The Mechanism Study Of Mitochondrial Dynamin-related Protein Drp1 In Hepatocellular Carcinoma

Hepatocellular carcinoma(HCC)is one of the most common malignant tumors in China,with high incidence and mortality.Although significant progress of fundamental and clinical research have been made in recent years,the long-term survival and tumors recurrence of HCC patients do not improve well.The treatment of HCC is still a great challenge.Mitochondria are necessary for maintaining normal cellular functions.In physiological conditions,Mitochondrial dynamics maintain network homeostasis by constant fission and fusion.Dysregulated mitochondrial dynamics are implicated in numerous diseases such as tumors.Mitochondrial fission is mainly regulated by dynamin-related protein 1(Drpl).Several studies have reported that the expression of Drpl is upregulated in human cancers of the breast,lung,and pancreas.In addition,Drp1 is associated with cell proliferation and apoptotic.,that may contribute to tumorigenesis.Part? Mitochondrial Dynamin-related Protein Drpl expression is upregulated in HCC and associated with liver tumorigenesisAims:Dysregulated mitochondrial dynamics are implicated in numerous diseases such as tumors.Compared with mitochondrial fusion proteins,there is few researches about dynamin-related protein Drp1.The exact molecular mechanism by which Drp1 contributes to tumorigenesis remains unclear.In this part,we aim to explore the relationship between Drpl expression and tumorigenesis.Then we will evaluate the function of Drp1 in cell proliferation and apoptotic,exploring the potential mechanism.Methods:The expression level of Drpl in tumor tissues and adjacent normal tissues was detected by RT-qPCR,immunohistochemistry and Western Blot,analyzed with clinicopathological features.We knocked down Drpl expression in HCC cell lines HCCLM3,Huh7 with siRNAs,and overexpressed Drpl in HCC cell HepG2 and normal liver cell QSG-7701 with lentivirus vectors.The cell proliferation was detected by EdU assay,CCK-8 assay and clonogenic survival assay.The cell apoptotic was examined by flow cytometry.Results:The results of RT-qPCR,immunohistochemistry and Western Blot showed a significantly increase of Drpl expression in tumor tissues compared with that in adjacent normal tissues.We also found that the expression levels of Drpl were associated with tumor size and histopathologic grading,with regard to the association with clinicopathological features.The results of EdU assay,CCK-8 assay,clonogenic survival assay and flow cytometry showed that Drp1 knockdown inhibited HCC cell proliferation and increases apoptosis,while Drpl overexpression promoted HCC cell proliferation and made no difference in cell apoptosis.Conclusion:Drpl expression is upregulated in liver cancer tissues compared with that in adjacent normal tissues.Drpl is associated with liver cell tumorigenesis.Part ? Drpl-mediated limited mitochondrial permeabilization contributes to liver tumorigenesis through DNA damage and genomic instabilityAims:Mitochondrial dynamics are implicated in cell apoptosis.Mitochondrial fission mediated by Drp1 regulates mitochondrial outer membrane permeabilization(MOMP).The exact relationship between mitochondrial fission and liver tumorigenesis needs to be explored.In this part,we aim to confirm the relationship between Drpl expression and limited mitochondrial permeabilization.Then we will explore the mechanism by which limited mitochondrial permeabilization contributes to liver tumorigenesis.Methods:We overexpressed Drpl in HCC cell HepG2 and normal liver cell QSG-7701 with lentivirus vectors.The limited mitochondrial permeabilization was detected by Western Blot and immunofluorescence.Next,we quantified caspase-3 activity using assay kits and captureed activity-dependent precipitation of caspase-3.The cell proliferation was detected by EdU assay,CCK-8 assay and clonogenic survival assay after treated with caspase inhibitors.Then we used comet assay to measure DNA damage.The DNA damage in tumor tissues was determined by immunohistochemistry.Finally,We investigated Drp1 expression with HCC patient prognosis in the TCGA data set.Results:The results of Western Blot and immunofluorescence showed Drpl overexpression induced limited mitochondrial permeabilization and Cytochrome c release.Cytochrome c engages caspase activity.Treatment with the caspase inhibitor had adverse effects on cell proliferation.Caspase activity induces CAD activation through cleavage of its inhibitor ICAD,that trigger caspase-dependent DNA damage.The investigation of Drpl expression in the TCGA data set showed HCC patients withigher levels of Drpl underwent a higher frequency of copy number alterations and a worse prognosis than cancer patients with lower Drp1 expression.Conclusion:Drp1 overexpression induces limited mitochondrial permeabilization,engages limited caspase activity related to liver cell tumorigenesis.Drpl overexpression induces caspase-dependent DNA damage,contributes to genomic instability and liver tumorigenesis.