| The Galectins are a family of β-galactoside-binding animal lectins. Galectins are widelydistributed in many animals and localized in the nucleus, cytoplasm and extra cellular matrix.They have many functions, such as cell adhesion, proliferation, cell apoptosis, modulation ofinflammation and immune regulation. Galectin-9, as a member of Galectin family, which hasa typical conservative structure of the Galectin family is widely distributed in the inside andoutside of cells. Galectin-9has been found to regulate different biological functions, such asimmune regulation, inflammation, cell apoptosis, angiognesis and matastasis of tumors.Recently the relationship of Galectin-9on tumors, such as melanomas, cervical squamous cellcarcinomas and hepatocellular carcinomas, has attracted more attention, and Galectin-9maybe a new therapeutic field of study related to the treatment of tumors.The T-cell immunoglobulin domain and mucin domain (Tim) family, mainly expressed inthe surface of immune cells, regulates the immune response which is mediated by Th1andTh2cells. Tim-3, a Th1-specific type1membrane protein, emerges on the cell surface of fullyactivated Th1cells. Binding to its ligand, Galectin-9, to regulate the immune system.At present, few articles about the expression of Galectin-9and Tim-3in gastric cancerhave been reported. The aim of this study has been to observe the expression and distributionof Galectin-9and Tim-3in gastric tissues and gastric cancer cell lines, and to explore its rolein the genesis and development of gastric cancer and its possible mechanism.In gastric tissues and gastric cancer cell lines, the Galectin-9has been localized in bothcytomembrane and cytoplasm, and Tim-3has been localized in both the nucleus andcytoplasm by the immunohistochemical method. To investigate the involvement of theGalectin-9mRNA and protein expression in the pathogenesis of primary gastric cancer,52clinical gastric cancer tissues and matched adjacent (<2cm away from the tumor) and/ornormal (>5cm away from the tumor) tissues were collected in pairs from September2008upuntil July2013in the Department of Gastrointestinal Surgery of The First Hospital of JilinUniversity. All cases were confirmed to have gastric cancer by postoperative pathology.Galectin-9mRNA and protein expression levels were measured by qPCR and Western blot.Compared to matched normal or adjacent tissues, the mRNA expression of Galectin-9wassignificantly decreased in gastric cancer tissues (P<0.001, P<0.001). To determine whether the reduction of Galectin-9mRNA expression resulted in decreasing of Galectin-9proteinlevels, gastric cancer and corresponding normal or adjacent tissues were analyzed by Westernblotting. Similar to our expected results, significant reduction of Galectin-9protein in gastriccancer (0.999±0.435) compared to those matched normal tissues (1.796±0.684) were detected(P<0.05). To extend above observations and to know the relationship between Galectin-9mRNA levels and clinicopathological parameters, we analyzed qPCR results according to theclinical characteristics of gastric cancer. Compared to the mRNA expression of Galectin-9inT1stage, Galectin-9mRNA levels in T3and T4stage were significantly decreased (P<0.05,P<0.05). However, there were no differences in the T2and T1stages, which suggest thatGalectin-9may be related to the degree of tumor invasion. N0-N3stage cancer Galectin-9mRNA levels were not significantly different. With no distant metastasis group (M0)compared with distant metastasis group (M1), Galectin-9mRNA expression decreasedobviously (P<0.05). Compared to stageⅠ, Galectin-9mRNA expression levels weredecreased in stage II、III、IV gastric tumors, and there was a significant difference in stages IIand IV (P<0.05). The survival time after surgery with high Galectin-9mRNA expression waslonger than that with low Galectin-9mRNA expression, which suggests Galectin-9expressionis associated with survival time. In summary, Galectin-9mRNA expression levels have acertain correlation with the degree of malignancy found in the tumor. However, there was nosignificant difference between age and gender (P>0.05). Compared to matched normal tissues,Tim-3mRNA and protein expression were decreased in the tumors. Statistical analysisconfirmed that there was no significant difference between the tumor and matched normaltissues.The expressions of Galectin-9and Tim-3in gastric cancer cell lines (SGC-7901andMGC-803) and the normal gastric mucosa cell line (GES-1) were measured by qPCR andWestern blot. Compared to GES-1, the mRNA expression of Galectin-9was significantlydecreased in SGC-7901and MGC-803（P<0.05, P<0.01）, and significant reduction ofGalectin-9protein in SGC-7901and MGC-803to GES-1were detected (P<0.05, P<0.01) byWestern blot. Expression of Tim-3mRNA and protein in the SGC-7901and MGC-803cellsare not significantly lower than in GES-1cells. The expression trend of Tim-3and Galectin-9in gastric cell lines is consistent with that in gastric tissues.For the further study of Galectin-9in the carcinogenesis, development of gastric cancerand the mechanism, and to further clarify whether there is a correlation regulatory relationship between Galectin-9and Tim-3in cells, we constructed the pcDNA3.1(-)-Galectin-9plasmid,transiently transfected into MGC-803cells and established a transient transfection expressionsystem. We observed that over expression of Galectin-9can inhibit the proliferation ofMGC-803cells, and DAPI staining showed that over expression of Galectin-9can induce theapoptosis of MGC-803cells. The result of Western blot to detect the apoptosis signalingpathway related protein showed that transfection of Galectin-9can down regulate theexpression of Bcl-2protein, but Bax protein and Caspase-3protein expression increase, whichsuggests that over expression of Galectin-9may induce cell apoptosis through themitochondrial pathway. To determine whether the Tim-3expression was regulated byGalectin-9, MGC-803cells were transiently transfected with Galectin-9DNA, the resultshowed that the expression of Tim-3protein had not been changed. The immunoprecipitationcomplexes only detected Galectin-9protein, and no Tim-3protein precipitation, whichsuggests that the two had no direct effect on MGC-803cells, the effects of Galectin-9ongastric cancer cells may not be played by Galectin-9/Tim-3pathway.In summary, the normal stomach mucosa and gastric cancer tissues can show Galectin-9and Tim-3protein expression. In gastric cancer tissues and gastric cancer cell lines, theexpression of Galectin-9mRNA and protein were significantly lower than that of normaltissues. The over expression of Galectin-9in gastric cancer cell lines can inhibit tumor cellgrowth, promote the apoptosis of tumor cells, Galectin-9can down regulate Bcl-2andupregulate the expression of Bax and Caspase-3protein, and then promote apoptosis.Galectin-9may not interact with Tim-3directly in MGC-803cells, but the mechanism ofGalectin-9and Tim-3in the occurrence and development of gastric carcinoma should besubject to further studies. |
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