| Background and AimHepatocellular carcinoma (HCC) is one of the most common malignant tumors, there are about 75 million new cases and nearly 70 million deaths every year. Although surgical resection is the best treatment for liver cancer treatment, but radical resection and postoperative recurrence rate is still very high, and more liver cancer patients lost the opportunity to effect a radical cure due to tumor metastasis. Because the recurrence and metastasis after resection of hepatocellular carcinoma is still the main obstacle of improvement of prognosis, the research on invasion and metastasis of hepatocellular carcinoma will become the focus of cancer research.The invasion and metastasis of HCC is a kind of very complex process.It involves the participation of a variety of factors. HCC cell invasion and metastasis ability is mainly determined by the genotype. Oncogene activation and suppressor genes inactivation can lead liver cancer cells to invasion and metastasis phenotype. Tumor protein 53-induced nuclear protein 1(TP53INP1)gene localizes to human chromosome 8q22.It almost 20 kb pairs in length with five exons. TP53INP1 is expressed in many tissues upon exposure to various stress agents, and e HepG2-NCodes two nuclear isoforms, TP53INP1α and TP53INP1β. It has been found that TP53INP1 present in non-malignant human pancreatic lesions, but significantly or completely lost in the majority of primary pancreatic ductal adenocarcinomas and absent in metastatic tumors. Similarly,TP53INPl-positive rate decreases with the progression of gastric cancer. TP53INP1 protein negativity is significantly associated with aggressive pathological phenotypes of gastric cancer. TP53INP1 is related to the apoptosis of gastric cancer cells. Ito Y et al investigated TP53INP1 expression by immunohistochemical in 81 cases of breast carcinoma. It was found that diffuse and intense TP53INP1 expression was observed in the normal mammary gland. Decreased TP53INP1 expression was found in 45 cases (55.6%) of breast carcinoma. Weng W et al found that the expression of TP53INP1 was downregulated in 28%(10/36 cases) of ESCC lesions. The expression of TP53INP1 in colon cancer tissue was also significantly decreased. These observations suggest that decreased expression of TP53INP1 may be a general feature of tumor progression. It was found that secreted protein acidic and rich in cysteine (SPARC) gene expression is up-regulated in normal pancreas in the TP53INP1-deficient animals, and in pancreatic intraepithelial neoplasia lesions in a mouse model of pancreatic adenocarcinoma. TP53INP1 transcriptionally blocks SPARC gene expression, and silencing of TP53INP1 increases cell migration in mouse embryonic fibroblasts and pancreatic cancer cells. It indicates TP53INP1 can work as a tumor suppressor by repressing tumor cell migration during metastasis. But recently TP53INP1 expression has been found to been enhanced in some cancers.Ito Y et al found 36 of the 38 (94.7%) anaplastic carcinomas expressed high levels of TP53INP1.And TP53INP1 expression has recently been found to be enhanced in prostate cancer cells after treatment with the proinflammatory mediators tumor necrosis factor a and interleukin 6,indicating that TP53INP1 overexpression could be involved in inflammation-mediated prostatic carcinogenesis. In this scenario, overexpression of TP53INP1 actually results in increased tumorigenesis,contradicting with the tumor suppressor characteristic of TP53INP1 as described invarious other cancers, suggesting a kind of tissue specific function.Perhaps,TP53INPlcan act either as a tumor suppressor gene or an Oncogene depending on the tissue type or the tumor microenvironment.Until now,the expression of TP53INP1, its relationship to clinicopathogical characteristics and prognostic value in hepatocellular carcinoma (HCC) have not been reported. In this study, we studied the effects of TP53INP1 on proliferation, apoptosis, invasion and metastasis of hepatocellular carcinoma from the tissue, cell and in vivo by using clinical samples, chronic viral overexpression and nude mice transplanted tumor model.Cell apoptosis and autophagy is in close relationship with tumor.They can interact with each other, which form complex influence on the development of tumor.It was showed that TP53INP1 is also able to interact with ATG8-family proteins and to induce autophagy-dependent cell death. But in hepatocellular carcinoma, Wether TP53INP1 can promote apoptosis through this pathway is not yet known, We will further do research on it.Methods1. The expressions of TP53INP1 in the cancer tissues and perinunor normal liver tissues:65 pairs of cancer tissues and perinunor normal liver tissues (>5cm) were the collected from the operating room, department of hepatobiliary surgery, Nanfang hospital.The TP53INP1 expression in hepatocellular carcinoma tissues was detected by using quantitative real-time PCR(qRT-PCR), Western blot and immune-histochemical staining (IHC). The relationship between the expression with clinical and pathological parameters such as age, gender, hepatitis infection, serum AFP level, tumor size, tumor number, pathological grading, liver cirrhosis, AJCC staging, vascular invasion, intrahepatic metastasis and prognosis were analysed.2. TP53INP1 expression in liver cell lines and HCC cell lines:a collection of human liver cell line hl7702, HCC cell line Huh-7, SMMC7721, HepG2, qRT-PCR were used to detect TP53INP1 expression in different cell lines.3.Construction of TP53INP1-lentiviral vector and packaged lentivirus:TP53INP1 target gene was amplificated by use of cDNA as template and lentiviral recombinant plasmid was constructed by linking enzyme digestion (PLVX-MCMV-ZSGREEN-PURO-TP53INP1, PLVX-MCMV-ZSGREEN-PURO as negative control), enzyme digestion was used to verify and sequencing.Lipo2000 transfection recombinant plasmids and packaging plasmids (phelperl.O, phelper 2.0) to 293T cells, and lentivirus was packaged.4.Develop a stably overexpression TP53INP1 cell lines:The lentivirus packaged was used to infect HepG2 cell. Puromycin culture medium was used to screen and qPCR was used to detect.5.TP53INP1 effects on cell proliferation:the overexpression TP53INP1 cell was examed by MTS assay on the cell growth rate.6.TP53INP1 effects on cell cycle:flow cytometry was used to exam the effect of overexpression TP53INP1 cell on cell cycle.7.TP53INP1 effects on cell invasion and metastasis:transwell assay was applied to test the effect of overexpressing TP53INP1 cells on invasion and metastasis.8.TP53INP1 effects on cells apoptosis:overexpression TP53INP1 cells apoptosis was analysed by flow cytometry.The percent of apoptotic cells were compared.9.Establish the nude mice orthotopic transplantation tumor model. Mice were purchased,fed and observed about one week. Each in the right side of the abdomen was inoculated liquid cell suspension after a week. Then they were fed after inoculation,tumor size was measured every one or two days, and tumor growth curve was plotted.10.TP53INP1 effects on autophagy:Transmission electron microscopy and Western blot were used to detect the effect of TP53INP1 cells on autophagy.11. Statistical assay:Using the statistical software SPSS13.0, the measurement data with the is χ±SD description. Paired t test was used for comparison of hepatocellular carcinoma and adjacent normal liver tissue. Cell proliferation, apoptosis, invasion metastasis by single factor variance analysis (ANOVA); nude mouse tumor volume compared with group multiple compared with LSD. Univariate and multivariate analyses were performed using the Kaplan meire and Cox proportional hazard model. There was significant difference as P<0.05.Resultsl.The relationship among the expression of TP53INP1 in hepatocellular carcinoma with its clinicopathological parameters and prognosis.(1) Expression of TP53INP1 in hepatocellular carcinoma tissuesThe experimental results by qRT-PCR show that TP53INP1 in hepatocellular carcinoma tissues and matched normal adjacent tissues relative expression quantity were 0.4103±0.03674 and 0.6851±0.05825, the difference was significant.Randomly selecting well differentiation,moderate differentiation, poorly differentiated hepatocellular carcinoma specimens for Western Blot experiment, β-actin as the internal reference. The results show that compared with adjacent normal liver tissue,various degrees of differentiation HCC tissues were low expression of TP53INP1 protein.Immunohistochemistry experiment results show that, TP53INP1 was mainly located in the cytoplasm of epithelial cells. Some nuclei were also stained for TP53INP1. In HCC tissues,Expression of TP53INP1 protein was positive in 30 cases whereas 35 cases of TP53INP1 protein expression was negative. In paracancerous tissues,60 cases of TP53INP1 protein expression was positive whereas 5 cases TP53INP1 protein expression was negative.the difference was significant(P=0.000).(2)The expression of TP53INP1 were significant different with,AJCC stage(P=0.014) and vascular invasion (P=0.024),but there were no difference with age,gender,hepatitis infection, serum AFP level, tumor size, tumor number, the degree of pathological differentiation, cirrhosis and liver metastasis(p>0.05).(3)In univariate analysis, the degree of pathological differentiation(RFS:P=0.003, HR95%CI=0.521[0.338-0.801];OS:P=0.002,HR95%CI=0.432[0.251-0.744]), AJCC stage(RFS:P=0.045, HR 95%CI=2.224[1.016-4.869]; OS:P=0.020,HR 95%CI=2.943[1.182-7.327]),vascularinvasion(RFS:P=0.004,HR95%CI=3.004[1.416-6.373];OS:P=0.001,HR95%CI=4.275[1.848-9.889])and the expression of TP53INP1 are the important factor related to RFS and OS. Multivariate analysis of risk ratio in Cox, vascular invasion(RFS:P=0.034, HR 95%CI=2.310[1.065-5.012]; OS:P=0.021,HR 95%CI=2.841[1.172-6.884])and the expression of TP53INP1(RFS:P=0.017, HR 95%CI= 2.284[1.157-4.511]; OS:P=0.032,HR 95%CI=2.680[1.087-6.608]are the independent prognostic factors of DFS and OS in patients with hepatocellular carcinoma. And the lower of the TP53INP1 expression level, the poorer of the prognosis.2.TP53INP1 expression in liver cell lines and HCC cell lines:Among HL7702, Huh-7, SMMC7721, HepG2 cell lines, the expression of HL7702 cell was the highest than the others by qPCR. Among HCC cell lines, the expression of Huh7 cells was the highest whereas in HepG2 cells TP53INP1 expression was the lowest.3.Construction of TP53INP1-lentiviral vector and packaged lentivirus:The recombinant lentiviral vector was analysed by enzyme digestion electrophoresis and sequencing.It proved that TP53INP1 overexpression vector was successfully constructed. The viral titer of the lentivirus packaged was determined by GFP.4. Harvest a overexpression TP53INP1 cell line:The infected hepatoma cells with packaged lentivirus was screened through Puromycin culture medium. It was showed by qPCR that transfection TP53INP1 overexpression of lentivirus stable cell lines of target gene expression level was significantly higher than that of the negative control group and blank control group. It also confirmed by fluorescencence microscopythat the obtained stable cell line can stably express GFP.The successful expression of TP53INP1 stable cell line was gained by the verification of qPCR and fluorescencence. The lowest expression cell line was selected to the next research.5.TP53INP1 gene inhibits the proliferation of hepatocellular carcinoma cells:The cell viability of 24h,48h,72h in TP53INP1 overexpression cell lines and control cell lines was detect by MTS assay.lt also confirmed TP53INP1 overexpression inhibits cell growth (day 2,3,4, P<0.05). The proliferation capacity between TP53INP1 overexpression cell line and the control cell line were significantly different after cells were cultured 24 hours.6. TP53INP1 prolongs cell cycle:It was showed by flow cytometry assay that The cell number of TP53INP1 overexpression HepG2 stable cell lines in the Gl phase was significantly higher than control cell line. The overexpression of TP53INP1 can prolong HepG2 cells from G1 phase into S phase, and then extend the cell cycle.7.TP53INP1 inhibits the invasion and metastasis of liver cancer cell:Transwell experiment showed that overexpression TP53INP1 cell strains through the cells of the chamber decreased significantly than blank group and negative control group (P<0.01,six independent experiments), indicating TP53INP1 suppress the cell invasion. 8. TP53INP1 promotes the apoptosis of hepatoma cells:It was showed by flowcytometry that TP53INP1 overexpression cell line early apoptotic cells (LR) and late apoptotic cells (UR) were more than those of the control group.And it confirmed that the expression TP53INP1 cell line apoptosis rate (UR+LR) was higher than that of blank group and negative control group.9.TP53ENP1 overexpression inhibits the growth of orthotopic transplantation tumor in nude mice:Nude mouse transplantation tumor experiment shows that the tumor size was gradually increased with overexpression TP53INP1 cell line, negative control group and blank group, which explaining that TP53INP1 expression significantly inhibit the growth of tumor.10. TP53INP1 promotes autophagy:It was showed by western blot that the expression of Beclinl and LC3-protein in TP53INP1 overexpression HepG2 cell lines was significantly higher than that of control cell lines, which indicated that TP53INP1 overpression promoted autophagy.And it was confirmed by transmission electron microscope that TP53INP1 overexpression cells were pyknotic nuclei and cells appear double membraned autophagosomes and lysosomes, mitochondrial cristae and dissolution of loose, ribosomal particles fuzzy distribution disorder. Whereas chromatin were loose and nuclear membrane were clear and prominent nucleoli, mitochondria arranged neatly in the control cells.lt confirmed that overexpression of TP53INP1 has obvious autophagosomes and lysosomes of cells, which promoting autophagy and apoptosis.Conclusion1. It is showed by Immunohistochemistry that TP53INP1 was mainly located in the cytoplasm of epithelial cells. Some nuclei were also stained for TP53INP1.TP53INP1 expression level is closely related to AJCC staging and vascular invasion, it has nothing to do with the other pathological parameters. Cox proportional hazards analysis shows that TP53INP1 is the independent prognostic factors of HCC.2.Real time fluorescencent quantitative PCR in the level of mRNA, Western blot and immunohistochemical experiments from the protein expression level detected in hepatic carcinoma and adjacent normal liver tissue, there are differences between the expression of TP53INP1.The expression of TP53INP1 in perinunor normal liver tissues is higher than in tumor tissues, suggesting that it may play a role in the process of tumor suppressor in hepatocellular carcinoma development.3. After the successful construction of TP53INP1 overexpression cell lines by the use of the lentiviral vector, The vitro cellular function tests such as cell proliferation, cell invasion, cell cycle and apoptosis detection were used to confirm that TP53INP1 can inhibit cancer cell growth and invasion,prolong cell cycle and promote apoptosis.4. After HepG2 cells overexpressing TP53INP1, dorsal subcutaneously transplanted tumor grew slowly and tumor mass became small. It indicated that TP53INP1 may inhibits tumor cell growth in nude mice. TP53INP1 negatively regulates invasion and metastasis of hepatocellular carcinoma cells.5.TP53INP1 is involved in the autophagy pathway, affecting autophagic apoptosis. |
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