免疫负调控分子TIPE2在肝细胞肝癌中的作用及其机制
摘要
研究目的
肝细胞肝癌是最常见的人类恶性肿瘤之一,在全世界范围内肝癌发病率位于世界第五位,死亡率位于世界第三位。肝细胞肝癌一个显著特点是容易发生侵袭和转移,是影响病人预后的重要因素。因此,寻找能抑制肝癌生长和转移的新分子,对肝癌的治疗具有十分重要的价值。
TIPE2是新发现的免疫负调控分子,是TNFAIP8家族的重要成员。目前关于TIPE2的研究主要集中在调控固有免疫和适应性免疫应答,维持免疫平衡。TIPE2在小鼠体内主要表达于免疫细胞,TIPE2-/-小鼠发生多器官炎症,表现为脾大,白细胞增多,对TLR和TCR过度敏感。TIPE2缺失还会加重LPS诱导的内毒素休克,实验性中风和动脉粥样硬化。人TIPE2表达水平在多种慢性炎症病人表达下调,如系统性红斑狼疮,慢性乙型肝炎,儿童哮喘等,并且与疾病进程呈负相关关系。我们在前期研究中发现人TIPE2的表达谱与小鼠的TIPE2不同,人TIPE2除表达于免疫细胞外,也表达于肝细胞等非免疫细胞,并且人TIPE2在肝细胞肝癌中表达下调或缺失,提示TIPE2可能在肝癌的发生发展中发挥重要作用。
为了明确TIPE2在人肝细胞肝癌发生发展中的作用,阐明其作用机制,为以TIPE2为靶点设计合成抗肿瘤药物提供研究基础。本文拟从以下几个方面进行研究：一、TIPE2对肝细胞肝癌恶性行为的研究；二、TIPE2抑制肝癌细胞侵袭转移和血管形成机制的研究；三、TIPE2对于肝癌细胞自噬的影响及其效应研究方法
一.TIPE2对肝细胞肝癌恶性行为的研究
1.免疫组化方法检测112例肝癌患者肝癌组织中TIPE2的表达,并且分析其与临床病理特征之间的相关性。
2.将Mock和TIPE2表达质粒分别转染入肝癌细胞系,用CCK8的方法检测细胞增殖,用平板集落形成实验检测克隆形成能力,用台盼蓝染色的方法检测细胞死亡,用Transwell的方法检测细胞迁移和侵袭,取肿瘤细胞条件培养上清培养内皮细胞,然后用CCK8的方法检测内皮细胞的增值,用小管形成实验观察内皮细胞环化。
3.建立裸鼠皮下肝癌移植瘤模型,分别给予Mock和TIPE2质粒治疗,治疗过程中根据肿瘤体积绘制肿瘤生长曲线,测量肿瘤体积和重量,同时取血清检测AFP含量。免疫组化的方法检测瘤体中TIPE2表达情况。
4.建立裸鼠肝脏原位移植瘤模型。取肝脏,肺等可疑的转移器官,HE染色观察转移灶情况,测量肿瘤体积和重量,取血清检测AFP含量。
二.TIPE2抑制肝癌侵袭转移和血管形成的机制
1.用western blot的方法检测四种肝癌细胞系(BEL-7402, HepG2, SMMC-7721, HCCLM3)中Rac1的表达。用Rac1的抑制剂NSC23766处理BEL-7402,然后用Transwell的方法检测细胞的迁移和侵袭。
2.免疫共沉淀方法检测Thp-1中内源性TIPE2能否与Racl结合。构建人TIPE2突变载体。将野生型TIPE2载体和突变载体分别转入BEL-7402细胞进行免疫共沉淀,检测外源性TIPE2能否与内源性Racl结合。用PAK-PBD pull down实验检测Racl的活性。
3.在TIPE2过表达体系中加入Racl抑制剂NSC23766或Racl的小干扰,用Transwell的方法检测肝癌细胞的迁移和侵袭。
4.将野生型TIPE2载体和突变载体分别转入肝癌细胞系,用Transwell的方法检测细胞的迁移和侵袭,CCK8的方法检测内皮细胞的增殖,用小管形成实验检测内皮细胞环化。
5.将野生型TIPE2载体和突变载体分别转入肝癌细胞系,用免疫荧光的方法检测微丝的聚合。用PCR和western blot的方法检测MMP9, uPA和VEGF的表达。
三.TIPE2对肝癌细胞自噬的影响及其效应
1.在TIPE2过表达体系中,用western blot的方法检测凋亡相关分子caspase3和PARP的变化。
2.在TIPE2过表达体系中,用western blot的方法检测自噬相关分子LC3B和P62的表达,用免疫荧光的方法检测自噬小体的形成。
3. TIPE2过表达体系中加入凋亡阻断剂Z-VAD,自噬抑制剂wortmanin, Rac1抑制剂NSC23766或ATG5小干扰,用CCK8的方法检测细胞存活。结果
一.TIPE2对肝细胞肝癌恶性行为的影响
1. TIPE2表达降低或缺失与肝癌的侵袭转移有关
我们课题组在前期研究中发现,相比于癌旁组织,TIPE2在肝癌组织中表达下调。为了阐明TIPE2在肝癌发生发展中的作用,我们详细分析了TIPE2表达与肝癌临床病理特征之间的关系。结果显示,TIPE2表达与性别、年龄、病理分级、血清AFP含量以及是否有肝硬化和乙型肝炎无关,但是与TNM分期有显著相关性。Ⅲ-Ⅳ期病人中TIPE2低表达或缺失的比例显著高于Ⅰ-Ⅱ期病人。提示我们TIPE2表达降低或缺失与肝癌的侵袭转移有关。
2. TIPE2抑制肝癌细胞在体外的生长,转移和血管形成
(1) TIPE2抑制肝癌细胞的生长和克隆形成能力
为了探究TIPE2对肝癌细胞生长的作用,我们选取两种肝癌细胞系BEL-7402和HepG2,瞬时转染Mock和TIPE2重组质粒,用CCK8的方法检测细胞增殖。结果发现,转染TIPE2后细胞生长明显减缓。克隆形成实验的结果显示,过表达TIPE2组肝癌细胞克隆形成数明显减少。表明TIPE2能够抑制肝癌细胞的生长和克隆形成能力。
(2) TIPE2促进饥饿诱导的细胞死亡
为了研究TIPE2对细胞生存的影响,我们采用台盼蓝染色的方法检测细胞死亡,发现转染TIPE2后细胞死亡率增加。说明TIPE2能够加速饥饿诱导的细胞死亡。
(3) TIPE2抑制肝癌细胞的迁移和侵袭能力
为了研究TIPE2对肝癌细胞迁移和侵袭的作用,我们采用Transwell的方法,结果发现,转染TIPE2后细胞迁移和侵袭的数量均明显降低。表明TIPE2能够抑制肝癌细胞的迁移和侵袭能力。
(4) TIPE2抑制肝癌细胞促血管形成能力
为了研究TIPE2对血管形成的影响,我们采用了两种方法-内皮细胞增殖实验和毛细血管形成实验。用肿瘤细胞条件培养上清(TCM)培养脐静脉内皮细胞(HUVEC),通过CCK8的方法检测HUVEC的活性。结果显示,来自Mock组的TCM能够维持HUVEC的生长,而用TIPE2组TCM培养的HUVEC随着时间延长细胞活力明显降低。毛细血管形成实验结果显示,来自Mock组的TCM促进HUVEC形成较多的管腔样结构,而用TIPE2组TCM培养的HUVEC比较分散,管腔样结构明显减少。以上结果表明TIPE2抑制肝癌细胞促血管形成能力。
3. TIPE2抑制肝细胞肝癌在体内的生长和转移
(1) TIPE2抑制裸鼠皮下肿瘤的生长
为了进一步研究TIPE2在体内的作用,我们对荷瘤的裸鼠给予外源性质粒治疗。结果显示,TIPE2抑制皮下肿瘤的生长。TIPE2组皮下瘤重量和体积以及血清中AFP的含量明显均低于Mock组。免疫组化结果显示,TIPE2组瘤体中TIPE2表达升高。HE染色结果显示,TIPE2组肿瘤组织相比于Mock组细胞形状更规则,分化程度更高。以上结果表明,TIPE2能够抑制裸鼠皮下瘤的生长。
(2) TIPE2抑制裸鼠肝脏原位瘤的生长和转移
为了模拟肝细胞肝癌原位生长的情况,我们构建了肝脏原位移植模型。结果显示,TIPE2组原位肿瘤的重量和体积以及血清AFP含量均低于Mock组。对可疑转移器官进行连续切片并进行组织病理分析,结果显示TIPE2组裸鼠发生肺转移比例降低,而且Mock组裸鼠有胰腺、膈肌和腹壁侵犯的情况,而TIPE2组则没有。说明TIPE2能够抑制肝脏原位肿瘤的生长和转移。
二.TIPE2抑制肝癌细胞侵袭转移和血管形成的机制
1. Racl的表达和活性与肝癌细胞的转移能力呈正相关
最新的研究显示小鼠TIPE2能与Racl结合并且抑制其活性,而Racl在肿瘤的发生发展中也起着十分重要的作用。因此,我们提出了假设,人TIPE2是否是通过Racl通路影响肝癌的侵袭转移?为了验证这一假设,我们首先用western blot的方法检测了四种肝癌细胞系中Racl的表达。结果发现所有肝癌细胞系均高表达Racl,并且与肝癌细胞系的转移能力呈正相关。接下来,我们用Racl的抑制剂NSC23766处理BEL-7402,然后用Transwell的方法检测细胞的迁移和侵袭,结果显示,NSC23766处理后细胞迁移和侵袭的数量明显降低,并且具有浓度依赖性。以上结果表明,Racl的表达和活性与肝癌细胞的转移能力呈正相关。
2. TIPE2能够与Racl结合并抑制其活性
为了进一步证实TIPE2与Racl的相互关系,我们用TIPE2高表达细胞系Thp-1进行免疫共沉淀,结果显示人内源性TIPE2能够与Racl结合。同时我们构建了人TIPE2突变载体,然后将野生型TIPE2和突变载体分别转入BEL-7402细胞进行免疫共沉淀。结果显示,野生型TIPE2能够与Racl结合,而突变的TIPE2与Racl的结合能力降低。接下来,我们用PAK-PBD pull down实验检测Racl的活性,结果显示转染野生型TIPE2后Racl的活性降低,而突变的TIPE2能逆转其抑制效应。以上结果表明人TIPE2也能与Racl结合并抑制其活性。
3. TIPE2通过Racl通路抑制肝癌细胞的迁移和侵袭
为了研究Racl在TIPE2调节肝癌迁移和侵袭中的作用,我们在TIPE2过表达体系中加入NSC23766抑制Racl的活性。结果发现,过表达TIPE2后细胞迁移和侵袭的数量明显降低,而NSC23766处理后Mock组和TIPE2之间的差异消失,同时我们用Racl的小干扰,得到了与Racl抑制剂类似的结果。接下来,我们将野生型TIPE2载体和突变载体分别转入肝癌细胞系,用Transwell的方法检测细胞的迁移和侵袭。结果发现,转染野生型TIPE2后细胞迁移侵袭数量均降低,而突变载体的抑制效应消失。以上结果均表明TIPE2抑制肝癌细胞的迁移和侵袭依赖于Racl通路。
4. TIPE2通过Racl通路抑制肝癌细胞促血管形成能力
为了研究Racl在TIPE2抑制血管形成中的作用,我们用TCM培养HUVEC,结果发现,来自野生型TIPE2组的上清能够显著抑制内皮细胞的增值和小管形成,来自突变载体转染组的上清则能够逆转这一抑制效应。以上结果表明,TIPE2抑制血管形成的作用依赖于Racl通路。
5. TIPE2抑制Racl下游的效应分子
(1) TIPE2通过Racl通路抑制微丝的聚合
为了进一步研究TIPE2抑制细胞迁移的机制,我们用免疫荧光的方法检测细胞微丝的聚合。Mock组细胞中微丝丰富、排列规则,并且有伪足形成,而转染TIPE2后微丝发生了明显的解聚,但是转染突变载体后这一效应得以逆转。说明TIPE2通过Racl通路抑制微丝聚合。
(2) TIPE2通过Racl通路抑制MMP9和uPA的表达
为了进一步探究TIPE2抑制细胞侵袭的机制,我们用real time PCR和western blot的方法检测MMP9和uPA的表达。结果发现,转染野生型TIPE2后MMP9和uPA的mRNA水平和蛋白水平均降低,而突变的TIPE2则丧失了抑制效应。说明TIPE2通路Racl通路抑制MMP9和uPA的表达。
(3) TIPE2通过Racl通路抑制VEGF的表达
为了进一步探究TIPE2抑制血管形成的机制,我们用RT-PCR和western blot的方法检测VEGF的表达。结果显示,野生型TIPE2抑制VEGF的表达,而突变的TIPE2抑制作用消失。说明TIPE2通过Rac1通路抑制VEGF的表达。
三.TIPE2对肝癌细胞自噬的影响及其效应
1. TIPE2通过凋亡非依赖的途径促进细胞死亡
我们在第一节的结果中提到TIPE2能够促进细胞死亡,但是这种死亡的具体形式尚不清楚。为了研究TIPE2对凋亡通路的影响,我们用western blot的方法检测凋亡相关分子的变化。结果显示转染TIPE2后caspase3和PARP的总量以及剪切体都没有明显的变化。CCK8结果显示TIPE2能够抑制细胞的活性,加入caspase抑制剂Z-VAD后TIPE2的抑制作用依然存在,说明TIPE2促进细胞的死亡可能不依赖于凋亡。
2. TIPE2促进肝癌细胞的自噬
为了研究TIPE2对自噬的影响,我们用western blot的方法检测自噬水平的变化。结果显示过表达TIPE2后自噬标志分子LC3BII水平升高,自噬底物P62水平降低。同时,我们用免疫荧光的方法检测自噬小体的形成,发现转染TIPE2后自噬小体的数量升高。说明TIPE2能够促进自噬的发生和自噬小体的形成。
3. TIPE2促进自噬性细胞死亡
为了研究自噬在TIPE2诱导细胞死亡中的作用,我们在TIPE2过表达体系中加入自噬抑制剂wortmanin,然后用CCK8的方法检测细胞活性。结果显示,过表达TIPE2后细胞活性降低,而加入wortmanin后Mock和TIPE2组之间的差异消失。ATG5的小干扰也得到了同样的结果。以上结果说明TIPE2诱导自噬性细胞死亡。
4. TIPE2通过Racl通路促进细胞死亡
为了探究Racl在TIPE2促进细胞死亡中的作用,我们在TIPE2过表达体系中加入Racl抑制剂NSC23766,然后用CCK8的方法检测细胞存活。结果显示,过表达TIPE2后细胞活性降低,加入Racl抑制剂NSC23766后Mock组和TIPE2组之间的差异消失。表明TIPE2通过Racl通路促进细胞死亡。
结论
1. TIPE2在人肝癌组织中表达降低或缺失与肝细胞肝癌的转移密切相关。
2. TIPE2在体内外抑制肝细胞肝癌的生长、转移和血管形成。
3. TIPE2通过Racl通路抑制细胞骨架的聚合,MMP9. uPA和VEGF的表达,进而抑制肝癌细胞的迁移,侵袭和促血管形成能力。
4. TIPE2促进自噬性细胞死亡,并且不依赖于凋亡,其机制可能是通过抑制Racl通路发挥作用。
创新性和意义
1.首次提出TIPE2与人肝细胞肝癌的关系,发现TIPE2在肝癌中表达降低或缺失与肝癌的转移有关,TIPE2在体内外均能抑制肝癌的生长和转移。为以TIPE2作为靶点设计肝癌治疗的药物提供理论依据和实验基础。
2.首次提出人TIPE2能够与Racl结合并抑制其活性,证实TIPE2通过Racl通路抑制肝癌的侵袭和转移,丰富了人TIPE2的功能和分子机制研究。
3.首次提出TIPE2与自噬的关系,明确了TIPE2促进自噬性细胞死亡而非凋亡,对于研究自噬和肿瘤的关系具有十分重要的意义。
局限性
1.扩大病例标本,对临床资料的相关性进行更加准确的分析。
2. TIPE2促进自噬的通路还需要进一步研究
Objective
Hepatocelluar carcinoma (HCC) is one of the most common cancers in the world. The morbidity and mortality of HCC were ranked fifth and third among all the cancers respectively. Lack of effective technique for HCC early diagnosis and curative therapy leads to extremely poor prognosis of patients. The primary characters of HCC are invasion and metastasis, accordingly the overall lifetime of HCC patients on last stage is only6months. Therefore, searching for mechanism of development and progression of HCC will provide new technology of HCC therapy.
TIPE2is a newly described immunity negative molecule and belongs to TIPE family. TIPE2maintain negatively both innate and adaptive immunity. It is preferentially expressed in immune organ and lymphoid tissue in mice. Its deficiency in mice leads to multi-organ inflammation, splenomegaly and premature death. TIPE2deficient animals are hypersensitive to septic shock, experimental stroke, and atherosclerosis. TIPE2is down-regulated in patients with chronic inflammatory diseases such as systemic lupus erythematosus (SLE) and hepatitis, and its expression inversely correlated with disease progression. Unlike murine TIPE2, human TIPE2is expressed in multiple non-hematopoietic cell type, including hepatocytes, indicating more functions of human TIPE2. Recently, our previous research is shown that TIPE2is down-regulated or completely lost in HCC compared with adjacent tissue. However, the role and underling mechanism of TIPE2in progression of HCC remain unclear.
In this study, we explore the effect of TIPE2on development and progression of HCC, and search for the mechanism.
Methods
Ⅰ. The effect of TIPE2on malignant phenotype of HCC
1. IHC was used to detect TIPE2level in tumor tissue of112HCC cases. The correlation between TIPE2and clinical pathological parameters was analyzed by wilcoxon test.
2. Mock and TIPE2plasmid was transfected into HCC cell. Cell proliferation was detected by CCK8. Colony formation capacity was detected by colony assay. Trypan blue staining was used for cell death and transwell assay for cell migration and invasion ability. And proliferation and tube formation of HUVEC were also detected.
3. To provide in vivo evidence that TIPE2suppresses HCC, we established subcutaneous xenograft tumor model and treated with Mock and TIPE2plasmid respectively. The size and weight of tumor and AFP in serum were detected.
4. We established orthotopic tumor in liver of mice. The size and weight of primary tumor were detected. All possible metastasizing visceral organs or tissues were processed for standard histopathological study.
Ⅱ. The mechanism of TIPE2on metastasis and angiogenesis of HCC
1. Western blot was applied to detect Racl expression in four different HCC cell lines. And cells pretreated with Racl inhibitor NSC23766were applied for transwell assay.
2. Co-IP was used to find interaction between endogenous TIPE2and Racl in Thp-1. Wild TIPE2and mutant TIPE2were transfected into HCC cells respectively and interaction between exogenous TIPE2and Racl was detected by co-IP. And activity of Racl was detected by PAK-PBD pull down assay.
3. Cells pretreated with NSC23766or siRNA for Rac1were overexpressed with TIPE2and applied for transwell assy.
4. HCC cell transfected with Wild and mutant TIPE2plasmids were applied for transwell assay. Proliferation and tube formation were detected also. Furthermore, MMP9, uPA and VEGF expression were detected by PCR and western blot.
III. The effect of TIPE2on autophagy and its function
(1) Apoptosis associated molecules (caspase3and PARP) were detected by western blot.
(2) Autophagy associated molecules (LC3BⅡ and P62) were detected by western blot and autophagosomes were detected by IF.
(3) Caspase inhibitor Z-VAD, autophagy inhibitor wortmannin, Racl inhibitor NSC23766and siRNA for ATG5was added to cells and cell proliferation was detected by CCK8.
Results
I. The effect of TIPE2on malignant phenotype of HCC
1. Loss or reduction of TIPE2expression in HCC was associated with metastasis
In order to explore effect of TIPE2on HCC, we first analyze association between TIPE2expression and clinical pathological characters. We found that there was no significant correlation of TIPE2to age, gender, cirrhosis, hepatitis B, serum AFP and pathological grade. However, TIPE2was related to TNM stage. The results indicated that loss or reduction of TIPE2expression in HCC was associated with metastasis.
2. TIPE2suppressed HCC cell growth, metastasis and angiogenesis in vitro
(1) TIPE2suppressed proliferation and colony formation of HCC cells
In order to explore the effect of TIPE2on proliferation of HCC cells, we detected cell viability using CCK8. We found that overexpression of TIPE2suppressed cell viability from48h and colony formation numbers compared with Mock. The results indicated that TIPE2inhibited HCC cell proliferation and colony formation capacity.
(2) TIPE2accelerated cell death of HCC cells
To determine the effect of TIPE2on cell death, we used trypan blue staining assay and found that overexpression of TIPE2increased cell death rate. It was indicated that TIPE2accelerated cell death induced by serum free medium.
(3) TIPE2suppressed HCC cell migration and invasion ability.
To determine effect of TIPE2on HCC cell metastasis, we used transwell assay. Overexpression of TIPE2decreased HCC cell migration and invasion numbers. The results indicated that TIPE2suppressed HCC cell migration and invasion ability.
(4) TIPE2inhibited angiogenesis of HCC
To determine effect of TIPE2on angiogenesis of HCC, we used HUVEC proliferation assay and tube formation assay. Supermatant from HCC cell with TIPE2suppressed HUVEC proliferation and capillary formation. The results indicated that TIPE2inhibited angiogenesis of HCC.
3. TIPE2suppressed HCC growth and metastasis in vivo
(1) TIPE2suppressed subcutaneous tumor growth.
To determine effect of TIPE2on tumor in vivo, we established subcutaneous tumor model. We found that treatment of TIPE2plasmid attenuated the tumor growth. The tumor volume and weight and AFP level of TIPE2group were decreased compared with Mock group. The results showed that TIPE2suppressed subcutaneous tumor growth.
(2) TIPE2suppressed orthotopic tumor growth and metastasis.
To determine effect of TIPE2on metastasis of HCC, we established orthotopic tumor model. The tumor volume and weight and AFP level of TIPE2group were decreased compared with Mock group. Significantly, the lung metastasis of TIPE2group reduced. Tumor had invaded into pancreas, diaphragm and abdominal wall in Mock group but not in TIPE2group. All the results demonstrate that TIPE2markedly suppressed metastasis of orthotopic tumor.
II. The mechanism of TIPE2on invasion, metastasis and angiogenesis of HCC
1. Expression and activity of Racl were correlated with metastasis
In order to explore mechanism of inhibitory effect of TIPE2on HCC metastasis, we detected Racl expression in different HCC cell lines. Racl was upregulated in HCC cell line and was positively correlated with metastasis capacity. Racl inhibitor NSC23766suppressed HCC cell migration and invasion ability in dose dependent manner. All the results indicated that expression and activity of Racl was correlated with HCC metastasis.
2. TIPE2could bind to Racl and blocked its activity
To further determine the association between TIPE1and Rac1, we used co-IP assay. We found that endogenous TIPE2could bind to Racl in Thp-1cell. Furthermore, wild type TIPE2and mutant TIPE2were transfected into HCC cell. We found that exogenous wild type TIPE2could bind to Racl while the mutant lost the ability. PAK-PBD pull down assay showed that wild type TIPE2decreased the Rac1activity while mutant TIPE2reversed the inhibitory effect. All the results indicated that human TIPE2could bind to Rac1and block its activity.
3. TIPE2suppressed HCC cell migration and invasion via Racl pathway
In order to confirm association between Rac1and inhibitory effect of TIPE2on metastasis, we added NSC23766into HCC cell. Pretreatment with Racl inhibitor NSC23766or siRNA diminished the difference between Mock and TIPE2group. And siRNA for Racl had similar effect. And we also found that wild type TIPE2inhibited cell migration and invasion while the mutant one reversed the inhibitory effect. The results indicated that TIPE2inhibited HCC cell migration and invasion via Racl pathway.
4. TIPE2suppressed angiogenesis via Racl pathway
Furthermore we detected association between Rac1and inhibitory effect of TIPE2on angiogenesis. The TCM from wild type TIPE2cell decreased proliferation and tube formation of HUVEC, however the TCM from mutant reversed the inhibitory effect. It was indicated that TIPE2suppressed angiogenesis via Rac1pathway.
5. The molecular targets of TIPE2on metastasis and angiogenesis
(1) TIPE2inhibited F-actin polymerization via Racl pathway
To determine mechanism that TIPE2inhibited migration, we detected microfilament using IF. The results showed that wild TIPE2decreased F-actin polymerization while the mutant TIPE2inversed the inhibitory effect. It was indicated that TIPE2inhibited F-actin polymerization via Rac1pathway.
(2) TIPE2inhibited MMP9and uPA expression via Racl pathway
To determine mechanism that TIPE2inhibited invasion, we detected MMP9and uPA expression by real time PCR and western blot. The results showed that wild TIPE2decreased MMP9and uPA expression, however the mutant TIPE2inversed the inhibitory effect. It was indicated that TIPE2inhibited MMP9and uPA expression via Rac1pathway.
(3) TIPE2inhibited VEGF expression via Racl pathway
To determine mechanism that TIPE2inhibited angiogenesis, we detected VEGF expression by RT-PCR and western blot. The results showed that wild TIPE2decreased VEGF expression, however the mutant TIPE2inversed the inhibitory effect. It was indicated that TIPE2inhibited VEGF expression via Racl pathway.
III. The effect of TIPE2on autophagy and its function
1. TIPE2enhanced cell death via apoptosis-independent pathway
In the previous results, we proposed that TIPE2increased cell death. But the underlying mechanism was unknown. We detected apoptosis associated molecules by western blot. The results showed that TIPE2had no effect on the activity of caspase3and PARP. CCK8results showed that TIPE2decreased cell viability while Z-VAD could not reverse it. The results indicated that TIPE2enhanced cell death independent of apoptosis.
2. TIPE2enhanced autophagy
In order to determine effect of TIPE2on autophagy, we detected autophagy associated molecule by western blot. Overexpression of TIPE2increased LC3BII and decreased P62level. And autophagosomes numbers increased in TIPE2group by IF. The results indicated that TIPE2enhanced autophagy.
3. TIPE2induced autophagic cell death
To determine the effect of TIPE2induced autophagy, we detected cell viability via CCK8. We found that TIPE2decreased cell viability and wortmanin and siRNA for ATG5diminished the inhibitory effect of TIPE2. The results indicated that TIPE2induced autophagic cell death.
4. TIPE2induced cell death via Racl pathway
To determine effect of Rac1on TIPE2induced cell death, we used Rac1inhibitor NSC23766. TIPE2decreased cell viability while Racl NSC23766diminished the difference induced by TIPE2. The results indicated that TIPE2induced cell death via Rac1pathway.
Conclusions
1. Loss or reduction of TIPE2expression in tumor tissue of HCC is associated with metastasis.
2. TIPE2inhibits growth and metastasis of HCC in vivo and in vitro.
3. TIPE2suppresses migration, invasion and angiogenesis of HCC cell mediated by F-actin polymerization and MMP9, uPA and VEGF expression via Racl pathway.
4. TIPE2induced autophagic cell death via Racl pathway.
Originality and Significance
1. For the first time, we demonstrate the correlation between TIPE2and HCC. We find that TIPE2is lost or decreased in HCC and associated with TNM stage.TIPE2inhibits growth and metastasis of HCC in vivo and in vitro. The results are significant for new technology of HCC therapy.
2. Significantly, human TIPE2binds with Racl and block its activity. The finding provides new understanding of human TIPE2function.
3. TIPE2induced autophagic cell death that is important for tumor suppression.
Limitations
1. More cases are needed to confirm the relationship between TIPE2expression and clinopathological features.
2. The signaling pathway that TIPE2enhanced autophagy should be studied in the future.
肝细胞肝癌是最常见的人类恶性肿瘤之一,在全世界范围内肝癌发病率位于世界第五位,死亡率位于世界第三位。肝细胞肝癌一个显著特点是容易发生侵袭和转移,是影响病人预后的重要因素。因此,寻找能抑制肝癌生长和转移的新分子,对肝癌的治疗具有十分重要的价值。
TIPE2是新发现的免疫负调控分子,是TNFAIP8家族的重要成员。目前关于TIPE2的研究主要集中在调控固有免疫和适应性免疫应答,维持免疫平衡。TIPE2在小鼠体内主要表达于免疫细胞,TIPE2-/-小鼠发生多器官炎症,表现为脾大,白细胞增多,对TLR和TCR过度敏感。TIPE2缺失还会加重LPS诱导的内毒素休克,实验性中风和动脉粥样硬化。人TIPE2表达水平在多种慢性炎症病人表达下调,如系统性红斑狼疮,慢性乙型肝炎,儿童哮喘等,并且与疾病进程呈负相关关系。我们在前期研究中发现人TIPE2的表达谱与小鼠的TIPE2不同,人TIPE2除表达于免疫细胞外,也表达于肝细胞等非免疫细胞,并且人TIPE2在肝细胞肝癌中表达下调或缺失,提示TIPE2可能在肝癌的发生发展中发挥重要作用。
为了明确TIPE2在人肝细胞肝癌发生发展中的作用,阐明其作用机制,为以TIPE2为靶点设计合成抗肿瘤药物提供研究基础。本文拟从以下几个方面进行研究：一、TIPE2对肝细胞肝癌恶性行为的研究；二、TIPE2抑制肝癌细胞侵袭转移和血管形成机制的研究；三、TIPE2对于肝癌细胞自噬的影响及其效应研究方法
一.TIPE2对肝细胞肝癌恶性行为的研究
1.免疫组化方法检测112例肝癌患者肝癌组织中TIPE2的表达,并且分析其与临床病理特征之间的相关性。
2.将Mock和TIPE2表达质粒分别转染入肝癌细胞系,用CCK8的方法检测细胞增殖,用平板集落形成实验检测克隆形成能力,用台盼蓝染色的方法检测细胞死亡,用Transwell的方法检测细胞迁移和侵袭,取肿瘤细胞条件培养上清培养内皮细胞,然后用CCK8的方法检测内皮细胞的增值,用小管形成实验观察内皮细胞环化。
3.建立裸鼠皮下肝癌移植瘤模型,分别给予Mock和TIPE2质粒治疗,治疗过程中根据肿瘤体积绘制肿瘤生长曲线,测量肿瘤体积和重量,同时取血清检测AFP含量。免疫组化的方法检测瘤体中TIPE2表达情况。
4.建立裸鼠肝脏原位移植瘤模型。取肝脏,肺等可疑的转移器官,HE染色观察转移灶情况,测量肿瘤体积和重量,取血清检测AFP含量。
二.TIPE2抑制肝癌侵袭转移和血管形成的机制
1.用western blot的方法检测四种肝癌细胞系(BEL-7402, HepG2, SMMC-7721, HCCLM3)中Rac1的表达。用Rac1的抑制剂NSC23766处理BEL-7402,然后用Transwell的方法检测细胞的迁移和侵袭。
2.免疫共沉淀方法检测Thp-1中内源性TIPE2能否与Racl结合。构建人TIPE2突变载体。将野生型TIPE2载体和突变载体分别转入BEL-7402细胞进行免疫共沉淀,检测外源性TIPE2能否与内源性Racl结合。用PAK-PBD pull down实验检测Racl的活性。
3.在TIPE2过表达体系中加入Racl抑制剂NSC23766或Racl的小干扰,用Transwell的方法检测肝癌细胞的迁移和侵袭。
4.将野生型TIPE2载体和突变载体分别转入肝癌细胞系,用Transwell的方法检测细胞的迁移和侵袭,CCK8的方法检测内皮细胞的增殖,用小管形成实验检测内皮细胞环化。
5.将野生型TIPE2载体和突变载体分别转入肝癌细胞系,用免疫荧光的方法检测微丝的聚合。用PCR和western blot的方法检测MMP9, uPA和VEGF的表达。
三.TIPE2对肝癌细胞自噬的影响及其效应
1.在TIPE2过表达体系中,用western blot的方法检测凋亡相关分子caspase3和PARP的变化。
2.在TIPE2过表达体系中,用western blot的方法检测自噬相关分子LC3B和P62的表达,用免疫荧光的方法检测自噬小体的形成。
3. TIPE2过表达体系中加入凋亡阻断剂Z-VAD,自噬抑制剂wortmanin, Rac1抑制剂NSC23766或ATG5小干扰,用CCK8的方法检测细胞存活。结果
一.TIPE2对肝细胞肝癌恶性行为的影响
1. TIPE2表达降低或缺失与肝癌的侵袭转移有关
我们课题组在前期研究中发现,相比于癌旁组织,TIPE2在肝癌组织中表达下调。为了阐明TIPE2在肝癌发生发展中的作用,我们详细分析了TIPE2表达与肝癌临床病理特征之间的关系。结果显示,TIPE2表达与性别、年龄、病理分级、血清AFP含量以及是否有肝硬化和乙型肝炎无关,但是与TNM分期有显著相关性。Ⅲ-Ⅳ期病人中TIPE2低表达或缺失的比例显著高于Ⅰ-Ⅱ期病人。提示我们TIPE2表达降低或缺失与肝癌的侵袭转移有关。
2. TIPE2抑制肝癌细胞在体外的生长,转移和血管形成
(1) TIPE2抑制肝癌细胞的生长和克隆形成能力
为了探究TIPE2对肝癌细胞生长的作用,我们选取两种肝癌细胞系BEL-7402和HepG2,瞬时转染Mock和TIPE2重组质粒,用CCK8的方法检测细胞增殖。结果发现,转染TIPE2后细胞生长明显减缓。克隆形成实验的结果显示,过表达TIPE2组肝癌细胞克隆形成数明显减少。表明TIPE2能够抑制肝癌细胞的生长和克隆形成能力。
(2) TIPE2促进饥饿诱导的细胞死亡
为了研究TIPE2对细胞生存的影响,我们采用台盼蓝染色的方法检测细胞死亡,发现转染TIPE2后细胞死亡率增加。说明TIPE2能够加速饥饿诱导的细胞死亡。
(3) TIPE2抑制肝癌细胞的迁移和侵袭能力
为了研究TIPE2对肝癌细胞迁移和侵袭的作用,我们采用Transwell的方法,结果发现,转染TIPE2后细胞迁移和侵袭的数量均明显降低。表明TIPE2能够抑制肝癌细胞的迁移和侵袭能力。
(4) TIPE2抑制肝癌细胞促血管形成能力
为了研究TIPE2对血管形成的影响,我们采用了两种方法-内皮细胞增殖实验和毛细血管形成实验。用肿瘤细胞条件培养上清(TCM)培养脐静脉内皮细胞(HUVEC),通过CCK8的方法检测HUVEC的活性。结果显示,来自Mock组的TCM能够维持HUVEC的生长,而用TIPE2组TCM培养的HUVEC随着时间延长细胞活力明显降低。毛细血管形成实验结果显示,来自Mock组的TCM促进HUVEC形成较多的管腔样结构,而用TIPE2组TCM培养的HUVEC比较分散,管腔样结构明显减少。以上结果表明TIPE2抑制肝癌细胞促血管形成能力。
3. TIPE2抑制肝细胞肝癌在体内的生长和转移
(1) TIPE2抑制裸鼠皮下肿瘤的生长
为了进一步研究TIPE2在体内的作用,我们对荷瘤的裸鼠给予外源性质粒治疗。结果显示,TIPE2抑制皮下肿瘤的生长。TIPE2组皮下瘤重量和体积以及血清中AFP的含量明显均低于Mock组。免疫组化结果显示,TIPE2组瘤体中TIPE2表达升高。HE染色结果显示,TIPE2组肿瘤组织相比于Mock组细胞形状更规则,分化程度更高。以上结果表明,TIPE2能够抑制裸鼠皮下瘤的生长。
(2) TIPE2抑制裸鼠肝脏原位瘤的生长和转移
为了模拟肝细胞肝癌原位生长的情况,我们构建了肝脏原位移植模型。结果显示,TIPE2组原位肿瘤的重量和体积以及血清AFP含量均低于Mock组。对可疑转移器官进行连续切片并进行组织病理分析,结果显示TIPE2组裸鼠发生肺转移比例降低,而且Mock组裸鼠有胰腺、膈肌和腹壁侵犯的情况,而TIPE2组则没有。说明TIPE2能够抑制肝脏原位肿瘤的生长和转移。
二.TIPE2抑制肝癌细胞侵袭转移和血管形成的机制
1. Racl的表达和活性与肝癌细胞的转移能力呈正相关
最新的研究显示小鼠TIPE2能与Racl结合并且抑制其活性,而Racl在肿瘤的发生发展中也起着十分重要的作用。因此,我们提出了假设,人TIPE2是否是通过Racl通路影响肝癌的侵袭转移?为了验证这一假设,我们首先用western blot的方法检测了四种肝癌细胞系中Racl的表达。结果发现所有肝癌细胞系均高表达Racl,并且与肝癌细胞系的转移能力呈正相关。接下来,我们用Racl的抑制剂NSC23766处理BEL-7402,然后用Transwell的方法检测细胞的迁移和侵袭,结果显示,NSC23766处理后细胞迁移和侵袭的数量明显降低,并且具有浓度依赖性。以上结果表明,Racl的表达和活性与肝癌细胞的转移能力呈正相关。
2. TIPE2能够与Racl结合并抑制其活性
为了进一步证实TIPE2与Racl的相互关系,我们用TIPE2高表达细胞系Thp-1进行免疫共沉淀,结果显示人内源性TIPE2能够与Racl结合。同时我们构建了人TIPE2突变载体,然后将野生型TIPE2和突变载体分别转入BEL-7402细胞进行免疫共沉淀。结果显示,野生型TIPE2能够与Racl结合,而突变的TIPE2与Racl的结合能力降低。接下来,我们用PAK-PBD pull down实验检测Racl的活性,结果显示转染野生型TIPE2后Racl的活性降低,而突变的TIPE2能逆转其抑制效应。以上结果表明人TIPE2也能与Racl结合并抑制其活性。
3. TIPE2通过Racl通路抑制肝癌细胞的迁移和侵袭
为了研究Racl在TIPE2调节肝癌迁移和侵袭中的作用,我们在TIPE2过表达体系中加入NSC23766抑制Racl的活性。结果发现,过表达TIPE2后细胞迁移和侵袭的数量明显降低,而NSC23766处理后Mock组和TIPE2之间的差异消失,同时我们用Racl的小干扰,得到了与Racl抑制剂类似的结果。接下来,我们将野生型TIPE2载体和突变载体分别转入肝癌细胞系,用Transwell的方法检测细胞的迁移和侵袭。结果发现,转染野生型TIPE2后细胞迁移侵袭数量均降低,而突变载体的抑制效应消失。以上结果均表明TIPE2抑制肝癌细胞的迁移和侵袭依赖于Racl通路。
4. TIPE2通过Racl通路抑制肝癌细胞促血管形成能力
为了研究Racl在TIPE2抑制血管形成中的作用,我们用TCM培养HUVEC,结果发现,来自野生型TIPE2组的上清能够显著抑制内皮细胞的增值和小管形成,来自突变载体转染组的上清则能够逆转这一抑制效应。以上结果表明,TIPE2抑制血管形成的作用依赖于Racl通路。
5. TIPE2抑制Racl下游的效应分子
(1) TIPE2通过Racl通路抑制微丝的聚合
为了进一步研究TIPE2抑制细胞迁移的机制,我们用免疫荧光的方法检测细胞微丝的聚合。Mock组细胞中微丝丰富、排列规则,并且有伪足形成,而转染TIPE2后微丝发生了明显的解聚,但是转染突变载体后这一效应得以逆转。说明TIPE2通过Racl通路抑制微丝聚合。
(2) TIPE2通过Racl通路抑制MMP9和uPA的表达
为了进一步探究TIPE2抑制细胞侵袭的机制,我们用real time PCR和western blot的方法检测MMP9和uPA的表达。结果发现,转染野生型TIPE2后MMP9和uPA的mRNA水平和蛋白水平均降低,而突变的TIPE2则丧失了抑制效应。说明TIPE2通路Racl通路抑制MMP9和uPA的表达。
(3) TIPE2通过Racl通路抑制VEGF的表达
为了进一步探究TIPE2抑制血管形成的机制,我们用RT-PCR和western blot的方法检测VEGF的表达。结果显示,野生型TIPE2抑制VEGF的表达,而突变的TIPE2抑制作用消失。说明TIPE2通过Rac1通路抑制VEGF的表达。
三.TIPE2对肝癌细胞自噬的影响及其效应
1. TIPE2通过凋亡非依赖的途径促进细胞死亡
我们在第一节的结果中提到TIPE2能够促进细胞死亡,但是这种死亡的具体形式尚不清楚。为了研究TIPE2对凋亡通路的影响,我们用western blot的方法检测凋亡相关分子的变化。结果显示转染TIPE2后caspase3和PARP的总量以及剪切体都没有明显的变化。CCK8结果显示TIPE2能够抑制细胞的活性,加入caspase抑制剂Z-VAD后TIPE2的抑制作用依然存在,说明TIPE2促进细胞的死亡可能不依赖于凋亡。
2. TIPE2促进肝癌细胞的自噬
为了研究TIPE2对自噬的影响,我们用western blot的方法检测自噬水平的变化。结果显示过表达TIPE2后自噬标志分子LC3BII水平升高,自噬底物P62水平降低。同时,我们用免疫荧光的方法检测自噬小体的形成,发现转染TIPE2后自噬小体的数量升高。说明TIPE2能够促进自噬的发生和自噬小体的形成。
3. TIPE2促进自噬性细胞死亡
为了研究自噬在TIPE2诱导细胞死亡中的作用,我们在TIPE2过表达体系中加入自噬抑制剂wortmanin,然后用CCK8的方法检测细胞活性。结果显示,过表达TIPE2后细胞活性降低,而加入wortmanin后Mock和TIPE2组之间的差异消失。ATG5的小干扰也得到了同样的结果。以上结果说明TIPE2诱导自噬性细胞死亡。
4. TIPE2通过Racl通路促进细胞死亡
为了探究Racl在TIPE2促进细胞死亡中的作用,我们在TIPE2过表达体系中加入Racl抑制剂NSC23766,然后用CCK8的方法检测细胞存活。结果显示,过表达TIPE2后细胞活性降低,加入Racl抑制剂NSC23766后Mock组和TIPE2组之间的差异消失。表明TIPE2通过Racl通路促进细胞死亡。
结论
1. TIPE2在人肝癌组织中表达降低或缺失与肝细胞肝癌的转移密切相关。
2. TIPE2在体内外抑制肝细胞肝癌的生长、转移和血管形成。
3. TIPE2通过Racl通路抑制细胞骨架的聚合,MMP9. uPA和VEGF的表达,进而抑制肝癌细胞的迁移,侵袭和促血管形成能力。
4. TIPE2促进自噬性细胞死亡,并且不依赖于凋亡,其机制可能是通过抑制Racl通路发挥作用。
创新性和意义
1.首次提出TIPE2与人肝细胞肝癌的关系,发现TIPE2在肝癌中表达降低或缺失与肝癌的转移有关,TIPE2在体内外均能抑制肝癌的生长和转移。为以TIPE2作为靶点设计肝癌治疗的药物提供理论依据和实验基础。
2.首次提出人TIPE2能够与Racl结合并抑制其活性,证实TIPE2通过Racl通路抑制肝癌的侵袭和转移,丰富了人TIPE2的功能和分子机制研究。
3.首次提出TIPE2与自噬的关系,明确了TIPE2促进自噬性细胞死亡而非凋亡,对于研究自噬和肿瘤的关系具有十分重要的意义。
局限性
1.扩大病例标本,对临床资料的相关性进行更加准确的分析。
2. TIPE2促进自噬的通路还需要进一步研究
Objective
Hepatocelluar carcinoma (HCC) is one of the most common cancers in the world. The morbidity and mortality of HCC were ranked fifth and third among all the cancers respectively. Lack of effective technique for HCC early diagnosis and curative therapy leads to extremely poor prognosis of patients. The primary characters of HCC are invasion and metastasis, accordingly the overall lifetime of HCC patients on last stage is only6months. Therefore, searching for mechanism of development and progression of HCC will provide new technology of HCC therapy.
TIPE2is a newly described immunity negative molecule and belongs to TIPE family. TIPE2maintain negatively both innate and adaptive immunity. It is preferentially expressed in immune organ and lymphoid tissue in mice. Its deficiency in mice leads to multi-organ inflammation, splenomegaly and premature death. TIPE2deficient animals are hypersensitive to septic shock, experimental stroke, and atherosclerosis. TIPE2is down-regulated in patients with chronic inflammatory diseases such as systemic lupus erythematosus (SLE) and hepatitis, and its expression inversely correlated with disease progression. Unlike murine TIPE2, human TIPE2is expressed in multiple non-hematopoietic cell type, including hepatocytes, indicating more functions of human TIPE2. Recently, our previous research is shown that TIPE2is down-regulated or completely lost in HCC compared with adjacent tissue. However, the role and underling mechanism of TIPE2in progression of HCC remain unclear.
In this study, we explore the effect of TIPE2on development and progression of HCC, and search for the mechanism.
Methods
Ⅰ. The effect of TIPE2on malignant phenotype of HCC
1. IHC was used to detect TIPE2level in tumor tissue of112HCC cases. The correlation between TIPE2and clinical pathological parameters was analyzed by wilcoxon test.
2. Mock and TIPE2plasmid was transfected into HCC cell. Cell proliferation was detected by CCK8. Colony formation capacity was detected by colony assay. Trypan blue staining was used for cell death and transwell assay for cell migration and invasion ability. And proliferation and tube formation of HUVEC were also detected.
3. To provide in vivo evidence that TIPE2suppresses HCC, we established subcutaneous xenograft tumor model and treated with Mock and TIPE2plasmid respectively. The size and weight of tumor and AFP in serum were detected.
4. We established orthotopic tumor in liver of mice. The size and weight of primary tumor were detected. All possible metastasizing visceral organs or tissues were processed for standard histopathological study.
Ⅱ. The mechanism of TIPE2on metastasis and angiogenesis of HCC
1. Western blot was applied to detect Racl expression in four different HCC cell lines. And cells pretreated with Racl inhibitor NSC23766were applied for transwell assay.
2. Co-IP was used to find interaction between endogenous TIPE2and Racl in Thp-1. Wild TIPE2and mutant TIPE2were transfected into HCC cells respectively and interaction between exogenous TIPE2and Racl was detected by co-IP. And activity of Racl was detected by PAK-PBD pull down assay.
3. Cells pretreated with NSC23766or siRNA for Rac1were overexpressed with TIPE2and applied for transwell assy.
4. HCC cell transfected with Wild and mutant TIPE2plasmids were applied for transwell assay. Proliferation and tube formation were detected also. Furthermore, MMP9, uPA and VEGF expression were detected by PCR and western blot.
III. The effect of TIPE2on autophagy and its function
(1) Apoptosis associated molecules (caspase3and PARP) were detected by western blot.
(2) Autophagy associated molecules (LC3BⅡ and P62) were detected by western blot and autophagosomes were detected by IF.
(3) Caspase inhibitor Z-VAD, autophagy inhibitor wortmannin, Racl inhibitor NSC23766and siRNA for ATG5was added to cells and cell proliferation was detected by CCK8.
Results
I. The effect of TIPE2on malignant phenotype of HCC
1. Loss or reduction of TIPE2expression in HCC was associated with metastasis
In order to explore effect of TIPE2on HCC, we first analyze association between TIPE2expression and clinical pathological characters. We found that there was no significant correlation of TIPE2to age, gender, cirrhosis, hepatitis B, serum AFP and pathological grade. However, TIPE2was related to TNM stage. The results indicated that loss or reduction of TIPE2expression in HCC was associated with metastasis.
2. TIPE2suppressed HCC cell growth, metastasis and angiogenesis in vitro
(1) TIPE2suppressed proliferation and colony formation of HCC cells
In order to explore the effect of TIPE2on proliferation of HCC cells, we detected cell viability using CCK8. We found that overexpression of TIPE2suppressed cell viability from48h and colony formation numbers compared with Mock. The results indicated that TIPE2inhibited HCC cell proliferation and colony formation capacity.
(2) TIPE2accelerated cell death of HCC cells
To determine the effect of TIPE2on cell death, we used trypan blue staining assay and found that overexpression of TIPE2increased cell death rate. It was indicated that TIPE2accelerated cell death induced by serum free medium.
(3) TIPE2suppressed HCC cell migration and invasion ability.
To determine effect of TIPE2on HCC cell metastasis, we used transwell assay. Overexpression of TIPE2decreased HCC cell migration and invasion numbers. The results indicated that TIPE2suppressed HCC cell migration and invasion ability.
(4) TIPE2inhibited angiogenesis of HCC
To determine effect of TIPE2on angiogenesis of HCC, we used HUVEC proliferation assay and tube formation assay. Supermatant from HCC cell with TIPE2suppressed HUVEC proliferation and capillary formation. The results indicated that TIPE2inhibited angiogenesis of HCC.
3. TIPE2suppressed HCC growth and metastasis in vivo
(1) TIPE2suppressed subcutaneous tumor growth.
To determine effect of TIPE2on tumor in vivo, we established subcutaneous tumor model. We found that treatment of TIPE2plasmid attenuated the tumor growth. The tumor volume and weight and AFP level of TIPE2group were decreased compared with Mock group. The results showed that TIPE2suppressed subcutaneous tumor growth.
(2) TIPE2suppressed orthotopic tumor growth and metastasis.
To determine effect of TIPE2on metastasis of HCC, we established orthotopic tumor model. The tumor volume and weight and AFP level of TIPE2group were decreased compared with Mock group. Significantly, the lung metastasis of TIPE2group reduced. Tumor had invaded into pancreas, diaphragm and abdominal wall in Mock group but not in TIPE2group. All the results demonstrate that TIPE2markedly suppressed metastasis of orthotopic tumor.
II. The mechanism of TIPE2on invasion, metastasis and angiogenesis of HCC
1. Expression and activity of Racl were correlated with metastasis
In order to explore mechanism of inhibitory effect of TIPE2on HCC metastasis, we detected Racl expression in different HCC cell lines. Racl was upregulated in HCC cell line and was positively correlated with metastasis capacity. Racl inhibitor NSC23766suppressed HCC cell migration and invasion ability in dose dependent manner. All the results indicated that expression and activity of Racl was correlated with HCC metastasis.
2. TIPE2could bind to Racl and blocked its activity
To further determine the association between TIPE1and Rac1, we used co-IP assay. We found that endogenous TIPE2could bind to Racl in Thp-1cell. Furthermore, wild type TIPE2and mutant TIPE2were transfected into HCC cell. We found that exogenous wild type TIPE2could bind to Racl while the mutant lost the ability. PAK-PBD pull down assay showed that wild type TIPE2decreased the Rac1activity while mutant TIPE2reversed the inhibitory effect. All the results indicated that human TIPE2could bind to Rac1and block its activity.
3. TIPE2suppressed HCC cell migration and invasion via Racl pathway
In order to confirm association between Rac1and inhibitory effect of TIPE2on metastasis, we added NSC23766into HCC cell. Pretreatment with Racl inhibitor NSC23766or siRNA diminished the difference between Mock and TIPE2group. And siRNA for Racl had similar effect. And we also found that wild type TIPE2inhibited cell migration and invasion while the mutant one reversed the inhibitory effect. The results indicated that TIPE2inhibited HCC cell migration and invasion via Racl pathway.
4. TIPE2suppressed angiogenesis via Racl pathway
Furthermore we detected association between Rac1and inhibitory effect of TIPE2on angiogenesis. The TCM from wild type TIPE2cell decreased proliferation and tube formation of HUVEC, however the TCM from mutant reversed the inhibitory effect. It was indicated that TIPE2suppressed angiogenesis via Rac1pathway.
5. The molecular targets of TIPE2on metastasis and angiogenesis
(1) TIPE2inhibited F-actin polymerization via Racl pathway
To determine mechanism that TIPE2inhibited migration, we detected microfilament using IF. The results showed that wild TIPE2decreased F-actin polymerization while the mutant TIPE2inversed the inhibitory effect. It was indicated that TIPE2inhibited F-actin polymerization via Rac1pathway.
(2) TIPE2inhibited MMP9and uPA expression via Racl pathway
To determine mechanism that TIPE2inhibited invasion, we detected MMP9and uPA expression by real time PCR and western blot. The results showed that wild TIPE2decreased MMP9and uPA expression, however the mutant TIPE2inversed the inhibitory effect. It was indicated that TIPE2inhibited MMP9and uPA expression via Rac1pathway.
(3) TIPE2inhibited VEGF expression via Racl pathway
To determine mechanism that TIPE2inhibited angiogenesis, we detected VEGF expression by RT-PCR and western blot. The results showed that wild TIPE2decreased VEGF expression, however the mutant TIPE2inversed the inhibitory effect. It was indicated that TIPE2inhibited VEGF expression via Racl pathway.
III. The effect of TIPE2on autophagy and its function
1. TIPE2enhanced cell death via apoptosis-independent pathway
In the previous results, we proposed that TIPE2increased cell death. But the underlying mechanism was unknown. We detected apoptosis associated molecules by western blot. The results showed that TIPE2had no effect on the activity of caspase3and PARP. CCK8results showed that TIPE2decreased cell viability while Z-VAD could not reverse it. The results indicated that TIPE2enhanced cell death independent of apoptosis.
2. TIPE2enhanced autophagy
In order to determine effect of TIPE2on autophagy, we detected autophagy associated molecule by western blot. Overexpression of TIPE2increased LC3BII and decreased P62level. And autophagosomes numbers increased in TIPE2group by IF. The results indicated that TIPE2enhanced autophagy.
3. TIPE2induced autophagic cell death
To determine the effect of TIPE2induced autophagy, we detected cell viability via CCK8. We found that TIPE2decreased cell viability and wortmanin and siRNA for ATG5diminished the inhibitory effect of TIPE2. The results indicated that TIPE2induced autophagic cell death.
4. TIPE2induced cell death via Racl pathway
To determine effect of Rac1on TIPE2induced cell death, we used Rac1inhibitor NSC23766. TIPE2decreased cell viability while Racl NSC23766diminished the difference induced by TIPE2. The results indicated that TIPE2induced cell death via Rac1pathway.
Conclusions
1. Loss or reduction of TIPE2expression in tumor tissue of HCC is associated with metastasis.
2. TIPE2inhibits growth and metastasis of HCC in vivo and in vitro.
3. TIPE2suppresses migration, invasion and angiogenesis of HCC cell mediated by F-actin polymerization and MMP9, uPA and VEGF expression via Racl pathway.
4. TIPE2induced autophagic cell death via Racl pathway.
Originality and Significance
1. For the first time, we demonstrate the correlation between TIPE2and HCC. We find that TIPE2is lost or decreased in HCC and associated with TNM stage.TIPE2inhibits growth and metastasis of HCC in vivo and in vitro. The results are significant for new technology of HCC therapy.
2. Significantly, human TIPE2binds with Racl and block its activity. The finding provides new understanding of human TIPE2function.
3. TIPE2induced autophagic cell death that is important for tumor suppression.
Limitations
1. More cases are needed to confirm the relationship between TIPE2expression and clinopathological features.
2. The signaling pathway that TIPE2enhanced autophagy should be studied in the future.
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