The Effect Of MiRNA-362-5P On Hepatocellular Cancer Cells’ Malignant Biological Behavior Through Target CYLD, The Negative Regulatory Factor Of NF-κB Pathway

Hepatocellular carcinoma is one of the clinical common malignant tumor, its incidenceis the third of the world. In the causes of death to malignant tumors, it is also come outin front, it has a high degree of malignancy, easy to metastasis and recurrence and othercharacteristics, is a serious human harm health disease. Among them, the highmetastasize rate and recurrence rate are important reasons for its high mortality.Inrecent years, a growing number of studies suggest that, abnormal expression ofmicroRNA is closely related with the occurrence and development of many solidtumors, which can be adjusted through the influence of many pathways to the malignantbiological behavior of tumor cells.But his influence on the biological behavior ofmalignant liver cancer development and metastasis of hepatocellular carcinoma is stillunknown。MiRNAs is a tiny ribonucleotide generation which transcribed by RNA polymerase II,usually consists of19-25bases, and play an important role in body growth、developmentand the process of metabolism. Study found that, it is mainly through target the mRNA3’-UTR region to completely or partial binding, to make the target gene degradation orsilence, then influence the tumor’s occurrence and development, play a similar rolesuch as the oncogenes or tumor suppressor genes, One miRNA possibly can regulatedseveral different mRNAs, the same mRNA can also be regulated by several differentmiRNAs, matching mechanism is similar to the codon, involved in the expression ofabout30%protein in cellular, forming a huge network. We through the biologicalinformation prediction and luciferase reporter gene assays showed that CYLD may be apotential target for miRNA-362-5p function。CYLD is a tumor suppressor gene reported in Nature magazine2003, its function as adeubiquitinating enzyme,can inhibit NF-κ B, JNK, Wnt/β-catenin pathway activation,so play an importantrole in the regulation of cell cycle,cell proliferation,inhibit the molecule mechanism of tumor formation.Study found that that it has a closerelationship with many kinds of solid tumor formation, such as cervical cancer, coloncancer, renal cell carcinoma.But the miRNA-362-5p’s function in hepatocellularcarcinoma through target CYLD to affect the biological behavior of hepatoma cellshave not been clearly reported.To investigate the abnormal expression of miRNA-362-5p how to function in thedevelopment of HCC, to using the reverse transcription polymerase chain reaction(RT-PCR) amplification of mirR-362-5p gene in hepatocellular carcinoma tissues andcell lines, use transient transfection method to transfecte HepG2and normal liver cellsHL-7702with Anti-mirR-362-5p,RT-PCR to investgate the transfection efficiency.Western-blot to investgate the activation of NF-kB signal pathway, explore themalignant biological behavior change of hepatoma cells, the experimental results are asfollows:First of all, we success extracted the total RNA from liver cancer tissues andparacancerous tissues, using polymerase chain reaction (RT-PCR) amplified themiRNA-362-5p, gray analysis showed that the expression of miRNA-362-5p in HCCtissues is2-3times higher than paracancerous tissues, the RNA RT-PCR results from thecell culture were consistent with the situation.Then to find the target of miRNA-362-5p, through the biological information predictionand luciferase reporter gene assays we found that CYLD may be a potential target formiRNA-362-5p work, in order to prove this finding, we use miRNA-362-5p inhibitors(Anti-miRNA-362-5p) and agonist (miRNA-362-5p-mimics) transfected293A (humanembryonic kidney cells) cells, culture48h, then extract the total cellular protein,Western-blot experiments found that CYLD levels are directly affected by themiRNA-362-5p inhibitor and activator, initially confirmed that CYLD is a potentialtarget for miRNA-362-5p function.Further experiments to confirm the CYLD isimmediately targeted by miRNA-362-5p in liver cell lines, we transfected themiRNA-362-5p inhibitors and agonists into the miRNA-362-5p high expression cellline HepG2and miRNA-362-5p low expression HL-02cell line, then found that inthese two kinds of cell lines has the same condition. Moreover, HepG2cell lines’malignant biological behavior which transfected with Anti-miRNA-362-5p wasinhibited. To further explore the molecular mechanism of the malignant tumor biological behaviorchange, we extracted HL-02cytoplasmic protein and nuclear protein transfection withmiRNA-362-5p inhibitor of HepG2cell transfection with agonist, study found that NF-kappa B signaling pathway activation is weakened, and the pathway is an importantsignaling pathway closely related with the malignant biological behavior which hasbeen even known,such as cell proliferation, apoptosis, also suggests that themiRNA-362-5p through down-regulation or silent the NF-к B negative regulator toenhance the cells’ proliferation, anti-apoptosis ability and so on.