Effects Of Silencing RRS1 Expression By Lentiviral-mediated RNA Interence On The Biological Behavior In Human Liver Cancer SMMC-7721 Cells

The hepatocellular carcinoma(HCC) is one of most common malignant tumors in the world, which is the fifth most common cancer and the second cause of death, and its incidence and mortality have increased year by year. HCC has occult process, rapid progress, high mortality, and serious threat to the patient’s health and life.RNA interference(RNAi) is a gene silencing technology mediated by double-stranded RNA(ds RNA), which can specifically block the target gene functional expression by joining exogenous or endogenous short hairpin RNA to a variety of biological cells, causing specifically endogenous mRNA degradation and post-transcriptional gene silencing. RNAi technology will continue to provide a new method for developping the study of human gene function, cancer early diagnosis and targeted therapy.In recent years, the researchers found that the synthesis and biological activity of ribosome and the tumor cells have a close relationship. Ribosome biogenesis regulatory protein 1(RRS1) is a conserved eukaryotic nuclei protein with a 203 amino acid. RRS1 can be adjusted ribosome biogenesis speed according to the state of cell, thereby maintaining the homeostasis of cells, and it is an important protein in signal transduction pathway of protein secretion and ribosome synthesis. Previous studies demonstrated RRS1 protein expression was increased in HCC patients’ tissue, whether RRS1 is involved in the regulation of liver cancer malignant behavior and possible regulatory mechanism is unclear.In this study, we constructed the RRS1-siRNA expression vector mediated lentivirus, transfected the vector into human hepatoma cell SMMC-7721 after successfully gene sequencing test. We have adopted Western blot and RT PCR technology to detect the change of the expression levels of RRS1 mRNA and RSS1 protein after transfection. We used Cellomics and flow cytometry(FCM) to detect the change of the proliferation, cell cycle and apoptosis of SMMC7721 cell after RRS1 gene interference. The purpose of this study is to investigate the effect of RRS1 mRNA and RRS1 protein expression levels and biological behavior proliferation and apoptosis of human hepatoma SMMC7721 cell after lentivirus mediated RRS1 gene silencing.Experimental results show that: 1. We successfully constructed pGCSIL-GFP-RRS1 vector,and DNA sequencing confirmed the insertion sequence met the design requirements. After co-transfected 293 T cells and packaged Lentiviral Particles containing LV-RRS1-shRNA, the virus titer was 8x108 TU / mL. 2. After infected SMMC-7721 cell lines by RRS1 shRNA lentiviruses, compared with that of Lv-shCon, the RRS1 mRNA and RRS1 protein expression levels in SMMC-7721 of Lv-shRRS1 group were significant decline, respectively falling 68% and 73%. 3. Compared with that of the Lv-shCon group,the cell proliferation ability of SMMC-7721 in Lv-shRRS1 group was significantly decreased. 4. The cell apoptotic rate of the group of Lv-shCon was(8.15±0.33)%, significantly lower than Lv-sh RRS1 group’s cell apoptotic rate(36.5±0.19)%, the difference was statistically significant difference(P<0.01). 5. Compared with that of Lv-sh Con group, the cell cycle of Lv-shRRS1 group was changed that the proportion of cells in G1 phase were significantly increased(P <0.01), and the proportion of cells in S and G2 / M phase were decreased, the difference was significant(P <0.05).In this study, the following conclusions: 1. We successfully constructed siRNA lentiviral vector targeting of RRS1 mRNA, constructed the SMMC-7721 cell lines infected by RRS1 shRNA lentiviruses,that were tested significantly inhibited RRS1 gene expression,which can be provide the experimental basis for the further mechanisms Research. 2. Targetly suppressed RRS1 gene,the proportion G1 phase of SMMC-7721 cells was increased and the proportion of S and G2 / M phase was decreased so that the cell cycle arrest in G1 phase, significantly decreased cell proliferation and promote apoptosis. It implies that RRS1 can involve in regulation of hepatocellular proliferation activity, cell cycle and apoptosis ability,which could to be provided the theoretical support for gene-targeted therapy in patients with liver cancer, and provide a experimental basis for further research that the action of RRS1 gene during the progression, invasion and metastasis, tumor recurrence of HCC.