The Role Of Tumor Necrosis Factor Alpha-Induced Protein8-Like1in The Development Of Hepatocelluar Carcinoma

Hepatocellular carcinoma, as the sixth most common cancer worldwide with the third highest mortality rate, is one of the most fatal human malignancies, and its incidence rate is increasing. HCC remains a highly complex disease. The alteration of tumor related genes is one of the most important causes, of which methylation of tumor suppressor gene is one of the major changes. HCC is resistant to commonly used chemotherapy and radiotherapy, while surgical resection and radiofrequency ablation are only effective in early tumors, so new treatment strategies are urgently needed. Recently, molecular targeted therapy has gradually become a hotspot of HCC treatment. The multikinase inhibitor sorafenib is the first molecular targeted drug in HCC that leads to significant survival benefit in patients with advanced tumors, which supports the importance of molecular therapies in the treatment of this cancer. Understanding the molecular mechanisms of HCC and identification of effective targets in HCC are foundation of molecular therapies. Therefore, it is urgent to explore the genetic and epigenetic alterations as well as different molecular pathways involved in the development of HCC.TEPE1(tumor necrosis factor alpha-induced protein8-like1, TNFAIP8L1) is a newly identified gene in2008, belonging to TIPE (tumor necrosis factor alpha-induced protein8, TNFAIP8) family, which consists of four members, TIPE, TIPE1, TIPE2and TIPE3. The identities shared between human TIPE1and those of the following proteins are as follows:human TIPE,57%; human TIPE2,57%; human TIPE3,55%; murine TIPE1,82%. There are only three published papers related with TIPE1. Microarray analysis indicated that TIPE1may be related with the cardiac decompensation associated with diabetes. TIPE1is widely distributed in various tissues of mice except mature T or B lymphocytes, but there is no research about TIPE1expression pattern in human. A genome-wide siRNA screen for regulators of necroptosis in Cell in2008found that knockdown of TIPE1could respectively inhibit apoptosis and necroptosis, indicated that TIPE1might be involed in regulation of both apoptosis and necroptosis. However, it is completely unknown if TIPE1involves in cell death in human cells.Here, in this study, we aim to describe the distribution of TIPE1in human tissues and human cells and explore the role of TIPE1in HCC development.Ⅰ.TIPE1expression pattern in human tissues1. The distribution of TIPE1mRNA in normal human tissues and embryonic tissues. The TIPE1expression in human tissues is not clear yet. Firstly, Real-time PCR was used to detect the distribution of TIPE1mRNA in the cDNA library of normal human tissues and embryonic tissues. Results showed that TIPE1mRNA was detected in various human tissues with relative higher expression in liver, but only highly expressed in fetal brain.2. The TIPE1protein expression in normal human tissues. To further research the distribution of TIPE1in human tissues, we detect TIPE1protein in various normal human tissues by immunohistochemistry. Immunohistochemistry results of normal human tissues using this antibody showed that TIPE1protein was detected in various human tissues, especially highly expressed in skeletal muscle, cardiac muscle and liver.3. TIPE1is mainly expressed in the cytoplasm in liver cells. According to the above expression pattern of TIPE1, we choose liver as our research target. Results of immunofluorescence study in the paracancerous tissue derived liver cell line QSG7701showed that TIPE1located in the cytoplasm in hepatocytes. Ⅱ. TIPE1was significantly downregulated in HCC tissues, which was negative related to lymphatic/venous metastasis and positive related to pathological grade and prognosis1. Compared with adjacent noncancerous tissues, TIPE1was significantly downregulated in HCC tissues, and positive related to tumor pathological grade while negative related to lymphatic/venous metastasis.26HCC tissues and matched adjacent noncancerous tissues and6hepatic cavernous hemangioma tissues (used as normal liver tissues control) were collected for immunohistoochemical staining with anti-TIPE1antibody. The results showed that, normal liver tissues in hepatic cavernous hemangioma and noncancerous tissues expressed relatively higher level of TIPE1. On the contrary, TIPE1expression was significantly downregulated in HCC tissues compared with matched adjacent noncancerous tissues (p<0.001). In order to confirm the results, tissue microarray was stained with anti-TIPE1antibody, and the same results were obtained. Further statistic analysis showed that TIPE1expression was positive related to tumor pathological grade (p<0.001) and negative related to metastasis (p=0.0191):HCC tissues with lower differentiation grade had lower TIPE1expression, and HCC tumors with lower TIPE1expression were more prone to lymphatic/venous metastasis.2. TIPE1expression was significantly positive related to HCC progression. Tissue microarray was stained with anti-TIPE1antibody, and correlation analysis between TIPE1expression and survival time of HCC patients was performed. Statistical analysis demonstrated that TIPE1expression was positive related to the HCC progression:patients with positive TIPE1expression survived much longer than those with negative TIPE1expressionⅢ. TIPE1evidently inhibited the growth of H22homograft in Balb/c miceThe above results strongly indicated that TIPE1might act as a tumor suppressor in human hepatocellular carcinoma. To further validate the suppression effect of TIPE1on HCC cells, we designed the following in vivo experiments.Murine liver cancer cell line H22(2×105) cells were transplanted subcutaneously into Balb/c mice. After reaching a diameter of0.5cm, tumors were randomly divided into two groups and injected with mTIPE1-pRK5or pRK5plasmids (20μg) every four days for4injections.Tumor size was monitored every other day. Mice were sacrificed four days after the final injection and the tumors were isolated, photographed and weighed. The results showed that overexpression of mTIPEl could significantly inhibit H22homograft growth (p<0.001). Consistently, the average tumor weight of mTIPEl-injected group at the time of sacrifice was evidently less than pRK5-injected control group. Both RT-PCR assay and immunohistochemistry results were used to verify the effect of plasmid injection.IV. TIPE1significantly inhibited the growth of HCC cell lines and induced cell deathClinical studies and animal experiments have shown that TIPE1has obvious inhibitory effects on HCC cell growth and cell survival. To further investigate the tumor suppression mechanism of TIPE1, we designed the following in vitro experiment.1. TIPE1inhibited the growth of HCC cell lines. pcTIPEl was transfected into HCC cell lines (Be17402, SMMC7721and HepG2) with low endogenous TIPE1expression. Cell growth was analyzed by CCK-8at24h intervals before and after transfection. TIPE1overexpression significantly reduced the growth of Be17402, SMMC7721and HepG2cells (p<0.001). To verify the above results, siRNAs (siRNA199, siRNA313and siRNA999) targeted to TIPE1were transfectedinto HCC cell lines (QSG7701and HepG2.2.15) with high endogenous TIPE1expression. Expectedly, reducing TIPE1levels by siRNAs (siRNA313and siRNA999) in QSG7701and HepG2.2.15cells significantly enhanced cell growth (p<0.001).2. TIPE1decreased the colony formation ability of HCC cell lines. TIPE1overexpression and knockdown cell models were obtained as above.24h after transfection, the transiently transfected cells were placed into6-well plates and cultured for7-12days. Colonies were stained with crystal violet and counted under a microscope. TIPE1overexpression led to a significant decrease in the number of colonies formed in Be17402, SMMC7721and HepG2cell lines (p<0.001), while the siRNA-mediated knockdown of TIPE1significantly increased the number of colonies formed in QSG7701and HepG2.2.15cell lines (p<0.001).3. TIPEl induced cell death of HCC cell line. It has been reported that knockdown of TIPE1inhibits both apoptosis and necroptosis, indicating that TIPE1may induce cell death. In order to confirm the hypothesis, Be17402cells were transfected with pcTIPE1-HA or control pcDNA3.0plasmids.24h later, cells were stained with anti-HA antibody by immunofluorescence and photographed using a microscope to observe the morphologic changes. Be17402cells transfected with pcTPE1-HA or control pcDNA3.0plasmids were then digested and divided into two parts. One part of cells was counted by trypan blue staining, and the rest cells were labeled with Annexin V/PI for cell apoptosis analysis by flow cytometry. After transfected with pcTTPEl-HA, Be17402cells became shrinking, rounding and swelling, and the nucleus became karyopyknosis and nuclear fragmentation. The results of trypan blue staining and flow cytometry showed more dead cells in pcTIPE1transfected group, and the dead cells were mainly Annexin V/PI double positive cells. These results showed that TIPEI could induce HCC cell death.Ⅴ. The mechanism of TIPE1inhibiting HCC cell growth and migration as well as inducing HCC cell death was related to Racl1. HCC cell death induced by TIPE1was caspase and RIP1independent cell death. To explore the mechanism of TIPE1inducing cell death, Be17402cells were pretreated with zVAD-fmk (broad spectrum caspase inhibitor) or/and Nec-1(RIP1inhibitor), and then transfected with pcTIPE1or control pcDNA3.0plasmids. Cell viability was measured48h after transfection. The results showed that neither single zVAD-fmk, single Nec-1nor combined with the two inbibitors could reverse the cell death induced by TIPE1.2. TIPE1could bind to Racl and inhibit its activity, therefore influence the following signal pathway. According to the phylogenetic analysis, TIPE1has a more close relationship with TIPE2than TIPE, indicated that TIPEI and TIPE2may have an identical molecular binding pattern. A recent study found that TEPE2could bind to Racl through TIPE2N-terminal lysine residues, Lys-15, Lys-16, and Lys-24, therefore inhibit the activity of Racl. Coincidentally, TIPE1has the same lysine residues as TIPE2, indicated that TIPE1can also bind to Racl and inhibit its activity. To testify this hypothesis, co-immunoprecipitation and pull-down using PAK-GST protein beads were used to study the interaction of TIPE1and Racl as well as Racl activity. Co-IP results identified the TIPEI-Racl interaction. Pull-down assay confirmed that TIPE1overexpression could significantly inhibit the activity of Racl. Western blot results showed that phosphorylated p65, phosphorylated JNK and antiapoptotic Bcl-2were reduced in TIPE1overexpressed cells, indicating that TIPE1could inhibit the activation of NF-κB and JNK pathway. Reduced Bcl-2in TIPE1overexpressing cells suggested that TIPE1might influence the mitochondrial apoptotic pathway by the reducing the Bcl-2protein expression.3. Knockdown of Racl could reverse the cell growth induced by TIPE1siRNA. The above results indicated that overexpressed TIPE1could inhibit the activation of NF-κB and reduce Bcl-2level to induce cell death by suppressing the activity of Racl. In order to verify this hypothesis, HepG2.2.15cells were transfected with Racl siRNA and/or TIPE1siRNA. Cell viability was measured by CCK-872h after transfection. Results showed that knockdown of Racl could reverse the cell growth induced by TIPE1siRNA, indicated that the effects of TIPE1on cell growth might be associated with Rac1.4. TIPE1inhibited the migration of HCC cells. It has been confirmd that NF-κB and JNK pathway are not only related to cell death, but also strongly connected with cell migration and tumor metastasis. Our previous study showed that TIPE1was associated with HCC metastasis, so we designed the migration assay. Be17402cells were transfected with pcTIPE1or control pcDNA3.0plasmids.24h after transfection, cells were placed into transwell chamber and96-well plates simultaneously at the same density, and the living cells in96-well plates were measured by CCK-8immediately.12h later, transwell chambers were fixed and stained with crystal violet, at the same time, the living cells in96-well plates were measured again by CCK-8. Results showed that cell growth was not suppressed obviously by TIPE1within12h, but the inhibition of cell migration was distinct (p<0.0001). Ⅵ. Promoter methylation of TIPE1in both HCC tissues and matched noncancerous tissuesBoth in vitro and in vivo studies proved that TIPE1might serve as a tumor suppressor gene in HCC tumorigenesis. Lots of researches demonstrate promoter hypermethylation of tumor suppressor genes leads to their decreased expression in tumors. To identify whether downregulation of TIPE1in HCC tissues results from promoter hypermethylation, the following experiments have been carried out. 1.5-Aza enhanced the level of TIPE1mRNA in HepG2cells. HepG2cells were treated with5-Aza and its control.48h later, cells were collected for RT-PCR. Results showed that5-Aza significantly increased the level of TIPE1mRNA in HepG2cells.2. Promoter methylations of TIPE1in HCC tissues were the same as matched noncancerous tissues Methylation-specific primers were desiged and tested in two positive controls, and unmethylation primers were then desiged according to the confirmed methylation primers. MS-PCR was involved to evaluate the promoter methylation of TIPE1in5HCC cell lines and26HCC tissues and matched noncancerous tissues. Results showed that promoter methylation of TIPE1existed in all detected samples, including5HCC cell lines as well as both HCC tissues and matched noncancerous tissues. Moreover, there were no significant difference between HCC tissues and matched noncancerous tissues, indicating that the promoter methylation might not be the reason of TIPEl-downregulation in HCC tissues. The mechanism remained to be further studied.In conclusion, we for the first time identify the expression pattern of TIPE1in human tissues, and investigate its function and mechanism in human liver tumorogenesis. This is the first paper to study the relationship between TIPE1and diseases. TIPE1is widely distributed in various human tissues, but only highly expressed in fetal brain. TIPE1binds to Racl and inhibits its activity, thereby suppresses the activation of downstream NF-κB and JNK pathway, which affects HCC cell growth, cell death and cell migration. TIPE1is obviously downregulated in HCC tissues compared with matched noncancerous tissues, and significantly associated with tumor pathological grade, lymphatic and venous metastasis, as well as survival time of patients, indicating that TIPE1plays an important role in HCC.5-Aza can enhance the expression of TIPE1in HCC cells, but promoter methylation of TIPE1exists in both HCC tissues and matched noncancerous tissues, indicating the promoter methylation might not be the reason of TIPE1-downregulation in HCC tissues, and its mechanism needs to be further explored.