Decreased Mitochondrial-associated Endoplasmic Reticulum Membrane Induced By Dysregulated Mitochondrial Dynamics Promotes The Survival Of HCC Cells

Objectives:1.To determine whether mitochondrial division affects the structure and function of mitochondria associated ER membranes?MAMs?;2.To explore the effects of MAMs structure on the growth of HCC cells;3.To analyze the effects of mitochondrial division on structure of MAMs and the survival of HCC cells via Rab32.Methods:1.The changes of MAMs structure in liver cancer and adjacent tissues were observed by transmission electron microscopy.The number of MAMs and the prognosis of patients with liver cancer were analyzed by Kaplan-Meier curve.Drp1interference/overexpression cell was constructed by liposome transfection method,and the transfection effect was observed by Western blot and laser confocal microscopy.ER and mitochondria were labeled with ER-RFP and Mitotacker Green dyes respectively.Super-resolution microscope was used to observe the co-localization of mitochondria and ER after overexpression of Drp1,and the effect of Drp1 overexpression on Ca²⁺concentration in mitochondria was detected by Rhod-2 dye;2.The cell line of PACS-2 interference was constructed by liposome transient transfection method.The interference effect was observed by Western blot,and the effect of PACS-2 interference on MAMs structure was observed by electron microscope.EDU and flow cytometry were used to detect effect of PACS-2 interference on proliferation and apoptosis of HCC Cells.3.The labeled proteins of these organelles were detected by Western blot to verify the extraction purity of MAMs components,and the obtained MAMs component proteins were detected by a proteomics techniques,unlabeled liquid chromatography tandem mass spectrometry,which also screened out the proteins that affect MAMs formation;An expression vector of Rab32 molecule was construct and was used to transfect Drp1overexpressing-liver cancer SNU-739 cells by liposome transfection method.The successfully constructed cell lines were screen out by Western blot.The changes of MAMs structure after the overexpression of Rab32 in Drp1 overexpressing-cells was observed by transmission electron microscope and Rhod-2 dye was used to detect its effect on Ca²⁺concentration in the mitochondria.Finally,EDU flow cytometry was used to analyze its effect on HCC cell proliferation and apoptosis.Results:1.MAMs number was significantly reduced in liver cancer tissues.Kaplan-Meier curve analysis showed that the smaller the number and the worse the prognosis;Overexpression of Drp1 inhibited the co-localization of mitochondria and the ER and reduced the concentration of Ca²⁺in the mitochondria;2.Interfering with PACS-2 could inhibit the formation of MAMs structure,could promote the proliferation of HCC cells and inhibit the apoptosis of HCC cells;3.After identification,it was found that the MAMs component proteins we extracted had high purity.A total of 3043 proteins were detected by mass spectrometry,of which 190 proteins had significant differences?more than 2 folds of changes and P<0.05?between them.Subcellular localization and function analysis of differential proteins were performed,and Rab32 molecules were screened for further research.Rab32 expression vector was successfully constructed and it was found that overexpression of Rab32 in Drp1 overexpressing-cells could promote the formation of MAMs structure,increase concentration of Ca²⁺in mitochondria,inhibit the proliferation of liver cancer cells and promote apoptosis of liver cancer cells.Conclusions:This study revealed the impact of mitochondrial division on mitochondrion-endoplasmic reticulum interaction and the potential molecular mechanisms that regulate tumor cell survival,provided more evidence for the promoting mechanism of mitochondrial division to liver cancer cell proliferation,and also furnish new ideas for the search for molecular targets for Drp1 gene-related liver cancer.