Research On Expression Changes And Tumor Inhibition Of SIRT6in The Hepatocellular Carcinoma

Background and objectivesHepatocellular carcinoma is one of the world’s most widespread tumors with high degree of malignancy, and exerts a serious threat to human health. In ten tumors affecting human health, digestive system tumors account for5seats close to the fronts, in which the liver cancer ranks the third one only after gastric cancer, esophageal cancer. Our country is a high incidence area of liver cancer, each year the number of died from liver cancer accounts for nearly50%of all liver cancer deaths worldwide.International medical experts generally expected that by2015the global death population caused by cancer will reach9millions, and reached11.4millions in2030. After years of research, although the treatment of liver cancer has made considerable progress, but including the multi-kinase inhibitor sorafenib, there is no fundamental therapeutic interventions appearing. The incidence of the liver cancer is often concealed, without obvious symptoms in early stage, and together the disease progresses rapidly, causing liver cancer in difficulty of diagnosis. To most patients,they have reached the advanced stage of cancer or with poor liver function,which all means the lost of chance for surgery. More than90%of HCC in China is known as hepatocellular carcinoma, its cause in our country is mainly associated with hepatitis B virus infection, suggesting the occurrence of liver cancer have direct relationship with unstable gene interferenced by hepatitis B virus DNA. Hepatitis, liver cirrhosis and liver cancer is often the three steps in patients with liver disease, so that many patients with diagnosis of hepatocellular carcinoma lost the chance of operation because of the followed liver cirrhosis and the poor hepatic function on that time. Therefore active treatment of hepatitis B, improving liver function and regular physical examination are main priority measures for those patience with liver disease. The reason why the majority of hepatocellular carcinoma secondary to hepatitis, is mainly decided by the molecular mechanism caused by the hepatitis B virus. A plurality of HBV gene coding for the activation of various signal transduction pathways and kinase in liver cells, such as PE3K-Akt, MAPK, SAPK/JNK, NF-κB and other aspects stimulated, these links are activated and influenced each other, so that normal liver cell growth conversion effect progressively enlarged, and ultimately lead to formation of HCC. Signal transduction pathway is very important, whether in the process of liver cancer’s occurrence and development, or in the future for the biological treatment of hepatocellular carcinoma.These years, molecular biology, genomics and proteomics research has made a spurt of progress of development, a number of molecular targeted drugs gradually are developed and put into clinical application.The so-called molecular targeted drugs target the overexpressed key genes, tumor cell receptors or some proprietary marker molecule, and chooses forantagonists or inhibitors for effective intervention to the above key genes, marker molecule regulation or closely related signal transduction pathway, thereby inhibiting tumor growth, progression or metastasis. For example, sorafenib is a multi-kinase inhibitor successfully developed in recent years, it has its unique advantage of in the control of tumor growth,preventing and delaying recurrence, metastasis and improve the quality of life, which has most significance for patients in advanced liver cancer who losing the chance of operation, sorafenib has greatly encouraged the medical workers, but its effect is limited to some extent. One study showed that, in patients with advanced cancer who taking sorafenib extend their lifespan only for about1.4months contrast to to placebo control group. The limited effectiveness indicates that persistent search for the signal transduction network and its key control node in the development of HCC is worth of our continuous efforts in the direction for new drugs of biological treatment to block the initiation and development of liver cancer. The new mode of treatment of liver cancer brings bright prospects of the cancer curing.Low biological creature ke fly. yeast having the silent information regulator protein2(silent information regulator2, Sir2), is considered to be an important factor of regulating species aging. It also has analogues in mammals, namely Sirtuin (SIRT)family. Sirtuins is nicotinamide adenine dinucleotide (NAD+) dependent histone deacetylase. The Sirtuin family consists of7members (SIRT1-7), each member’s position in the cells and the function of each oen are not identical. They are widely distributed, complex and diverse in function, and play an important role on cell proliferation, various material metabolism, aging regulation, tumor growth control and other aspects, among which the SIRT1and SIRT2is researched more than others. SIRT6is a Sirtuin member less studied locating in nuclear, however it is one of the most important one of trhem and shows more and more potential. Nowadays, the present research is also gradually more up. SIRT6and SIR2have only25%homologic rate in common, so the functional differences may also be large, so it is likely that its function is different from the SIR2identified by the deacetylation action. At that time, people generally think that SIRT6’s acetylation ability is weak or not available, with ADP-nucleic acid transferase activity only. But with the SIRT6study in depth step by step, theacetylation substrate found gradually, it has also gradually been recognizedt as the deacetylation function. So, SIRT6is a versatile and important factor, having both acetyltransferase activity and ADP-nucleic acidtransferase. In particular of growth regulation in cancer, its importance is considered second only to SIRT1, which has an important role in the genetic damage repair, maintenance of homeostasis, in inhibiting the occurrence of some tumor. At the same time, SIRT6also has an important role in the regulation of metabolism, such as glucose metabolism and lipid metabolism, and even has important contribution to bone metabolism. The existing data has proved that SIRT6is a master of a hepatic glucose homeostasis which maintains its influence by achieving genomic stability through DNA damage repair in the process. According to the different genetic damage mode, such as double strand DNA damage and single strand DNA damage, SIRT6can have a beneficial treatment through different ways, such as the BER or NER genes-repairing involved in the way. The mechanism of of this series actions play an important role in the prevention of aging and body prevention of neoplasia tendency. Therefore, SIRT6plays a certain role in the prevention and treatment of cancer, and the effect and mechanism of these is still not fully explained. A study has confirmed that knockout of mice SIRT6in vivo increased the number of tumors, size, and aggressive. And Min et al found that SIRT6has inhibitory effect on tumor, mechanism may be that survivin depending on SIRT6is inhibited by binding sites induced tumor suppression by providence of an implementation of AP-1. However, there are also some different conclusions. From some laboratory evidence, SIRT6may have tumorigenic effect on pancreatic cancer and breast cancer. Therefore,the exact role of SIRT6in cancer is still under discussion. In this study, in order to understand whether the SIRT6in hepatocellular carcinoma having regulation function and mechanism, we do something in study of SIRT6expression, regulation and its regulation mechanism in hepatocellular carcinoma through Scientific experiments in vitro and in vovo.ObjectiveTo investigate the expression change of SIRT6in primary hepatocellular carcinoma tissue in contrast of normal para cancer tissues, and to analyze its significance in hepatocellular carcinoma; To observe the effects of SIRT6on HepG2cell growth and reproduction caused by SIRT6expression changes; Through in vitro cell research method, to analyze the molecular mechanism and to explore the potential clinical value.MethodsFirst of all, using RT-PCR and Western blot experiments to detect and analyze of SIRT6mRNA and SIRT6protein levels in liver cancer tissues contrast of adjacent normal liver tissue, in order to understand the SIRT6expression change in HCC both in initial stage and afterwards, thus understanding the pathogenesis of hepatocellular carcinoma affected by SIRT6.Secondly, we extract SIRT6mRNA from mouse tissue, and then make reverse transcription to cDNA library. Afterwards we acquire the full-length of SIRT6DNA by PCR amplification. The complete SIRT6PCR product are cloned into pacAd5CMV-IRES vector. Then, thepacAd5CMV-IRES SIRT6and backbone vector pacAd5are linearized by PacI, and the purified linearized plasmid is transfected into HEK293cells through transfection reagent,.recombinant adenovirus from HEK293cells acquired and we get Ad-SIRT6. In the same way to get the SIRT6RNAi products of Ad-SIRT6-shRNA and control Ad-GFP. Using the combinant gene product to infect HepG2cells which help us to understand the effect of SIRT6on HepG2cell proliferation.At last, we performed TUNEL analysis in the case of recombinant HepG2cells in order to understand the effects of SIRT6on apoptosis of HepG2cells. And, we detected ROS and superoxide anion levels for over-expression SIRT6effect on the oxidative stress of HepG2hepatocellular carcinoma cells. At the same time, through study relationship between SIRT6and extracellular signal regulated kinase (ERK1/2) we explore the molecular mechanisms that regulate the growth of hepatocellular carcinoma.Results1.RT-PCR test results:Comparising the expression of SIRT6in4cases, of carcinoma and adjacent tissue mRNA, mRNA expression in cancer tissues was significantly down regulated (35～40%, P<0.05), with statistical significance.2.Western blot results:Comparison of SIRT6protein expression in4cases of hepatocellular carcinoma and its adjacent tissues (SIRT6/beta-actin), the amount of SIRT6protein in cancer tissues was significantly lower than that of adjacent tissues, the difference was statistically significant.3.The success of construction of eukaryotic expression vector of SIRT6exert a solid basis for the trial of play.4.We tested if significantly reduced expression or overexpression of SIRT6levels would affect the growth of hepatocellular carcinoma cells in vitro environment. The sh-RNA effect mediated by adenovirus decreased SIRT6level which promote the growth of hepatoma cells, and the adenovirus mediated overexpression of SIRT6significantly inhibited the growth of HepG2cells. 5.We study the effects of overexpression of SIRT6on apoptosis of hepatocellular carcinoma cells. First of all, we analyzed Ad-GFP and Ad-SIRT6transfected HepG2cells with TUNEL method. Apoptotic cells (TUNEL positive cells, green) in Ad-SIRT6infected cells can be detected, but in control Ad-GFP cells did not change. We also analyzed the activation of protein expression level of caspase-3, it is an important medium and labeled protein in cell apoptosis. The activated caspases-3in overexpression of SIRT6showed positive but no expression in the control cells. These results clearly show that, over expression of SIRT6in HepG2induced hepatocarcinoma cell apoptosis.6. We study the effects of SIRT6overexpression on oxidative stress of HepG2hepatocellular carcinoma cells. The DCF assay indicates that, the over expression of SIRT6significantly decreased the total ROS level in HepG2cells. Furthermore, overexpression of SIRT6down regulated the level of superoxide anion in HepG2cells.7.Extracellular signal-regulated kinase (ERK1/2), is a major extracellular Pro mitogenic signal transducer, which can promote cell proliferation. We analyze the effect of overexpression of SIRT6on ERK1/2phosphorylation. The results showed, the over expression of SIRT6in hepatoma cells significantly inhibited ERK1/2phosphorylation.8.1n order to know whether SIRT6regulating ERK1/2growth by inhibiting hepatoma cell signaling pathways, we used chemical inhibitors of U0126in HepG2cells treated with ERK1/2pathway. The U0126significantly attenuated the inhibitory effect of SIRT6on growth of hepatocellular carcinoma cells.ConclusionsAfter down regulation of SIRT6gene can promote the growth of liver cancer cell line HepG2, and the over expression of S1RT6on HepG2cell line growth inhibition. Overexpression of SIRT6induced apoptosis in HepG2cells, has been demonstrated in the TUNEL method and the activation of caspases-3western blot. Furthermore, overexpression of SIRT6reduced intracellular reactive oxygen free radical and superoxide anion levels. Finally, SIRT6overexpression inhibits the phosphorylation of ERK1/2, and the application of chemical specific inhibitor U0126blocked the inhibitory effect of ERK1/2pathway can be greatly attenuated tumor overexpression of SIRT6.The results of the study show that, SIRT6expression in HCC was significantly decreased in the present, and the corresponding para carcinoma tissue expressed in normal. This suggests that SIRT6plays a regulatory role to some extent in the process of carcinogenesis and development. SIRT6is considered the possible treatment of hepatic carcinoma for inhibitory effect in the future.