| Background and Objection:Primary hepatocellular carcinoma(HCC) is one of the most forms of malignancy which seriously affect the body health of human being.In our country HCC has the incidence rate of 20.37/10 ten thousand and the second mortality rate in the total cancer mortality.Though many methods including operation,liver transplantation, blood intervention,ablation technique,chemotherapy and radiotherapy,can be performed in HCC therapy,clinically more than one half of HCC patients had the micrometastasis failing to detect before radical surgery,which may be the direct reason of metastasis or recurrence of HCC after surgery.Thus,to predict HCC metastasis tendency,identify the prognosis markers,find the effective way for prevention and therapy and raise five-year survival rate of HCC patients have been the hot spots for HCC studies,and also been an emphasis and difficult point in treatment and prevention of tumor.Tiam1 is one member of guanine nucleotide exchange factors(GEFs),which mainly act to regulate the Rho GTPase activity.Rho GTPase family is members of Ras superfamily including Rho,Rac and Cdc42.Tiam1 could specifically activate Racl in vivo and vitro,and beside this,recent studies found Tiam1 directly interacted cytoplastic and epimembranal proteins,and then coupled the complex into Tiam1-Rac signaling pathway,thus affecting the specificity of Rac signaling pathway. Tiam1 was initially separated and identified from the variant cell line of mouse T lymphoma with high invasive ability.After that,related studies on breast cancer,lung cancer and skin cancer induced by Ras confirmed that Tiam1 could accelerate tumor progression and metastasis significantly.In the Tiam1-knockout animal model,the incidence rate of mouse skin cancer obviously reduced and tumors were grown slowly. Malliri et al suggested that Tiam1 played the vital role in the initial and progressive stages of induced carcinogenesis of skin,which had the obvious relationship with the amount of Tiam1 expression.The above studies showed that Tiam1 might accelerate carcinogenesis and metastasis in many tumors.In our study,by using tissue chip of normal tissues and common malignant tumors which was designed and produced by ourselves and immunohistochemistry, Tiam1 protein expressions were detected in human normal tissues and common malignant tumors,152 cases of hepatocellular carcinoma tissues,8 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell line.Then the relationship between Tiam1 expressions and various clinopathologic parameters was analyzed,and the relationship between Tiam1 expressions and the life spans of hepatocellular carcinoma patients was also retrospectively analyzed combined with clinical follow-up data in order to find the way to improve the sensitivity and specificity of prognosis evaluation of hepatocellular carcinoma patients.After that, RNA interfere was performed to explore Tiam1 functions in proliferation and invasion of hepatocellular carcinoma.Our study aimed to explore the possibility of Tiam1 as the molecular target of hepatocellular carcinoma and lay the potent theoretical basis on diagnosis,prognosis evaluation and combined therapy design of hepatocellular carcinoma patients.Methods:1.The distributions and expressions of Tiam1 protein in human normal tissues and common malignant tumorsThe tissue chip including 18 normal tissues and 19 common malignant tumors in the whole body was designed and produced by ourselves.Common malignant tumors included nasopharyngeal carcinoma,squamous carcinomas of lung and esophagus, adenocarcinoma of esophagus,gastric carcinoma,colorectal carcinoma, hepatocellular carcinoma,pancreatic carcinoma,clear cell carcinoma of kidney, uterine cervix cancer,ovary carcinoma,prostatic carcinoma,transitional cell carcinoma of bladder,neurogliocytoma,skin squamous carcinoma,diffuse large B lymphoma.Normal tissues included nasopharynx,lung,esophagus,stomach,colon, rectum,fiber and smooth muscle,liver,pancreas,mammary gland,cervix,ovary, kidney,prostate,lymph node,skin and brain.2.The expressions of Tiam1 gene/protein in human normal liver cells and liver cancer cells.Real time PCR,immunocytochemistry,Western blot and cellular immunofluorescence were used to examine the expressions of Tiam1 in 8 hepatocellular carcinoma cell lines named HepG2,Hep3B,SMMC-7721,QGy-7701,QGy-7703, BEL-7402,BEL-7404,BEL-7405 and 1 normal one named HL-7702.3.The expressions of Tiam1 protein in hepatocellular carcinoma tissues and its clinical significance.The expressions of Tiam1 protein in 152 cases of hepatocelluar carcinoma tissues were detected by EnVisionTM two-step immunohistochemistry.The results were analyzed with SPSS13.0 software.The relationship of Tiam1 protein expressions with clinical-pathologic parameters was analyzed with x2 test. Kaplan-Meier method was used to retrospectively analyze the relationship of Tiam1 protein expressions with life span of hepatocellular carcinoma patients.The relationship of single factor of each index or multi-variable combination with life span was analyzed by COX regression.4.The effect of Tiam1 gene silencing on the biological characters of hepatocellular carcinoma cells.Tiam1 gene fragments for silencing were designed by online database and software and the fragments were then transiently transfected into QGy-7701 hepatocellular carcinoma cell line.Real time PCR and Western blot were performed to detect the interference efficiency of Tiam1 gene.The effects of Tiam1 gene silencing on cell proliferation in vitro were detected by MTT method and flow cytometry,while those on cell invasion were detected by invasive chamber in vitro.Results:1.The distributions and expressions of Tiam1 protein in human normal tissues and common malignant tumorsNo signals of Tiam1 were observed in fiber and fat of breast,pancreatic gland and liver,while positive signals were observed in 15 normal tissues including nasopharynx,lung,esophagus,stomach,colon,rectum,fiber and smooth muscle, bladder,cervix,ovary,kidney,prostate,lymph node and brain,among which Tiam1 were highly expressed in glandular epithelium of hyperplastic prostate,hyperplastic smooth muscle,distal convoluted tubule of kidney and smooth muscle of vessel wall. Tiam1 proteins were up-regulated in nasopharyngeal cancer,breast cancer,lung squamous cancer,lung adenocarcinoma,colon cancer,rectal cancer,hepatocellular carcinoma,prostate cancer,ovary cancer and diffuse large B cell lymphoma,while low-regulated in adenocarcinoma and squamous carcinoma of esophagus,gastric carcinoma,pancreatic carcinoma,clear cell carcinoma of kidney,cervical carcinoma, transitional cell carcinoma of bladder,glioma and skin squamous carcinoma.In addition,we found that liver sclerotic tissues or liver tissues with obviously hyperplastic fibers besides liver cancer showed Tiam1 expressions at the low or moderate degree.Their expression degree and scope were lower than those in liver cancer.2.The expressions of Tiam1 gene in human normal liver cell line and liver cancer cell linesTiam1 gene expressions in 8 human hepatocellular carcinoma cell lines and 1 human normal hepatic cell line were detected by real time PCR.The results of one-factor analysis of variance showed that the expressions of Tiam1 in 9 cell lines had significant difference(F=80.514,P＜0.001).The expressions of Tiam1 in 8 hepatocellular carcinoma cell lines were higher than those in normal hepatic cell line with significant difference.3.The expressions of Tiam1 protein in human normal hepatic cell and hepatocellular carcinoma cellsNo signals of Tiam1 expression were observed in normal hepatic cell line while positive signals in 8 hepatocellular carcinoma cell lines by immunocytochecmistry. The above result was consistent with those of Western Blot and cellular immunofluorescence.4.The expressions of Tiam1 protein in hepatocellular carcinoma tissues and their clinical significanceThe total positive expressive rate of Tiam1 protein in 152 cases of hepatocellular carcinoma was 88.1%,but no expression in normal hepatic tissues by immunohistochemistry.The ratio of every grade of hepatocellular carcinoma tissues was respectively that:"-" was 11.9%(18/152);"+" was 24.3%(37/152);"++" was 39.5%(60/152);"+++" was 24.3%(37/152).Tiam1 expressions had no association with sex,age,tumor size,differentiation,cirrhosis,metastasis,recurrence,serum HBsAg,serum AFP(P＞0.05).By follow-up analysis,hepatocellular carcinoma patients with high Tiam1 expression had 27.0 months of median survival time,and the survival time had significant difference between patients with high Tiam1 expression and those with low Tiam1 expression(P=0.008＜0.05).The expressions of Tiam1 protein,tumor size,metastasis,recurrence and serum AFP value were important factors affecting the survival time of hepatocellular carcinoma patients(P value was 0.009,0.002,0.030,0.035,0.002 respectively).Multi-factor analysis showed that the high expression of Tiam1 protein,tumor size or serum AFP value were independent factors affecting the survival prognosis of hepatocellular carcinoma (P value was 0.042,0.008,0.001 respectively).5.The effect of Tiam1 gene silencing on the biological characters of hepatocellular carcinoma cellsThe specific interference fragments of Tiam1 gene were produced and transiently transfected into QGy-7701 cell line,with 60%-70%of the transfective efficiency detected by fluorescence microscope.The results of real time PCR and Western blot showed that the interference efficiency of Tiam1 was 72%(named QGy-7701/Tiam1- cell line).MTT method was carried out to observe the cell proliferation in vitro after Tiam1 gene silencing.Compared to QGy-7701 cell line,QGy-7701/Tiam1- cell line had slower proliferative rate in the time-dependent manner(F=237.790,P＜0.001). The cell cycle of QGy-7701/Tiam1- was obviously reduced compare with QGy-7701 by flow cytometry(t=14.177,P＜0.001),indicating that cell proliferation became slowly after Tiam1 gene silencing.The results showed that growth in vitro of tumor cells were obviously inhibited after the expressive level of Tiam1 decreased.The changes of cell invasive abilities after Tiam1 gene silencing were detected by invasive chamber experiment in vitro and the results showed that compared to QGy-7701 cell,the invasive abilities of QGy-7701/Tiam1- cell decreased obviously (t=18.054,P＜0.001),suggesting that Tiam1 silencing inhibited the invasive abilities of hepatocellular carcinoma cell.Conclusions:1.Tiam1 protein was highly expressed in many tumors and it can be acted as one important tumor maker for tumorigenesis and development;2.Tiam1 protein was up-regulated in hepatocellular carcinoma tissues,expressed lowly in liver sclerotic tissues and had no expression in normal hepatic tissue, indicating that Tiam1 may be regarded as the important marker for clinical detection of hepatocellular carcinoma;3.Combined evaluation of Tiam1 protein high expression and clinical follow-up data analysis showed that Tiam1 had close relationship with prognosis of hepatocellular carcinoma patients.Tiam1 may act as the important marker for judging prognosis of hepatocellular carcinoma;4.Tiam1 gene silencing can inhibit the proliferation and invasion of hepatocellular carcinoma cell,indicating Tiam1 can promote the proliferation and invasion of hepatocellular carcinoma cell and suggesting the important role of Tiam1 gene in proliferation and invasion of hepatocellular carcinoma cell.Innovations of our study:1.Tiam1 gene had relationship with proliferation,invasion and prognosis of hepatocellular carcinoma,which laid theoretical basis on targeted therapy of hepatocellular carcinoma;2.The establishment of a hepatocellular carcinoma cell line with Tiam1 stable silencing will be provide a valuable tool for Tiam1 functional studies.
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