Abstract: Objective To observe the effect of the Hps1 and Hps4 subunits of biogenesis of lysosome⁃related organelles complex⁃3(BLOC⁃3) on the mitochondrial localization of Rab32 in liver cancer cells and the role of  liver cancer growth. Methods The interaction of Hps1 and Hps4 with Rab32 was analyzed by public databases GenDoma, String and InBio Discover. Human liver cancer cell lines SNU⁃739 and Hep⁃3B were cultured in vitro and transfected with Hps1 and Hps4⁃related siRNAs and plasmids, respectively, by using the liposome transfection method. The effect of Hps1 and Hps4 on the mitochondrial localization of Rab32 was observed by immunofluorescence and Western blot. Cell scratch, clonogenesis, EdU, MTS and Transwell invasion assays were used to detect the changes of liver cancer cell migration, proliferation and invasion after the regulation of Rab32 mitochondrial localization by Hps1 and Hps4. The expression difference of Rab32 protein in liver cancer and normal liver tissues was analyzed by using dataset CPTAC in the public database UALCAN. Results Database analysis showed that both Hps1 and Hps4 could interact with Rab32. Rab32 protein expression was significantly decreased in liver cancer tissues compared with normal liver tissues (P<0.001). After interference with Hps1 or Hps4 or both Hps1 and Hps4 in liver cancer cell line SNU⁃739, the mitochondrial localization of Rab32 were decreased (all P<0.01), mitochondrial Rab32 protein expression were decreased (allP<0.001), and cell proliferation, migration and invasion were enhanced (all P<0.05). After overexpression of Hps1 and Hps4 in liver cancer cell line Hep⁃3B, the mitochondrial localization of Rab32 was increased (P<0.01), the protein expression of mitochondrial Rab32 was increased (P<0.001), and the proliferation, migration and invasion of cells were inhibited (all P<0.01), while the overexpression of Hps1 or Hps4 alone had no significant inhibitory effect (all P>0.05).  Conclusions Both Hps1 and Hps4, the subunits of BLOC⁃3, can interact with Rab32 and increase the mitochondrial localization of Rab32, thereby inhibiting the growth of liver cancer  cells.

Key words: Liver cancer , BLOC-3, Rab32, Mitochondria, Migration, Invasion