The Role Of AIM2 In The Progress Of HCC And Its Molecular Mechanism

AimsAbsent in melanoma (AIM2), a member of the interferon-inducible HIN-200 family, is recognized as a cytoplasmic DNA sensor playing important role in both innate immunity and tumor pathology. Over-expression of AIM2 can inhibit melanoma phenotype, and delay the mice fibroblast proliferation, which leads to the recognition of its role as a tumor suppressor.Hepatocellular carcinoma is the fifth cancer in the world, but its fatality rate is in the third. The cause and pathogenesis of HCC have not been completely clarified so far. Here we investigate the role of AIM2 in the progression of hepatocellular carcinoma, and explore its molecular mechanism.Materials and Methods1. Detection of AIM2 expression in HCC and corresponding non-cancerous liver tissues1.1 In order to dectect the expression of AIM2 in HCC,49 pairs of matched cancer tissue samples and their corresponding non-cancerous liver tissues were used for immunohistochemistry (IHC) assay.1.2 64 pairs of matched cancer and non-cancerous liver tissue were used for western blot to detect the protein level of AIM2 in HCC.1.3 64 pairs of matched cancer and non-cancerous tissue were used for Real-time PCR to detect the mRNA level of AIM2 in HCC.1.4 Statistical analysis of the relation between AIM2 protein expression level in HCC tissue and clinical parameters.2. Overexpression and knockdown of AIM2 in HCC cells2.1 Transfection of AIM2 plasmid or Si-AIM2SMCC7721 and HUH7 cells were transfected with AIM2 expression plasmid to investigate the effect of exogenous overexpression of AIM2 in HCC cells. HepG2 and BEL7402 cells were transfected with SiRNA specifically targeting AIM2(Si-AIM2) to investigate the effect of AIM2-knockdown on HCC cells.2.2 Effect of AIM2 on the proliferation, colony formation and invasion of HCC cellsAfter exogenous overexpression or konckdown of AIM2, cell proliferation and colony formation capability were analyzed by CCK-8 kit and colony formation assay, respectively.3. Molecular mechanisim of AIM2 in regulation of HCC progressionAfter exogenous overexpression or knockdown of AIM2, western blot was used to detect the activation of mTOR,AKT,NF-kappaB pathway,et al; wih the intension to screening out the involved signaling transduction pathway..4. Nude mouse tumorigenicity assay was used to confirm the role of AIM2 in HCC progressionXenograft tumor models were constructed by subcutaneous injection of BEL7402 and SMMC7721 cells in nude mice. Visible tumor appeared on day 5 after the transplantation. The formed tumors were then transfected with AIM2 expression plasmid or the empty vector control every other day before the mice were sacrificed on day 25 after the injectionResults1. Expression of AIM2 in HCC tissues was significantly decreased compared with distal non-cancerous liver tissues1.1 Immunohistochemical staining showed that expression of AIM2 in the liver cancer cells was significantly decreased compared with corresponding distal non-cancerous liver tissues. Statistical analysis further confirmed that the expression level of AIM2 in HCC patients was significantly negatively correlated with tumor volume, Edmonson grade and TNM stages.1.2 Real-time PCR and western blot assay further verified that there were significant reductions in both mRNA and protein level of AIM2 expression in HCC tissues compared with corresponding distal non-cancerous liver tissues. Further analysis showed that loss of AIM2 expression in HCC tissues was significantly negatively correlated with later TNM stages, larger tumor volumes and more advanced metastasis status.2. AIM2 inhibited proliferation, colony formation and invasion of HCC cells2.1 Construction of cell modelsSMCC7721 and HUH7 cells were transfected with AIM2 expression plasmid to investigate the effect of exogenous overexpression of AIM2 on HCC cells. HepG2 and BEL7402 cells were transfected with siRNA specifically targeting AIM2 to investigate the effect of AIM2-knockdown on the HCC cells2.2 AIM2 inhibited proliferation, colony formation and invasion of HCC cellsAfter exogenous overexpression of AIM2, SMMC7721 and HUH7 cells had significantly decreased capabilities of proliferation, colony formation and invasion compared with mock control group.While after knockdown of AIM2 expression, the proliferation, colony formation and invasion capabilities of HepG2 and BEL7402 were significantly enhanced. Thus these data indicated that AIM2 could significantly suppress the malignant behaviors of HCC cells, and loss of AIM2 expression in HCC cells might contribute to HCC progression.3. AIM2 exerted its effect by forming an inflammasome and inducing pyroptosisAfter overexpression of AIM2 in HCC cells, both of the caspase-1 activation and IL-1β cleavage were significantly upregulated, while knockdown of AIM2 in HCC cells dramatically suppressed the cleavage of caspase-1 and IL-1β, which indicated that AIM2 inflammasome was formed and activated in these AIM2 overexpressed cells.Overexpression of AIM2 induced a time-dependent activation of caspase-1 and IL-1β as detected, by Caspase-1 activation assay and IL-1β ELISA; meanwhile, the release of LDH was also increased in a time-dependent manner.After blocking the effect of inflammasome formation by its downstream inflammatory caspase inhibitors, suppression of malignant behaviors, including the proliferation, colony formation and invasion of AIM2-overexpressed HCC cells were significantly rescued, which indicated that AIM2 exerted its anti-tumor effect on HCC cells in an inflammasome-dependent manner.4. AIM2 inflammasome formation induced suppression of mTOR-S6K1 pathway After overexpression of AIM2 in these HCC cells, the mTOR-S6K1 pathway was significantly suppressed as defined by the activation level of the key signaling molecules in this pathway, including p-AKT, p-mTOR, p-S6K1, p-S6, p-4E-BP1, and p-eIF4E. Furthermore, its downstream molecule hypoxia induced factor-la (HIF-la) was also significantly suppressed. On the contrary, block of AIM2 by its specific siRNAs significantly upregulated mTOR-S6K1 pathway activation and its downstream HIF-la.To further define whether suppression of mTOR-S6K1 pathway was dependent of AIM2 inflammasome, we detected the mTOR pathway activation after block of the effect of inflammsome formation by its downstream caspase inhibitors. Our data showed that suppression of mTOR pathway was significantly rescued after block of inflammasome formation.These data indicated that AIM2 induced suppression of mTOR-S6K1 pathway in an inflammasome-dependent manner.5. AIM2 inhibited malignant behaviors of HCC cells through suppression of mTOR-S6Kl pathwaySMCC7721 and HUH7 cells were transfected with AIM2 expression plasmid, after treatment with rapamycin, the malignant behaviors of these AIM2 deficient HCC cells, including proliferation, colony formation and invasion capabilities were significantly suppressed. These data indicated that loss of AIM2 in the HCC cells could promote cancer progression through activation of mTOR pathway.6. Exogenous overexpression of AIM2 suppressed the tumorigenecity in nude miceBoth of the average size and weight of the AIM2 transfected tumors were significantly decreased compared with the mock control group. Western blot assay showed that mTOR-S6K1 pathway was significantly suppressed in the AIM2 transfected tumors. These in vivo data further verified that AIM2 inhibited HCC growth through suppression of mTOR-S6K1 pathway.Conclusions1. Data from IHC, western blot and real-time PCR assay confirmed that expression of AIM2 was significantly down-regulated at both mRNA and protein level in the HCC tissue compared with matched non-cancerous liver tissue. Further statistical analysis showed that the expression of AIM2 was significantly negatively correlated with clinical indicators of HCC progression.2. AIM2 inhibited proliferation, colony formation and invasion of HCC cells.3. AIM2 suppressed mTOR-S6K1 pathway and inhibited malignant behaviors of HCC in an AIM2 inflammasome-dependent manner.Innovations and significances1. The expression and role of AIM2 in HCC progression were investigated for the first time, and our data pave a new way for elucidation the molecular mechanism of HCC progression.2. Effect of AIM2 on mTOR-S6K1 pathway was investigated for the first time.3. This is the first time to show that AIM2 suppressed HCC progression in an inflammasome-depedent manner.