HAb18G/CD147 Promotes Cell Cycle Progression Via Ca²⁺ Dependent ERK1/2 Activation In Human Hepatocellular Carcinoma Cells

Aims:Relationships between cancer and cell cycle regulation are extremely critical research field. It is now accepted that cancer is more accurately described as being the product of malfunctions within the regulation of the cell cycle. Cancer cells are characteristically independent of growth stimulus due to mutation of intracellular signal pathways. Such independence facilitates re-entry into the cell cycle. Cancer was thought to arise when cell growth exceeds the rate at which cells die, so that cells are proliferating at an uncontrollable rate. Under normal conditions, cell cycle-regulating mechanisms endeavour to maintain homeostasis. Disturbance in the homeostasis between cell growth and apoptosis, may result in hyperplasia or neoplasia. In addition, the survival of cancer cells following radiotherapy or chemotherapy will depend on cell cycle phase. Regulation mechanism of cell cycle offer huge potential targets for chemotherapeutic and biotherapy. Greater understanding of molecular components involved in cell cycle is needed to fully manipulate and exploit the mechanisms.HAb18G, a transmembrane glycoprotein of the immunoglobulin superfamily , cloned by anti- hepatocellular carcinoma monoclonal antibody HAb18 screening of hepatocellular carcinoma cDNA library has an identical amino acid sequence as CD147 family. HAb18G/CD147 is extensively expressed in various cell types, especially at high levels in cancer cells. Our previous studies have proved that HAb18G/CD147 may enhance the invasion and adhesion potential of human hepatocellular carcinoma cells by promoting fibroblast cells to secrete elevated levels of MMPs. In addition, HAb18G/CD147 enhances metastatic potentials in human hepatoma cells by disrupting the regulation of store-operated Ca2+ entry by NO/cGMP. Although, some researches demonstrate important role for HAb18G /CD147 on proliferation potential of cancer progression, but the particular research on the exact mechanism of cell cycle regulation by HAb18G /CD147 has not been reropted. The aim of this study is to recognize the precise mechanism of HAb18G/CD147 promoting cell cycle progression of human hepatocellular carcinoma cells. This research not only develop the of acknowledge of HAb18G/CD147,but also provide possible strategy for therapeutics of human hepatocellular carcinoma.Methods: HAb18G/CD147 cDNA plasmid, its extracellular fragment plus transmembrane domain plasmid, its intracellular fragment plus transmembrane domain plasmid, and specific siRNA target HAb18G/CD147 were transfected into SMMC-7721 individually by Lipofectamine,as assayed by Real Time PCR and Indirect Immunofluorescence.In addition,cell proliferation potency was assayed by BrdU incorporation analysis. G0/G1 phase,S phase and G2/M phase individual proportion in whole cell cycle assayed by flow cytometer(FCM). Furthermore, Western-blot assay was carried out to identify protein variation of ERK1/2, phosphorylated ERK1/2 and CyclinD1. Moreover, pretreated with Tg and PI3K inhibitor wortmannin or LY294002, Ca2+ entry and Ca2+ release was analyzed by laser scanning confocal microscope.Results: In this study, we obtained stably up-regulation and transiently down-regulation of HAb18G/CD147 expression via transfect specific plasmid and siRNA, then we found that up-regulation of HAb18G/CD147 resulted in significant growth enhancement, G0/G1 phase proportion reduction, ERK1/2 phosphorylation increase and overexpression of cyclinD1 . Moreover, blockade of ERK1/2 function by U0126 inhibited HAb18G/CD147-dependent cyclinD1 expression and cell cycle progression. In addition, up-regulation of HAb18G/CD147 increased both Ca2+ release and ERK1/2 phosphorylation. Furthermore, both ERK1/2 phosphorylation and cyclinD1 expression are Ca2+ dependent. LY294002 and Wortmannin, specific inhibitor of PI3K, suppressed HAb18G/CD147 induced Ca2+ release dependent ERK1/2 activation. By transfecting truncated HAb18G/CD147 fragments into human hepatoma cells,we found that no effects on G0/G1 to S transition, Ca2+ release, ERK1/2 phosphorylation increase and cyclinD1 expression were observed by either C or N terminus truncation.Conclusion : HAb18G/CD147 promotes G0/G1 to S transition mediated by activating PI3K, enhancing Ca²⁺ release and induction of ERK1/2 activity dependent cyclinD1 synthesis, and both ectodomain and intracellular domains of HAb18G/CD147 are required to mediate the effect of HAb18G/CD147 on the G0/G1 to S transition processes in human hepatoma cells.