新基因CIAPIN1的功能研究

附件2
论文中英文摘要格式
作者姓名：李晓华
论文题目：新基因CIAPIN1的功能研究
作者简介：李晓华，男，1981年09月出生，2004年09月师从于第四军医大学樊代明教授，于2009年06月获博士学位。

中文摘要
CIAPIN1是本实验室2000年通过抑制性消减杂交（SSH）、差异显示技术（DD-PCR）及双向电泳（2-DE）的方法筛选胃癌多药耐药（MDR）相关分子时得到的一个结构与功能均未明确的新基因，当时命名为V62。

本实验室前期研究发现V62基因在SGC7901/VCR耐药细胞中表达上调(1)，提示其可能在胃癌MDR中发挥重要作用。

2004年，Shibayama等报道了V62在小鼠体内同源分子Anamorsin的研究。

Anamorsin可介导小鼠的前B细胞系Ba/F3抵抗细胞因子IL-3撤除所致凋亡(2)。

V62与小鼠Anamorsin在氨基酸序列上有88 % 的同源性，为不同种属的同源分子。

2004年，国际基因命名委员会将该基因统一命名为CIAPIN1，即细胞因子诱导的凋亡抑制因子1，V62亦即获得了正式的命名——CIAPIN1。

目前新基因CIAPIN1的基本生物学功能尚未明确，为了后续实验顺利进行，本实验室于2005年率先在国际上成功制备了CIAPIN1鼠源性单克隆抗体，并申请专利保护。

本课题将对CIAPIN1蛋白在人体多器官组织内的表达分布，CIAPIN1基因抗凋亡的机制、与恶性肿瘤发生的关系及如何参与体内复杂的信号转导网络等进行初步探讨，以明确新基因CIAPIN1的基本生物学功能。

【目的】
1、研究CIAPIN1蛋白在人和小鼠发育不同阶段多器官组织中表达分布的特点及趋势，为进一步研究其基本生物学功能提供新的思路和实验依据；
2、检测验证CIAPIN1分子的亚细胞定位，为其生物学功的阐明提供重要线索；
3、探讨CIAPIN1介导胃癌MDR的机制；
4、构建并包装CIAPIN1腺病毒系列载体及病毒，以利于下一步细胞学实验；
5、研究CIAPIN1与胃癌发生的相关性；
6、筛选CIAPIN1下游效应分子；
7、初步筛选CIAPIN1相互作用分子。

【方法】
1、用抗CIAPIN1单克隆抗体，对小鼠出生后P0、P7、P14、P21和P28天心、脑、肝、肾和骨骼肌的组织石蜡连续切片、人孕5个月胚胎多器官组织石蜡连续切片进行免疫组织化学检测；分析CIAPIN1蛋白在成年鼠及成人多器官组织中的表达趋势；
2、采用免疫组织化学和免疫荧光检测、EGFP-CIAPIN1融合蛋白表达及亚细胞器分离等方法观察CIAPIN1在多种体外培养细胞系内的亚细胞定位；
3、利用体外药物敏感试验、阿霉素蓄积与潴留试验、阿霉素诱导凋亡试验、Western Blot等研究CIAPIN1对胃癌MDR的影响；
4、构建CIAPIN1正义及siRNA腺病毒载体，利用包装的腺病毒感染胃癌SGC7901和MKN45细胞系，检测CIAPIN1对胃癌恶性生物学行为的影响；
5、利用2-DE和质谱技术鉴定CIAPIN1上调和下调的蛋白质；
6、通过基因芯片技术筛选CIAPIN1相关的差异表达基因；
7、利用串联亲和纯化（TAP）和质谱技术检测CIAPIN1相互作用分子。

【结果】
1、CIAPIN1蛋白在出生后不同时间点小鼠多器官组织内的表达
在刚出生小鼠P0的心、脑、肝、肾和骨骼肌中，可见不同程度的CIAPIN1阳性免疫反应物，尤其在心肌和骨骼肌中最明显，脑和肾脏次之，肝脏最弱；CIAPIN1阳性反应物主要定位于心肌细胞、骨骼肌细胞，几乎所有的脑细胞、肾小管上皮细胞（主要在髓质肾小管）及部分肝细胞。

总体来看，从P0到P7，CIAPIN1阳性反应物强度在心、脑、肝、肾和骨骼肌中变化逐渐加强。

从P7到P14，CIAPIN1阳性反应物在肝、肾、骨骼肌中略有降低；在心肌、中脑和小脑中有增强。

从P14到P21，CIAPIN1阳性反应物在心、脑、肝、肾和骨骼肌中变化略有减弱。

从P21到P28，CIAPIN1阳性反应物在心、脑、肝和骨骼肌中进一步减弱；此时，随着肾皮质进一步增厚，在髓质和皮质肾小管均可见明显的CIAPIN1阳性反应物，CIAPIN1阳性反应物在肾脏中增加；在这段时期，CIAPIN1阳性反应物在皮质肾小管比髓质肾小管更明显。

然而，在3个月大的成年鼠中，CIAPIN1阳性反应物在心、脑、肝和肾小管中的表达强度要低于P28小鼠；但CIAPIN1阳性反应物在成年鼠骨骼肌中较P28小鼠高。

2、CIAPIN1蛋白在人5个月胚胎及成人多器官组织内的表达
在人5月胚胎多器官组织中，CIAPIN1阳性反应物见于心脏、胆囊单层柱状上皮和粘膜、结肠粘膜、小肠粘膜和绒毛、肝脏、直肠腺体、胃粘膜、肾上腺束状带、甲状腺滤泡、脾索、胸腺小叶间隔、皮肤真皮层和汗腺、睾丸白膜和间质、脑组织内神经元和神经胶质、肺小支气管和肺泡、骨骼肌、肾脏皮髓质和肾小管、子宫内膜、胰腺腺泡和胰岛、卵巢、输卵管粘
膜等绝大多数组织细胞。

与CIAPIN1在5个月胚胎器官组织中的表达水平相比，几乎所有被检测的内、外胚层发育而来的成人器官组织内CIAPIN1蛋白的表达水平相对其5个月胚胎器官组织中的表达水平有不同程度下降，如肝、胰腺、脑、神经系统等；在中胚层发育而来的成人器官组织内CIAPIN1蛋白的表达水平相对其5个月胚胎器官组织中的表达水平有不同程度增强，如肾、肌肉、结缔组织等；但极个别器官组织例外，有待进一步证实。

3、CIAPIN1在细胞内广泛分布于胞浆、胞核并富集于核仁
我们采用免疫荧光染色、免疫组织化学染色及EGFP-CIAPIN1融合蛋白表达等方法观察了CIAPIN1在多种体外培养细胞系包括小鼠成纤维细胞系NIH3T3、人肿瘤细胞系和永生化细胞系内的分布，发现CIAPIN1在胞浆、胞核均有分布，而核仁为CIAPIN1的浓集部位。

为进一步确证以上结果，通过HepG2亚细胞成分的分离，用Western Blot方法检测了CIAPIN1在各亚细胞成分内的分布，结果与免疫学方法和EGFP融合表达所得到的结果一致。

上述结果提示：CIAPIN1在细胞内可能经历胞浆－胞核－核仁转位过程，而核仁可能是CIAPIN1发挥生物学功能的最终部位或部位之一。

另一种可能是，核仁是CIAPIN1的重要储存场所，通过储存和释放调节CIAPIN1在不同时期的作用。

4、CIAPIN1介导胃癌MDR
前期本实验室研究发现CIAPIN1基因在胃癌SGC7901/VCR细胞内表达上调(1)。

我们进而采用Western Blot的方法证实CIAPIN1能上调P-gp的表达和Bcl-2/Bax的相对比率；基因转染、siRNA技术、阿霉素蓄积与潴留试验、药敏实验显示：CIAPIN1的特异性siRNA转染后可有效逆转胃癌MDR表型。

这些结果提示抑制细胞对抗癌药物外排，抵抗化疗药物诱导的凋亡，可能是CIAPIN1介导肿瘤MDR的重要机制。

5、CIAPIN1正义及siRNA腺病毒
腺病毒是目前最常用的基因运载工具，有安全、高效、稳定等优点，为了进一步研究CIAPIN1在恶性肿瘤中的功能和具体的分子机制；我们应用腺病毒载体试剂盒，成功构建并包装了CIAPIN1及CIAPIN1-siRNA系列腺病毒，实验证明它可以有效感染肿瘤细胞，干预细胞内CIAPIN1表达，为下一步实验奠定良好基础。

6、CIAPIN1抑制胃癌生长和成瘤
本实验室前期通过免疫组化法检测了CIAPIN1蛋白在胃癌组织与其癌旁组织中的表达，结果显示：CIAPIN1在胃癌组织中的表达显著低于相应的癌旁组织及正常胃粘膜；CIAPIN1在胃癌细胞内的表达水平低于永生化人胃粘膜细胞系GES-1；其中以SGC7901和MKN45细胞系比较显著。

我们分别将腺病毒Ad‑CIAPIN1、Ad‑CIAPIN1siRNA及其对照空病毒分别感染SGC7901和MKN45细胞，观察CIAPIN1对胃癌SGC7901和MKN45细胞恶性生物学行
为的影响。

结果显示：(1) Ad‑CIAPIN1可以上调CIAPIN1在SGC7901和MKN45细胞中的表达，Ad‑CIAPIN1siRNA可以显著抑制CIAPIN1在上述两种细胞系中的表达；(2)上调CIAPIN1表达可抑制SGC7901和MKN45细胞的生长，下调CIAPIN1表达可促进SGC7901和MKN45细胞的生长；(3) CIAPIN1可以抑制胃癌SGC7901和MKN45细胞体外锚定非依赖成长能力；(4) CIAPIN1可抑制SGC7901和MKN45细胞的体内成瘤；(5) CIAPIN1抑制胃癌SGC7901和MKN45细胞增殖的机制，部分是通过下调CyclyinD1的表达，上调P27的表达，抑制细胞周期G1进展实现的。

上述结果提示，CIAPIN1在肿瘤发生过程中发挥重要作用。

7、CIAPIN1下游信号分子的筛选
在相似条件下分别对来自SGC7901-pSi CIAPIN1及其对照SGC7901-pSi的总蛋白样品重复进行了3次2-DE，从肉眼观察发现相同样品的3次2-DE图谱基本一致。

图谱中蛋白质分子大小主要集中在20kd至100kd之间。

利用Melanie3 2-D分析软件分析，发现CIAPIN1能够上调SGC7901细胞内13个蛋白的表达，下调其中7个蛋白的表达。

我们对这20个蛋白质斑点进行了胶内酶解和MALDI-TOF-MS肽指纹图谱分析，结果成功确定了8个差异表达蛋白。

它们分别是P66、If-A20、Twist、IGHD、CACNA1B、GSDM1、IFT20和RGS，其中P66、RGS、Twist、CACNA1B、GSDM1和IGHD为CIAPIN1所上调，IFT20和If-A20为CIAPIN1所下调。

然而，这一结果还需要Western Blot进行进一步验证。

经过Trizol法抽提SGC7901-pc和SGC7901-p CIAPIN1细胞的总RNA，琼脂糖检测和Lab-on-Chip分析表明：所提取的RNA无明显降解，RNA的质和量符合基因芯片测定的要求。

经过RNA纯化、荧光探针的制备纯化及定量、芯片杂交、图像采集及数据标准化等步骤，发现芯片的杂交信号强度较高，片内信号均一，14,000个杂交点的质控点变异系数（CV）均值为12.4 %，芯片杂交点的检出率为84.6 %。

以两种细胞信号强度的比值小于0.5或者大于2为标准判定差异表达基因，结果发现，与对照的SGC7901-pc细胞相比，SGC7901-p CIAPIN1细胞中上调表达的基因有411个，下调表达的基因有288个。

8、CIAPIN1相互作用分子的筛选
Rigaut等于1999年描述了一种新型的用于纯化蛋白复合物的亲和层析策略（TAP）策略。

在此，我们采用TAP与质谱技术连用检测CIAPIN1相互作用的蛋白复合物，我们成功构建了CIAPIN1分子C末端融合表达TAP标签的真核表达载体，瞬时转染SGC7901细胞，通过质谱分析首次检测到了一个与CIAPIN1相互作用的分子TXNL2。

虽然TXNL2作为另外一个功能未知的新基因未能为CIAPIN1基本功能的阐明提供有力支持，但是我们在本研究中建立的CIAPIN1特异的哺乳动物TAP系统为进一步采用TAP方法寻找CIAPIN1相互作用分子奠定了有利的基础。

【结论】
本研究揭示了新基因CIAPIN1的基本生物学功能。

主要结论如下：
1、C IAPIN1蛋白在哺乳动物小鼠和人发育不同阶段的组织与器官中广泛分布，其表达水平与
胚层发育相关；
2、C IAPIN1蛋白在细胞胞浆、胞核和核仁均有分布，并富集于核仁；
3、C IAPIN1通过上调P-gp的表达，增加药物泵出率；上调Bcl-2/Bax的相对比率抵抗凋亡，
介导胃癌MDR；
4、C IAPIN1可抑制胃癌细胞增殖、成瘤，其机制部分是通过下调CyclyinD1、上调P27的表
达，抑制细胞G1周期进展实现的；
5、C IAPIN1蛋白可与新的分子——TXNL2相互作用。

【关键词】
CIAPIN1；组织分布；亚细胞定位；MDR；腺病毒；胃癌；2-DE；基因芯片；TAP；相互作用分子
The functional characterization of a novel gene CIAPIN1
Li Xiaohua
ABSTRACT
CIAPIN1, a novel antiapoptotic molecule, which does not show any homology to known apoptosis regulatory molecules such as Bcl-2 family, Caspase family, or signal transduction molecules, is proven to be a mediator of the RAS signaling pathway and plays a vitally important role in fetal liver hematopoiesis. Microarray analysis showed that Bcl-xL and Jak2were downregulated in fetal liver of mCIAPIN1null mice, suggesting that mCIAPIN1 might exert its function by up-regulating the expression of Bcl-xL and Jak2. In addition, mCIAPIN1 protects Ba/F3 cells a gainst etoposide, γ radiation and stauroporine in vitro. Our previous studies revealed that CIAPIN1was upregulated in MDR gastric cancer cell subline SGC7901/VCR at mRNA level. Furthermore, CIAPIN1 was found to act as an antiapoptotic molecule in vivo because mCIAPIN1-/-
mice died in late gestation from defective definitive hematopoiesis in the fetal liver. However, although mCIAPIN1 as an anti-apoptosis molecule has been observed in the mouse cell lines, the basic biological function of CIAPIN1 is still unclear. Here, we aim to explore the basic biological function of CIAPIN1 and to examine its effects on the embryogenesis, multidrug resistance, cancer development and so on. These studies would reveal the basic biological function of this novel gene-CIAPIN1.
【Objectives】
In order to explore the basic biological function of CIAPIN1, we first examined the expression and location of CIAPIN1 in mammal development and its subcellular location in differential cell lines. Then, we focused on the effect and mechanism of CIAPIN1 on gastric cancer MDR, cancer development and process. Finally, by the approaches of the 2-DE, gene chip, TAP and MALDI-TOF-MS technologies, we tried to understand the possible signal pathways and interaction molecules of this novel gene-CIAPIN1.
【Methods】
(1) Immunohistochemistry was used to analyze the expression trend of CIAPIN1 in heart, liver, brain, kidney and skeletal muscle during mouse postnatal development from P0 to P28 and the various tissues of adult mouse. Immunochistochemistry was also used to analyze the expression distribution of CIAPIN1 in the various tissues from five month human embryos and adult human.
(2) Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. Here, we want to examine the distribution of CIAPIN1 in normal fetal and adult human tissues by immunohistochemical (IHC) staining with an in-house-produced we further performed immunofluorescence, 6×His-tagged fusion protein expression, and analytical cell fractionation in our present study. (3) CIAPIN1was found to be overexpressed at the mRNA level in the vincristine induced MDR gastric cancer cell SGC7901/VCR compared to its parental cell line. In this study, with the gene transfected, in vitro drug sensitivity assay, flow cytometry detect apoptosis, RT-PCR, Western Blot and Luciferase reporter assay methods, we investigated the role of CIAPIN1in multidrug resistance (MDR) of gastric cancer cells and the possible underlying mechanisms. (4) CIAPIN1, CIAPIN1-siRNA and their control vectors and adenovirus were constructed and packaged. In order to explore the
relationship of CIAPIN1 and gastric carcinoma, CIAPIN1 related adenoviruses were used to infect gastric cancer cells. (5) The downstream effectors were screened by two-dimensional electrophoresis and MALDI-TOF-MS. (6) The downstream effectors were screened by microarray.
(7) The interaction molecules were screened by TAP.
【Results】
1．T he distribution of CIAPIN1 in tissues of mouse postnatal of differential time point During mouse postnatal development, CIAPIN1 immunoreactions in the heart, brain, liver, kidney and skeletal muscle have some upregulated between P0 and P7. CIAPIN1 expression becomes slightly low in the liver, kidney and skeletal muscle, but higher between P7 and P14 in the heart and brain. Between P14 and P21, CIAPIN1 expression in the brain, heart and liver becomes much lower. However, between P21 and P28, CIAPIN1 expression in the heart, brain, liver and skeletal muscle becomes much lower, while with the kidney development, CIAPIN1 expression in the kidney becomes higher. In various tissues from adult mouse, CIAPIN1 has been found in the cardiac muscle, brain, liver, epithelium of renal tubule, skeletal muscle, lung tissue, gastric mucosa and gland, acinus lienalis.
2. Distribution of CIAPIN1 in normal fetal and adult human tissues
To reveal the possible physiological role of CIAPIN1, we examined the expression and distribution of CIAPIN1 in fetal and adult human tissues using immunohistochemistry. We found that CIAPIN1 was ubiquitously expressed in fetal and adult tissues, and localized in both the cytoplasm and the nucleus. The expression patterns of CIAPIN1 were similar in fetal and adult tissues and correlated with the previously described expression pattern of Ras. These observations suggested that CIAPIN1 expression appears to be involved in the cell differentiation, and it might exert some universal and possibly important physiological functions in the regulation of Ras in humans.
3. Subcellular localization of CIAPIN1
Our previous studies have demonstrated that CIAPIN1 is ubiquitously expressed in normal fetal and adult human tissues. However, fundamental biological functions of CIAPIN1 have not been elucidated. In this study, we first predicted the subcellular localization of CIAPIN1 with bioinformatic approaches and then characterized the intracellular localization of CIAPIN1 in both human and mouse cells by a combination of techniques including (a) immunohistochemistry and immunofluorescence, (b) His-tagged CIAPIN1 expression, and (c) subcellular fractionation and
analysis of CIAPIN1 in the fractions by Western Blot. All methods produced consistent results: CIAPIN1 localized in both the cytoplasm and the nucleus and accumulated in the nucleolus. Bioinformatic prediction disclosed a putative nuclear localization signal and a putative nuclear export signal within both human and mouse CIAPIN1. These findings suggested that CIAPIN1 may undergo a cytoplasm–nucleus–nucleolus translocation.
4. CIAPIN1 confers MDR of gastric cancer cells
The overexpression of CIAPIN1 gene has been previously reported in SGC7901/VCR cell line compared with its parental cell line SGC7901 by our team. In the present study, we constructed the siRNA eukaryotic expression vectors of CIAPIN1 and transfected them into SGC7901/VCR cells to examine whether the downregulation of CIAPIN1 increased cell sensitivity towards chemotherapeutic drugs. After transfection, the expression of CIAPIN1 was dramatically decreased in CIAPIN1-siRNA transfectants compared with that in parental cells and vector control cells. The downregulation of CIAPIN1 significantly enhanced the sensitivity of SGC7901/VCR cells to vincristine (VCR), adriamycin (ADR) and etoposide (VP-16), but not to 5-fluorouracil (5-Fu) and cisplatin (CDDP). The capacity to efflux adriamycin decreased markedly in CIAPIN1-siRNA transfectants, and the correlation between CIAPIN1 downregulation and decreased MDR-1 transcriptional activity was obtained. Inhibition of the CIAPIN1expression could significantly downregulate the expression of Bcl-2, and upregulate the expression of Bax, but does not alter the expression of PTEN in gastric cancer cells. These observations suggested that the CIAPIN1-siRNA could effectively downregulate the expression of CIAPIN1 and reverse the multi-drug resistance of gastric cancer cells. Further study of the biological functions of CIAPIN1may be helpful for understanding the mechanisms of multidrug resistance of gastric cancer and developing possible strategies for the treatment gastric cancer.
5. CIAPIN1 inhibits gastric cancer cell proliferation and cell cycle progression
CIAPIN1 was found to be downregulated in human gastric cancer tissues as compared to the matched adjacent nonneoplastic tissues. In this study, we investigated the effect of CIAPIN1 on the proliferation of gastric cancer cells in vitro and in vivo. Ectopic expression and knockdown of CIAPIN1 in gastric cancer cells SGC7901 and MKN45 were achieved by transduction with recombinant adenoviruses expressing human CIAPIN1(Ad‑CIAPIN1) and human CIAPIN1‑specific small interference RNA adenovirus (Ad‑CIAPIN1siRNA). Gastric cancer cells with upregulated expression of CIAPIN1 significantly decreased proliferation, while cells with
downregulated CIAPIN1 expression showed significantly quicker growth rate as compared with their respective controls in both in vitro and in vivo tests. Also, CIAPIN1could significantly suppress the colony formation of SGC7901 and MKN45 cells in soft agar cloning test. Furthermore, cell cycle distribution analysis revealed that CIAPIN1induced cell cycle arrest at G1/S phase. Downregulation of CyclyinD1 and upregulation of P27 might contribute, at least in part, to this altered cell cycle distribution. This study demonstrated that CIAPIN1 is a suppressor of the proliferation of gastric cancer cell and suggested that CIAPIN1 might play an important role in the development of human gastric cancer.
6. Screening of the downstream effectors of CIAPIN1
The proteomic maps of SGC7901-pSi CIAPIN1 and SGC7901-pSi were successfully acquired through two-dimensional electrophoresis. Most of the protein spots were distributed in the center of the map. Melanie3 2D software was used to analyze the two proteomic maps. Compared to the map of SGC7901-pSi, there were 13 protein spots of interest in SGC7901-pSi CIAPIN1which were strongly stained and 7 spots weakly stained. All the spots were treated by destaining of silver, in-gel digestion and then analyzed by MALDI-TOF-MS. After MASCOT database searching, eight proteins were identified. They were P66、If-A20、Twist、IGHD、CACNA1B、GSDM1、IFT20 and RGS, among which P66、RGS、Twist、CACNA1B、GSDM1 and IGHD were downregulated, IFT20 and If-A20 were upregulated by CIAPIN1. The total RNA of SGC7901-p CIAPIN1and SGC7901-pc were extracted. The quality of extracted RNA was evaluated by agarose electrophoresis and analysis of Lab-on-chip. After microarray hybridization and data normalization, 84.6 % spots out of 14,000 genes were positively detected, among which in gastric cancer cell lines 411 genes were up-regulated while 288 genes were down-regulated by CIAPIN1.
7. Screening of the interaction molecules of CIAPIN1
Identification of protein–protein interactions is at the core of understanding biological processes occurring in living cells. The tandem affinity purification (TAP) combines purification of a protein complex of interest using affinity purification tags with subsequent mass spectrometry identification of unknown protein complex components. The key feature of this technology is the use of two different affinity purification tags that are fused to at least one known component of the protein complex of interest by genetic methods. Performing two consecutive purification steps using affinity purification tags that have gentle washing and elution conditions allows for isolation without disrupting the targeted complex. In this study, we constructed the pCTAP expression vector
of CIAPIN1 and trasfected the vectors into SGC7901 cells. With the TAP and SDS-PAGE, the spots were treated by destaining of Coomassie brilliant blue, in-gel digestion and then analyzed by MALDI-TOF-MS. After MASCOT database searching, one protein molecule – TXNL2 interacted with CIAPIN1 was identified.
【Conclusion】
In conclusion, we provide evidence of several new biological function of novel gene CIAPIN1.
(1) CIAPIN1 was ubiquitously expressed in fetal and adult tissues of the different development stages in mammalian mouse and human; (2) CIAPIN1 localized in both the cytoplasm and the nucleus and accumulated in the nucleolus; (3) CIAPIN1 confers multidrug resistance by upregulating the expression of P-gp and Bcl-2/Bax ratio in gastric cancer cells; (4) CIAPIN1 inhibits gastric cancer cell proliferation and cell cycle progression by downregulating CyclinD1 and upregulating P27; (5) One new protein molecule – TXNL2 interacted with CIAPIN1 was identified. 【Keywords】
CIAPIN1; embryonic development; tissue distribution; subcellular localization; MDR; adenovirus; gastric cancer; 2-DE; gene chip; TAP; interaction molecule.。