumor-bearing mice model was established by subcutaneously inoculating with xenografts of GIST-T1 cell into the left armpit of BALB/c nude mice.CCK8 results showed that cell viability were decreased,so as thecolony formation. Implanted tumor mouse model was successively established.The tumor weight and sizes of PHB-knockdown group were markedly lower than control group.The third part,in mitochondria structure, we used confocal microscope and electron transmission microscope to observe mitochondria and inspected the mitochondria fusion protein OPA-1.In the area of mitochondrial function,analysis of mitochondrial membrane potential and the activity of respiratory chain Complex I were checked to see whether changed mitochondria structure affect function.And further studying whether the abnormal membrane potential and respiratory chain had a negative impact on reactive oxygen species and ATP synthesis.Fragment of mitochondrial reticulum and loss of micochondrial cristae were observed upon PHB-silencing in GIST-T1 cells.Deletion of PHB lead to the selective loss of long isoforms of OPA1. An aberrant cristae morphogenesis and impaired mitochondrial structure result in a decline in mitochondrial membrane potential and respiratory chain enzyme complex I.This rusults affect the electron transference of respiratory chain and increased production of reactive oxygen species.However,ATP synthesis didn't obviously change.