The Role Of Sulfatase-1(Sulf-1)on Hepatocellular Carcinoma Lymphatics Metastasis Cells And Its Relation With Mesothelin(Msln)Gene In Vitro And In Vivo

BackgroundIn order for tumor cells to grow and to able to move from one place to another,they need to be able to facilitate the formation of new blood vessels,these blood vessels will surround the tumor cells in order to provide them with adequate oxygen supply and nutrient for tumor growth.The migratory process of the tumor cells is acquired by the lymphatic vessels.It is an important route for the tumor cells to move from primary site directly into the lymph nodes,this movement will help the tumor cells to metastasize to other distance organs in the body.Metastasis to reginal lymph node tend to have important role of cancer prognosis and lesion metastasize through lymphatic system.Though many expression factors have been mention to contribute in providing adequate environment in metastasis process but still to date,few reports have been documented in the literature concerning hepatocellular carcinoma lymph node metastasis(LNM).Also,still to date,few researches have been done concerning the mechanism of signaling pathway in HCC involving in the lymph node metastasis by which malignant cells tumor move from their primary site and invade regional lymph node.Worldwide hepatocellular carcinoma is the 6th most common cancer and it is the 3rd leading cancer with high morbidity and mortality.In 2016,a study reported that,75%of HCC incidence cases was diagnosed in developing countries,and?50%of the HCC mortality and incidence occurred in China.HCC is characterized by extensive carcinoma infiltration and metastasis of the tumor cells.The signaling molecular mechanism for cancer metastasis are complex interconnected molecular pathways.Understanding of molecular mechanisms which causing the dissemination of the cancer cell metastasis will help in the development of effective antimetastatic therapy.Sulf-I is one of the sulfatases which is known to function as a tumor suppressor gene and it is also known as anti metastatic gene in various malignant cancer cell lines.Sulf-1 reported to decrease the expression of several metastatic lymphangiogenic 5 factors in malignant cancer cells.These factors reported to have important function in tumor cell survival,tumor growth,invasion and metastasis.Sulf-1 exert its action through proteoglycans such as heparin sulfate proteoglycan(HSPG)by removing 6-O-moeties from the heparin sulfate chains and this leads to inhibit all of the heparin sulfate dependent signaling molecules and prevent the stimulation of the downward signaling involved in proliferation,migration,cell-invasion and metastasis.Msln is a membrane glycoprotein which known to act as a tumor promoter gene in various cancers,and its expression reported to increased cancer cell infiltration,cell-invasion and cell-metastasis.The expression of msln was reported to be facilitated by highly sulfated HSPG by allowing it to bind to its receptors and activates the signaling pathway involve in tumor growth metastasis.For the past few years,our laboratory has been conducting research concerning lymph node metastasis by using syngeneic two types of mouse hepatocarcinoma cell lines(Hca-F with>75%LNM potential and Hca-P with<25%LNM potential).These two cell lines were generated by our laboratory and have shown to be an ideal cell lines in studying liver cancer metastasis in the mouse model.In these works,Sulf-1 have been found to be lowly expressed in Hca-F cells compared to the Hca-P cells,while msln found to be highly expressed in the Hca-F compared to the Hca-P cells.So,the research question to this study is that:what is the role played by Sulf-1 in hepatocellular carcinoma metastatic cell lines and if there is any relation with msln,as Sulf-1 known to inhibits HSPG action while msln expression facilitated by HSPG?Based on the various research work done on these two genes,we hypothesized that,Sulf-1 is a tumor suppressor gene and msln is a tumor promoter gene,so up-regulating Sulf-1 gene in Hca-F cell lines could down regulates msln and leads to the reduction in lymphatic metastasis both in vitro and in vivo,and down regulation of Sulf-1 in Hca-P cell line could up-regulated msln expression and leads to the increase in lymphatic metastasis both in vitro and in vivto.So far there are no studies investigating the role of Sulf-1 in hepatocellular carcinoma lymphatic metastasis and its relation with msln gene.This is the first study involving Sulf-1 and msln gene in hepatocellular carcinoma lymphatic metastatic cell lines.Objectives1.The first objective of this study is to get stably up regulation of Sulf-1 gene in Hca-F cell line and then to measure the changes in protein and mRNA level of Sulf-1 and msln gene expression with the respect to the changes in the cell proliferation,migration,invasion in vitro and to evaluate the rate of tumor growth and metastasis to the lymph node in vivo.2.My second objective is to stably down regulate Sulf-1 gene in Hca-P cells line and followed by measuring the changes in protein and mRNA level on Sulf-1 and msln gene expression with the respect with the changes in the cell proliferation,migration,invasion in vitro and to evaluate the rate of tumor growth and metastasis of the lymph node in vivo.Methods1.Achievement of stable up-regulation of Sulf-1 was done by using plasmid expressed Sulf-1 for up-regulation in Hca-F cell line.The Hca-F cells were divided into three groups;(1)Sulf-1 expression plasmid in Hca-F cells(Sulf-l-Hca-F),(2)nonspecific sequence control plasmid in Hca-F cells(Nc-Hca-F)and(3)non-transfected group Hca-F cell line(Hca-F)2.In a Hca-P cell line,the stable down regulation of Sulf-1 was achieved by using Sulf-1 down regulation plasmid(shRNA-Sulf-1).The cells were also divided into three groups;(1)shRNA-Sulf-1-Hca-F cell line,(2)nonspecific sequence control plasmid(shRNA-Nc-Hca-P)and the non-transfected group was labelled as Hca-P cells(Hca-P).3.Western blot and qRT-PCR were applied to measure both protein and mRNA level for Sulf-1 and msln expression level after stable up-regulation and down regulation of Sulf-1 in Hca-F cell lines(Hca-F,Sulf-Hca-F and Nc-Hca-F)and Hca-P(Hca-P,shRNA-Sulf-l-Hca-P and shRNA-Nc-Hca-P)cell lines both in vitro and in vivo.4.In order to measure the cell ability to proliferate in all of the cell line groups,I performed cell proliferation assay by using both the manual cell count with hemocytometer and CCK8 in vitro both in Hca-F(Hca-F,Sulf-Hca-F and Nc-Hca-F)and Hca-P(Hca-P,shRNA-Sulf-1-Hca-P and shRNA-Nc-Hca-P)cell lines.5.In order to measure the cell number in G0-G1 phase,S-phase and G2-M-phase,I performed the cell cycle assay by using Propidium iodide staining followed by flow cytometry in vitro both in Hca-F(Hca-F,Sulf-Hca-F and Nc-Hca-F)and Hca-P(Hca-P,shRNA-Sulf-1-Hca-P and shRNA-Nc-Hca-P)cell lines6.The ability of the cells to migrate from one place to another is measured by using migration assay using Boyden's transwell plates in vitro both in the Hca-F(Hca-F,Sulf-Hca-F and Nc-Hca-F)and Hca-P(Hca-P,shRNA-Sulf-1-Hca-P and shRNA-Nc-Hca-P)cell lines.7.The ability of the of these cancer cells to invade the surrounding area were measured by using cell invasion assay,this assay was assessed by using the same transwell plates with additional of ECM coating of the inner chamber in vitro both in Hca-F(Hca-F,Sulf-Hca-F and Nc-Hca-F)and Hca-P(Hca-P,shRNA-Sulf-1-Hca-P and shRNA-Nc-Hca-P)cell lines.8.Immunohistochemistry(IHC)and Hematoxylin and Eosin staining were used to assess genes expression level in the tumor tissues and lymph node metastatic rate in vivo both in mice inoculated with Hca-F(Hca-F,Sulf-Hca-F and Nc-Hca-F)and Hca-P(Hca-P,shRNA-Sulf-1-Hca-P and shRNA-Nc-Hca-P)cell lines.ResultsSulf-1 is one of the tumor suppressor gene which inhibits the expression of genes which are associated in the promotion of lymphatic metastasis in the cancer cells.Forced expression of Sulf-1 in Sulf-1-Hca-F cells both in in vitro and in vivo showed to have higher significant expression of Sulf-1 protein level with a significant lower expression of msln protein level respectively,compared to the two control cell lines,while both in Hca-F and Nc-Sulf-1-Hca-F cells the protein expression level for Sulf-1 gene was lower with the higher protein expression level of msln both in vitro and in vivo.The stable up regulation of Sulf-I gene was further confirmed by performing qRT-PCR in order to detect the mRNA expression level of Sulf-1 and msln.The quantified collected data,were normalized by using the house keeping gene Gapdh,and I used the Act method to calculated the mRNA expression level of each gene.The results showed that,both in Hca-F and Nc-Hca-F cells the mRNA expression level for Sulf-1 was lower while the mRNA expression level for msln was higher both in vitro and in vivo.In a stable transfected Sulf-1-Hca-F cells showed to have significantly higher Sulf-1 mRNA expression level and lower msln mRNA expression level both in vitro and in vivo.These findings confirmed that Sulf-1 was successful up regulated in Hca-F cells,and that its up regulation leaded to down regulation of msln expression both in vitro and in vivo.After stable down regulation of Sulf-1 in Hca-P cells,I run western blot and qRT-PCR in order to confirm the down regulation of Sulf-1 gene.In western blot analysis the result showed that,compared to the Hca-P and shRNA-Nc-Hca-P cell lines,Sulf-1 was successful down regulated in shRNA-Sulf-1-Hca-P and leaded to increase msln expression level both in vitro and in vivo.Then I run qRT-PCR assay and the results showed that in shRNA-Sulf-1-Hca-P cells,the mRNA level for Sulf-1 was significantly lower after down regulation of Sulf-1 while the mRNA level for msln significantly increased both in vitro and in vivo.These results showed that Sulf-I was successful down regulated in Hca-P cells,and its down regulation leaded to up regulate msln expression bot in vitro and in vivo.Over proliferation of the cells is one of the properties of the malignant cells,and this is due to the deletion in the chromosome site which leaded to mutation in the receptor site or to the gene responsible to control proliferation process.Another cause of over proliferation in cancer cells is due to the loss of function mutation in the tumor suppressor gene.In this study,I observed that the proliferation rate of Hca-F and Nc-Hca-F was higher,but after forced expression of Sulf-1 in Sulf-l-Hac-F cells the proliferation rate was dramatically reduced by 60%with statistical significant of P<0.05.In Hca-P cells lines the trend was difference after Sulf-1 down regulation,the result showed that in shRNA-Sulf-1-Hca-P cells the cell proliferation rate increased by 40%compared to the Hca-P and shRNA-Nc-Hca-P cells.These results confirmed that Sulf-1 played a major role in proliferation of hepatocellular carcinoma cells.As reported that,one of the characteristic of the cancer cell is to have the ability to speed up the process of the cell circle and this condition is correlate with the situation where cancer cells start to over proliferate.In this study,I analyzed the percentage cell distribution in each cell cycle phases after up regulation and down regulation of the Sulf-1 gene both in(Hca-F,Sulf-1-Hca-F and Nc-Hca-F)and(Hca-P,shRNA-Sulf-1-Hca-P and shRNA-Nc-Hca-P).After successful up regulation of Sulf-1 in Sulf-l-Hca-F cells I observed that,the results for the cell cycle analysis showed significant increase in number of cell accumulate at G0-G1 phase whilst at the same time there was a reduction in the S-phase cell population compared to Hca-F and Nc-Hca-F cells where there were decreased in number of cells in G0-G1 phase and increase cell number accumulation in S-phase.The pattern was strikingly difference after Sulf-1 down regulation in Hca-P cell line,where there was decrease in cell accumulation in G0-G1 phase while at the same time there was increased in S-phase and G2-M phase cell population.This is a further confirmation of the cell proliferation role of Sulf-1 to hepatocellular carcinoma and cell cycle arrest reported to inhibits the process of DNA synthesis in cancer cells.Migration and invasion are characteristics of a malignant cancer metastatic cells and these behavior is achieved by the over expression of the genes associated with the progression of cancer metastasis.In this study,I observed that,compared to the Hca-F and Nc-Hca-F,Sulf-l-Hca-F showed to acquire the migration and invasion inhibitory properties by significantly reduced the cell number invaded or migrated through in vitro.The results were different in shRNA-Sulf-l-Hca-P,in this cell line there were increased in cell number migrated or invaded through,compared to the Hca-P and Nc-Hca-P cell lines in vitro.These findings implying that Sulf-I play significant role in reduction of metastasis of hepatocellular carcinoma.Moreover,the in vivo study showed that,there were significantly increased in tumor growth in mice inoculated with Hca-F and Nc-Hca-F while I observed reduction in tumor growth in mice inoculated with Sulf-1-Hca-F cell respectively.Moreover,the lymph node metastatic rate in mice inoculated with Hca-F and Nc-Hca-F cell were 86%(12/14)and 92%(13/14)respectively,while the lymph node metastatic rate was 7%(1/14)in mice inoculated with Sulf-l-Hca-F cells.On the other hand,significantly increased in lymph node metastasis observed which was 78%(11/14)in mice inoculated with shRNA-Sulf-1-Hca-P cells,compared to Hca-P 28.6%(4/14)and shRNA-Nc-Hca-P 35.7%(5/14)respectively.Furthermore,IHC results showed that,the tumor tissues from the mice inoculated with Sulf-1-Hca-F cells to have significant higher intensity of Sulf-1 expression and significantly lower intensity of msln expression,compared to the tissues sample from the mice inoculated with Hca-F and Nc-Hca-F which shown to have low intensity of Sulf-1 expression and higher intensity of msln expression level.The results were quite difference after down regulation of Sulf-1,the tissue sample form the mice inoculated with shRNA-Sulf-1-Hca-P cells showed to have significantly lower intensity Sulf-1 expression and higher intensity of msln level respectively,compared to the tissues sample from mice inoculated with Hca-P and shRNA-Nc-Hca-P cells which shown to express both Sulf-1 and msln.This finding was consistent with my western blot and qRT-PCR results in vitro and in vivo.H&E results showed that,lymph nodes from Sulf-l-Hca-F bearing mice showed to have less tumor cells with less mitotic figures with no necrosis,compared to the lymph nodes came from mice inoculated with Hca-F and Nc-Hca-F which show to have tumor cells with mitotic figures in the nodal marginal sinus with necrosis formation.The lymph nodes from shRNA-Sulf-1-Hca-P bearing mice showed to have more tumor cells with mitotic figures in the nodal marginal sinus with necrotic tissues compared to the Hca-P and shRNA-Nc-Hca-P bearing mice which shown to have less tumor cells with necrosis in the lymph nodes.Base on my findings,the role of Sulf-1 played in the inhibition of metastasis in hepatocellular carcinoma were confirmed in this study both in in vitro and in vivo.ConclusionSulf-1 is one the gene reported to control the expression of the metastatic associated gene by inhibiting their expression.Few study have been reported to explore the function of Sulf-1 in the metastatic condition of the Hepatocellular carcinoma.Sulf-1 reported to control the signaling molecules which were over expressed by proteoglycan especial HSPG The main function of Sulf-1 is to remove 6-0-sulfate groups from heparin sulfate chain of HSPG,and this leaded to decrease receptors expression which are important for binding of the signaling molecules.Msln is one of the signaling molecules which its expression is facilitated by highly sulfated HSPG.In this study,I up regulated Sulf-1 gene in Hca-F cell and dawn regulated in Hca-P cells in order to explore to functional relationship between Sulf-1 and msln in proliferation,migration and invasion in vitro and tumor growth and metastasis of lymph node in vivo.Confirmation of the genes expression both in protein and mRNA level results showed decreased in msln expression after up-regulation of Sulf-1 in Sulf-1-Hca-F cells whilst it's expression increased after down regulation of Sulf-1 in shRNA-Sulf-1-Hca-P cells both in in vitro and in vivo.This interaction could be due to the fact that Sulf-1 inhibits proteoglycans receptors protein expression level and this will inhibit proteoglycan to stimulated msln expression in the cancer cells.Cell proliferation is one of mechanism affected in carcinogenesis.This mechanism consists of multiple event which regulate virous kind of genes in our body.Any fault in this mechanism is directed to special genes repair at a checkpoint position and repair the fault on the DNA.This check point system found at different phases of the cell cycle and provided with DNA repair and prevent the cells to transform into the cancer cells.Though,in carcinogenesis cancer cells undergo multiple proliferation process which create problem in the repair system due to the over load of the damaged DNA and this resulted into the formation cancer.Tumor cell over proliferation is one of the factor causes frequency DNA damage in tumor evolution,and this happens when there is a gain of function mutation in the genes controlling cell proliferation or loss of function mutation in tumor suppressors gene.In this study,I have seen that Sulf-1 decreased cell proliferation rate in the Hca-F after up regulation and also increased proliferation of Hca-P cells after down regulation of Sulf-1.This means that Sulf-1 have the tumor suppressor effects on metastatic hepatocellular carcinoma cells.I also observed more proliferation effect on cell cycle where here most of the cancer cells in Hca-F were arrested in the G0-G1 phase with the decreased in number of cells in S-phase.This means that Sulf-1 inhibits DNA synthesis in the hepatocellular carcinoma cells,loss of sulf-1 gene in Hca-P cells showed to promote DNA synthesis in cancer cells by promoting S-phase and G2-M phase.Migration and invasion are characteristics of a malignant cancer metastatic cells.These behavior is achieved by the over expression of the genes associated with the progression of cancer metastasis.In this work,Sulf-1 expression inhibits migratory and invasion properties of Hca-F cells this result were compared with the one from the down regulation of Sulf-1 in Hca-P cells and showed that loss of Sulf-1 expression increased in cell migration and invasion of Hca-P cells.These findings showed that Sulf-1 to be an important gene in controlling the migration and invasion of the HCC cancer cells.My in vivo results confirm the tumor suppression function of Sulf-1 in HCC cancer cells.Sulf-1 expression shown to inhibits tumor growth from the mice inoculated with Sulf-1-Hca-F while lack of Sulf-1 expression leaded to promote tumor growth in mice inoculated with shRNA-Sulf-1-Hca-P cells.In addition to this,reduction in lymph node metastasis observed in Sulf-1-Hca-F expressed group and promotion of lymph node metastasis observed in a group which lack Sulf-1 in shRNA-Sulf-1-Hca-P.All put together I conclude that my findings show that Sulf-1 is an important tumor suppressor gene in metastatic hepatocellular carcinoma cells,and this functional relationship between Sulf-1 and Msln could be exploited for the development of a novel liver cancer therapy.