【作者】 邓燕；

【导师】 刘思德；

【作者基本信息】 南方医科大学 ， 消化内科（专业学位）， 2016， 博士

【摘要】 背景和目的肝细胞癌(HCC)是常见的恶性肿瘤之一,每年大约有75万的新发病例及接近70万的死亡病例。虽然手术切除是治疗肝癌的最佳治疗方案,但是很多的肝癌患者初诊时即发现肿瘤转移,而失去了根治的机会,即使是行根治性切除术,其术后复发率仍然很高。肝癌的转移与切除术后的复发仍是决定预后的主要障碍,因此,对肝癌侵袭转移的研究一直是肝癌研究关注的重点。肝癌的侵袭与转移是一个涉及多种因子的参与十分复杂的过程。肝癌细胞侵袭与转移的能力主要是由其基因型决定,促癌基因的激活与抑癌基因的失活均可导致肝癌细胞向侵袭转移表型转化。肿瘤蛋白p53诱导核蛋白1(TP53INP1)定位于人染色体8q22,具有5个外显子和超过2万个碱基对。TP53INP1在压力因素诱导下或在应激因子作用下会在组织中大量表达,其编码两种蛋白异构体,分别为TP53INP1α和TP53INP1β。研究发现,在非恶性胰腺病变中,存在TP53INP1表达；但在原发性胰腺导管腺癌和转移性肿瘤中,TP53INP1的表达明显减少或者缺失。与之一致,TP53INP1的表达随着胃癌的进展而显著降低,表明TP53INP1与胃癌的侵袭性病理表型显著相关,并与胃癌细胞凋亡相关。Ito Y等对81例乳腺癌及癌旁正常肝组织行免疫组化检查。结果发现正常乳腺组织中可观察到TP53INP1的表达。81例乳腺癌中,45例(55.6%)TP53INP1表达下降。Weng W等发现36例食管癌中,10例TP53INP1表达下调。结肠癌组织中TP53INP1的表达也明显降低。这些观察结果表明,TP53INP1表达降低可能是肿瘤进展的一般特征。另有研究表明,在正常胰腺TP53INP1缺失的动物,以及胰腺癌小鼠模型中胰腺上皮内瘤样病变时,富含半胱氨酸的酸性蛋白(SPARC)基因的表达上调。而TP53INP1转录能够阻止SPARC基因的表达,在小鼠胚胎成纤维细胞和胰腺癌细胞中沉默TP53INP1后可增加细胞迁移。这提示TP53INP1能够抑制肿瘤细胞迁移。但是近期研究发现,TP53INP1在少数肿瘤组织中为高表达。Ito Y等发现38例甲状腺未分化癌中,有36例(94.7%)高表达TP53INP1。Giusiano S等使用炎症介质肿瘤坏死因子α和白细胞介素6治疗后,前列腺癌细胞中TP53INP1表达增强。这提示TP53INP1过度表达可能参与炎症介导的前列腺癌。在这种情况下,TP53INP1的过表达实际上增强了其致癌作用,这与之在许多其它肿瘤中的抑癌特性相互矛盾。TP53INP1是否作为抑癌基因亦或促癌基因可能与组织类型或者肿瘤微环境相关。关于TP53INP1在原发性肝癌中的表达情况,其表达与临床病理特征及生存预后的关系,笔者进行了文献检索,暂未检索到公开发表的相关文章。本实验从临床标本、TP53INP1过表达慢病毒细胞转染以及裸鼠原位移植瘤模型等几个方面入手进行研究,在组织水平、细胞水平以及动物实验中探讨了TP53INP1对肝癌发生、侵袭转移及细胞凋亡的影响。细胞凋亡、自噬与肿瘤的关系十分密切,二者之间能够发生相互作用,从而对肿瘤的发展产生复杂的影响。有研究表明,在人骨肉瘤U20S细胞中,TP53INP1通过与ATG8家族蛋白相互作用,诱导自噬过程,从而促进细胞凋亡。但在肝细胞癌中,TP53INP1能否通过此种途径促进肝癌细胞凋亡目前尚未可知,本文将加以研究探讨。材料和方法1.检测TP53INP1在癌旁正常组织和肝癌组织中的表达：收集南方医院肝胆外科手术室保存的65例肝癌患者的组织标本。每例患者分别取肝癌组织及癌旁正常肝组织(>5cm)标本,运用实时定量PCR、western迹实验、免疫组化法检测TP53INP1在肝癌组织的表达情况,及其与临床病理参数(年龄、性别、有无肝炎感染、血清AFP水平、肿瘤大小、肿瘤数量、病理分化程度、肝硬化、AJCC分期、有无血管浸润、有无肝内转移)以及预后的关系。2.TP53INP1在正常肝细胞株及肝癌细胞株中的表达：收集人正常肝细胞株HL7702,人肝癌细胞株Huh-7、SMMC7721、HepG2,应用qRT-PCR方法检测TP53INP1在不同细胞株中的表达情况。3.构建TP53INP1-慢病毒载体并包装慢病毒：用人cDNA作模板扩增TP53INP1目的基因,经酶切连接构建慢病毒重组质粒(PLVX-MCMV-ZSGREEN-PURO-TP53INP1,PLVX-MCMV-ZSGREEN-PURO为阴性对照),酶切验证并测序。Lipo2000转染重组质粒及包装质粒(pHelper1.0、 pHelper 2.0)至293T细胞,包装慢病毒。4.建立TP53INP1稳定过表达细胞株：用包装的慢病毒感染HepG2细胞,Puromycin培养基筛选,qPCR检查和荧光观察进行验证。5. TP53INP1对肝癌细胞增殖的影响：MTS法检测肝癌细胞稳定过表达TP53INP1对细胞的增长速度的影响。6. TP53INP1对肝癌细胞周期的影响：流式细胞技术检测过表达TP53INP1细胞对细胞周期的影响。7.TP53INP1对肝癌细胞侵袭转移的影响：Transwell实验检测过表达TP53INP1细胞对侵袭转移能力的影响。8.TP53INP1对细胞凋亡的影响：流式细胞技术分析凋亡细胞比例,探讨细胞过表达TP53INP1对细胞凋亡影响。9.建立裸鼠移植瘤模型：小鼠购进后,正常饲养观察一周,在每只小鼠腹部右侧接种细胞悬液,接种后正常饲养,每隔一到二天测量一次肿瘤的大小,绘制成肿瘤增长曲线。10.TP53INP1影响自噬：采用自噬相关蛋白Western blot检测以及透视电镜观察过表达TP53INP1细胞对细胞自噬的影响。11.统计学分析：采用SPSS13.0统计软件,计量资料用χ±SD描述,配对t检验用于比较肝癌和癌旁正常肝组织。细胞增殖、凋亡、侵袭转移采用单因素方差分析(one-way ANOVA);裸鼠肿瘤体积的比较采用组间多重比较LSD法；采用Kaplan-Meire和Cox比例风险模型进行单因素和多因素分析。以P<0.05为差异具有显著性。结果1.肝癌组织中TP53INP1的表达情况及其与临床病理参数、预后的关系：(1)TP53INP 1在肝癌组织中的表达情况实时荧光定量PCR的实验结果表明,TP53INP1在肝癌组织的表达量为0.4103±0.03674,癌旁正常肝组织中的表达量0.6851±0.05825,两者相比,差异具有显著性(p=0.0001)。随机选取不同分化程度的肝癌患者组织标本,分组行Western Blot实验,β-actin作为内参,结果表明,TP53INP1在高分化、中分化及低分化等不同分化程度肝癌组织中的表达均较癌旁正常肝组织低,差异具有显著性。免疫组织化学实验结果表明,]TP53INP1在肝癌组织和癌旁正常肝组织的细胞质与细胞核中均有表达,细胞质中表达强于细胞核。65例肝癌组织标本中TP53INP1蛋白表达阳性为30例,TP53INP1蛋白表达阴性为35例；癌旁正常肝组织中TP53INP1蛋白表达阳性为60例,阴性仅5例,差异具有显著性(P=0.000)。(2)TP53INP1的表达与AJCC分期(P=0.014)、血管浸润(P=0.024)存在显著差异,而与年龄、性别、有无肝炎感染、血清AFP水平、肿瘤大小、肿瘤数量、病理分化程度、肝硬化、有无肝内转移等参数无显著性差异(P>0.05)。(3)单因素分析中,病理分化程度(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 分期(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))、血管浸润(RFS:P= 0.004,HR95%CI=3.004(1.416-6.373);OS:P=0.001,HR95%CI=4.275(1.848-9.889))和TP53INP1表达水平(RFS:P=0.005,HR 95%CI= 2.604(1.345-5.039);OS:P=0.005,HR95%CI=3.403(1.436-8.063))是RFS和OS相关的重要因素。Cox比例风险多因素分析中,血管浸润(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))和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)表达水平是影响肝癌患者DFS和OS的独立预后因素。且TP53INP1表达水平越低,预后越差。2.TP53INP1在正常肝细胞株及肝癌细胞株中的表达：qPCR证实在HL7702、Huh-7、SMMC7721、HepG2四种细胞株中,人正常肝细胞株HL7702较肝癌细胞株表达量最高：肝癌细胞株中,Huh7细胞株表达量最高,HepG2细胞株TP53INP1表达量最低。3.构建TP53INP1-慢病毒载体并包装慢病毒：慢病毒重组载体经酶切电泳及测序分析,证明过表达TP53INP1载体成功构建。包装的慢病毒通过GFP进行病毒滴度测定。4.成功建立TP53INP1过表达细胞株：包装好的慢病毒感染后的肝癌细胞经Puromycin培养基筛选,通过qPCR检测结果显示转染TP53INP1过表达慢病毒的稳定细胞株目的基因的表达水平显著高于阴性对照组和空白对照组。通过荧光显微镜观察也证实,获得稳定的细胞株能够稳定表达GFP。证实已成功获得稳定细胞株,选择TP53INP1表达最低的细胞株进行下一步研究。5. TP53INP1抑制肝癌细胞增殖：MTS分别检TP53INP1过表达细胞株和对照细胞株培养24h、48h、72h后的细胞活力。实验证实TP53INP1过表达抑制细胞生长(第2,3,4天,P<0.05),细胞培养24小时后TP53INP1过表达细胞株与其对照细胞株增殖能力有显著性差异。6. TP53INP1延长肝癌细胞周期：流式细胞技术检测显示,TP53INP1过表达HepG2稳定细胞株处于G1期的细胞数明显比对照细胞株多,表明过表达TP53INP1可延长HepG2细胞从G1期进入S期,进而延长细胞周期。7.TP53INP1抑制肝癌细胞侵袭转移：Transwell实验显示,TP53INP1过表达的细胞株穿过小室的细胞比空白组和阴性对照组明显减少(P<0.01,6次独立实验),表明TP53INP1抑制细胞侵袭。8.TP53INP1促进肝癌细胞凋亡：流式细胞技术检测显示,TP53INP1过表达细胞株早期凋亡细胞(LR)及晚期凋亡细胞(UR)均较对照组凋亡细胞多。TP53INP1过表达细胞株的总细胞凋亡率(UR+LR)也明显高于空白组和阴性对照组。9. TP53INP1过表达抑制裸鼠肿瘤生长：裸鼠成瘤实验显示,TP53INP1过表达细胞株形成的肿瘤最小,阴性对照组次之,空白组最大,说明过表达TP53INP1对肿瘤的生长有明显抑制作用。10.TP53INP1促进细胞自噬：Western blot检测显示在TP53INP1过表达HepG2细胞株中beclinl和LC3-Ⅱ蛋白的表达量明显较对照细胞株多,表明TP53INP1过表达促进细胞自噬。透视电镜显示TP53INP1过表达细胞株细胞核固缩,细胞内出现双层膜自噬体及溶酶体,线粒体嵴疏松溶解,核糖体颗粒模糊分布紊乱。对照细胞株染色质松散,核膜清晰,核仁明显,线粒体排列整齐。TP53INP1过表达细胞株有明显的自噬体和溶酶体,促进细胞自噬性凋亡。结论1.免疫组织化学方法检测表明TP53INP1在肝脏细胞的细胞质与细胞核中均有表达,其表达主要位于细胞质。TP53INP1表达水平与AJCC分期及血管浸润密切相关,与其他病理参数无关。通过Cox风险比例分析发现,TP53INP1是影响肝癌RFS以及OS的独立预后因素。2.实时荧光定量PCR从nRNA水平、免疫蛋白印迹及免疫组化实验从蛋白表达水平检测显示TP53INP1表达在肝癌和癌旁正常肝组织之间存在差异,TP53INP1在癌旁正常肝组织中高表达,肝癌细胞中低表达,提示其可能在肝癌发展的过程中发挥抑癌作用。3.使用慢病毒载体成功构建TP53INP1过表达细胞株后,通过细胞增殖、细胞侵袭、细胞周期及细胞凋亡检测等体外细胞学功能实验,证实TP53INP1抑制肝癌细胞生长及侵袭,延长细胞周期,促进细胞凋亡。4.HepG2细胞过表达TP53INP1后,裸鼠背侧皮下移植成瘤生长缓慢、瘤块变小,提示TP53INP1抑制裸鼠肿瘤细胞的生长,说明TP53INP1负向调控肝癌细胞的侵袭转移能力。5.TP53INP1通过参与自噬过程,促进自噬性凋亡。更多还原

【Abstract】 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.更多还原