Effect Of Liposomal Transfection Of UCH-L1 SiRNA On Proliferation And Apoptosis Of H157 Cell

Objective:Lung cancer is the most common malignancy worldwide and is the leading cause of cancer death in men.60% of lung cancer have been in the advanced stage,when they are discoved.Five-year survival rate of non-small cell lung cancer (NSCLC) is Less than 10%,and small cell lung cancer (SCLC) Less than 3%,though many therapeutic measures have been taken such as operation,radiotherapy,and chemotherapy.Because of metastasis and multidrug resistance of lung cancer,it is urgent to find new diadynamic criteria and effective therapeutic method.The development of cancer is a complex process that many factors,levels and genes participate it.Because of the change of gene is the key point,it has been the hot spot and nodus how to clarity the mechanism of cancer and find the effective target point. What's more,Molecular Biology has open up new path.Several reports demonstrated that the expression of Ubiquitin C terminal hydrolase-L1 (UCH-L1) has been found to be an oncogene in malignant tumor such as esophageal carcinoma,lung cancer and breast cancer.UCH-L1 whose anothor name is protein gene product 9.5(PGP9.5) belongs to the UCH proteases family that deubiquitinates ubiquitin-protein conjugates in the ubiquitin-proteasome system.Covalent conjugation of ubiquitin to proteins plays a crucial role in a wide variety of biological processes,including the cell cycle,cell proliferation,development,apoptosis,signal transduction,and me-mbrane protein internalization.Ubiquitination is reversible,i.e. ubiquitin is recycled by proteolytic removal from its conjugating protein by deubiqui-tinating enzymes (DUBs),a family of proteases with exquisite specificity for ubiquitinated sustrates.Deubiquitination is widely recognized as an important component of regulatory mechanisms in all ubiquitin-dependent pathways.Ubiquitin carboxy terminal hydrolase (UCH) is a subclass of DUBs,which catalyzes the hydrolysis of COOH-terminal ubiquityl esters and amides.It removes ubiquitin from ubiquitinated cellular proteins, thereby preventing them from targeted degradation via the proteasome pathway.One member of the UCH family is represented by UCH-Ll,which is selectively expressed in the testis/ovary and brain.Hibi K showed that the PGP9.5 gene had no detectable expression in normal lung tissues but was frequently overexpressed in primary non-small-cell lung tumors.They also suggested that the expression of PGP9.5 in lung cancer might play a causative role in the oncogenic transformation of human lung epithelial cells,because 1) PGP9.5 shares conserved domains with the members of ubiquitin hydrolase family,several of which are potential oncogenes;2) PGP9.5 expression is not present in the normal lung epithelium,but becomes activated sometimes during the course of neoplastic transformation;and 3) expression of PGP9.5 is closely associated with advanced stages of NSCLC. This evidence suggests that UCH L1 may possess tumorigenic properties and promote tumor progression,although the mechanism is largely unknown. We wanted to investigate whether UCH-L1 affects known oncogenic processes by utilizing the application of RNAi to gain insight into genes regulated by UCH L1 in lung cancer H157 cells.Methods:UCH-L1 siRNA was synthetized and transfected into H157 cell by liposome.Cell morphological change was observed with microscope, cell proliferation and apoptosis index detected by flow cytometry,UCH-Ll mRNA expression determined by RT-PCR and protein level of UCH-Ll revealed by western blot analysis.Results:Cell morphological change was observed with microscope. Before transfection,the cells with round caryon were polygon, thin and flat, and they were fast clingy.There were one or more nucleoles within the caryon.But after 48 h of transfection,the cells became contraction and not good at clingy.What's more,the ecphyma of cells disappeard.To obtain a quantitative characterization of the apoptotic effect,we examined the above cells for apoptotic change by measuring DNA degradation using flow cytometry.The results indicate that loss of UCH L1 expression by siRNA leads to induction of apoptosis due to arrest in the G0/G1 phase of cell-cycle.Compared with their negative controls,up-regulation of UCH-L1 expression in H157 cells markedly decreased the fraction of apoptotic cells, while down-regulation of UCH-L1 expression in H157 cells led to the opposite effect.The apoptotic ratio of the UCH-L1 silencing transfectants is (28.06±1.58)%,the blank control is (0.45±0.09)%, and the negative control is (0.52±0.07)%.There is no obvious difference between the blank control and the negative control(P>0.05),but there is obvious difference between the siRNA group and the two controls(P=0.000).This shows that UCH-Ll siRNA can suppress UCH-Ll greatly,and induce the apoptosis of the lung cancer H157 cells.RT-PCR analysis shows that there are two straps of 272 bp (UCH-Ll) and 540 bp(β-actin) respectively.The straps ofβ-actin are similar and clear.The strap of the UCH-L1 siRNA group is weaker than the other groups.The Labworks revealed that mRNA levels of H157 were notably lower in UCH-Ll silencing transfectants than in their blank and negative controls.The IOD ratio of UCH-L1/β-actin of the blank control is 0.523,4±0.090, the negative control is 0.520,3±0.108,5,There is no obvious difference between the blank control and the negative control (P>0.05).The siRNA group is 0.259,6±0.037,6,and there is obvious difference between the siRNA group and the two controls (P=0.009).This shows that UCH-L1 siRNA can induce the degradation of UCH-L1 mRNA. Western blot analysis shows that there are two straps of 25 KD (UCH-Ll) and 43 KD(β-actin) respectively.The straps of P-actin are similar and clear. The strap of the UCH-Ll siRNA group is weaker than the other groups.The Labworks revealed that the protein levels of H157 were notably lower in UCH-L1 silencing transfectants than in their blank and negative controls. The IOD ratio of UCH-L1/β-actin of the blank control is 0.739,4±0.053,3, the negative control is 0.660,7±0.065,4,There is no obvious difference between the blank control and the negative control(P>0.05).The siRNA group is 0.253,7±0.041, 1,and there is obvious difference between the siRNA group and the two controls (P=0.000).This shows that UCH-Ll siRNA can induce the special degradation of the protein of UCH-L1.Conclusions:UCH-L1 siRNA is able to inhibit the proliferation of lung adenocarcinoma cell lines H157 and induce the apoptosis.UCH-L1 might become a new target for lung carcinoma gene therapy.