| Objective: The present study aimed to detect the expression of T-cell immunoglobulin and mucin domain-containing protein 3(TIM-3)in human esophageal squamous cell carcinoma(ESCC)tissues and matched adjacent normal tissues,and to evaluate the clinicopathologic and prognostic significance of it in ESCC.Furthermore,RNA interference was used to explore the effects of TIM-3 on the proliferation,migration,invasion,apoptosis and cell cycle distribution of ESCC cells,and the possible molecular mechanisms involved in promoting ESCC metastasis.Methods:1 The expression levels of TIM-3mRNA in ESCC and matched adjacent normal tissues were detected by qRT-PCR.2 The expression levels of TIM-3 protein in ESCC and matched adjacent normal tissues were detected by immunohistochemistry.In addition,the correlations between TIM-3 expression and clinicopathological parameters or prognosis were analyzed.3 The intensity of tumor infiltrating CD8+T lymphocytes in ESCC tissues were evaluated by immunohistochemistry.In addition,the correlation between TIM-3 expression and the intensity of tumor infiltrating CD8+T lymphocytes were analyzed.4 The expression levels of TIM-3 mRNA and protein in various ESCC cell lines,including Eca109,KYSE30,KYSE170,TE-1,and TE-13 were evaluated by qRT-PCR and Western blotting analysis,respectively.5 The TIM-3 eukaryotic expression vector(psi-U6-TIM-3)were transfected into human esophageal cancer cell line Eca109 and TE-1 cells.We screened the cells with puromycin for two weeks and picked monoclonal cells to expand culture.Finally,we obtained shRNA-mediated stable Eca109-shTIM-3 and TE-1-shTIM-3 cells,and their respective negative controls(Eca109-NC and TE-1-NC cells).Transfection efficiency was evaluated by flow cytometry and Western blotting analysis.6 Effects of TIM-3 konckdown on proliferation and colony formation potential of esophageal cancer cells were detected by MTS and colony formation assays,respectively.7 Effects of TIM-3 konckdown on migratory ability of esophageal cancer cells were detected by wound scrape assay and Transwell migration assay.8 Effects of TIM-3 konckdown on invasive ability of esophageal cancer cells were detected by Matrigel invasion assay.9 Effects of TIM-3 konckdown on cell apoptosis and cell cycle distribution in ESCC were detected by flow cytometry.10 Effects of TIM-3 konckdown on metastasis-related molecules(MMP-9,TIMP-1,E-cadherin,N-cadherin and Vimentin)expression of esophageal cancer cells were detected by qRT-PCR and Western blotting analysis.11 Effects of TIM-3 konckdown on Akt/GSK-3β/Snail signaling pathway in ESCC were detected by Western blotting analysis.Results:1 qRT-PCR analysis revealed that the relative expression level of TIM-3 mRNA was significantly higher in tumor tissues than that in adjacent normal tissues(0.002±0.0007 vs.0.001±0.0004;P<0.001).2 Immunohistochemical analysis revealed that TIM-3 protein localized predominantly in cellular membrane and cytoplasm of tumor cells.TIM-3 positive staining(overall score>1)was present in 55 of 64 ESCC tissue sections(85.9%),compared to only 7 of 64 normal tissue sections(10.9%).Of the 55 TIM-3-positive ESCC samples,19(34.5%)showed low TIM-3 expression(overall score ≤3),and 36(65.5%)showed high TIM-3 expression(overall score >3).Whereas all 7 of the TIM-3-positive normal tissue samples showed low TIM-3 expression.Statistical analysis of these results indicated that TIM-3 protein expression was significantly higher in ESCC tissues than that in adjacent normal tissues(P<0.001).In addition,the expression of TIM-3 was significantly related to TNM stage(P=0.008),lymph node metastasis(P=0.042)and depth of tumor invasion(P=0.042).However,no significant differences were observed between the TIM-3 expression and the other clinicopathologic parameters.Kaplan–Meier survival analysis indicated that patients in TIM-3 high expression group had a significantly shorter overall survival than patients in TIM-3 low expression group(P=0.001).The overall 5-year survival rate for patients in TIM-3 high expression group was 9% with median survival time 22 months,while the overall 5-year survival rate for patients in TIM-3 low expression group was 19.2% with median survival time 41 months.3 Immunohistochemical analysis revealed that CD8+T lymphocyte slight infiltration was present in 25 ESCC tissue sections and CD8+T lymphocyte severe infiltration was present in 39 ESCC tissue sections.Spearman correlation analysis showed that the expression of TIM-3 in tumor cells was negatively correlated with the intensity of tumor infiltrating CD8+T lymphocytes in 64 ESCC tissue sections(P=0.008).4 qRT-PCR analysis revealed that TIM-3 mRNA in five kinds of esophageal cancer cell line,including Eca109,KYSE30,KYSE170,TE-1,and TE-13 were constitutively expressed.The expression level of TIM-3 mRNA exhibited the relatively highest in Eca109 and TE-1 cell lines.Consistent with the TIM-3 mRNA expression,Western blotting analysis revealed that the expression level of TIM-3 protein exhibited the relatively highest in Eca109 and TE-1 cell lines.5 We selected Eca109 and TE-1 cell lines to knockdown TIM-3 expression and explore the effects on biological characteristics of esophageal cancer cells.Flow cytometry analysis revealed that the transfection efficiency of Eca109-shTIM-3,Eca109-NC,TE-1-shTIM-3 and TE-1-NC were 91.5%,93.2%,87.3% and 85.7%,respectively.qRT-PCR and Western blotting analysis revealed that the mRNA and protein levels of TIM-3 were markedly decreased in Eca109-shTIM-3 and TE-1-shTIM-3 cells than those in their corresponding parental or negative control cells(P<0.001).These results suggest that TIM-3shRNA-mediated stable transfection can effectively downregulate the expression level of TIM-3 in Eca109 and TE-1 cell line.6 MTS assay demonstrated that proliferation ability was significantly decreased in Eca109-shTIM-3 and TE-1-shTIM-3 cells than that in their corresponding parental or negative control cells at 24,48 and 72h(P<0.01).Colony formation assays demonstrated that colony formation potential was significantly decreased in Eca109-shTIM-3 and TE-1-shTIM-3 cells than that in their corresponding parental or negative control cells(P<0.001).7 Wound scrape assay results shown that compared with their corresponding parental or negative control cells,crawling speed and wound healing of Eca109-shTIM-3 and TE-1-shTIM-3 cells was significantly slowed down at 24 and 48 h post-wounding.Ultimately,scratch spacing was relatively wide.The differences were statistically significant(P<0.01).Transwell migration assay demonstrated that the number of Eca109-shTIM-3 and TE-1-shTIM-3 cells which migrated through the Transwell was dramatically decreased compared with the number of their corresponding parental or negative control cells which migrated(P<0.001).8 Matrigel invasion assay demonstrated that the number of Eca109-shTIM-3 and TE-1-shTIM-3 cells which invaded through the Transwell was dramatically decreased compared with the number of their corresponding parental or negative control cells which invaded(P<0.001).9 Flow cytometry analysis revealed that no obvious effects of TIM-3 knockdown on cell apoptosis in Eca109 and TE-1 cells.In addition,compared with their corresponding parental or negative control cells,there was an increased relative proportion of Eca109-shTIM-3 and TE-1-shTIM-3 cells in the G0/G1 phase,and a reduced relative proportion in S phase(P<0.001).10 qRT-PCR and Western blotting analysis revealed that TIM-3 knockdown up-regulated TIMP-1,E-cadherin and down-regulated MMP-9,N-cadherin and Vimentin in both Eca109 and TE-1 cells(P<0.001).11 Western blotting analysis revealed that the protein level of the active form of Akt(phosphorylated on Ser473),but not total Akt,the active form of GSK-3β(phosphorylated on Ser9),but not total GSK-3β was dramatically decreased by knocking down TIM-3.In addition,EMT-related transcription factor Snail was downregulated after TIM-3 knockdown.Conclusion:1 TIM-3 is abnormal overexpressed in ESCC,and its expression was correlated with tumor progression and prognosis.TIM-3 may become a novel therapeutic target and prognostic biomarker for ESCC.2 TIM-3 expression in ESCC negatively correlated with the intensity of tumor infiltrating CD8+T lymphocytes,which may be involved in tumor immune escape.3 Knockdown of TIM-3 inhibited ESCC cell proliferation probably through the induction of G0/G1 phase cell cycle arrest.4 TIM-3 promotes metastasis of ESCC by inducing EMT via,at least partially,the Akt/GSK-3β/Snail signaling pathway. |
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