The Involvement Of Microrna-212in The Pathogenesis Of Hepatocellular Carcinoma By Directly Regulating Histone Demethylase RBP2

Objective:We want to investigate the mechanism of demethylase regulation by microRNA molecule. This helps us to know more about the involvement of microRNA molecule in the pathogenesis of human cancer, which will provide some reference for the microRNA molecules as therapeutic targets in the cancer treatments.Methods:1. In the tissue level, we detennined the expression of hsa-microRNA-212and RBP2in the liver cancerous tissues and normal ones with qRT-PCR method and initially analyzed the relationship between them. We also detected the expression of the direct RBP2targets, p16ink4a and p27kip1with the same methods.2. In the molecular level, we transfected the hsa-microRNA-212overexpression plasmid, hsa-microRNA-212inhibitor and their respective control plasmid into HepG-2and SMMC-7721cell lines and determined the expression of RBP2and p16ink4a as well as p27kip148hours later. We also analyzed cellular senescence and cell proliferation after hsa-microRNA-212overexpression plasmid in these cells. Luciferase reporter gene assay was also used to analyse the binding of hsa-microRNA-212to the RBP23’-UTR. The following are the specific methods in this part:(1). After hsa-microRNA-212overexpression plasmid and its control plasmid transfection into HepG-2and SMMC-7721cell lines for48hours, we determined the expression of RBP2 (2). After hsa-microRNA-212inhibitor and its control plasmid transfection into HepG-2and SMMC-7721cell lines for48hours, we determined the expression of RBP2(3). We first transfected HepG-2cells with hsa-microRNA-212overexpression plasmid, and24hours later we transfected RBP2overexpression plasmid into these cells to see the change of the ability to form clones48hours later.(4). We first transfected SMMC-7721cells with hsa-microRNA-212overexpression plasmid, and24hours later we transfected RBP2siRNA into these cells to see the change of the ability to form clones48hours later.(5). Luciferase reporter gene with normal3’-UTR as well as the mutated3’-UTR of RBP2were co-transfected with has-microRNA-212overexpression plasmid to see the directed binding.3. In the organic level, we divided10nude mice (7weeks old) into2groups (n=5each) for subcutaneous injection with HepG-2cells with hsa-miR-212overexpression, and their matched control cells. The size of tumor was measured every5days. Tumor volume was calculated as longest tumor diameter×(shortest tumor diameter)2/2.5weeks later, the tumors were harvested and one half was stored at-80for RNA detection and the other half was stored at formaldehyde for Immunohistochemistry staining.Result:1. The expression of RBP2was stronger in cancerous than non-cancerous tissues, but that of its binding microRNA, hsa-miR-212, showed an opposite pattern.The expression of p16"ik4aand p27kip1was also down-regulated in the cancerous tissues.2. After hsa-microRNA-212overexpression plasmid transfection into HepG-2and SMMC-7721cell lines for48hours, the expression of RBP2was significantly suppressed, but the expression of p16ink4a and p27kip1was enhanced accordingly.3. After hsa-microRNA-212inhibitor plasmid transfection into HepG-2and SMMC-7721cell lines for48hours, the expression of RBP2was significantly increased, but the expression of p16mk4aand p27kip1was suppressed accordingly. 4. Cellular senescence was triggered in HepG-2and SMMC-7721cell lines transfected with hsa-microRNA-212overexpression plasmid for48hours.5. Cell proliferation was inhibited in HepG-2and SMMC-7721cell lines transfected with hsa-microRNA-212overexpression plasmid for48hours.6. In the reversion experiment, the transfection of RBP2overexpression plasmid could reverse the suppression of cell proliferation induced by hsa-microRNA-212overexpression plasmid transfection in HepG-2cells. And the transfection of RBP2siRNA could reverse the enhancement of cell proliferation induced by hsa-microRNA-212inhibitor plasmid transfection in SMMC-7721cells.7. The luciferase activity dramatically decreased with hsa-microRNA-212overexpression plasmid and normal3’-UTR of RBP2co-transfection compared with their contol group. But the luciferase activity partly reversed after hsa-microRNA-212overexpression plasmid and mutated3’-UTR of RBP2co-transfection.8. Both the tumor incidence and the tumor size were smaller in the nude mice group injected into HepG-2cells with hsa-microRNA-212overexpression plasmid transfection than their matched control group. The tumor index (tumor weight/mice weight) was the same case. The expression of RBP2was decreased in the group injected into HepG-2cells with hsa-microRNA-212overexpression plasmid transfection with qRT-PCR method and immunohistochemistry staining. Of key importance, we found that where the expression of RBP2was stronger, the expression of p21CIP2and p27kip1was lower and vice verse in the serial sections with immunohistochemistry staining.Conclusion:Hsa-microRNA-212participated in the pathogenesis of HCC by directly regulating RBP2expression, affecting the expression of its downstream targets CDKI. So the hsa-microRNA-212-RBP2-CDKI pathway may be important in the initiation and development of HCC.