Study On The Mechanism Of Tim-3 In The Metastasis Of Hepatocellular Carcinoma

BACKGROUND AND OBJECTIVEHepatocellular carcinoma(HCC)is the most common type of malignancy worldwide and has been a serious worldwide public health problem.The fatality rate ranks third among all malignancies.In China,the mortality rate induced by HCC ranks highest,particularly in economically underd eveloped areas.The recurrence and metastasis of HCC is the concentrated expression of its biological characteristics and the main cause of the high mortality of HCC.It was reported that>60%of HCC cases had encountered metastasis at the time of diagnosis.the five-year survival rate of patients with HCC and metastasis is markedly lower compared with patients with HCC without metastasis.Although the precise liver resection has been widely used in clinical application,combined with radiotherapy and interventional chemotherapy and other methods of comprehensive treatment,the therapeutic effect of liver cancer has made great improvement,However,the high metastasis and recurrence rate after hepatic resection make the prognosis of HCC still not complete.Therefore,investigations into the molecular mechanism of liver metastasis is of great significance for understanding liver cancer.Epithelial-mesenchymal transition(EMT),a biological process during whereby epithelial cells transform into mesenchymal cells under a specific program,the cells lose their cell polarity and cell adhesion ability,and its ability to obtain migration and invasion,with interstitial cell morphology and the characteristics of the cells,with the ability to move.EMT serves an important role in numerous physiological and pathological processes,including embryogenesis,organ development,tissue repair,organ fibrosis,and tumor metastasis.In epithelial malignancies,tumor cells acquire potent migratory and invasive abilities,transfer to a different site via the blood,and form further tumor metastasis through mesenchymal-epithelial transition.The mechanism underlying EMT in a number of solid tumors has been investigated extensively,including HCC.However,the treatment of HCC requires further study on the regulation mechanism of EMT.T-cell immunoglobulin and mucin molecule 3(Tim-3)is an important member of the TIM gene family,and is thought to be involved in the development of various types of cancer,including HCC.In the progression of hepatocellular carcinoma,Tim-3 is considered a new participant to regulate the biological behavior of HCC.Tim-3 plays an important role in tumor immunity and is considered to be a negative immunomodulatory molecule involved in immune regulation of multiple pathophysiological processes.It has been reported that Tim-3 is involved in the immunoregulation of T cells in cervical cancer,hepatocellular carcinoma,colorectal and ovarian cancer,it also can suppress the proliferation of autologous CD8+ T cells in vitro significantly.TIM-3 is also involved in the pathogenesis of human osteosarcoma,and TIM-3-triggered tumor cells have been observed to acquire the characteristics of aggressive EMT,indicating the possible role of Tim-3 in EMT occurrence.However,to the best of our knowledge,no study has been performed regarding the function of TIM-3 in the EMT progression of HCC.The present study aimed to investigate the mechanism of TIM-3 in the metastasis of HCC and the relationship between Tim-3 and epithelial mesenchymal transition of HCC cells.It was report that epithelial mesenchymal transition was the key step in the metastasis of hepatocellular carcinoma.The related biomarkers of EMT were:E-cadherin,N-cadherin,MMP-9,Twistl,Slug,Snail,Smad.This study interfered with the expression of Tim-3 by Tim-3 siRNA and transfect Tim-3 overexpression plasmids to construct Tim-3 high and low expression model.Change the expression level of Tim-3,and then use Rreal-time PCR and Western blot to detect EMT-related biomarkers:E-cadherin,N-cadherin,MMP-9,Twistl,Slug,Snail and Smad and to detect the proliferation,migration and invasion of hepatocarcinoma cells.This study described the important role of Tim-3 in the metastasis of HCC and the relationship between Tim-3 and EMT in HCC cells.These results suggest that Tim-3 may induce the occurrence of EMT and promote the metastasis of HCC.Interfering with the expression of Tim-3 may be an effective intervention method to block the occurrence of EMT and to treat the metastasis of liver cancer.Part I.Establishment of low expression and over expression cell model of Tim-3 in hepatocellular carcinoma cellsObjective:To investigate the role of Tim-3 in the metastasis of HCC,and detect the expression of Tim-3 in hepatocellular carcinoma and normal liver cell,to construct Tim-3 high expression and low expression model and test model.Methods:1.Cultured human hepatocellular carcinoma SMMC-7721,normal human hepatocyte L02.2.The expression of Tim-3 mRNA and protein in hepatocellular carcinoma cell SMMC-7721 and normal human hepatocyte L02 was detected by real-time fluorescent polymerase chain reaction(Real-time PCR)and Western blot.3.PEX-3-hTim-3 high expression plasmid and Tim-3 inhibitor(siRNA)were transfected into human hepatocellular carcinoma cell SMMC-7721 by liposome transient transfection.After transfection,a high expression and a low expression cell model of Tim-3 were established.4.Through the inverted fluorescence microscope observation,transfection of successful cells to express GFP,microscopic can appear green fluorescence.Image-Pro Plus image analysis and processing software,image acquisition,analysis.At the same time,after 24 hours transfection,the model was identified by Real-time PCR and 72 hours by Western blot to detect Tim-3 expression.Result:1.The expression of Tim-3 was higher in the hepatocarcinoma cell MMC-7721 than that in the normal human hepatocyte L02,suggesting that Tim-3 may be involved in the invasion and metastasis of HCC cells.2.Fluorescence microscope observation of two groups of cells,two groups of cells can be green fluorescence,statistical analysis,transfection of siRNA and overexpression plasmid PEX-3-hTim-3 transfection efficiency of 81.25%and 83.36%,respectively.3.RT-PCR and Western blot results showed that the transfection of PEX-3-hTim-3 plasmid group high expression in hepatocellular carcinoma cells,the expression of Tim-3 was significantly higher than the control group and the blank vector group.The transfection of Tim-3 inhibitor(siRNA)Tim-3 expression was significantly lower than that of the control group and the transfection group siRNA-NC.Conclusion:Tim-3 is expressed in HCC cells and normal hepatocytes.The expression of Tim-3 in SMMC-7721 was higher than that in normal human hepatocyte cell L02(P<0.05).The high expression Tim-3 and low expression Tim-3 cell model of HCC were successfully established by transfection.Part ?.Effect of Tim-3 on invasion and metastasis of hepatocellular carcinoma cells and its mechanismObjective:Application of established high expression and low expression of Tim-3 cell model to investigate the effect of Tim-3 on the invasion and metastasis of HCC cells,and to explore the possible mechanism.Method:1.The experiment was divided into three groups:control group:HCC cells SMMC-7721 without transfection;experimental group:HCC transfected with Tim-3 siRNA,low expression of Tim-3;positive control group:HCC transfected with PEX-3-hTim-3 high expression plasmid,high expression of Tim-3.2.Three groups of HCC cells cells were cultured for 72 hours,and the cell characteristics of the three groups were observed under inverted fluorescence microscope.3.The proliferation ability of three groups of liver cancer cells was detected by MTT.4.The invasion and migration ability of HCC cells in three groups were detected by Transwell chambers test,cell scratch test and wound healing test.To reveal the effect of Tim-3 on metastasis and invasion of hepatoma cells.5.The expression of Tim-3 and EMT related genes in HCC cells:E-cadherin,N-cadherin,MMP-9,Twistll,Slug,Snail,Smad and mRNA were detected by real-time quantitative PCR;Western blot detection the expression of Tim-3,and the expression of EMT related protein:E-cadherin,N-cadherin,MMP-9,Twistll,Slug,Snail,Smad.Each experiment repeat three times.6.To analyze the correlation between the expression of Tim-3 and the expression of EMT related genes.7.Statistical analysis:The data is expressed by mean standard deviation and analyzed by SPSS 16.P<0.05 is considered to be statistically significant difference.Result:1.After 72 hours transfection,three groups cells were observed by phase contrast microscope.The positive control group showed high expression of Tim-3,The cell morphology showed more spindle-like morphology and connections between cells were fewer,which was previously revealed to be more conducive of migration and invasion.Simultaneously,the experimental group demonstrated epithelial and the adhesion showed stronger,with more antennas,indicating a lower aggressive type of cancer.2.There was no significant difference in the rate of increment in the first 12 hours in the three groups of HCC cells.After 12 hours,there was a significant difference between the three groups.In the observation 72 hours,the difference became more and more obvious with the extension of time?Compared with the control group,the proliferation ability of HCC cells in the experimental group was obviously decreased,and the proliferation rate of the experimental group was 71.71%of the control group(F=145.758,P<0.05).The cell proliferation in the positive control group was significantly higher than that in the control group and the experimental group,which was 260.12%in the control group,with a significant difference(F=195.428,P<0.05).3.Transwell assay were used to analyze migration and invasion of SMMC-7721 cells.Compared with the control group and the positive control group,the metastasis and invasion of HCC cells were significantly reduced in the experimental group.Taking the relative number of migrated and invaded cells in control group as 100,the migrated and invaded number of cells in the positive control group was 157± 10 and 179±11,and the number in the experimental group was 61 ±8 and 34±7,There was significant difference between the three groups(P<0.05).The experiments showed that the migration and invasion of HCC cells were significantly decreased by inhibiting the expression of Tim-3.Therefore,the change of the expression level of Tim-3 affects the migration and invasiveness of HCC.After 72 hours of cell scratch,the three groups of cells moved to the middle of the scratch,and in the positive control group,the liver cancer cells almost covered the middle scratch,and there was still a certain gap in the scratch area of the control group.In the experimental group,the liver cancer cells migrated slightly to the middle of the scratch,and the gap was larger than that of the positive control group,the difference was statistically significant(F=168.365,P<0.05).4.RT-PCR results showed that the positive control group,the expression of E-cadherin was lower than the control group,the difference was statistically significant(F=2830,P<0.05),whereas the expression of N-cadherin,MMP-9,Twistl,Snail,Slug,Smad was significantly higher than the control group,the differences were statistically significant(P<0.05).The experimental group decreased the expression of Tim-3,E-cadherin expression compared with the control group increased significantly,compared with the control group,the difference was statistically significant,(F=2932,P<0.05),while the expression of N-cadherin,MMP-9,Twistl,Snail,Slug,Smad was lower than the control group,the difference was statistically significant(P<0.05).The results of the Western blotting experiments were consistent with the PCR results?5.The expression of Tim-3 and E-cadherin correlated negatively (r=-0.870,P=0.00),while Tim-3 was positively related to the expression of N-cadherin,MMP-9,Twistl,Snail,Slug and Smad(P<0.05).Conclusion:1.Three groups of cells were cultured for 72 hours to observe the cell morphology,experimental group demonstrated epithelial and the adhesion showed stronger,with more antennas,indicating a lower aggressive type of cancer,the positive control group demonstrated a more spindle-like morphology and connections between cells were fewer,which was previously revealed to be more conducive of migration and invasion.The characteristics of EMT obtained from hepatoma cells in positive control2.The proliferation rate of the three groups was detected by MTT assay.The cell proliferation rate of the positive control group was higher than that of the experimental group after 12 hours,suggesting that Tim-3 expression may promote the proliferation of hepatocellular carcinoma cells.3.Transwell and cell scratches were used to test the migration and invasion of the three groups.The results indicated that the positive control group showed strong migration and invasion;while the experimental group inhibited the expression of Tim-3,the migration and invasion of the cells were significantly decreased.The result showed that Tim-3 promoted the migration and invasion of HCC cells.4.The expressions of EMT related genes and proteins:E-cadherin,N-cadherin,MMP-9,Twistl,Smad,1,Snail,Slug and bolt were detected by Real-time PCR and Western blot.The results suggest that the expression of E-cadherin in the positive control group decreased,However,the expression of N-cadherin,MMP-9,Twistl,1,Snail,Slug and Smad increased,this suggests that epithelial mesenchymal transition occurs in HCC cells.Inhibition of Tim-3 expression in the experimental group,the expression of E-cadherin increased significantly,while the expression of N-cadherin,MMP-9,Twistl,1,Snail,Slug and Smad decreased,suggesting that HCC cells inhibited epithelial mesenchymal transition.These results suggest that Tim-3 promotes the occurrence of EMT in HCC cells,resulting in increased migration and invasion.Those results were consistent with the results of MTT assay,cratch test,and Transwell chambers test.5.Aalysis of the expression of Tim-3 and EMT genes and proteins related to the correlation showed that the expression of Tim-3 and E-cadherin correlated negatively,while Tim-3 was positively related to the expression of N-cadherin,MMP-9,Twistl,Snail,Slug and Smad.These results suggest that Tim-3 induces the occurrence of EMT in hepatocellular carcinoma cells,thereby affecting the migration,invasion and proliferation of HCC cells.