| Background and ObjectiveHepatocellular carcinoma (HCC) is produced by the liver cells or intrahepatic bile duct epithelial cells of malignant tumor, one of the most common malignant tumor, and China is a high risk of liver cancer,45% of which occurred in our country, China is one of the most common malignant tumors in the digestive system malignant tumor, ranked third, second only to gastric cancer and esophageal cancer.the incidence and mortality of liver cancer in china is is the highest in the world.It is a serious threat to the health of the majority of patients. Epidemiological studies have shown that, with the changing of people’s living environment, in the whole world, liver cancer incidence rate is rising year by year, in the world each year to add 500000 to 1000000 cases, the disease is more common in middle-aged men, male to female ratio was 2-5:1.The incidence and treatment of liver cancer has been the focus of today’s medical research,with the in-depth study of clinical trials and basis for a large number of tests, significant advances in the diagnosis and treatment of liver cancer are taken, but a considerable number of liver cancer is discovered late, currently its clinical effect is still quite limited.5-year survival rate of patients with liver cancer worldwide is less than 15%, the mortality rate was 20.73/100,000, an average of about 25 million people worldwide each year die from liver cancer. Even five years postoperatively, patients receiving radical surgery of recurrence rates are still about 30% to 40%. At present, surgical operation is the main treatment of hepatocellular carcinoma, the most direct and effective means remains liver transplantation and liver resection, combined with the comprehensive treatment of radiotherapy, chemotherapy and other adjuvant therapy despite continued progress, but the overall treatment is not satisfactory, recurrence and metastasis is the leading cause of liver cancer affecting prognosis.Tumor metastasis and invasion of tumor cells are closely related, we can through the inhibition of cancer cell invasion to inhibit tumor metastasis. Conversely, if long-term exposure to a human body to promote cell invasive ability of the environment, the possibility of a tumor metastasis will be higher. Liver cancer is now widely recognized that multiple factors, multiple genes involved in multiple stages of development of the disease, but the exact mechanism of its occurrence and development has not yet been elucidated. In China, Researcher has carried on the long-term study found that in the high incidence area of hepatocellular carcinoma, there are dozens of factors associated with liver cancer. The most important reason is the hepatitis B virus (HBV) and hepatitis C virus (HCV) infection.cirrhosis of the liver, aflatoxin, water pollution, genetic factors are also closely related to the incidence of liver cancer.in addition, certain other substances such as nitrosamines, even nitrogen mustard, alcohol, trace elements and parasitic infections are caused by liver suspicious substances. A large number of epidemiological and basic experimental evidence that chronic infection is closely related to the development of certain serious diseases, including cancer.nine-kind of infectious agents has been identified as a human carcinogen, the world each year 17% of new cancer cases caused by infection, especially in developing countries, can reach 26%, while in developed countries, a relatively small proportion, is about 7.2%. Inflammation is an important part of the tumor in the development process. Many cancers arise from infection, chronic irritation and inflammation. Chronic inflammation of the tumor microenvironment provides conditions for the occurrence of tumor occurrence and growth,it is an indispensable participant in tumor invasion and metastasis. Nasopharyngeal carcinoma, gastric cancer, liver cancer, cervical cancer and infection-related cancers are higher incidence of malignant tumor in our country, especially cancer of the stomach and liver cancer, has been one of the incidence of malignant tumors in our country’s top three, and pay more attention to the effect of the onset of infection factors in malignant tumors have important significance on the prevention, diagnosis and treatment of malignant tumor.In these tumor-associated infection factors, the most important is viral infections, parasites and bacteria account for a certain percentage.since 1982 Warren et al. successfully separated Helicobacter pylori(Hp), the Hp infection has been studies by many scholars both at home and abroad, Hp is considered to be one of the highest bacterial infection rate in the world, as chronic gastritis and peptic ulcer the main cause has been affirmed and confirmed in many studies, the occurrence of Hp infection and gastric cancer are closely related, and in 1994 was listed as a Class I carcinogen, the first time human contact with the bacterial infection to cancer together. Since then, with the research of Helicobacter pylori in depth, more and more of the Helicobacter bacteria are identified and isolated, so far it has been found more than 20 kinds.. According to the settlement of different parts, Helicobacters divided into the stomach Helicobacter and the enterohepatic Helicobacter. Helicobacter hepaticus as a member of the enterohepatic Helicobacter, belonging to the one of the five species which have a clear relationship with liver diseases (H pullorum,H hepaticus, H bills,H cholecystus,H canis). In 1992, the National Cancer Institute USA Frederick cancer research and development center, found in a long-term toxicology studies, as a control group and aseptic rearing A/JCr mice Occurred with unexplained hepatitis and liver cancer. After the exclusion of chemical and other carcinogens, and the detection of a spiral bacteria in the small liver tissue, after scanning electron microscopy and biochemical traits and gene sequencing confirmed the identification of related new varieties of Helicobacter pylori, because the Helicobacte closely related to the development of ther liver disease, in 1994 officially named it as Helicobacter hepaticus(Hh). Several studies have shown that Hh can long-term engraft in rodents liver, cause chronic hepatitis, liver lesions, etc. Hh engrafted mainly in the lower digestive tract, including the caecum, colon and hepatic duct system, at present not found in the stomach. The natural host of the bacteria is mice, but also detected in human liver tissues reported.Many scholars at home and abroad, using the PCR DNA sequence analysis, immunohistochemistry confirmed HCC exist Helicobacter pylori infection, which fully shows that Helicobacter can survive in the human liver tissue. The liver damage caused by Hh is similar to its pathological features of human chronic liver disease in many ways.the study of Hh infection related diseases has become a hot research.The exact molecular etiology and mechanism of primary liver cancer are not entirely clear, now that the disease is a complex process of multiple factors and steps, and is affected by both environmental and genetic factors. Since the discovery of the Hp, many new Helicobacter members began from animal and poultry isolated. Among them, there are some clear pathogenicity of human or animal, some pathogenicity is not clear or have pathogenic potential.As we all know, Hp infection is closely associated with the development of gastric cancer, and later also in isolated the Hp from cat, thus prompting us to think about the development of liver cancer is also associated with Helicobacter infection. In recent years, many people through animal experiments confirmed that Hh infection can lead to some strains of mice liver cancer, whether Hh infection may also play a role in human hepatocarcinogenesis.Hh may be an independent risk factor for primary liver cancer or auxiliary factors, but it needs to be more powerful experimental evidence in support of Hh. the recent attention of Hh pathogenesis study also increased. But the research mostly stay at the organizational level, a direct action of Hh on the cell is scanty, therefore, in vitro, Hh can effect on liver cancer cell, which will contribute to a better understanding of the mechanism of the occurrence and development of Hh infection and hepatocellular carcinoma.Recent studies have found that, Toll-like receptors (Toll like receptor, TLR) can be combined with some endogenous molecules (ie endogenous ligands) produced by the body itself, the tumor in the development process can produce some that can be recognized by TLRs endogenous ligands,so TLRs may play a role in tumor immune surveillance. The TLR4 as a member of TLRs family, was confirmed the presence of abnormal expression in liver cancer, ovarian cancer, stomach cancer, breast cancer, pancreatic cancer and lung cancer and other malignant tumors, the gene and protein expression level was significantly related to the degree of malignancy. Functional expression of TLR4 may play a key role in the development of cancer. Many studies have shown that TLR4 can promote tumor development, mRNA and protein expression levels of TLR4 in HCC tissues and cells has increased, and the expression level of TLR4 was positively correlated with the degree of malignancy of liver cancer. Studies suggest that Helicobacter can trigger inflammation through direct activation of TLR4, and inflammation is closely related to the development of tumors. However, Hh whether can promote the invasive ability of liver cancer cells are not very clear, the related mechanism remains to be further research. This study aimed to explain the direct effect of Hh on the tumor cell itself, to study the effects of Hh on the invasion of hepatocellular carcinoma cells and its possible mechanism.Therefore, this paper takes Hh as the study factor,takes human hepatoma cell line HEPG2 and HCCLM3 as the research object, using Transwell invasion test, Western-blot, RealtimePCR and siRNA to analyze the impact of Hh to invasive ability of liver cancer cells and the level of related gene and protein expression, and to investigate the possible molecular mechanisms.Methods(1)effect of Hh on invasion of human hepatocellular carcinoma cells:① with different concentrations of Hh living bacterium effect on liver cancer cell HEPG2 and HCCLM3, divided into two groups, the experimental group with different concentrations of Hh (1×109CFU/ml,1×108CFU/ml, 1×107CFU/ml,1×106CFU/ml) groups, each group were observed by12h,24h,48h,72h; the other with only the addition of Hh blank liquid medium as the control group.② By Transwell experiments to detect the different time points the invasive cell number, in order to observe the different concentrations at different times under the influence of Hh on the invasion ability of liver cancer cells.And based on this, determine the optimal concentration and stimulate of subsequent experiments.(2) Hh induced human hepatoma cells to produce IL-8, TGF-β1,MMP-2 and MMP-9:① based on the first part to determine the Hh living bacterium optimal stimulation concentration and optimal stimulation time, Hh were incubated with liver cancer cells as the experimental group, no Hh blank liquid medium as control group② by ELISA method to detect changes in IL-8 and TGF-β1 concentration of each group cells secreted.③ using Western Blot method to detect the expression levels of MMP-2 and MMP-9 protein,④ through the real time PCR method detected IL-8, TGF-beta 1, MMP-2 and MMP-9 mRNA. expression.(3) Hh through effecting TLR4/MyD88 signaling pathways enhanced human hepatocellular carcinoma cell invasion:①the protein expression of TLR4, MyD88, TRIF was detected by Western Blot ②the mRNA level of TLR4, MyD88, TRIF was verified by rea time PCR in each group;③ the establishment of siRNA interference system, and downregulate the expression of TLR4 or MyD88 in HEPG2 and HCCLM3 cells, using Western Blot to detect TLR4, MyD88 expression level, to verify the effect of siRNA interference. TLR4 siRNA, MyD88 are respectively provided with siRNA interference group, negative control group (transfected with unrelated sequence) and blank control group (transfected with blank),④ Hh were incubated with cells in each group, by Transwell test detect whether there are changes in cell invasion.Results(1) effect of Hh on invasion of human hepatocellular carcinoma cells:① as the experimental group the ability of invasion was significantly higher than the control group (P<0.05), and in a certain range with the increase of the concentration of Hh,the invasion of hepatocellular carcinoma cells increased with time, concentration of 1×108CFU/ml reached to the peak in the invasion ability.② In each concentration group, in a certain range, with the increase of Hh action time on hepatocellular carcinoma cell,the invasion ability is increased, at the same concentration, when the stimulation time is 48h,the invasion reached the peak.(2) Hh induced human hepatcellular carcinoma cells produce IL-8, TGF-beta 1, MMP-2 and MMP-9:① the levels of IL-8 and TGF-beta 1 of the experimental group was significantly higher than the control group (P<0.05),② in protein levels of MMP-2 and MMP-9 of HEPG2 and HCCLM3 cells increased significantly, mRNA level certificate similar results (P<0.05).(3) Hh enhanced human hepatocellular carcinoma cell invasion through effecting TLR4/MyD88 signal pathway:① in the experimental group, TLR4 and MyD88 protein levels in HEPG2 and HCCLM3 cells were significantly higher than those in the control group (P<0.05), the expression level of TRIF of eath group have no significant difference (P>0.05). mRNA level also shows similar results.②siRNA interference system can successful interfere with the implementation of TLR4 or MyD88 molecule expression.in HEPG2 HCCLM3 cells and Western Blot results showed that in the human hepatoma cells transfected with siRNA hardly expressTLR4 or MyD88. compared with normal control group, Hh can not increase the invasive ability of the cells in the experimental group, in which case these invasive ability of liver cancer cells decreased.Conclusion(1) Hh living bacterium can increase hepatocellular carcinoma cell ivasion capacity in concentration and time dependent manner(2) Hh induced liver cancer cells secrete IL-8, TGF-betal and the expression of MMP-2 and MMP-9. These cytokines and proteins can increased liver cancer cells digest artificial membrane matrix, thus promotes the invasive ability of o the liver cancer cells. This may be one of the mechanisms of Hh enhanced the invasion ability to the liver cancer cells.(3) Hh living bacterium can raise HEPG2 and HCCLM3 cell TLR4 and its downstream molecules MyD88 protein and mRNA expression level, but not influence the expression of TRIF, indicating that Hh may Specifically activate TLR4/MyD88 signaling pathways in hepatocellular carcinoma cell line.(4) Hh living bacterium cannot increase the liver cancer cell invasion ability which do not express TLR4 or MyD88,liver cancer cell invasion ability significantly lower at this time. This shows that, Hh enhanced the invasion of human hepatocellular carcinoma cells is mediated through TLR4/MyD88 signal pathway, TLR4/MyD88 signaling pathways is very important to the invasion ability of human liver cancer cell. |
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