DcR3在食管鳞状细胞癌组织中的表达及相关机制研究
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摘要
背景
食管癌是人类八种最常见的恶性肿瘤之一。在全世界每年新增的30万病例中,约70 %发生在我国,并且其发病率仍呈上升趋势。食管癌总体预后不佳,总的2年和5年生存率分别为35%～42%和15%～24%。食管癌主要分为食管鳞状细胞癌(ESCC)和食管腺癌,而前者占了90%以上。因此,研究ESCC的发病机制,寻找其新的诊断和治疗靶点,已经成为提高食管癌预防和治疗效果的当务之急。在肿瘤的发生、发展中有一对矛盾贯穿始终,那就是机体的免疫监视和肿瘤的免疫逃逸。而诱导肿瘤特异性的细胞毒性T淋巴细胞(CTL)反应和瘤细胞自身生长停滞或凋亡是机体用以清除肿瘤细胞的主要手段。LIGHT及FasL介导的凋亡是体内最常见的凋亡模式,在免疫调节和肿瘤的免疫监视中起着重要作用。DcR3是TNFRSF的一个新成员。研究发现,DcR3通过竞争性结合可以阻断经由其配体FasL、LIGHT以及TL1A所介导的肿瘤细胞的凋亡;同时DcR3还可抑制免疫应答中T细胞的增殖并影响DCs的分化和成熟,并抑制CD4+T细胞在混合淋巴细胞反应(MLR)中的增殖;此外,DcR3还可抑制T细胞的趋化效应,降低T细胞与抗原提呈细胞间的相互作用。研究显示,DcR3 mRNA低表达于正常人体组织中的胃、脊髓、淋巴结、肺、脾、结肠组织及多数造血细胞中,而高表达于胃肠道恶性肿瘤、乳腺癌、结肠癌、肺癌、胶质细胞瘤和淋巴瘤等多种恶性肿瘤中。研究证实,高表达DcR3是肿瘤细胞逃避机体免疫监视和杀伤的一个重要机制,其表达水平同多种肿瘤的进展程度、转移情况、对化疗的抵抗性以及不良预后有明显的相关性。因此,作为一种候选的肿瘤标志物和基因治疗标靶,DcR3目前已经成为了肿瘤诊断和治疗相关研究的热点。研究DcR3 mRNA和蛋白在人ESCC细胞系和ESCC病人组织标本中的表达情况并分析DcR3的表达与疾病的侵袭、转移、复发乃至预后间的关联性,将有助于评价DcR3作为ESCC的临床及预后的分子标志的可能性;而探讨其表达调控的可能机制则将有助于ESCC肿瘤基因治疗靶点的筛选。因此,关于DcR3在ESCC病人中的高表达及其相关机制的研究对于该疾病的防治有相当重要的意义。
目的
研究DcR3基因在ESCC病人肿瘤组织和相应的远癌组织中的表达情况,分析DcR3基因的表达与病人临床病理特征之间的关系,为评价DcR3作为ESCC的临床及预后的分子标志提供实验依据;检测DcR3基因启动子和编码区的多态性位点在重庆地区汉族ESCC人群中的基因型和等位基因频率,探讨DcR3启动子多态性与ESCC患病风险之间的关联,筛查与ESCC肿瘤组织中DcR3基因高表达相关联的SNP位点,为进一步研究和分析影响其表达调控的分子机制奠定基础;检测DcR3基因启动子区的甲基化状态差异,探讨ESCC肿瘤组织DcR3启动子甲基化状态是否参与其表达调控;考察DcR3 RNAi对ESCC肿瘤细胞的生长能力、侵袭能力和凋亡等方面的影响,初步探讨DcR3在人ESCC细胞发生和发展过程中可能作用和机制。
方法
1.收集109例ESCC肿瘤组织和对应的远癌组织标本,通过半定量逆转录聚合酶链反应(Semi-quantitative RT-PCR)检测其中DcR3 mRNA表达情况;
2.运用免疫组织化学检测52例ESCC病人肿瘤组织中DcR3蛋白表达情况并行半定量分析;
3.选取DcR3基因启动子和编码区四个多态性位点(-369G/T、-323A/C、-321C/T、147C/T),通过PCR扩增产物测序的方法了解这些多态位点在重庆地区正常人群中的频率分布,检测上述多态位点在ESCC人群中的基因型和等位基因频率;
4.选择4对DcR3 mRNA表达状态有明显差异的癌及相对应的远癌组织标本,利用重亚硫酸盐修饰后测序法(BSP)检测DcR3基因启动子区的甲基化状态;
5.构建基于miR-30的、靶向DcR3的慢病毒干扰载体;
6.用干扰载体、包装质粒pMD2G及包膜质粒psAX2共同转染293FT细胞进行病毒包装;
7.通过流式细胞仪(FACS)感染后的KYSE150细胞,获得DcR3 RNAi的ESCC细胞模型;
8.利用Real-time PCR和Western Blotting检测DcR3 RNAi后瘤细胞上DcR3的表达情况;
9.绘制体外培养的感染细胞、对照细胞和亲本细胞的生长曲线;
10.通过Transwell侵袭小室实验检测DcR3 RNAi与LIGHT-Fc协同作用对瘤细胞体外侵袭能力的影响;
11.运用Tunel试剂盒检测DcR3 RNAi与LIGHT-Fc协同作用对瘤细胞凋亡的影响。
结果
1. ESCC肿瘤标本中DcR3 mRNA表达率为73.4%,而远癌组织为47.7%(p<0.01);分析发现DcR3 mRNA的表达与病人的性别、年龄、部位和组织分化程度没有明显的相关性,而与肿瘤组织的外侵程度(p<0.05)、区域淋巴结转移情况(p<0.05)和临床TNM分期(p<0.05)有较强的相关性;
2. ESCC肿瘤组织中DcR3蛋白的表达强弱与mRNA表达基本一致,DcR3的过表达率为28.8%;DcR3的过表达与ESCC病人区域淋巴结转移(p<0.05)和TNM分期(p<0.05)有明显的相关性;
3.重庆地区汉族人群中-369G/T、-323A/C、-321C/T三个位点不存在多态性;而147C/T位点等位基因C的频率为43.7%,略高于中国汉族人(39.0%)和日本人(40.0%)。147C/T多态位点是与重庆地区汉族人群ESCC易感性相关联的一个风险位点,而且该多态位点的等位基因型C具有显性遗传效应,携带等位基因C的基因型CC和CT的ESCC发病风险显著增加(p<0.05)。但该多态位点与ESCC肿瘤组织中DcR3蛋白的异常高表达无明显的相关性;
4. DcR3阳性的肿瘤组织和DcR3阴性的远癌组织,基因的启动子区的甲基化状态相同,即基因启动子区所有的CpG位点都呈非甲基化状态;
5.成功设计并构建了基于miR-30的、靶向DcR3的慢病毒干扰载体;包装获得的病毒上清在体外能有效地感染人KYSE150细胞;
6.经FACS分选,Western Blotting检测证实载体pPRIME-DcR3.3i能有效地抑制瘤细胞DcR3的蛋白表达,抑制率为82.6%;
7. DcR3 RNAi对KYSE150细胞的体外生长无明显影响;
8. DcR3 RNAi与LIGHT-Fc协同能有效地抑制瘤细胞的体外侵袭能力;
9. DcR3 RNAi明显促进了LIGHT-Fc对KYSE150细胞的凋亡诱导。
结论
ESCC肿瘤组织中DcR3在mRNA和蛋白质水平较之对应的远癌组织存在明显的高表达,其高表达与肿瘤的区域淋巴结转移情况和临床TNM分期有较强的相关性。重庆地区汉族人群中147C/T位点是与ESCC易感性相关联的一个风险位点,该位点的等位基因型C具有显性遗传效应;ESCC病人肿瘤组织中DcR3的异常高表达与该基因CpG岛的甲基化状态异常无关; DcR3 RNAi对肿瘤细胞的体外生长没有直接的抑制效应,但能与LIGHT-Fc协同,有效地抑制瘤细胞的体外侵袭能力并促进对瘤细胞的凋亡诱导;DcR3对于ESCC是一个潜在的肿瘤生物标记。
Background: Esophageal cancer is one of the eight most popular malignant tumors in human. Among 300,000 new diagnosed cases every year in the world, seventy percent were in China and the disease incidence still increases recently. One of the main problems of esophageal cancer is that long-term survival is very low, the totally 2-year survival rate is 35%-42% and only 15%-24% for 5-year survival. Esophageal cancer can be characterized into ESCC (esophageal squamous cell carcinoma) and esophageal adenocarcinoma, while ninty percent disease cases are ESCC. So it is very urgent to study the pathogenesis of ESCC and develop new target for diagnosis and treatment.
As we known, the balance between the immunological surveillance of host and the immune escape of tumor is tightly regulated during the tumorigenesis and tumor development. Inducing the tumor-specific CTL response and/or the apotosis of tumor cells are two important methods to eliminate tumor cells. LIGHT and FasL are two members of TNF super family (TNFRSF). They are the most common pathway for inducing the apotosis in vivo, and play essential role in immune regulation and the tumor immune monitoring. DcR3 is a new member of TNFRSF, which can competitively bind to Fas, LIGHT and TL1A and then inhibite the apotosis of tumor cells induced by these three molecular. In the other hand, during the immune response, DcR3 can inhibit the proliferation of T cells and the differentiation and maturation of dendritic cells (DCs), especially the proliferation of CD4+ T cells in MLR. Besides, DcR3 can also inhibit the chemotaxis of T cells and reduce the interact between T cells and antigen processing cells.
Previous studies showed that DcR3 mRNA expression level was very low in many normal human tissues, such as stomach, spinal cord, lymph node, lung, spleen, colon tissue and most hematopoietic cells, but contrarily, in many kinds of malignant tumor, including gastrointestinal cancer, breast cancer, carcinoma of colon, lung cancer, spongiocytoma and lymphoma, DcR3 mRNA expression level was very high. It was reported that overexpression of DcR3 was an important mechanism for tumor cells escaping from immune surveillance and destruction. In many different kinds of cancer, the expression level of DcR3 is clearly related with the progress of carcinogenesis, cancer metastasis, the resistance to chemotherapy and the prognosis of tumor. So, DcR3, as a candidate cancer biomarker and target for cancer gene therapy, becomes a very hot point in cancer research. Studying the expression of DcR3 on the mRNA and protein level in ESCC cell strains and patient biopsies, exploring the relationship between DcR3 expression and the invasion, metastasis, recurrence and prognosis of tumor are very helpful to evaluate the possibility of using DcR3 as a molecular marker in ESCC’s clinical diagnose and prognosis prediction. In the meantime, investigating the regulation mechanism behind DcR3 expression can help us develop a new gene therapy approach to treat ESCC. These facts suggested that studying the expression of DcR3 and how it is regulated are very important in the prevention and treatment of ESCC.
Aims: To identify the expression of DcR3 gene in ESCC patient’s samples and analyze its possible correlation between DcR3 expression and their clinicopathologic features. The polymorphic sites frequencies of DcR3 promoter and coding region in Chongqing normal population and ESCC patients would to be detected, in order to address the possible correlation between polymorphism of DcR3 promoter and ESCC risk, and screen the SNP sites associated with DcR3 high expression. For exploring whether the the methylation was involved in the regulation of DcR3 expression, the methylation difference in promoter region of DcR3 was to be tested. In addition, the effects of DcR3 RNAi on ESCC tumor metastasis, invasion and immune escape were to be detected, and the biological behavior and the mechanism of DcR3 in the progression of human ESCC cells were also to be investigated.
Methods: 1. Semi-quantitative RT-PCR assay was utilized to detect the expression of DcR3 mRNA in 109 ESCC tumor tissues and corresponding normal tissues far away from tumor.
2. Immunohistochemistry and semi-quantitative analysis were performed to test DcR3 protein expressions from 52 ESCC patients.
3. DcR3 promotor and four polymorphic sites (-369G/T、-323A/C、-321C/T、147C/T) in coding region were selected, these polymorphic sites frequencies in Chongqing normal population and their frequencies in ESCC patients were studied with PCR sequencing to address the possible correlation between polymorphism of DcR3 promoter and ESCC risk.
4. Based on their significant difference of DcR3 expression with each other, four pairs of sample from the tumor tissues and corresponding normal tissues far away from tumor were choosed, and their methylation status of the CpG in the promoter region of DcR3 were detected by bisulfite sequencing assay(BSA).
5. A DcR3 targeted RNA interfering lentivirus vector based on a new miR30 like was constructed to inhibit the expression of DcR3 in esophageal squamous cell line KYSE150.
6. The verified recombined vector was co-transfect with pMD2g and psAX2 into 293FT packing cell line to produce the virus.
7. ESCC cell model of DcR3 RNAi was obtained by FACS.
8. The DcR3 expression level of infected cells were evaluated by Real-time PCR and Western blotting.
9. The growth curves of infected cells, controlled cells and wild cells were drawn.
10. The effects of DcR3 RNAi collaborated with hLIGHT-Fc on invasion activity of infected ESCC cells were studied by Transwell loculus assay.
11. The effects of DcR3 RNAi collaborated with hLIGHT-Fc on apoptosis sensitivity of infected ESCC cells were investigated by Tunel assay.
Results: 1. The expression of DcR3 mRNA in 109 ESCC tumor tissues was 73.4% and 47.7% in the corresponding normal tissues far away from tumor. The expression of DcR3 mRNA was no close relation with gender, age, localization and tissue differential degree of patients, but strong related with tumor infiltration (p<0.05), regional lymph node metastasis (p<0.05) and clinical TNM stage (p<0.05).
2. DcR3 protein expression was consistent with DcR3 mRNA expression. The overexpression rate of DcR3 was 28.8%, and there was a correlation between DcR3 overexpression and regional lymph node metastasis (p<0.05) and clinical TNM stage (p<0.05).
3. Polymorphism was found only existe at 147C/T, instead of -369G/T、-323A/C or -321 C/T, on Chongqing Han population. The minor allel frequence (MAF) was 43.7%, which was a little bit higher than the whole Chinese Han population(39.0%) and Janpense population(40.0%). 147C/T polymorphism was a risk site of susceptibility to ESCC in Chongqing Han population, and the 147C had the dominant inheritance effect comparing with 147T. The risk of susceptibility to ESCC was significantly increased in CC or CT, comparing with TT (p<0.05). But no significant correlation between this site and the upregulation of DcR3 protein was found.
4. There was no difference of the methylation status between the cancer tissues and matched normal tissues. All the CpG sites in the promoter region were unmethylated.
5. The new miR30 like sequence based lentivirus interfering vector was successfully constructed and the virus-containing cell culture supernatants could infect KYSE150 cells efficiently.
6. FACS sorting and Western Blotting showed that the pPRIME-DcR3.3i vector could inhibit the expression of DcR3 protein with the 82.6% inhibition rate.
7. There was no significant suppressing effect of DcR3 RNAi on infected KYSE150 cells.
8. The invasion activity could be inhibited by the joint action of DcR3 RNAi and hLIGHT-Fc.
9. DcR3 RNAi obviously promoted the apoptosis of KYSE150 cell induced by hLIGHT-Fc.
Conclusion: The mRNA and protein expression level of DcR3 in ESCC tumor tissues are higher than that in matched normal tissues. Increased expression of DcR3 is significant related with regional lymph node metastasis and clinical TNM stage. 147C/T polymorphism is a risk site of susceptibility to ESCC in Chongqing Han population, and the 147C has the dominant inheritance effect. The abnormal high expression of DcR3 in ESCC patients is no relationship with methylation status of the CpG in the promoter region of DcR3. DcR3 RNAi has no direct inhibition on growth of ESCC, but it can inhibit invasion effectly and induce apoptosis with the collaboration of hLIGHT-Fc. Above all, DcR3 is a potential ESCC clinical biomarker.
食管癌是人类八种最常见的恶性肿瘤之一。在全世界每年新增的30万病例中,约70 %发生在我国,并且其发病率仍呈上升趋势。食管癌总体预后不佳,总的2年和5年生存率分别为35%～42%和15%～24%。食管癌主要分为食管鳞状细胞癌(ESCC)和食管腺癌,而前者占了90%以上。因此,研究ESCC的发病机制,寻找其新的诊断和治疗靶点,已经成为提高食管癌预防和治疗效果的当务之急。在肿瘤的发生、发展中有一对矛盾贯穿始终,那就是机体的免疫监视和肿瘤的免疫逃逸。而诱导肿瘤特异性的细胞毒性T淋巴细胞(CTL)反应和瘤细胞自身生长停滞或凋亡是机体用以清除肿瘤细胞的主要手段。LIGHT及FasL介导的凋亡是体内最常见的凋亡模式,在免疫调节和肿瘤的免疫监视中起着重要作用。DcR3是TNFRSF的一个新成员。研究发现,DcR3通过竞争性结合可以阻断经由其配体FasL、LIGHT以及TL1A所介导的肿瘤细胞的凋亡;同时DcR3还可抑制免疫应答中T细胞的增殖并影响DCs的分化和成熟,并抑制CD4+T细胞在混合淋巴细胞反应(MLR)中的增殖;此外,DcR3还可抑制T细胞的趋化效应,降低T细胞与抗原提呈细胞间的相互作用。研究显示,DcR3 mRNA低表达于正常人体组织中的胃、脊髓、淋巴结、肺、脾、结肠组织及多数造血细胞中,而高表达于胃肠道恶性肿瘤、乳腺癌、结肠癌、肺癌、胶质细胞瘤和淋巴瘤等多种恶性肿瘤中。研究证实,高表达DcR3是肿瘤细胞逃避机体免疫监视和杀伤的一个重要机制,其表达水平同多种肿瘤的进展程度、转移情况、对化疗的抵抗性以及不良预后有明显的相关性。因此,作为一种候选的肿瘤标志物和基因治疗标靶,DcR3目前已经成为了肿瘤诊断和治疗相关研究的热点。研究DcR3 mRNA和蛋白在人ESCC细胞系和ESCC病人组织标本中的表达情况并分析DcR3的表达与疾病的侵袭、转移、复发乃至预后间的关联性,将有助于评价DcR3作为ESCC的临床及预后的分子标志的可能性;而探讨其表达调控的可能机制则将有助于ESCC肿瘤基因治疗靶点的筛选。因此,关于DcR3在ESCC病人中的高表达及其相关机制的研究对于该疾病的防治有相当重要的意义。
目的
研究DcR3基因在ESCC病人肿瘤组织和相应的远癌组织中的表达情况,分析DcR3基因的表达与病人临床病理特征之间的关系,为评价DcR3作为ESCC的临床及预后的分子标志提供实验依据;检测DcR3基因启动子和编码区的多态性位点在重庆地区汉族ESCC人群中的基因型和等位基因频率,探讨DcR3启动子多态性与ESCC患病风险之间的关联,筛查与ESCC肿瘤组织中DcR3基因高表达相关联的SNP位点,为进一步研究和分析影响其表达调控的分子机制奠定基础;检测DcR3基因启动子区的甲基化状态差异,探讨ESCC肿瘤组织DcR3启动子甲基化状态是否参与其表达调控;考察DcR3 RNAi对ESCC肿瘤细胞的生长能力、侵袭能力和凋亡等方面的影响,初步探讨DcR3在人ESCC细胞发生和发展过程中可能作用和机制。
方法
1.收集109例ESCC肿瘤组织和对应的远癌组织标本,通过半定量逆转录聚合酶链反应(Semi-quantitative RT-PCR)检测其中DcR3 mRNA表达情况;
2.运用免疫组织化学检测52例ESCC病人肿瘤组织中DcR3蛋白表达情况并行半定量分析;
3.选取DcR3基因启动子和编码区四个多态性位点(-369G/T、-323A/C、-321C/T、147C/T),通过PCR扩增产物测序的方法了解这些多态位点在重庆地区正常人群中的频率分布,检测上述多态位点在ESCC人群中的基因型和等位基因频率;
4.选择4对DcR3 mRNA表达状态有明显差异的癌及相对应的远癌组织标本,利用重亚硫酸盐修饰后测序法(BSP)检测DcR3基因启动子区的甲基化状态;
5.构建基于miR-30的、靶向DcR3的慢病毒干扰载体;
6.用干扰载体、包装质粒pMD2G及包膜质粒psAX2共同转染293FT细胞进行病毒包装;
7.通过流式细胞仪(FACS)感染后的KYSE150细胞,获得DcR3 RNAi的ESCC细胞模型;
8.利用Real-time PCR和Western Blotting检测DcR3 RNAi后瘤细胞上DcR3的表达情况;
9.绘制体外培养的感染细胞、对照细胞和亲本细胞的生长曲线;
10.通过Transwell侵袭小室实验检测DcR3 RNAi与LIGHT-Fc协同作用对瘤细胞体外侵袭能力的影响;
11.运用Tunel试剂盒检测DcR3 RNAi与LIGHT-Fc协同作用对瘤细胞凋亡的影响。
结果
1. ESCC肿瘤标本中DcR3 mRNA表达率为73.4%,而远癌组织为47.7%(p<0.01);分析发现DcR3 mRNA的表达与病人的性别、年龄、部位和组织分化程度没有明显的相关性,而与肿瘤组织的外侵程度(p<0.05)、区域淋巴结转移情况(p<0.05)和临床TNM分期(p<0.05)有较强的相关性;
2. ESCC肿瘤组织中DcR3蛋白的表达强弱与mRNA表达基本一致,DcR3的过表达率为28.8%;DcR3的过表达与ESCC病人区域淋巴结转移(p<0.05)和TNM分期(p<0.05)有明显的相关性;
3.重庆地区汉族人群中-369G/T、-323A/C、-321C/T三个位点不存在多态性;而147C/T位点等位基因C的频率为43.7%,略高于中国汉族人(39.0%)和日本人(40.0%)。147C/T多态位点是与重庆地区汉族人群ESCC易感性相关联的一个风险位点,而且该多态位点的等位基因型C具有显性遗传效应,携带等位基因C的基因型CC和CT的ESCC发病风险显著增加(p<0.05)。但该多态位点与ESCC肿瘤组织中DcR3蛋白的异常高表达无明显的相关性;
4. DcR3阳性的肿瘤组织和DcR3阴性的远癌组织,基因的启动子区的甲基化状态相同,即基因启动子区所有的CpG位点都呈非甲基化状态;
5.成功设计并构建了基于miR-30的、靶向DcR3的慢病毒干扰载体;包装获得的病毒上清在体外能有效地感染人KYSE150细胞;
6.经FACS分选,Western Blotting检测证实载体pPRIME-DcR3.3i能有效地抑制瘤细胞DcR3的蛋白表达,抑制率为82.6%;
7. DcR3 RNAi对KYSE150细胞的体外生长无明显影响;
8. DcR3 RNAi与LIGHT-Fc协同能有效地抑制瘤细胞的体外侵袭能力;
9. DcR3 RNAi明显促进了LIGHT-Fc对KYSE150细胞的凋亡诱导。
结论
ESCC肿瘤组织中DcR3在mRNA和蛋白质水平较之对应的远癌组织存在明显的高表达,其高表达与肿瘤的区域淋巴结转移情况和临床TNM分期有较强的相关性。重庆地区汉族人群中147C/T位点是与ESCC易感性相关联的一个风险位点,该位点的等位基因型C具有显性遗传效应;ESCC病人肿瘤组织中DcR3的异常高表达与该基因CpG岛的甲基化状态异常无关; DcR3 RNAi对肿瘤细胞的体外生长没有直接的抑制效应,但能与LIGHT-Fc协同,有效地抑制瘤细胞的体外侵袭能力并促进对瘤细胞的凋亡诱导;DcR3对于ESCC是一个潜在的肿瘤生物标记。
Background: Esophageal cancer is one of the eight most popular malignant tumors in human. Among 300,000 new diagnosed cases every year in the world, seventy percent were in China and the disease incidence still increases recently. One of the main problems of esophageal cancer is that long-term survival is very low, the totally 2-year survival rate is 35%-42% and only 15%-24% for 5-year survival. Esophageal cancer can be characterized into ESCC (esophageal squamous cell carcinoma) and esophageal adenocarcinoma, while ninty percent disease cases are ESCC. So it is very urgent to study the pathogenesis of ESCC and develop new target for diagnosis and treatment.
As we known, the balance between the immunological surveillance of host and the immune escape of tumor is tightly regulated during the tumorigenesis and tumor development. Inducing the tumor-specific CTL response and/or the apotosis of tumor cells are two important methods to eliminate tumor cells. LIGHT and FasL are two members of TNF super family (TNFRSF). They are the most common pathway for inducing the apotosis in vivo, and play essential role in immune regulation and the tumor immune monitoring. DcR3 is a new member of TNFRSF, which can competitively bind to Fas, LIGHT and TL1A and then inhibite the apotosis of tumor cells induced by these three molecular. In the other hand, during the immune response, DcR3 can inhibit the proliferation of T cells and the differentiation and maturation of dendritic cells (DCs), especially the proliferation of CD4+ T cells in MLR. Besides, DcR3 can also inhibit the chemotaxis of T cells and reduce the interact between T cells and antigen processing cells.
Previous studies showed that DcR3 mRNA expression level was very low in many normal human tissues, such as stomach, spinal cord, lymph node, lung, spleen, colon tissue and most hematopoietic cells, but contrarily, in many kinds of malignant tumor, including gastrointestinal cancer, breast cancer, carcinoma of colon, lung cancer, spongiocytoma and lymphoma, DcR3 mRNA expression level was very high. It was reported that overexpression of DcR3 was an important mechanism for tumor cells escaping from immune surveillance and destruction. In many different kinds of cancer, the expression level of DcR3 is clearly related with the progress of carcinogenesis, cancer metastasis, the resistance to chemotherapy and the prognosis of tumor. So, DcR3, as a candidate cancer biomarker and target for cancer gene therapy, becomes a very hot point in cancer research. Studying the expression of DcR3 on the mRNA and protein level in ESCC cell strains and patient biopsies, exploring the relationship between DcR3 expression and the invasion, metastasis, recurrence and prognosis of tumor are very helpful to evaluate the possibility of using DcR3 as a molecular marker in ESCC’s clinical diagnose and prognosis prediction. In the meantime, investigating the regulation mechanism behind DcR3 expression can help us develop a new gene therapy approach to treat ESCC. These facts suggested that studying the expression of DcR3 and how it is regulated are very important in the prevention and treatment of ESCC.
Aims: To identify the expression of DcR3 gene in ESCC patient’s samples and analyze its possible correlation between DcR3 expression and their clinicopathologic features. The polymorphic sites frequencies of DcR3 promoter and coding region in Chongqing normal population and ESCC patients would to be detected, in order to address the possible correlation between polymorphism of DcR3 promoter and ESCC risk, and screen the SNP sites associated with DcR3 high expression. For exploring whether the the methylation was involved in the regulation of DcR3 expression, the methylation difference in promoter region of DcR3 was to be tested. In addition, the effects of DcR3 RNAi on ESCC tumor metastasis, invasion and immune escape were to be detected, and the biological behavior and the mechanism of DcR3 in the progression of human ESCC cells were also to be investigated.
Methods: 1. Semi-quantitative RT-PCR assay was utilized to detect the expression of DcR3 mRNA in 109 ESCC tumor tissues and corresponding normal tissues far away from tumor.
2. Immunohistochemistry and semi-quantitative analysis were performed to test DcR3 protein expressions from 52 ESCC patients.
3. DcR3 promotor and four polymorphic sites (-369G/T、-323A/C、-321C/T、147C/T) in coding region were selected, these polymorphic sites frequencies in Chongqing normal population and their frequencies in ESCC patients were studied with PCR sequencing to address the possible correlation between polymorphism of DcR3 promoter and ESCC risk.
4. Based on their significant difference of DcR3 expression with each other, four pairs of sample from the tumor tissues and corresponding normal tissues far away from tumor were choosed, and their methylation status of the CpG in the promoter region of DcR3 were detected by bisulfite sequencing assay(BSA).
5. A DcR3 targeted RNA interfering lentivirus vector based on a new miR30 like was constructed to inhibit the expression of DcR3 in esophageal squamous cell line KYSE150.
6. The verified recombined vector was co-transfect with pMD2g and psAX2 into 293FT packing cell line to produce the virus.
7. ESCC cell model of DcR3 RNAi was obtained by FACS.
8. The DcR3 expression level of infected cells were evaluated by Real-time PCR and Western blotting.
9. The growth curves of infected cells, controlled cells and wild cells were drawn.
10. The effects of DcR3 RNAi collaborated with hLIGHT-Fc on invasion activity of infected ESCC cells were studied by Transwell loculus assay.
11. The effects of DcR3 RNAi collaborated with hLIGHT-Fc on apoptosis sensitivity of infected ESCC cells were investigated by Tunel assay.
Results: 1. The expression of DcR3 mRNA in 109 ESCC tumor tissues was 73.4% and 47.7% in the corresponding normal tissues far away from tumor. The expression of DcR3 mRNA was no close relation with gender, age, localization and tissue differential degree of patients, but strong related with tumor infiltration (p<0.05), regional lymph node metastasis (p<0.05) and clinical TNM stage (p<0.05).
2. DcR3 protein expression was consistent with DcR3 mRNA expression. The overexpression rate of DcR3 was 28.8%, and there was a correlation between DcR3 overexpression and regional lymph node metastasis (p<0.05) and clinical TNM stage (p<0.05).
3. Polymorphism was found only existe at 147C/T, instead of -369G/T、-323A/C or -321 C/T, on Chongqing Han population. The minor allel frequence (MAF) was 43.7%, which was a little bit higher than the whole Chinese Han population(39.0%) and Janpense population(40.0%). 147C/T polymorphism was a risk site of susceptibility to ESCC in Chongqing Han population, and the 147C had the dominant inheritance effect comparing with 147T. The risk of susceptibility to ESCC was significantly increased in CC or CT, comparing with TT (p<0.05). But no significant correlation between this site and the upregulation of DcR3 protein was found.
4. There was no difference of the methylation status between the cancer tissues and matched normal tissues. All the CpG sites in the promoter region were unmethylated.
5. The new miR30 like sequence based lentivirus interfering vector was successfully constructed and the virus-containing cell culture supernatants could infect KYSE150 cells efficiently.
6. FACS sorting and Western Blotting showed that the pPRIME-DcR3.3i vector could inhibit the expression of DcR3 protein with the 82.6% inhibition rate.
7. There was no significant suppressing effect of DcR3 RNAi on infected KYSE150 cells.
8. The invasion activity could be inhibited by the joint action of DcR3 RNAi and hLIGHT-Fc.
9. DcR3 RNAi obviously promoted the apoptosis of KYSE150 cell induced by hLIGHT-Fc.
Conclusion: The mRNA and protein expression level of DcR3 in ESCC tumor tissues are higher than that in matched normal tissues. Increased expression of DcR3 is significant related with regional lymph node metastasis and clinical TNM stage. 147C/T polymorphism is a risk site of susceptibility to ESCC in Chongqing Han population, and the 147C has the dominant inheritance effect. The abnormal high expression of DcR3 in ESCC patients is no relationship with methylation status of the CpG in the promoter region of DcR3. DcR3 RNAi has no direct inhibition on growth of ESCC, but it can inhibit invasion effectly and induce apoptosis with the collaboration of hLIGHT-Fc. Above all, DcR3 is a potential ESCC clinical biomarker.
引文
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