Effects Of Argininosuccinate Synthase 1 On The Proliferation And Apoptosis In SMMC-7721

BackgroundHepatocellular carcinoma,the most common malignant tumor in the world,is the fifth malignant tumors in the world and is also known as the second-leading cause of cancer death in men.Every year,the incidence of HCC accounted for the fifth in the malignant tumor incidence ranking and the mortality of HCC accounted for the second in men.There are about 530,000 newly increasing clinical cases in men and 230,000 in women each year,respectively ranking 4th and 8th.460,000 men were dead due to hepatocellular carcinoma and 211,600 death cases in women each year.During the past decade,the incidence and mortality of HCC showed a clear upward trend in our country.With the rapid development of our society,demographic structure and people's lifestyle have been enormously changed,especially in the last five years.In city,metropolis in particular,HCC has become one of the most severe factor on people's physical and mental health and even be life-threatening.In spite of the medical technology advances and an in-depth understanding of HCC,such as transplant surgery,radical resection and chemotherapy,there isn't significant increase in the 5-year survival rate of HCC patients.In the current circumstance,the study of early diagnosis and how to reduce the recurrence rate should be the hot spot in the field of HCC treatment.It is confirmed that HCC is a multistep process which is involved in the molecular and signaling pathway chaos.Therefore,it is necessary to find out effective target and explore the mechanism beneath the HCC occurrence and progress,which would be the emphasis and difficulty for us.Liver,an important organ of human body,plays a vital role in metabolism,detoxification and excretion.Liver function would be seriously affected when liver diseases occur such as HCC,cirrhosis and severe hepatitis.Clinically,the concentration of blood ammonia catches more attention among doctors since it will not only indicate the severity of liver tumor but also contribute to diagnose hepaticencephalopathy.The concentration of blood ammonia in normal person is18-72?mol.Under normal condition,the liver convert ammonia to urea through a series of reactions known as the urea cycle then blood ammonia is eliminated through kidney,which is main metabolic pathway of blood ammonia in vivo.However,in other organs like brain,cardiac muscle and skeletal muscle,glutamine synthetase catalyzes amnonia and glutamate converting into glutamine.Then glutamine is transported into liver where glutaminase catalyzes glutamine into ammonia.The ornithine cycle(also known as the 'urea cycle')is a cycle of biochemical reactions occurring in many mammalian that produce urea from ammonia.The urea cycle consists of five reactions: two mitochondrial and three cytosolic.In the mitochondria,ornithine transcabamoylase catalyzes the condensation of ornithine with carbamoyl phosphate,producing citrulline.The energy for the OTC reaction is provided by the high-energy anhydride of carbamoyl phosphate.The synthesis of citrulline requires a prior activation of carbon and nitrogen as carbamoyl phosphate.The activation step requires two equivalents of ATP and the mitochondrial matrix enzyme carbamoyl phosphate synthetase-I.In the2-step reaction catalyzed by cytosolic argininosuccinate synthetase,citrulline and aspartate are condensed to form argininosuccinate.The reaction involves the addition of AMP to the amido carbonyl of citrulline,forming an activated intermediate on the enzyme surface,and the subsequent addition of aspartate to form argininosuccinate.In the final step of the cycle arginase cleaves urea from arginine,regenerating cytosolic ornithine,which can be transported to the mitochondrial matrix for another round of urea synthesis.The fumarate,generated via the action of arginiosuccinate lyase,is reconverted to aspartate for use in the argininosuccinate synthetase reaction.Argininosuccinate synthetase is rate-limiting enzyme in urea cycle.A lack of argininosuccinate synthetase expression has been observed in several types of cancer cells,including pancreatic cancer,liver cancer,and melanoma.For example,defects in ASS have been seen in 87% of pancreatic cancers.Cancer cells are therefore unable to synthesize enough arginine for cellular processes and so must rely on dietary arginine.ObjectiveTo study the effect of silencing ASS1,a rate-limiting enzyme in urea cycle,on the proliferation and apoptosis of liver cancer cell SMMC-7721 and its possible mechanism.Methods1.Real-time PCR and Western Blot were used to explore the expression of ASS1 mRNA and protein in Changliver,SMMC-7721,HepG2,HepG3 B and QGY-7703.2.To detect whether ASS1 gene expression was silenced or not after shRNA-ASS1 was transfected into SMMC-7721 cell,Western Blot was used to evaluated transfection efficiency.3.CCK-8 assay was performed to check the proliferation ability of shRNA-ASS1.4.Annexin V-APC/PI double staining flow cytometry was used to evaluated the cell apoptosis of shRNA-ASS1.5.The expressions of apoptosis related proteins such as Bax,Bcl-2 and Caspase-3 were detected by Western blotting.6.The results of experiment was analyzed by SPSS 21.0.All of the measurement data were expressed as mean ± standard deviation(x ± s),The Group-t test was used to analyze in two groups comparison.The comparison among different groups was tested by One-way analysis of variance.P < 0.05 showed statistically significant difference.Results1.Compared with Changliver,HepG2 and QGY-7703,SMMC-7721 andHepG3 B show significant high mRNA expression.At the same time,the expression of ASS1 in protein in different cell lines of liver cancer was detected,among which SMMC-7721 and HepG3 B also show significant higher protein expression than Changliver,HepG2 and QGY-7703.The difference was significant(P<0.05).2.After transfected with shRNA targeting ASS1 gene,the expression of ASS1 protein in liver cancer cell line SMMC-7721 were detected by Western blot.Compared with the mock group,ASS1 protein expression level in shRNA-ASS1 group was significantly reduced.The difference was significant(P<0.05).The difference of ASS1 protein level in between mock group and negative control group is not obvious(P>0.05).3.CCK-8 results suggested that the growth of SMMC-7721 after transfected with shRNA-ASS1 was significantly inhibited.Compared with the mock control group,the growth of shRNA-ASS1 group was significantly deceased.The difference was significant(P<0.05).There was not significant difference between mock group and negative group.(P>0.05)4.Flow cytometry analysis indicates that the cell apoptosis rate of mock,negative control group and shRNA-ASS1 group is(0.3±0.12)%,(3.0±1.08)% and(27.1±3.52)% in respective.The apoptosis rate increased obviously after down-regulation of ASS1.The difference was significant(P<0.05).There was not significant difference between mock group and negative group.(P>0.05)5.Western Blot analysis showed that shRNA-ASS1 group up-regulated the expressions of Bcl-2 and procaspase-3,and up-regulated the expression of Bax compared with mock group.The difference was significant(P<0.05).Conclusions1.Silencing ASS1 could significantly inhibit the proliferation of SMMC-7721 and also induced cell apoptosis which is dependent on the activation of the caspase family.2.ASS1 could be a potential target for the treatment and prevention of liver cancer.