The Expression Of Galectin-9 And Its Clinic Significance In Hepatocellular Carcinoma (HCC).

[Backgrounds]Primary liver cancer is the sixth most common malignant tumor and the third lethal killer in the world. There are nearly 626000 new cases of primary liver cancer each year worldwide, and China alone accounts for almost half of it. The incidence rate of primary liver cancer in some high incident area of China and other east Asian countries is much more times higher than that in western countries. It claims more than 100,000 victims each year; Besides, the incidence of primary liver cancer is mounting up, especially in the past 20 years and the average age of the patients has tended to younger. At present, Tumor resection is still thought to be the most important method of treatment of primary liver cancer; however, the rate of early diagnosis is so low due to primary liver cancer lack apparent symptoms in the early stage. When the patients feel uncomfortable, they are likely to miss the chance of the surgery treatment because most of whom have already been in advanced stage; furthermore, high degree of biological malignancy leads to overall 5-year survival rate so low that the overall prognosis is far from satisfactory. With the development of diagnostic imaging and molecular diagnostic techniques (AFP), the rate of early diagnosis of primary liver cancer has significantly improved. The previous treatment simply by surgery alone has been multiplied into comprehensive treatment such as TACE, chemotherapy, molecular targeted therapy (Sorafenib) and gene therapy,but the 3,5-year survival rate are not satisfied. As the improvement of molecular biology techniques facilitates us to deepen the understanding on tumor pathogenesis, we have known that the formation of the hepatocellular carcinoma is a complex process, triggered by multi-genes,but its molecular mechanisms are still not clear, Therefore, It is worth us to study further. The galectin-9 gene is located on human chromosome 17q 11.1. It contains 11 exons and its transcribed length 1.6Kb. Belonging to the conserved amino acids, the galectin-9 protein is a galactoside binding protein, It Consists of three isoforms, the molecular weight range of 36-39kDa,the protein main locates in the cytoplasm, and distributes in many tissues. Since it was be found, the galectin-9 has been actively involved in human physiological and pathological processes. It illustrates many key functions, such as eosinophil chemotaxis, cell differentiation and maturation, cell aggregation and adhesion, immune regulation, and apoptosis.At present, there are four mechanisms on apoptosis induced by galectin-9. Mechanism 1:the circuit of calcium-calpain-caspase-1 signaling pathway induces apoptosis by guiding the calcium influx to the intracellular; Mechanism 2:the galectin-9-Tim-3 (hepatitis A virus cellular receptor-2, HAVcr-2) signaling pathway, induces Tim-3+TH1, TH17 and other immune cells apoptosis and promotes Tim-3+ DC, CD8+T cell to increase IFN-γsecretion; Mechanism 3:the galectin-9 can induce apoptosis of myeloma cells through both JNK and p38 MAP kinase signaling pathways to inhibit the expression of IL-6 and vascular endothelial growth factor (VEGF); Mechanism 4:the galectin-9 can inhibit IκBαphosphorylation to suppress the nuclear factor-KB(NF-kB), which induces T, B leukemia cells arrest and apoptosis. The galectin-9 protein expression is associated with histopathology. A study reported that galectin-9 expression is inversely associated with melanoma progression,melanocytic nevi> Malignant melanoma primary lesions> metastasis. In addition, the positive rate of galectin-9 protein expression also declines with the descending trend in normal cervix squamous epithelium, low grade intraepithelial neoplasia (LGSIL), high degree of intraepithelial neoplasia (HGSIL) and invasive cervical squamous cell carcinoma (ISCC), the normal cervical epithelium> LGSIL> HGSIL> ISCC. The positive expression of galectin-9 in cancer is lower than adjacent tissues in breast cancer, colorectal cancer,so the galectin-9 can be used as a biomarker to differentiate neoplasia and cancer and to provide a reference for tumor pathological diagnosis.Galectin-9 can inhibit tumor invasion, distant metastasis. The clinical research on breast cancer, melanoma, colorectal cancer manifests that the level of galectin-9 expression is associated with tumor metastasis. The lower galectin-9 expression is, the higher the rate of distant metastasis becomes. The vitro experiments have found that the tumor cells with high expression of galectin-9 mRNA show colony-like growth phenomenon, while the tumor cells with low expression of galectin-9 mRNA show scattered growth phenomenon; however, the tumor cells with low expression of galectin-9 mRNA exhibit colony-like growth phenomenon as well after adding exogenous recombinant galectin-9 protein. Thus, it reveals the galectin-9 may play a relevant role in anti-tumor metastasis. In vivo experiments, the tumor cells with galectin-9 plasmid fragments transduction, transplanted into nude mice, were found gather the edge of the tumor; while the tumor cells without galectin-9 plasmid fragments transduction grow scatterly. These experiments further prove that galectin-9 can promote the aggregation of tumor cells in the body and have an important effect on tumor metastasis inhibition and spread, which provides some theoretical support for metastatic tumors treatment. So far, tumor cell adhesion has been widely recognized as a vital factor of the tumor invasion. Some studies demonstrate that galectin-9 can inhibit tumor cells adhesion by means of restraining tumor cell from adhering to extracellular matrix of normal cell and vascular endothelial cells. Thus, to some extent, explains that galectin-9 has the capability of anti-tumor invasion, metastasis. Although the mechanism of galectin-9 inhibits tumor cells from adhering to normal cells and the extracellular matrix of vascular endothelial cell is still not clear. Some suppose that galectin-9 might modulate E-selection to suppress the tumor cell to adhere to normal cells and vascular endothelial cells, so that perform the ability of Anti-tumor invasion and metastasis;Moreover, some report that galectin-9 may have the function of Anti-tumor invasion and metastasis by inhibiting CD44 in tumor cells from adhering to hyaluronic acid (HA), extracellular matrix (ECM) of vascular endothelial cells. (HA, ECM are play important roles in tumor cells adhesion to endothelial cells).Therefore, some scholars, on the basis of this mechanism, stimulate the expression of galectin-9 in vascular endothelial cells to inhibit tumor cell binding to vascular endothelial cells. They adopt some certain drugs or methods, such as IFN-γ, double-stranded RNA,15-deoxy-△(12,14)-prostaglandin J2, which provide some new theoretical basis for the treatment of tumor metastasis.It has reported that galectin-9 expression is related with prognosis of some tumors. Irie A reported that 5-year survival rate of the patients with positive expression of galectin-9 in breast tumor tissue was 93%, while 5-year survival rate of the patients with negative expression of galectin-9 was only 47%. The disease-free survival of the patients with positive expression of galectin-9 protein in breast cancer tissue is longer than the disease-free survival of patients with positive expression of galectin-9 protein, even with lymph node metastasis in patients, survival rate of the patients with positive expression of galectin-9 protein was 89%, while galectin-9 negative was only 19%, and the expression of galectin-9 was regarded as a prognostic indicator for breast cancer patients after receiving surgery. Except for the lymph node metastasis, the expression of galectin-9 level can be used as one of the indicators to decide whether the breast cancer patients need adjuvant chemotherapy or not after surgery. The similar phenomenon also found in melanoma, oral squamous cell carcinoma.There are some researches on galectin-9 in cancer at home and abroad, but only limited to some certain malignant tumors, such as breast cancer, colorectal cancer, and malignant melanoma. There is few research on the galectin-9 in the process of hepatocellular carcinoma (HCC) at home and broad, with regard that it is enourmously different from other tumors in the degree of malignant,biological properties. So the significance of studying the galectin-9 expression in hepatocellular carcinoma and its clinical significance is beyond doubt. Through this reseach study, we are on the way to exploring the participate of galectin-9 in the progress of tumor formation and development.[OBJECTIVE]Detects the expression of galectin-9 mRNA, protein in tumor tissues and adjacent tissues from patients with HCC,compared with galectin-9 mRNA, protein expression in tumor tissue and adjacent tissues, explores the difference of galectin-9 expression in tumor tissue and adjacent tissues, and analyzes the relationships between the expression of galectin-9 mRNA, protein and the clinicopathological features of patients with hepatocellular carcinoma. To investigates expression of galectin-9 in tumor tissues and adjacent tissues from patients with hepatocellular carcinoma and its corresponding clinical significance.[MATERIALS AND METHODS] 1. All tissues are collected from the Department of Hepatobiliary Surgery of Nanfang Hospital in 2009.8-2010.8, All cases are confirmed to be hepatocellular carcinoma by postoperative Pathology.2. The expression of Galectin-9 mRNA was detected by the Real-time fluorescence quantitative RT-PCR in resectable cancer tissues and adjacent tissues from 89 patients. The difference of galectin-9 mRNA expression between in liver cancer tissue and adjacent tissues was analysised, also we analysised the expression of galectin-9 mRNA correlated with clinical features simultaneously, initially revealed significance between the expression of galectin-9 mRNA and clinical features in hepatocellular carcinoma.3. The immunohistochemistry was used to detect the galectin-9 protein characterization, location and expression in tumor tissues and adjacent tissues from 60 patients, analysis the differences of galectin-9 protein expression in liver cancer tissue and adjacent tissues and the relationship between galectin-9 protein and clinical clinicopathological features.while the Western Blot was used to detect the expression of galectin-9 protein in tumor tissues and adjacent tissues from 43 patients, futher evdence the result that analysis by the immunohistochemistry.4. Statistical methods Using SPSS 13.0 software to analyze,Experimental data expressed as mean士SD and Median,the expression of galectin-9 mRNA and protein in tomour tissues and adjacent tissues was test by paired-samples t-Test; the relation between 2**-△△Ct and clinical pathological features was analysised by Kruskal-Wallis; the rate of the galectin-9 (mRNA,protein)expess in tumor were tested by the Chi-square test and kendall-b statistical bivariate correlation analysis were used to estimated, when P<0.05,the difference was considered statistically significant.[RESULTS] 1. The expression of galectin-9 mRNA in tumor tissues is 0.504-fold,lower than that in adjacent tissues (p<0.001), and the level of galecin-9mRNA expression in patients with HCC is assotiated with tumor size, histological differentiation, TNM classification, recurrence and metastasis (p< 0.05),but it is not related with gender, age, HBsAg, AFP, liver cirrhosis, the integrity of live capsule.2. The rate of the patients with galectin-9mRNA express in tumor higher than that in adjacent tissues is correlated with pathology, TNM classification, recurrence and metastasis (r:0.249,0.272,0.286,p:0.015,0.007,.007), p<0.05 are statistically significant.3. It is show that galectin-9 protein in tissues is localize in the cytoplasm by Immunohistochemistry, the positive rate of galectin-9 express (70%) in tumor tissue is lower than in adjacent tissues (93.3%),x2=-10.909,p=0.001,p<0.05 are statistically significant; the positive rate of galectin-9 express in hepatocellular carcinoma is positively correlated with pathological differentiation, recurrence and metastasis (r:0.556,0.258, p:0.001,0.048).4. The level of galectin-9 protein express in tumor (1.986±1.927) is lower than in adjacent-tissues(2.975±2.243), t=-2.393,p=0.021, p<0.05 are statistically significant, And further evidence that the expression of galectin-9 protein is correlate with histological differentiation, postoperative recurrence and metastasis.[CONCLUSION]1. The expression of galectin-9 mRNA in tumor tissues are lower than that in adjacent tissues,and it is correlate with tumor size, histological differentiation, TNM classification, recurrence and metastasis.2. The expression of galectin-9 protein in tumor tissues are lower than that in adjacent tissues,and it is correlate with histological differentiation, recurrence and metastasis.3. The expression of galectin-9 mRNA, protein in tumor tissues are correlate with the pathological differentiation, recurrence and metastasis, indicating that galectin-9 may play role in the process of the formation, progression and prognosis in hepatocellular carcinoma.4. The mechanisms that galectin-9 participates in the formation, progression and prognosis of hepatocellular carcinoma are worth us to further study.