分泌蛋白FSTL1在鼻咽癌中的抑瘤作用及其机制的研究
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摘要
据统计,世界上80%的鼻咽癌(Nasopharyngeal carcinoma, NPC)发生在中国。在过去二十年中,鼻咽癌一直是广西壮族自治区肿瘤的主要死因之一,占全部恶性肿瘤死亡的11.63%,在恶性肿瘤死亡中居第3位。探索鼻咽癌的发病机理,对鼻咽癌开展防治,具有深远的社会和经济意义。
鼻咽癌的病因学目前认为主要有遗传易感性,EB病毒(Epstein-Barr Virus, EBV)感染和环境化学致癌因素,其发病遵循典型的多基因—环境交互作用模式。有别于其它常见人类恶性肿瘤,在NPC的发生和演进过程中,癌基因的激活或过度表达并不是一个主要因素。而肿瘤抑制基因的失活却更为普遍,启动子CpG岛的DNA甲基化是大部分肿瘤抑制基因失活的主要机制。肿瘤抑制基因的表观遗传学改变参与了包括EBV的潜伏感染、细胞周期调控、DNA修复、细胞凋亡及肿瘤的浸润转移等鼻咽癌发生发展的全过程。因此,鼻咽癌的发生与演进和表观遗传学改变密切相关。
在本课题组前期研究中,我们利用表达谱芯片技术高通量筛选并建立了鼻咽癌的高甲基化谱式,发现FSTL1基因在鼻咽癌细胞系CNE2及HONE1中经去甲基化处理后平均表达上调了约20倍,并且FSTL1基因在鼻咽癌中转录表达明显降低,其差异具有统计学意义。这些结果提示我们FSTL1在鼻咽癌中可能受表观遗传学机制调控而下调。
因此,本研究的目的为：1.探索FSTL1基因在鼻咽癌中失活的表观遗传学机制；2.探讨FSTL1基因在鼻咽癌发生发展过程中的肿瘤生物学功能；3.初步探索分泌蛋白FSTL1对巨噬细胞功能的调控作用及其在鼻咽癌细胞免疫逃避中发挥的作用。通过以上研究评价FSTL1基因作为新的鼻咽癌候选肿瘤抑制基因的可能性,挖掘FSTL1在鼻咽癌中的免疫调控机制,还可为寻找和筛选鼻咽癌相关的分子标记物和治疗新靶点提供有力的实验基础数据和理论依据。
首先,我们通过甲基化DNA富集结合高通量测序和亚硫酸氢盐测序法,对鼻咽癌细胞株和原发肿瘤组织中FSTL1基因的DNA甲基化状态进行了进一步研究。我们将基因组DNA片段化后,通过MBD-生物素蛋白-磁珠捕获并富集甲基化DNA片段,利用SOLiD高通量测序平台对7例鼻咽癌和4例鼻咽上皮组织进行全基因组甲基化图谱描绘。对测序结果进行FSTL1基因的检索,发现FSTL1基因在鼻咽癌肿瘤组织中呈现高频的DNA甲基化。另外,还利用亚硫酸氢盐测序法对鼻咽癌细胞株CNE2和HONE1、2例鼻咽癌肿瘤组织和1例正常鼻咽上皮组织进行了甲基化状态检测。测序范围覆盖了FSTL1基因启动子区和第一外显子区的29个CG位点,发现鼻咽癌肿瘤组织和细胞中大多数CG位点均有60-100%的高频甲基化。结果提示我们FSTL1基因在鼻咽癌中因启动子区DNA高甲基化而表达失活,FSTL1基因很可能是鼻咽癌相关的肿瘤抑制基因。
因此,为了探讨FSTL1基因在鼻咽癌中的生物学功能,我们通过RT-PCR技术克隆了FSTL1基因编码区全长,构建了pCMV-Tag3A-FSTL1真核表达载体。并经双酶切法和Sanger测序法对此载体进行鉴定,酶切产物电泳结果及测序对比结果同时说明此表达载体已构建成功。然后,我们利用pCMV-Tag3A-FSTL1载体转染鼻咽癌细胞系CNE2,建立FSTL1稳定表达CNE2细胞系FSTL1-CNE2。利用RT-PCR技术和Western-blot可在FSTL1-CNE2细胞中分别检测到FSTL1在mRAN水平及蛋白水平的表达,说明该稳转FSTL1的鼻咽癌细胞系已成功建立,为研究FSTL1基因在鼻咽癌中可能的肿瘤生物学功能打下了基础。
下一步,我们通过细胞增殖抑制实验、细胞克隆形成实验、细胞周期实验、细胞划痕实验、Transwell实验及裸鼠体内实验从细胞增殖、克隆形成、细胞周期、细胞凋亡和细胞迁移运动和侵袭、体内成瘤等方面分析了FSTL1基因的肿瘤生物学功能。我们发现：1.外源性表达FSTL1基因可显著抑制鼻咽癌细胞增殖；重组人FSTL1蛋白也可有明显的细胞抑制作用。2.外源性表达FSTL1基因可抑制鼻咽癌细胞的迁移及浸润能力。3.外源性表达FSTL1基因可抑制鼻咽癌细胞的克隆形成效率。相对于Empty vector-CNE2, FSTL1-CNE2细胞的克隆数目减少,克隆体积也较小。4.外源性表达FSTL1基因可引起鼻咽癌细胞周期阻滞。经流式细胞仪检测,FSTL1-CNE2处于G0／G1期细胞比例为(72.03+1.27)%,而Empty vector-CNE2处于G0／G1期的细胞比例为(63.68±0.91)%(P=0.001)；FSTL1-CNE2处于S期的细胞仅为(1.34±0.83)%,而Empty vector-CNE2细胞处于S期的比例达(18.31±5.20)%(P=0.001); FSTL1-CNE2处于G2／M期的细胞为(26.624±1.59)%,而Empty vector-CNE2处于G2／M期的细胞比例为(18.01±4.28)%(P<0.05)。5.外源性表达FSTL1基因可诱导鼻咽癌细胞发生凋亡。Annexin V-FITC细胞凋亡检测结果显示,Empty vector-CNE2细胞凋亡率为(19.05±2.18)%,而FSTL1-CNE2的凋亡率为(37.13±3.13)%,发生凋亡的细胞数目明显增多。通过免疫蛋白印迹法,我们在FSTL1-CNE2细胞中检测到Caspase-3剪切后产生的19和17kD的cleaved Caspase-3片段,而在Empty vector-CNE2细胞中,仅有少量的cleaved Caspase-3被检测到。6.外源性表达FSTL1基因可抑制鼻咽癌细胞在裸鼠体内的生长。我们发现,FSTL1-CNE2细胞在裸鼠体内形成的肿瘤生长速度明显缓慢,在接种后第14天,FSTL1-CNE2组形成的肿瘤体积为(91.35±16.06)mm3,而Empty vector-CNE2细胞形成的肿瘤体积为(329.09±89.90)mm3,两组肿瘤体积的差别具有统计学意义(P＜0.05)；接种14天后处死裸鼠,对肿瘤进行解剖后拍照记录并称重,结果显示FSTL1-CNE2细胞接种组形成的肿瘤重量为(0.155±0.037)g,而Empty vector-CNE2细胞接种组的肿瘤重量(0.378±0.212)g(P<0.05)；经计算,FSTL1基因的抑瘤率为59%。本部分实验结果验证了FSTL1可作为鼻咽癌的候选肿瘤抑制基因。
为评价分泌蛋白FSTL1能否作为鼻咽癌血清学方面的检测指标,我们采用ELISA方法对鼻咽癌病例和健康对照组血浆中FSTL1蛋白的含量水平进行检测。结果提示,与健康对照组[(0.669±0.382) ng/ml]相比,鼻咽癌病例组血清中FSTL1蛋白的水平[(0.846±0.504) ng/ml](P>0.05)无明显改变。
文献报道FSTL1是一个致炎因子,可调控巨噬细胞的功能。因此,我们通过免疫荧光染色,对比分析鼻咽癌组织中FSTL1的表达水平、肿瘤间质中巨噬细胞的数目及产物的表达水平,慢性黏膜炎症组织则用作正常对照。我们对15例鼻咽癌组织和15例鼻咽部慢性FSTL1、巨噬细胞标记CD68、巨噬细胞的产物TNF-α和IL1进行了免疫荧光染色。结果发现,FSTL1在鼻咽上皮组织中正常表达；而在鼻咽癌肿瘤组织中明显降低,这与我们在前期研究结果一致；我们还发现,对比鼻咽黏膜炎症组织,鼻咽癌间质中的巨噬细胞数目并未明显减少,但是巨噬细胞的分泌产物TNF-α和IL1在肿瘤间质中的表达水平明显降低。这些结果提示,鼻咽癌组织中FSTL1的低表达与肿瘤间质中巨噬细胞功能降低相关。
为进一步证实FSTL1对巨噬细胞的调控作用,我们首先在体外利用重组人巨噬细胞集落刺激因子(rhM-CSF)处理人外周血单核细胞。处理7天后,细胞呈贴壁生长,由不规则状逐渐变椭圆,部分长梭形,细胞伸出板状伪足和突起,细胞无明显增殖。且细胞粘附牢固,在细胞传代过程中,贴壁细胞不易被胰酶消化。从形态学上观察,诱导的细胞具有典型的巨噬细胞特征。另外,我们通过免疫荧光细胞化学染色确定,未经rhM-CSF诱导的细胞无CD14(巨噬细胞系统的特征性标记物)表达,而经rhM-CSF诱导后的细胞胞质中表达CD14。通过形态学和CD14免疫荧光染色,我们确立人外周血巨噬细胞诱导培养成功。
进一步的,我们用重组人FSTL1蛋白处理体外诱导的人巨噬细胞,通过ELISA检测了巨噬细胞产物IL-1β和TNF-α。以0.75μg/ml的重组人FSTL1蛋白处理体外诱导的人巨噬细胞,发现随着处理时间的延长,细胞培养液上清中巨噬细胞产物IL-1β和TNF-α的水平呈上升趋势。FSTL1处理第7天,细胞培养液上清中IL-1β和TNF-α的水平均高于空白处理组。实验结果进一步证实了细胞外分泌蛋白FSTL1可通过调控巨噬细胞分泌IL-1β和TNF-α的水平,对巨噬细胞起正向调控作用。
综上所述,FSTL1基因是新的鼻咽癌相关的候选肿瘤抑制基因,鼻咽癌细胞通过表观遗传学机制,沉默或下调FSTL1基因的表达以促进自身的生长,通过减少FSTL1蛋白的旁分泌以削弱巨噬细胞的免疫作用而实现鼻咽癌细胞的免疫逃避。
It was documented that nasopharyngeal carcinoma (NPC) is endemic in Southern China. All over the World,80%of NPC cases occured in China. In the past two decades, NPC remains the main cause of cancer deaths in Guangxi Zhuang Autonomous Region, and the third cause of deaths in malignancy, accounts for11.63%of all cancer deaths. Exploring the pathogenesis of NPC is of great significance.
The aetiology of NPC involves three major factors:genetic susceptibility, Epstein-Barr virus (EBV) infection and exposure to chemical carcinogens, which follow typical multiple gene-environment interaction model. Unlike other human malignancy, activation or over-expression of oncogenes is not a main factor during the development and progression of NPC. However, the inactivation of tumor suppressor genes (TSGs) is more common. Promoter methylation has been proposed as a mechanism for inactivation of TSGs in human malignancies. The epigenetic changes influenced cellular pathways including EBV latent infection, cell cycle control, DNA repair system, apoptosis and tumor invasion and metastasis in NPC. Epigenetic changes play a crucial role in the tumourigenesis of NPC.
In previous study, we have estabilished a global hypermethylation profile in NPC by a high-throughput method-cDNA microarray. In which the transcription of FSTL1gene were up-regulated almost20folds after pharmacologic inhibition of DNA methyltransferase in both CNE2and HONE1cells. We also identied that the transcription of FSTLl was significantly downregulated in NPC compared with the normal controls. These results indicated that FSTL1could be a target gene for epigenetic siliencing in NPC.
In this study, we aimed to explore the mechanism of FSTL1epigenetic inactivated in NPC; to identify the biological functions of FSTL1in the tumorigenesis of NPC; to investigate the function of FSTLl in the regulation of macrophages and in the immune evasion of NPC cells. The results will address the role of FSTL1as a functional tumor suppressor gene in NPC, and its role in immune regulation, which in turn provide basic experimental data and theoretic evidences for disvovering diagnostic molecular markers and theraputic targets for NPC.
First, we analyzed the methylation status of FSTL1gene in NPC cell lines and primary tumors by methyl-CpG domain-based capture (MBDCap) sequencing, and bisulfite genomic sequencing (BGS). Genomic DNA from7NPC primary tumors and4NPC cell lines were sheared to fragment double strand DNA, and the methylated DNA were captured by MBD protein-biotin-magnetic beads, then the captured DNA were used to construct SOLiD fragment libraries and sequenced on a SOLiD system and performed a genome-wide scale methylation profiling. The methlation profiling showed that FSTL1is hypermethylation in NPC. The promoter and first exon of FSTL1including29CpG sites were covered in the BGS.60%-80%of the29CpG sites were hypermethylated in2NPC cell lines (CNE2and HONE1),2NPC primary tumors, while normal nasopharyngeal epithelia showed was almost free of methylation. These results indicated that FSTL1is inactivated by promoter hypermethylation, might act as a functional tumor suppression gene in NPC.
To explore the biological function of FSTL1in NPC, the full-length coding sequence of FSTL1was amplified by RT-PCR, and constructed to a mammalian expression vector pCMV-Tag3A. The pCMV-Tag3A-FSTL1plasmid was identified by double endonuclease digestion and Sanger sequencing. The results supported that the construction of expression vector was successful. pCMV-Tag3A-FSTL1plasmid was transfected into CNE2cells. After that, FSTL1stable transfectance of:FSTL1-CNE2was selected. The transcription and protein of exogenous FSTL1was confirmed by RT-PCR and Western-blot, respectively.
Next, we performed a series of in vitro and in vivo experiments to analyze the biological function of FSTL1in NPC. The results showed that:1. Either the ectopic expression of FSTL1or the treatment of recombinant human FSTL1protein inhibits NPC cell proliferation;2. Ectopic expression of FSTL1could impede NPC cell migration and invasion;3. Ectopic expression of FSTL1could inhibit the colony formation of NPC cells;4. Ectopic expression of FSTL1could induce NPC cell cycle arrest;(72.03±1.27)%of FSTL1-CNE2cells were detected in G0/G1phase, while the proportion of empty vector-CNE2cells was (63.68±0.91)%(P=0.001);(1.34±0.83)%of FSTL1-CNE2cells were in S phase, while the corresponding proportion of empty vector-CNE2cells was (18.31±5.20)%(P=0.001);(26.62±1.59)%of FSTL1-CNE2were in G2/M phase, while the proportion of empty vector-CNE2was (18.01±4.28)%(P<0.05);5. ectopic expression of FSTL1could induce apoptosis in NPC cells;(37.13±3.13)%of FSTL1-CNE2cells was shown apoptosis, while (19.05±2.18)%in empty vector-CNE2; Cleaved Caspade-3fragments were detected by Western-blot in FSTL1-CNE2;6. Ectopic expression of the FSTL1could suppress the tumorignesis of NPC cells in nude mice;FSTL1-CNE2cells inoculated in nude mice grew slower than empty vector-CNE2; Tumor volumes from FSTL1-CNE2and empty vector-CNE2cells reached (91.35±16.06) mm3and (329.09±89.90) mm3respectively two weeks after injection,(P<0.05); the average weight of the tumor mentioned above were (0.155mg±0.037)g and (0.378mg±0.212) g for FSTL1-CNE2cells and empty vector-CNE2, respectively (P<0.05); the tumorigenesis suppression rate of FSTL1was worked out as59%. These data suggested that FSTL1might be a potential TSG in NPC.
To evaluate whether secreted FSTLl could be a serological biomarker for NPC, we detected FSTL1in serum from NPC patients and normal controls by ELISA. The results suggested that there are no significant differences in FSTL1levels between two groups.
FSTL1is reported to regulate the function of macrophage as a pro-inflammation cytokine. Then, we analyzed the expression of FSTL1and products of macrophage in NPC biopsies by immunofluorescence staining, chronic inflammation nasophayngeal tissue was used as normal control. FSTL1, one of the macrophage marker:CD68, macrophage products TNF-a and IL1were evaluated in15NPC tissues and15NP inflammation tissues. Consistent with our previous study, that the expression level of FSTLl was significantly lower in NPC primary tumors. Although the number of macrophage did not decreased in NPC stroma, the expression levels of two secretory products of macrophage, TNF-a and IL1, were significantly decreased, which suggests that downregulation of FSTL1might inturn suppresses of macrophage function.
To further demonstrate that FSTL1may play an important role in the regulation of macrophage, we induced the human macrophage from peripheral blood mononuclear cell by recombinant human macrophage colony stimulating factor (rhM-CSF) as the first step. On the7th days after treatment, macrophage was successfully induced, which confirmed by morphological observation and immunofluorescence staining of CD14, a macrophage specific marker.
Furthermore, we treated the macrophages by the recombination human FSTL1protein, and evaluated the levels of the macrophage products, IL1β and TNF-a in, in the supernatant by ELISA. We found that IL1β and TNF-a secreted by macrophage were upregulated gradually under the treatment of FSTL1. The levels of IL1β and TNF-a were higher that those in empty control group at the7th day after treatment.
In conclusion, FSTL1is a novel candidate TSG in NPC; epigeneticly inactivating FSTL1in NPC cells could promote cell proliferation and inhibit the functions of macrophages, which in turn lead to the immuno evasion of NPC cells.
鼻咽癌的病因学目前认为主要有遗传易感性,EB病毒(Epstein-Barr Virus, EBV)感染和环境化学致癌因素,其发病遵循典型的多基因—环境交互作用模式。有别于其它常见人类恶性肿瘤,在NPC的发生和演进过程中,癌基因的激活或过度表达并不是一个主要因素。而肿瘤抑制基因的失活却更为普遍,启动子CpG岛的DNA甲基化是大部分肿瘤抑制基因失活的主要机制。肿瘤抑制基因的表观遗传学改变参与了包括EBV的潜伏感染、细胞周期调控、DNA修复、细胞凋亡及肿瘤的浸润转移等鼻咽癌发生发展的全过程。因此,鼻咽癌的发生与演进和表观遗传学改变密切相关。
在本课题组前期研究中,我们利用表达谱芯片技术高通量筛选并建立了鼻咽癌的高甲基化谱式,发现FSTL1基因在鼻咽癌细胞系CNE2及HONE1中经去甲基化处理后平均表达上调了约20倍,并且FSTL1基因在鼻咽癌中转录表达明显降低,其差异具有统计学意义。这些结果提示我们FSTL1在鼻咽癌中可能受表观遗传学机制调控而下调。
因此,本研究的目的为：1.探索FSTL1基因在鼻咽癌中失活的表观遗传学机制；2.探讨FSTL1基因在鼻咽癌发生发展过程中的肿瘤生物学功能；3.初步探索分泌蛋白FSTL1对巨噬细胞功能的调控作用及其在鼻咽癌细胞免疫逃避中发挥的作用。通过以上研究评价FSTL1基因作为新的鼻咽癌候选肿瘤抑制基因的可能性,挖掘FSTL1在鼻咽癌中的免疫调控机制,还可为寻找和筛选鼻咽癌相关的分子标记物和治疗新靶点提供有力的实验基础数据和理论依据。
首先,我们通过甲基化DNA富集结合高通量测序和亚硫酸氢盐测序法,对鼻咽癌细胞株和原发肿瘤组织中FSTL1基因的DNA甲基化状态进行了进一步研究。我们将基因组DNA片段化后,通过MBD-生物素蛋白-磁珠捕获并富集甲基化DNA片段,利用SOLiD高通量测序平台对7例鼻咽癌和4例鼻咽上皮组织进行全基因组甲基化图谱描绘。对测序结果进行FSTL1基因的检索,发现FSTL1基因在鼻咽癌肿瘤组织中呈现高频的DNA甲基化。另外,还利用亚硫酸氢盐测序法对鼻咽癌细胞株CNE2和HONE1、2例鼻咽癌肿瘤组织和1例正常鼻咽上皮组织进行了甲基化状态检测。测序范围覆盖了FSTL1基因启动子区和第一外显子区的29个CG位点,发现鼻咽癌肿瘤组织和细胞中大多数CG位点均有60-100%的高频甲基化。结果提示我们FSTL1基因在鼻咽癌中因启动子区DNA高甲基化而表达失活,FSTL1基因很可能是鼻咽癌相关的肿瘤抑制基因。
因此,为了探讨FSTL1基因在鼻咽癌中的生物学功能,我们通过RT-PCR技术克隆了FSTL1基因编码区全长,构建了pCMV-Tag3A-FSTL1真核表达载体。并经双酶切法和Sanger测序法对此载体进行鉴定,酶切产物电泳结果及测序对比结果同时说明此表达载体已构建成功。然后,我们利用pCMV-Tag3A-FSTL1载体转染鼻咽癌细胞系CNE2,建立FSTL1稳定表达CNE2细胞系FSTL1-CNE2。利用RT-PCR技术和Western-blot可在FSTL1-CNE2细胞中分别检测到FSTL1在mRAN水平及蛋白水平的表达,说明该稳转FSTL1的鼻咽癌细胞系已成功建立,为研究FSTL1基因在鼻咽癌中可能的肿瘤生物学功能打下了基础。
下一步,我们通过细胞增殖抑制实验、细胞克隆形成实验、细胞周期实验、细胞划痕实验、Transwell实验及裸鼠体内实验从细胞增殖、克隆形成、细胞周期、细胞凋亡和细胞迁移运动和侵袭、体内成瘤等方面分析了FSTL1基因的肿瘤生物学功能。我们发现：1.外源性表达FSTL1基因可显著抑制鼻咽癌细胞增殖；重组人FSTL1蛋白也可有明显的细胞抑制作用。2.外源性表达FSTL1基因可抑制鼻咽癌细胞的迁移及浸润能力。3.外源性表达FSTL1基因可抑制鼻咽癌细胞的克隆形成效率。相对于Empty vector-CNE2, FSTL1-CNE2细胞的克隆数目减少,克隆体积也较小。4.外源性表达FSTL1基因可引起鼻咽癌细胞周期阻滞。经流式细胞仪检测,FSTL1-CNE2处于G0／G1期细胞比例为(72.03+1.27)%,而Empty vector-CNE2处于G0／G1期的细胞比例为(63.68±0.91)%(P=0.001)；FSTL1-CNE2处于S期的细胞仅为(1.34±0.83)%,而Empty vector-CNE2细胞处于S期的比例达(18.31±5.20)%(P=0.001); FSTL1-CNE2处于G2／M期的细胞为(26.624±1.59)%,而Empty vector-CNE2处于G2／M期的细胞比例为(18.01±4.28)%(P<0.05)。5.外源性表达FSTL1基因可诱导鼻咽癌细胞发生凋亡。Annexin V-FITC细胞凋亡检测结果显示,Empty vector-CNE2细胞凋亡率为(19.05±2.18)%,而FSTL1-CNE2的凋亡率为(37.13±3.13)%,发生凋亡的细胞数目明显增多。通过免疫蛋白印迹法,我们在FSTL1-CNE2细胞中检测到Caspase-3剪切后产生的19和17kD的cleaved Caspase-3片段,而在Empty vector-CNE2细胞中,仅有少量的cleaved Caspase-3被检测到。6.外源性表达FSTL1基因可抑制鼻咽癌细胞在裸鼠体内的生长。我们发现,FSTL1-CNE2细胞在裸鼠体内形成的肿瘤生长速度明显缓慢,在接种后第14天,FSTL1-CNE2组形成的肿瘤体积为(91.35±16.06)mm3,而Empty vector-CNE2细胞形成的肿瘤体积为(329.09±89.90)mm3,两组肿瘤体积的差别具有统计学意义(P＜0.05)；接种14天后处死裸鼠,对肿瘤进行解剖后拍照记录并称重,结果显示FSTL1-CNE2细胞接种组形成的肿瘤重量为(0.155±0.037)g,而Empty vector-CNE2细胞接种组的肿瘤重量(0.378±0.212)g(P<0.05)；经计算,FSTL1基因的抑瘤率为59%。本部分实验结果验证了FSTL1可作为鼻咽癌的候选肿瘤抑制基因。
为评价分泌蛋白FSTL1能否作为鼻咽癌血清学方面的检测指标,我们采用ELISA方法对鼻咽癌病例和健康对照组血浆中FSTL1蛋白的含量水平进行检测。结果提示,与健康对照组[(0.669±0.382) ng/ml]相比,鼻咽癌病例组血清中FSTL1蛋白的水平[(0.846±0.504) ng/ml](P>0.05)无明显改变。
文献报道FSTL1是一个致炎因子,可调控巨噬细胞的功能。因此,我们通过免疫荧光染色,对比分析鼻咽癌组织中FSTL1的表达水平、肿瘤间质中巨噬细胞的数目及产物的表达水平,慢性黏膜炎症组织则用作正常对照。我们对15例鼻咽癌组织和15例鼻咽部慢性FSTL1、巨噬细胞标记CD68、巨噬细胞的产物TNF-α和IL1进行了免疫荧光染色。结果发现,FSTL1在鼻咽上皮组织中正常表达；而在鼻咽癌肿瘤组织中明显降低,这与我们在前期研究结果一致；我们还发现,对比鼻咽黏膜炎症组织,鼻咽癌间质中的巨噬细胞数目并未明显减少,但是巨噬细胞的分泌产物TNF-α和IL1在肿瘤间质中的表达水平明显降低。这些结果提示,鼻咽癌组织中FSTL1的低表达与肿瘤间质中巨噬细胞功能降低相关。
为进一步证实FSTL1对巨噬细胞的调控作用,我们首先在体外利用重组人巨噬细胞集落刺激因子(rhM-CSF)处理人外周血单核细胞。处理7天后,细胞呈贴壁生长,由不规则状逐渐变椭圆,部分长梭形,细胞伸出板状伪足和突起,细胞无明显增殖。且细胞粘附牢固,在细胞传代过程中,贴壁细胞不易被胰酶消化。从形态学上观察,诱导的细胞具有典型的巨噬细胞特征。另外,我们通过免疫荧光细胞化学染色确定,未经rhM-CSF诱导的细胞无CD14(巨噬细胞系统的特征性标记物)表达,而经rhM-CSF诱导后的细胞胞质中表达CD14。通过形态学和CD14免疫荧光染色,我们确立人外周血巨噬细胞诱导培养成功。
进一步的,我们用重组人FSTL1蛋白处理体外诱导的人巨噬细胞,通过ELISA检测了巨噬细胞产物IL-1β和TNF-α。以0.75μg/ml的重组人FSTL1蛋白处理体外诱导的人巨噬细胞,发现随着处理时间的延长,细胞培养液上清中巨噬细胞产物IL-1β和TNF-α的水平呈上升趋势。FSTL1处理第7天,细胞培养液上清中IL-1β和TNF-α的水平均高于空白处理组。实验结果进一步证实了细胞外分泌蛋白FSTL1可通过调控巨噬细胞分泌IL-1β和TNF-α的水平,对巨噬细胞起正向调控作用。
综上所述,FSTL1基因是新的鼻咽癌相关的候选肿瘤抑制基因,鼻咽癌细胞通过表观遗传学机制,沉默或下调FSTL1基因的表达以促进自身的生长,通过减少FSTL1蛋白的旁分泌以削弱巨噬细胞的免疫作用而实现鼻咽癌细胞的免疫逃避。
It was documented that nasopharyngeal carcinoma (NPC) is endemic in Southern China. All over the World,80%of NPC cases occured in China. In the past two decades, NPC remains the main cause of cancer deaths in Guangxi Zhuang Autonomous Region, and the third cause of deaths in malignancy, accounts for11.63%of all cancer deaths. Exploring the pathogenesis of NPC is of great significance.
The aetiology of NPC involves three major factors:genetic susceptibility, Epstein-Barr virus (EBV) infection and exposure to chemical carcinogens, which follow typical multiple gene-environment interaction model. Unlike other human malignancy, activation or over-expression of oncogenes is not a main factor during the development and progression of NPC. However, the inactivation of tumor suppressor genes (TSGs) is more common. Promoter methylation has been proposed as a mechanism for inactivation of TSGs in human malignancies. The epigenetic changes influenced cellular pathways including EBV latent infection, cell cycle control, DNA repair system, apoptosis and tumor invasion and metastasis in NPC. Epigenetic changes play a crucial role in the tumourigenesis of NPC.
In previous study, we have estabilished a global hypermethylation profile in NPC by a high-throughput method-cDNA microarray. In which the transcription of FSTL1gene were up-regulated almost20folds after pharmacologic inhibition of DNA methyltransferase in both CNE2and HONE1cells. We also identied that the transcription of FSTLl was significantly downregulated in NPC compared with the normal controls. These results indicated that FSTL1could be a target gene for epigenetic siliencing in NPC.
In this study, we aimed to explore the mechanism of FSTL1epigenetic inactivated in NPC; to identify the biological functions of FSTL1in the tumorigenesis of NPC; to investigate the function of FSTLl in the regulation of macrophages and in the immune evasion of NPC cells. The results will address the role of FSTL1as a functional tumor suppressor gene in NPC, and its role in immune regulation, which in turn provide basic experimental data and theoretic evidences for disvovering diagnostic molecular markers and theraputic targets for NPC.
First, we analyzed the methylation status of FSTL1gene in NPC cell lines and primary tumors by methyl-CpG domain-based capture (MBDCap) sequencing, and bisulfite genomic sequencing (BGS). Genomic DNA from7NPC primary tumors and4NPC cell lines were sheared to fragment double strand DNA, and the methylated DNA were captured by MBD protein-biotin-magnetic beads, then the captured DNA were used to construct SOLiD fragment libraries and sequenced on a SOLiD system and performed a genome-wide scale methylation profiling. The methlation profiling showed that FSTL1is hypermethylation in NPC. The promoter and first exon of FSTL1including29CpG sites were covered in the BGS.60%-80%of the29CpG sites were hypermethylated in2NPC cell lines (CNE2and HONE1),2NPC primary tumors, while normal nasopharyngeal epithelia showed was almost free of methylation. These results indicated that FSTL1is inactivated by promoter hypermethylation, might act as a functional tumor suppression gene in NPC.
To explore the biological function of FSTL1in NPC, the full-length coding sequence of FSTL1was amplified by RT-PCR, and constructed to a mammalian expression vector pCMV-Tag3A. The pCMV-Tag3A-FSTL1plasmid was identified by double endonuclease digestion and Sanger sequencing. The results supported that the construction of expression vector was successful. pCMV-Tag3A-FSTL1plasmid was transfected into CNE2cells. After that, FSTL1stable transfectance of:FSTL1-CNE2was selected. The transcription and protein of exogenous FSTL1was confirmed by RT-PCR and Western-blot, respectively.
Next, we performed a series of in vitro and in vivo experiments to analyze the biological function of FSTL1in NPC. The results showed that:1. Either the ectopic expression of FSTL1or the treatment of recombinant human FSTL1protein inhibits NPC cell proliferation;2. Ectopic expression of FSTL1could impede NPC cell migration and invasion;3. Ectopic expression of FSTL1could inhibit the colony formation of NPC cells;4. Ectopic expression of FSTL1could induce NPC cell cycle arrest;(72.03±1.27)%of FSTL1-CNE2cells were detected in G0/G1phase, while the proportion of empty vector-CNE2cells was (63.68±0.91)%(P=0.001);(1.34±0.83)%of FSTL1-CNE2cells were in S phase, while the corresponding proportion of empty vector-CNE2cells was (18.31±5.20)%(P=0.001);(26.62±1.59)%of FSTL1-CNE2were in G2/M phase, while the proportion of empty vector-CNE2was (18.01±4.28)%(P<0.05);5. ectopic expression of FSTL1could induce apoptosis in NPC cells;(37.13±3.13)%of FSTL1-CNE2cells was shown apoptosis, while (19.05±2.18)%in empty vector-CNE2; Cleaved Caspade-3fragments were detected by Western-blot in FSTL1-CNE2;6. Ectopic expression of the FSTL1could suppress the tumorignesis of NPC cells in nude mice;FSTL1-CNE2cells inoculated in nude mice grew slower than empty vector-CNE2; Tumor volumes from FSTL1-CNE2and empty vector-CNE2cells reached (91.35±16.06) mm3and (329.09±89.90) mm3respectively two weeks after injection,(P<0.05); the average weight of the tumor mentioned above were (0.155mg±0.037)g and (0.378mg±0.212) g for FSTL1-CNE2cells and empty vector-CNE2, respectively (P<0.05); the tumorigenesis suppression rate of FSTL1was worked out as59%. These data suggested that FSTL1might be a potential TSG in NPC.
To evaluate whether secreted FSTLl could be a serological biomarker for NPC, we detected FSTL1in serum from NPC patients and normal controls by ELISA. The results suggested that there are no significant differences in FSTL1levels between two groups.
FSTL1is reported to regulate the function of macrophage as a pro-inflammation cytokine. Then, we analyzed the expression of FSTL1and products of macrophage in NPC biopsies by immunofluorescence staining, chronic inflammation nasophayngeal tissue was used as normal control. FSTL1, one of the macrophage marker:CD68, macrophage products TNF-a and IL1were evaluated in15NPC tissues and15NP inflammation tissues. Consistent with our previous study, that the expression level of FSTLl was significantly lower in NPC primary tumors. Although the number of macrophage did not decreased in NPC stroma, the expression levels of two secretory products of macrophage, TNF-a and IL1, were significantly decreased, which suggests that downregulation of FSTL1might inturn suppresses of macrophage function.
To further demonstrate that FSTL1may play an important role in the regulation of macrophage, we induced the human macrophage from peripheral blood mononuclear cell by recombinant human macrophage colony stimulating factor (rhM-CSF) as the first step. On the7th days after treatment, macrophage was successfully induced, which confirmed by morphological observation and immunofluorescence staining of CD14, a macrophage specific marker.
Furthermore, we treated the macrophages by the recombination human FSTL1protein, and evaluated the levels of the macrophage products, IL1β and TNF-a in, in the supernatant by ELISA. We found that IL1β and TNF-a secreted by macrophage were upregulated gradually under the treatment of FSTL1. The levels of IL1β and TNF-a were higher that those in empty control group at the7th day after treatment.
In conclusion, FSTL1is a novel candidate TSG in NPC; epigeneticly inactivating FSTL1in NPC cells could promote cell proliferation and inhibit the functions of macrophages, which in turn lead to the immuno evasion of NPC cells.
引文
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