In The Interaction Of Hepatic Cacinoma Ang Stellate Cells, NDRG2 Has Effects On Metastasis Of Hepatic Cacinoma

Background The interaction of epithelial and endothelium, mesenchymal basal cells, fibroblasts, neuroendocrine cells, smooth muscle cells, inflammatory cells and extracellular matrix (ECM) which composes microenvironment regulates cells activities as proliferation, differentiation, and apoptosis as well as secretion and activation of soluble factors and/or deposition of extracellular matrix (ECM) components. The balance of these components results the homeostasis of cells and tissues. But, in tumor microenvironment, the increase of matrix metalloproteinase, growth factors, chemotactic factors induced by many cells promotes cancer progression, metastasis. Our study focused on the interaction of hepatoma carcinoma and stellate cells in tumor microenvironment. Hepatoma carcinoma cells can induce the activation of hepatoma stellate cells which can promote the invasion and metastasis of HCC.NDRG2 belongs to the NDRG (N-myc downstream-regulated genes) family where it has been reported to function as a tumor suppressor gene. NDRG2, a candidate of tumor suppressor gene (TSG), is expressed in many normal tissues. The aggressive cancers have Low and undetectable levels of NDRG2, along with the poor prognosis of cancer patients. NDRG2 is a gene inhibiting proliferation of tumors can enhance apoptosis of some tumor cell lines. Immunohistochemistry showed that NDRG2 expression was reduced at a late stag. This provoked the attention of the connection of metastasis and NDRG2. In 100 HCC patient tissues, Immunohistochemical analysis of NDRG2 protein indicated that loss of NDRG2 expression is significantly correlated with aggressive tumor behaviors such as late tumor-node-metastasis stage, differentiation grade, portal vein thrombi, infiltrative growth pattern, nodal/distant metastasis, and recurrent tumor, as well as shorter patient survival rates. NDRG2 could antagonize transforming growth factorβ1–mediated tumor cell invasion by specifically down-regulating the expression of matrix metalloproteinase 2 and PAI-1, with concomitant suppression of Rho GTPase activity. It has been demonstrated that loss of NDRG2 expression in hepatocellular carcinomas is significantly correlated with aggressiveness and NDRG2 expression contributes to the suppression of metastatic potential in Hepatic Carcinoma cells. In our study, we want to confirm that low expression levels of NDRG2 increased invasion, metastasis, adhesion ability of hepatic cacinoma cells.NDRG2 significantly suppresses tumor invasion by inhibiting MMPs activities. NDRG2 playing an major role in suppressing HCC metastasis can inhibit extracellular matrix– based, Rho-driven tumor cell invasion and migration. BMP-4 induced by NDRG2 expression inhibits the metastatic potential of breast cancer cells, especially via suppression of MMP-9 activity. Our study also suggested loss of NDRG2 expression in HCC is correlated with the increase of metastatic potential. NDRG2 expression will be a useful and functionally relevant biomarker to predict aggressive behavior in patients with meningioma,colorectal carcinogenesis and melanoma. It is urgent to study NDRG2 in metastasis of HCC.CD44, a widely expressed cell surface glycoprotein, plays a major role in cell-cell adhesion, cell-substrate interaction, lymphocyte homing, and tumor metastasis. CD44, a hyaluronic acid receptor, can promote or inhibit motogenic signaling in tumor cells. It may be possible to suppress CD44 by effective means, and thus potentially cause a reduction in invasive capability and metastasis.CD24, an adhesion molecule for P-selectin, was related with tumor growth and metastasis. CD24 expression increased tumor cell proliferation, adhesion to fibronectin, collagens I and IV, and laminin through the activation of alpha3beta1 and alpha4beta1 integrin activity. Moreover, CD24 expression supported rapid cell spreading and strongly induced cell motility and invasion. We want to know if NDRG2 can control tne expression of CD44 and CD24.Objecyives Studies indicated that NDRG2 have relationship with MMPs, PAI-1 and integrins. There isn't a study revealed the relationship between NDRG2 and adhesion molecules influencing metastasis of HCC. NDRG2 influences not only tumorigenesis, progression, metastasis of cancer cells but also differentiation, stress of cells. So, we want to know if NDRG2 effects the activation of HSC. If NDRG2 participates gene regulation of HSC activation.Methods 1) hepatic cacinama cells have defferent metastasis ability. The NDRG2 expression of hepatic cacinama cells were detected by RT-PCR and Western blot. We also detected invasion, metastasis, adhesion ability of defferent hepatic cacinoma cells. Subsequently, we investigated the change of invasion, metastasis, adhesion ability of defferent hepatic cacinama cells using Ad-NDRG2, siRNA-NDRG2 to overexpress, knockdown NDRG2 expression.2) We investigated the adhesion molecules influencing metastasis of hepatic cacinoma cells infected by Ad-NDRG2, transfected by NDRG2 siRNA by Real Time PCR and Western blot.3) We investigated molecules having relationship with HSC activation of HSC infected by Ad-NDRG2, transfected by NDRG2 siRNA by Real Time PCR and Western blot.4) This study investigated whether NDRG2 contributes to the tumor microenviroment. A model of inoculation of tumor cells and activated HSC of mixing glum (including Fibxinogen, Thrombin and Aprotinin) and (nu/nu) mice were created. Changing NDRG2 by Ad-NDRG2 and siRNA-NDRG2, we investigated the ashesion, chemotaxis and gene regulation of two kinds of cells.Results 1) hepatic cacinoma cells have defferent metastasis ability. Decrease of NDRG2 expression was compared with increase of invasion, metastasis and adhesion ability by RT-PCR and Western blot. Invasion, metastasis and adhesion ability decreased when the defferent hepatic cacinoma cells were infected by Ad-NDRG2. Invasion, metastasis and adhesion ability increased when the different hepatic cacinoma cells were tansfected by siRNA-NDRG2. Overexpression NDRG2 suppressed adhesion, invasion and migration of different invasive cell line, whereas small interfering RNA-mediated knockdown resulted in increased invasion and migration of different cell lines.2)CD44 and CD24 mRNA and protein decreased when the hepatic cacinoma cells were infected by Ad-NDRG2. CD44 and CD24 mRNA and protein increased when the hepatic cacinoma cells were tansfected by siRNA-NDRG2 by Real Time PCR and Western blot. The down-regulation of NDRG2 and increase of CD44, CD24 expression in the HCC cases were detected by immunohistochemisty.3)α-SMA,CD44 are activation marker of HSC.α-SMA,CD44 mRNA and protein decreased when the HSC were infected by Ad-NDRG2. At the same time, invasion ability was reduced.α-SMA,CD44 mRNA and protein increased when the hepatic stellate cells were infected by Ad-NDRG2. At the same time, the invasion ability was raised by siRNA-NDRG24)We investigated that overexpression or knockdown NDRG2 of hepatic cacinoma cells decreased or increased their chemotaxis and adhesion to HSC comparing with reduction or augmentation ofα-SMA and CD44. At the same time, the result indicated that overexpression of NDRG2 could reduce hyaluronic acid(HA) induced by TGF-β1.Conclusion As NDRG2 is reported to be a candidate tumor-suppressor gene in a wide variety of cancers, NDRG2 has effect on tumor microenviroment. Our study investigated that the decrease of NDRG2 raised the expression of CD44 and CD24 on hepatic cacinoma cells. NDRG2 Overexpression could decrease CD44 and CD24 on hepatic cacinoma cells. On HSC, reduction or augmentation ofα-SMA and CD44 were detected by NDRG2 Overexpression or knockdown. NDRG2 overexpression could reduce HA induced by TGF-β1 too. These results suggest that NDRG2 can inhibit hepatic cacinoma cells, activated HSC adhesion, invasion, migration and thereby play important role in suppressing HCC metastasis.