EN2在人膀胱尿路上皮癌中的表达及其作用机制初步研究
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摘要
膀胱癌是最常见的泌尿系统恶性肿瘤,严重威胁着人类健康。膀胱癌的病理组织学类型主要是尿路(移行)上皮癌。膀胱癌的病理分级与膀胱癌的复发和侵袭行为密切相关,对膀胱癌预后的评估有重要的参考价值。按病理分级程度可把膀胱癌分为低度恶性倾向的尿路上皮乳头状肿瘤、低分级尿路上皮癌以及高分级尿路上皮癌。膀胱癌的病理分期指肿瘤浸润深度及转移的情况,既是判断膀胱肿瘤预后最有价值的参数,也是制定膀胱癌治疗方案的重要依据。根据国际抗癌联盟制定的第7版TNM分期法,可把膀胱癌分为非肌层浸润性膀胱癌(包括Tis、Ta和T1期膀胱癌)和肌层浸润性膀胱癌(包括等于或大于T2期的膀胱癌)。
目前,非肌层浸润性膀胱癌的治疗手段仍以经尿道行膀胱肿瘤电切术及术后行膀胱灌注化疗；而肌层浸润性膀胱癌的治疗手段主要为根治性的膀胱肿瘤切除术,同时行盆腔淋巴结清扫及尿流改道术。然而,由于膀胱癌细胞群落呈现复杂的异质性,生物学行为多变,尽管多数肿瘤都表现为NMIBC,却仍然有多达80%的患者会在最初治疗后的1～2年内复发。对于MIBC,即使进行根治性的膀胱切除并系统的放化疗后,也会有50%的患者死于肿瘤转移。因此,进一步探索膀胱癌的发生、发展机制,进而开发新的有效的治疗手段,仍是今后的重要课题。
RNA干扰(RNA interference, RNAi)是一种由小的双链RNA分子诱导,并能够和与其序列互补的mRNA结合,从而使后者发生降解的现象。RNAi具有高效沉默特定基因的作用,是细胞本身固有的对抗外源基因侵害的一种自我保护现象,是一种转录后基因沉默现象。利用RNAi技术,将具有特定序列的小干扰RNA (small interfering RNA, siRNA)导入肿瘤细胞内,可以引起同源序列mRNA的特异性降解,从而高效并特异地阻断特定基因的表达,使细胞出现特定基因缺失的表型,并进而达到治疗的目的,有希望广泛应用于肿瘤的基因治疗。因此,利用RNAi技术封闭与膀胱癌的发生、发展密切相关的基因或因子的表达,必将改变膀胱癌治疗的现状,并成为更加高效和特异的手段。
同源异型盒基因是进化中高度保守的脱氧核糖核酸(deoxyribonucleic acid, DNA)序列。它们编码的蛋白质家族在动物胚胎发育过程中作为转录调控因子,继母系基因、受精卵基因和分节基因之后,在每个节旁(parasegment)中的各种特定类型的细胞分化之前表达,控制身体前后轴线上组成结构中的基因表达,从而形成严格的时空特异性表达模式,对于生物体胚胎体节和器官的发生及其相对位置的定位起着关键作用。除了控制胚胎细胞的分化和增殖以外,同源异型盒基因还在成人组织的发育和分化调控中起着重要作用。近年来的研究发现,同源异型盒基因在癌症发生发展中具有重要的作用。当同源异型盒基因表达异常时,在胚胎期可导致个体的发育和组织器官的形成出现异常,而在成年期可诱发细胞的恶性增殖,导致肿瘤的发生。
Engrailed (EN)是同源异型盒基因之一,普遍存在于包括环节动物、软体动物、昆虫、棘皮动物、脊索动物及脊椎动物等各种动物门中。在脊椎动物中,由于特定功能的不同,EN又分为EN1和EN2；在发育过程中,两种EN功能具有互补性。
EN2是一个高度保守的同源异型盒基因,广泛存在于各种动物中,在胚胎发育过程中,特别是神经系统发育过程中起着重要的作用,可能参与了发育中组织特定区域的空间完整性的建立与维持。近年来研究发现,EN2在乳腺癌、前列腺癌、肺癌、卵巢癌等肿瘤的发生、发展中具有重要作用,可能具有原癌基因的作用。
然而,目前对于EN2在人膀胱癌发生、发展中的作用尚研究不多。本课题采用本课题通过采用免疫组织化学、Western blot、反转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)及实时定量聚合酶链式反应(quantitative real-time polymerase chain reactionq, qRT-PCR)等技术研究EN2蛋白和基因在膀胱尿路上皮癌组织、膀胱尿路上皮癌细胞系中的表达；并通过RNAi技术抑制EN2基因的表达,进一步研究EN2对膀胱癌细胞增殖、凋亡及侵袭等方面的影响；从而探讨EN2在膀胱尿路上皮癌发生、发展中的作用,并为膀胱尿路上皮癌的治疗提供新思路。第一部分EN2在膀胱尿路上皮癌组织中的表达
目的：EN2是一种同源异型盒基因,在一些肿瘤中呈高表达,可能具有癌基因的作用,但在膀胱癌中的作用如何,目前研究较少。本研究旨在探讨EN2蛋白和基因在人膀胱尿路上皮癌组织和正常膀胱粘膜组织中的表达情况,初步明确EN2在膀胱尿路上皮癌发生发展中的作用。
方法：采集2012年1月至2012年12月手术切除的膀胱尿路上皮癌组织标本(n=60)作为实验组；因膀胱慢性黏膜炎行膀胱镜检而采集的膀胱粘膜组织作为对照组(n=10)。所获组织分为两部分,一部分用于免疫组织化学检测,另一部分用于western blot和RT-PC检测。
结果：免疫组织化学检测发现EN2蛋白主要表达于细胞浆,细胞核内也有少量表达。膀胱尿路上皮癌组织中EN2蛋白的表达强度明显高于正常膀胱组织(P<0.05),并且随病理分级和分期的增加,EN2蛋白的表达强度也明显增加,呈现出正相关的关系；另外,不同病理分期和病理分级的膀胱尿路上皮癌组织之间的EN2蛋白的表达强度与性别无明显关系(P>0.05)。Spearman等级相关分析显示病理分期为T2期的膀胱尿路上皮癌组织中,EN2蛋白的表达强度与年龄存在正相关关系,即在T2期膀胱尿路上皮癌患者中,EN2蛋白的表达强度有随年龄增大而增强的趋势(P<0.05)；而Ta及T1期膀胱尿路上皮癌患者的年龄则与EN2蛋白的表达强度无明显相关性(P>0.05)。另外,western blot和RT-PCI检测结果与免疫组织化学检测结果一致,即随病理分级、分期的增加,膀胱尿路上皮癌组织中的EN2蛋白和基因的相对表达量也明显增加(P<0.05)。
结论：EN2蛋白主要表达于膀胱尿路上皮细胞的细胞浆,细胞核内也有少量表达。膀胱尿路上皮癌组织中EN2蛋白和基因的表达明显高于正常膀胱组织,其蛋白的表达强度与膀胱尿路上皮癌的病理分级、分期及T2期膀胱尿路上皮癌的年龄呈正相关关系；而与性别及Ta和T1期膀胱尿路上皮癌患者的年龄没有明显无关。
第二部分EN2在人膀胱癌不同细胞系中表达的研究
目的：在初步明确EN2在膀胱尿路上皮癌组织中的表达情况后,进一步探讨EN2蛋白和基因在人三种人膀胱尿路上皮癌细胞系和人正常膀胱上皮细胞中的表达情况,筛选出EN2表达水平最高的尿路上皮癌细胞系。
方法：体外培养三种恶性程度不同的膀胱尿路上皮癌细胞系(EJ、T24及RT4)和一种正常膀胱尿路上皮细胞(SV-HUC-1),采用ELISA法、Western blot、 qRT-PCR方法检测膀胱尿路上皮癌不同细胞系的EN2表达情况。
结果：ELISA和Western blot检测发现,膀胱尿路上皮癌细胞的EN2蛋白表达明显高于正常膀胱尿路上皮细胞(P<0.05)。在三种膀胱尿路上皮癌细胞系中,以EJ细胞系的EN2蛋白水平表达最高,其次为T24细胞系,再次为RT4细胞系,正常膀胱尿路上皮细胞中也有少量EN2蛋白表达(P<0.05)。另外,ELISA法也在三种膀胱尿路上皮癌细胞系培养基的上清液中检测到不同浓度的EN2蛋白,其水平仍以EJ细胞系的培养基最高,其次为T24细胞系的培养基,再次为RT4细胞系的培养基；而正常膀胱尿路上皮细胞的培养基上清液中仅检测到微量水平的EN2蛋白(P<0.05)。进一步利用qRT-PCR技术检测EN2mRNA的表达情况,发现膀胱尿路上皮癌细胞系的EN2mRNA表达水平明显高于正常膀胱尿路上皮细胞系,而正常膀胱尿路上皮细胞中仅有少量EN2基因的表达；而且,不同的膀胱尿路上皮癌细胞系中,EN2mRNA的表达水平也明显不同,其中,以EJ细胞系的EN2mRNA表达水平最高,其次为T24细胞系,再次为RT4细胞系。
结论：与正常的膀胱尿路上皮细胞系相比,各种膀胱尿路上皮癌细胞系的EN2蛋白和基因的表达水平均明显上调；但在不同恶性程度的膀胱尿路上皮癌细胞系中,EN2蛋白和基因的表达水平不同,即恶性程度低的膀胱尿路上皮癌细胞系中EN2蛋白和基因的表达水平较低,而恶性程度高的膀胱尿路上皮癌细胞系中EN2蛋白和基因的表达水平较高。另外,EN2蛋白可能以分泌、漏出或其他的方式进入到细胞外环境,从而可能进一步进入尿液。
第三部分RNA干扰EN2基因表达对膀胱尿路上皮癌细胞生物学行为的影响
目的：在明确了EN2蛋白和基因在膀胱尿路上皮癌细胞系中的表达情况的基础上,进一步采用RNAi技术,以化学合成的特异性siRNA抑制EN2基因的表达,检测EN2基因受到抑制后,膀胱尿路上皮癌细胞的增殖、侵袭能力以及细胞凋亡等生物学行为的变化,从而进一步探讨EN2在膀胱尿路上皮癌发生、发展中的作用。
方法：选取第二部分实验中确定的EN2表达水平最高的膀胱尿路上皮癌细胞系(EJ细胞系)作为研究对象,以化学合成法合成针对EN2的三对特异性siRNA和一对阴性对照的siRNA,并成功转染至EJ细胞内。以倒置荧光显微镜观察转染效率,确定最佳转染浓度；以qRT-PCR检测成功转染三对siRNA后的EJ细胞的EN2mRNA表达水平,确定抑制效果最佳的一对siRNA。以激光共聚焦显微镜观察转染siRNA后的EN2蛋白表达情况；以Western blot、qRT-PCR技术分别检测转染siRNA后的EN2蛋白和基因的表达情况；同时,以MTS法、细胞侵袭实验检测EJ细胞的增殖和侵袭能力,并应用流式细胞术检测细胞凋亡情况。
结果：转然后48小时,激光共聚焦显微镜观察显示,EN2蛋白主要表达在EJ细胞的胞浆,胞核也有少量表达,阳性细胞可见胞浆和(或)胞核被染成绿色,空白对照组的细胞没有被染色；干扰组的荧光强度值明显低于未干扰组(P<0.05),即EN2蛋白表达明显降低,抑制率为(50.99±0.01)%。成功转染siRNA至EJ细胞系24小时和48小时后,qRT-PCR和Western blot检测结果分别显示,与阴性对照组和未干扰组相比,转染组EJ细胞的EN2基因和蛋白的表达水平出现明显下调(P<0.05)。MTS法检测细胞增殖能力发现,转染siRNA后24h,转染组EJ细胞的OD值与阴性对照组和未干扰组相比未见明显差异(P>0.05),其增殖能力未受明显影响(P>0.05)；转染siRNA后48h,干扰组EJ细胞的OD值明显低于阴性对照组和未干扰组,抑制率迅速上升到27.85%；转染72h后,抑制率进一步上升,达到43.02%；说明EJ细胞的增殖能力受到明显抑制(P<0.05)。侵袭实验结果显示,未干扰组和阴性对照组的EJ细胞的侵袭细胞数分别为29.27±4.08个和29.47±3.18个,两者相比,侵袭细胞数无明显差异(P>0.05)；而干扰组EJ细胞的侵袭细胞数则仅为5.13±1.68个,明显少于未干扰组和阴性对照组(P<0.05)；这说明转染siRNA后,EJ细胞的侵袭能力受到显著抑制。流式细胞术检测结果显示,转染48h后,与未转染组和阴性对照组相比,转染组的细胞凋亡率明显增加(P<0.05)。转染组EJ细胞早期凋亡率为9.72±0.58%；晚期凋亡率为8.46±0.62%；总凋亡率为18.19±×1.21%；与未转染组和阴性对照组相比,转染组的凋亡细胞所占比例明显增加(P<0.05)。相反,与未转染组和阴性对照组相比,转染组的活细胞比例明显降低(P<0.05)。
结论：以EN2的特异性siRNA转染膀胱尿路上皮癌细胞,可明显下调膀胱尿路上皮癌细胞中EN2的表达,从而明显抑制膀胱尿路上皮癌细胞的增殖和侵袭能力,并促进细胞的凋亡。因此,EN2有望成为膀胱癌治疗的新靶点,尤其为将来膀胱癌的基因治疗提供了一条新的研究途径。
Bladder cancer (BC) is the first most common cancer in urinary system in China, which is a serious threat to human health. With respect to the pathological type, most of BC belongs to bladder urothelial carcinoma (BUC). The recurrence and invasion of BC is closely related to the pathological grade which is very important to evaluate the prognosis of BC. According to the degree of pathological grade, BC can be divided into papillary urothelial neoplasms of low malignant potential (PUNLMP), low grade urothelial carcinoma and high grade urothelial carcinoma. Pathological stage of BC refers to infiltration depth of the tumor and the metastatic status, which is not only the most valuable parameter judging the prognosis of BC but also the important basis for the BC treatment. According to the seventh edition of TNM staging principle made by union for international cancer control (UICC), BC can be divided into nonmuscle-invasive bladder cancer (NMIBC) including Tis, Ta and T1stage of BC and muscle-invasive bladder cancer (MIBC) including equal to or greater than the T2stage of BC.
To date, transurethral resection of bladder tumor (TURBT) and postoperative adjuvant intravesical chemotherapy is still the main treatment for NMIBC, whereas the main treatment for NMIBC is radical cystectomy and pelvic lymphadencetomy with urinary diversion. However, because bladder cancer cell population present complex heterogeneity with various biological behavior, approximately80%of patients with NMIBC suffer from recurrence within1to2years and about8%will progress to MIBC although most of BC are characterized by NMIBC. For patients with MIBC, despite radical cystectomy and systemic therapy,50%of patients will die from metastasis. Therefore, to further explore the mechanism of the occurrence and development of BC and develop new effective treatments is still an important subject in the future.
RNA interference (RNAi) is a phenomenon which is induced by small double-stranded RNA which can combine with mRNA with complementary sequence and degrade the mRNA. RNAi can silence specific gene with high efficiency, which is a kind of self-protective mechanism intrinsic to the cells against the invasion of exogenous gene. Using RNAi technology, the siRNA imported into tumor cell can lead to mRNA with homologous sequence to degrade, block specific gene expression with high efficiency, make cell present phenotype of specific gene deletion, and then achieve the goal of treatment for some diseases. So, RNAi may be widely used in gene therapy of malignant tumor. Using RNAi technology to block expression of genes or factors closely related to the occurrence and development of BC will change the present status of treatment for BC with more efficient and specific characteristics.
Homeobox (HOX) genes are highly conserved clusters of DNA in evolution. The proteins coded by HOX genes are acted as transcriptional regulation factors in process of animal embryo development. Following maternal genes, fertilized egg genes and segmentation genes, these proteins present as expression before various specific types of cells in each parasegment differentiate and regulate the gene expression of the constituted structures in anterior-and posterior-axis of the body, thus forming expression pattern with strict space-time specificity, which play a key role in the development of embryonic somite and organs and the position of their corresponding location. In addition to regulate the differentiation and proliferation of embryonic cells, HOX genes play an important role in the regulation of the differentiation and development of tissues in adult. Recent studies indicated that the HOX genes may play an important role in occurrence and progression of some cancers. The changed expression of HOX genes makes the development of the individual and the formation of tissues and organs during the embryonal stages abnormal and induces malignant proliferation of cells and carcinogenesis in adult.
Engrailed (EN) is one of HOX genes and widely exists in various phyla including annelids, mollusca, insects, echinoderm, chordate and vertebrata. Due to different functions in vertebrata, EN is divided into EN1and EN2which are functionally complementary in development.
EN2is a highly conserved HOX gene and widely exists in various kinds of animals, which plays a key role in early embryonic development, particularly in nervous system development process. Additionally, EN2gene may be involved in establishment and maintenance of space integrity in the specific region of developing tissues and organs. Recent studies suggested that EN2play an important role in occurrence and progression of some cancers, such as breast cancer, prostate cancer, lung cancer and ovarian cancer, et al, which indicate that it may have the function of the proto-oncogene.
However, to date, there were rare studies about the expression of EN2and its significance in BC. In this study, we used immunohistochemistry (IHC), Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the expression of EN2in BUC tissues and cells. Moreover, RNAi technology was used to inhibit the expression of EN2gene, thus investigating the effect of EN2on cell proliferation, apoptosis and invasive ability of BUC in order to explore the role of EN2in occurrence and progression of BC and provide the treatment for bladder cancer with a new target.
Part1Expression of EN2in bladder urothelial cancer tissues
Objects:EN2is a member of HOX genes family, which is found to be over-expressed in a number of cancers and may have the function of the proto-oncogene. Nevertheless, there were rare studies about the expression of EN2and its significance in BC. This study aims to investigate the expression of EN2protein and gene in BUC tissues and normal bladder mucous membrane tissues in order to preliminarily identify its function in occurrence and progression of BUC.
Mthods:The tumor samples as experimental group (n=60) were collected from hospitalized patients with BUC between January2012and December2012while normal bladder mucous membrane tissues as control group (n=10) came from patients with chronic bladder mucosal inflammation. The harvested tissues were divided into two parts:one for IHC and another for western blot and RT-PCR.
Results:EN2protein was found to be present in both cytoplasm and nucleus, but mainly in cytoplasm by IHC. Compared with normal bladder urothelial tissues, the expression intensity of EN2protein in BUC tissues significantly increased (P<0.05). Moreover, there existed in positive correlation between the expression intensity of EN2protein and the pathological grade and stage of BUC. However, there was no correlation between patients'gender and the expression intensity of EN2protein in BUC tissues with different pathological grade and stage (P>0.05). Interestingly, there existed in positive correlation between the expression intensity of EN2protein in BUC tissues with T2stage and patients'age by Spearman rank correlation analysis, that is, the older patients'age was, the higher the expression intensity of EN2protein in BUC tissues with T2stage was (P<0.05). For patients with Ta and T1stage of BC, there was no correlation between patients'age and the expression intensity of EN2protein in BUC tissues (P>0.05). Additionally, the expression of EN2protein and mRNA detected by Western blot and RT-PCR were consistent with the expression of EN2protein detected by IHC (P<0.05).
Conclusions:EN2protein was mainly present in cytoplasm of urothelial cell with few in nucleus. Compared with normal bladder urothelial tissues, the expression intensity of EN2protein and gene in BUC tissues significantly increased. Furthermore, there existed in positive correlation between the expression intensity of EN2protein and the pathological grade and stage of BC without significant correlation with patients'gender and age with Ta and T1stage.
Part2Expression of EN2in various human bladder urothelial cancer cell lines
Objects:Based on preliminarily clarifying the expression of EN2in BUC tissues, the expression of EN2protein and gene in three kinds of human BUC cell lines and one kind of human normal bladder urothelial cell line was investigated and the cell line with the highest expression level of EN2was selected.
Methods:Three kinds of human BUC cell lines (EJ, T24and RT4, respectively) and one kind of human normal bladder urothelial cell line (SV-HUC-1) were cultured in vitro. The expression of EN2protien and gene in various cell lines was determined with ELISA, Western blot and qRT-PCR technology, respectively.
Results:Compared with normal bladder urothelial cell line, the expression of EN2protein in BUC cell lines significantly increased with ELISA and Western blot (P<0.05). In three kinds of human BUC cell lines, the expression of EN2protein in EJ cell line is the highest, followed by T24and RT4cell line whereas there was a little expression of EN2protein in normal bladder urothelial cell line (P<0.05). Additionally, the expression of EN2protein was also detected in the supernatant of culture medium for three kinds of BUC cell lines, the level of which presented the same trend as that in cell lines. Similarly, there was also a little expression of EN2protein in normal bladder urothelial cell line (P<0.05). The expression of EN2mRNA determined by RT-PCR was consistent with the expression of EN2protein detected by ELISA and Western blot.
Conclusions:Compared with normal bladder urothelial cell line, the expression of EN2protein and gene in various BUC cell lines significantly increased. However, the expression of EN2protein and gene was significantly different between BUC cell lines with different malignant degrees, that is, compared with BUC cell line with lower malignant degree the expression of EN2protein and gene in BUC cell line with higher malignant degree more significantly increased. Moreover, EN2protein may enter into the extracellular environment with secretion, leakage or other ways, thus further into the urine.
Part3Effects of interference of EN2gene expression by RNAi on biological behaviors of human BUC cell
Objects:Based on clarifying the expression of EN2proten and gene in BUC cell lines, the EN2gene was repressed by chemosynthetic siRNA against EN2with RNAi technology, and then the proliferation, invasion and apoptosis of human BUC cell were detected and the function of EN2in occurrence and progression of BUC was further investigated.
Methods:The BUC cell line (EJ cell line) with the highest expression level of EN2identified by part2experiment was selected. The siRNA against EN2with high specificity was chemically synthesized and then successfully transfected into EJ cell. Transfection efficiency was observed under the inverted flurescence microscopy to identify the best transfection concentration. The expression of EN2mRNA in EJ cell with successfully transfected-siRNAs was detected by qRT-PCR to identify the siRNA with the best inhibition efficiency. The expression of EN2protein was observed with the confocal laser scanning microscopy after the siRNA was transfected. The expression of EN2protein and gene was determined with Western blot and qRT-PCR after the siRNA was transfected, respectively. The apoptosis of EJ was detected with flow cytometry while the proliferation and invasion of EJ cell was determined MTS method and matrigel invasion assay, respectively.
Results:At the48th hour after transfection, the EN2protein was mainly present in the cytoplasm, rarely in the nuclear, under the confocal laser scanning microscopy, indicating that the cytoplasm and/or nuclear in the positive cells was dyed green whereas the cells in the blank group were not dyed. Furthermore, compared with the non-interfered group, the fluorescence intensity in the interfered group was significantly lower, indicationg that the expression of EN2protein was significantly downreulated with the inhibition rate of50.99±0.01%. At the24th and48th hour after the siRNA was successfully transfected into EJ cell, compared with the blank and non-interfered group, the expression of EN2protein and gene in EJ cells in the interfered group significantly decreased with qRT-PCR and Western blot, respectively (P<0.05). Compared with non-interference group and negative control group, the OD value in EJ cell in the transfected group had no significant difference (P>0.05) at the24th h after the transfection of siRNA, which indicated that the proliferation of EJ cell in the transfected group was not significantly affected at this moment (P>0.05). At the48th h after the transfection of siRNA, the OD value in EJ cell in the transfected group significantly decreased in comparison with non-interference group and negative control group, indicating that the inhibition rate rapidly increased to27.85%, and further increasing to43.02%at the72nd hour, which suggested that the proliferation of EJ cell in the transfected group was significantly inhibited (P<0.05). Matrigel invasion assay indicated that the invasive cell number of EJ cell had no difference between non-interference group and negative control group (29.27±4.08and29.47±3.18, respectively)(P>0.05) whereas the invasive cell number of EJ cell in the transfected group significantly decreased in comparison with non-interference group and negative control group (only5.13±1.68)(P<0.05), which suggested that invasive ability to EJ cell in transfected group was significantly inhibited. Flow cytometry analysis indicated that the apoptosis rate of EJ cell in transfected group significantly increased in comparison with non-interference group and negative control group at the48th h after the transfection of siRNA (P<0.05). For EJ cell in transfected group, the early, late and total apoptosis rate were9.72±0.58%,8.46±0.62%and18.19±1.21%, respectively. Compared with non-interference group and negative control group, the proportion of apoptotic cells in the transfected group significantly increased (P<0.05) whereas the proportion of living cells in the transfected group significantly decreased (P<0.05).
Conclusions:The siRNA against EN2with high specificity may significantly downregulate the expression of EN2in BUC cell, thus significantly inhibit the proliferation and invasion of BUC cell while promoting the apoptosis of BUC cell. Therefore, EN2is expected to become the new target for treatment of BC, particularly providing a new pathway with gene therapy of BC in the future.
目前,非肌层浸润性膀胱癌的治疗手段仍以经尿道行膀胱肿瘤电切术及术后行膀胱灌注化疗；而肌层浸润性膀胱癌的治疗手段主要为根治性的膀胱肿瘤切除术,同时行盆腔淋巴结清扫及尿流改道术。然而,由于膀胱癌细胞群落呈现复杂的异质性,生物学行为多变,尽管多数肿瘤都表现为NMIBC,却仍然有多达80%的患者会在最初治疗后的1～2年内复发。对于MIBC,即使进行根治性的膀胱切除并系统的放化疗后,也会有50%的患者死于肿瘤转移。因此,进一步探索膀胱癌的发生、发展机制,进而开发新的有效的治疗手段,仍是今后的重要课题。
RNA干扰(RNA interference, RNAi)是一种由小的双链RNA分子诱导,并能够和与其序列互补的mRNA结合,从而使后者发生降解的现象。RNAi具有高效沉默特定基因的作用,是细胞本身固有的对抗外源基因侵害的一种自我保护现象,是一种转录后基因沉默现象。利用RNAi技术,将具有特定序列的小干扰RNA (small interfering RNA, siRNA)导入肿瘤细胞内,可以引起同源序列mRNA的特异性降解,从而高效并特异地阻断特定基因的表达,使细胞出现特定基因缺失的表型,并进而达到治疗的目的,有希望广泛应用于肿瘤的基因治疗。因此,利用RNAi技术封闭与膀胱癌的发生、发展密切相关的基因或因子的表达,必将改变膀胱癌治疗的现状,并成为更加高效和特异的手段。
同源异型盒基因是进化中高度保守的脱氧核糖核酸(deoxyribonucleic acid, DNA)序列。它们编码的蛋白质家族在动物胚胎发育过程中作为转录调控因子,继母系基因、受精卵基因和分节基因之后,在每个节旁(parasegment)中的各种特定类型的细胞分化之前表达,控制身体前后轴线上组成结构中的基因表达,从而形成严格的时空特异性表达模式,对于生物体胚胎体节和器官的发生及其相对位置的定位起着关键作用。除了控制胚胎细胞的分化和增殖以外,同源异型盒基因还在成人组织的发育和分化调控中起着重要作用。近年来的研究发现,同源异型盒基因在癌症发生发展中具有重要的作用。当同源异型盒基因表达异常时,在胚胎期可导致个体的发育和组织器官的形成出现异常,而在成年期可诱发细胞的恶性增殖,导致肿瘤的发生。
Engrailed (EN)是同源异型盒基因之一,普遍存在于包括环节动物、软体动物、昆虫、棘皮动物、脊索动物及脊椎动物等各种动物门中。在脊椎动物中,由于特定功能的不同,EN又分为EN1和EN2；在发育过程中,两种EN功能具有互补性。
EN2是一个高度保守的同源异型盒基因,广泛存在于各种动物中,在胚胎发育过程中,特别是神经系统发育过程中起着重要的作用,可能参与了发育中组织特定区域的空间完整性的建立与维持。近年来研究发现,EN2在乳腺癌、前列腺癌、肺癌、卵巢癌等肿瘤的发生、发展中具有重要作用,可能具有原癌基因的作用。
然而,目前对于EN2在人膀胱癌发生、发展中的作用尚研究不多。本课题采用本课题通过采用免疫组织化学、Western blot、反转录聚合酶链式反应(reverse transcription-polymerase chain reaction, RT-PCR)及实时定量聚合酶链式反应(quantitative real-time polymerase chain reactionq, qRT-PCR)等技术研究EN2蛋白和基因在膀胱尿路上皮癌组织、膀胱尿路上皮癌细胞系中的表达；并通过RNAi技术抑制EN2基因的表达,进一步研究EN2对膀胱癌细胞增殖、凋亡及侵袭等方面的影响；从而探讨EN2在膀胱尿路上皮癌发生、发展中的作用,并为膀胱尿路上皮癌的治疗提供新思路。第一部分EN2在膀胱尿路上皮癌组织中的表达
目的：EN2是一种同源异型盒基因,在一些肿瘤中呈高表达,可能具有癌基因的作用,但在膀胱癌中的作用如何,目前研究较少。本研究旨在探讨EN2蛋白和基因在人膀胱尿路上皮癌组织和正常膀胱粘膜组织中的表达情况,初步明确EN2在膀胱尿路上皮癌发生发展中的作用。
方法：采集2012年1月至2012年12月手术切除的膀胱尿路上皮癌组织标本(n=60)作为实验组；因膀胱慢性黏膜炎行膀胱镜检而采集的膀胱粘膜组织作为对照组(n=10)。所获组织分为两部分,一部分用于免疫组织化学检测,另一部分用于western blot和RT-PC检测。
结果：免疫组织化学检测发现EN2蛋白主要表达于细胞浆,细胞核内也有少量表达。膀胱尿路上皮癌组织中EN2蛋白的表达强度明显高于正常膀胱组织(P<0.05),并且随病理分级和分期的增加,EN2蛋白的表达强度也明显增加,呈现出正相关的关系；另外,不同病理分期和病理分级的膀胱尿路上皮癌组织之间的EN2蛋白的表达强度与性别无明显关系(P>0.05)。Spearman等级相关分析显示病理分期为T2期的膀胱尿路上皮癌组织中,EN2蛋白的表达强度与年龄存在正相关关系,即在T2期膀胱尿路上皮癌患者中,EN2蛋白的表达强度有随年龄增大而增强的趋势(P<0.05)；而Ta及T1期膀胱尿路上皮癌患者的年龄则与EN2蛋白的表达强度无明显相关性(P>0.05)。另外,western blot和RT-PCI检测结果与免疫组织化学检测结果一致,即随病理分级、分期的增加,膀胱尿路上皮癌组织中的EN2蛋白和基因的相对表达量也明显增加(P<0.05)。
结论：EN2蛋白主要表达于膀胱尿路上皮细胞的细胞浆,细胞核内也有少量表达。膀胱尿路上皮癌组织中EN2蛋白和基因的表达明显高于正常膀胱组织,其蛋白的表达强度与膀胱尿路上皮癌的病理分级、分期及T2期膀胱尿路上皮癌的年龄呈正相关关系；而与性别及Ta和T1期膀胱尿路上皮癌患者的年龄没有明显无关。
第二部分EN2在人膀胱癌不同细胞系中表达的研究
目的：在初步明确EN2在膀胱尿路上皮癌组织中的表达情况后,进一步探讨EN2蛋白和基因在人三种人膀胱尿路上皮癌细胞系和人正常膀胱上皮细胞中的表达情况,筛选出EN2表达水平最高的尿路上皮癌细胞系。
方法：体外培养三种恶性程度不同的膀胱尿路上皮癌细胞系(EJ、T24及RT4)和一种正常膀胱尿路上皮细胞(SV-HUC-1),采用ELISA法、Western blot、 qRT-PCR方法检测膀胱尿路上皮癌不同细胞系的EN2表达情况。
结果：ELISA和Western blot检测发现,膀胱尿路上皮癌细胞的EN2蛋白表达明显高于正常膀胱尿路上皮细胞(P<0.05)。在三种膀胱尿路上皮癌细胞系中,以EJ细胞系的EN2蛋白水平表达最高,其次为T24细胞系,再次为RT4细胞系,正常膀胱尿路上皮细胞中也有少量EN2蛋白表达(P<0.05)。另外,ELISA法也在三种膀胱尿路上皮癌细胞系培养基的上清液中检测到不同浓度的EN2蛋白,其水平仍以EJ细胞系的培养基最高,其次为T24细胞系的培养基,再次为RT4细胞系的培养基；而正常膀胱尿路上皮细胞的培养基上清液中仅检测到微量水平的EN2蛋白(P<0.05)。进一步利用qRT-PCR技术检测EN2mRNA的表达情况,发现膀胱尿路上皮癌细胞系的EN2mRNA表达水平明显高于正常膀胱尿路上皮细胞系,而正常膀胱尿路上皮细胞中仅有少量EN2基因的表达；而且,不同的膀胱尿路上皮癌细胞系中,EN2mRNA的表达水平也明显不同,其中,以EJ细胞系的EN2mRNA表达水平最高,其次为T24细胞系,再次为RT4细胞系。
结论：与正常的膀胱尿路上皮细胞系相比,各种膀胱尿路上皮癌细胞系的EN2蛋白和基因的表达水平均明显上调；但在不同恶性程度的膀胱尿路上皮癌细胞系中,EN2蛋白和基因的表达水平不同,即恶性程度低的膀胱尿路上皮癌细胞系中EN2蛋白和基因的表达水平较低,而恶性程度高的膀胱尿路上皮癌细胞系中EN2蛋白和基因的表达水平较高。另外,EN2蛋白可能以分泌、漏出或其他的方式进入到细胞外环境,从而可能进一步进入尿液。
第三部分RNA干扰EN2基因表达对膀胱尿路上皮癌细胞生物学行为的影响
目的：在明确了EN2蛋白和基因在膀胱尿路上皮癌细胞系中的表达情况的基础上,进一步采用RNAi技术,以化学合成的特异性siRNA抑制EN2基因的表达,检测EN2基因受到抑制后,膀胱尿路上皮癌细胞的增殖、侵袭能力以及细胞凋亡等生物学行为的变化,从而进一步探讨EN2在膀胱尿路上皮癌发生、发展中的作用。
方法：选取第二部分实验中确定的EN2表达水平最高的膀胱尿路上皮癌细胞系(EJ细胞系)作为研究对象,以化学合成法合成针对EN2的三对特异性siRNA和一对阴性对照的siRNA,并成功转染至EJ细胞内。以倒置荧光显微镜观察转染效率,确定最佳转染浓度；以qRT-PCR检测成功转染三对siRNA后的EJ细胞的EN2mRNA表达水平,确定抑制效果最佳的一对siRNA。以激光共聚焦显微镜观察转染siRNA后的EN2蛋白表达情况；以Western blot、qRT-PCR技术分别检测转染siRNA后的EN2蛋白和基因的表达情况；同时,以MTS法、细胞侵袭实验检测EJ细胞的增殖和侵袭能力,并应用流式细胞术检测细胞凋亡情况。
结果：转然后48小时,激光共聚焦显微镜观察显示,EN2蛋白主要表达在EJ细胞的胞浆,胞核也有少量表达,阳性细胞可见胞浆和(或)胞核被染成绿色,空白对照组的细胞没有被染色；干扰组的荧光强度值明显低于未干扰组(P<0.05),即EN2蛋白表达明显降低,抑制率为(50.99±0.01)%。成功转染siRNA至EJ细胞系24小时和48小时后,qRT-PCR和Western blot检测结果分别显示,与阴性对照组和未干扰组相比,转染组EJ细胞的EN2基因和蛋白的表达水平出现明显下调(P<0.05)。MTS法检测细胞增殖能力发现,转染siRNA后24h,转染组EJ细胞的OD值与阴性对照组和未干扰组相比未见明显差异(P>0.05),其增殖能力未受明显影响(P>0.05)；转染siRNA后48h,干扰组EJ细胞的OD值明显低于阴性对照组和未干扰组,抑制率迅速上升到27.85%；转染72h后,抑制率进一步上升,达到43.02%；说明EJ细胞的增殖能力受到明显抑制(P<0.05)。侵袭实验结果显示,未干扰组和阴性对照组的EJ细胞的侵袭细胞数分别为29.27±4.08个和29.47±3.18个,两者相比,侵袭细胞数无明显差异(P>0.05)；而干扰组EJ细胞的侵袭细胞数则仅为5.13±1.68个,明显少于未干扰组和阴性对照组(P<0.05)；这说明转染siRNA后,EJ细胞的侵袭能力受到显著抑制。流式细胞术检测结果显示,转染48h后,与未转染组和阴性对照组相比,转染组的细胞凋亡率明显增加(P<0.05)。转染组EJ细胞早期凋亡率为9.72±0.58%；晚期凋亡率为8.46±0.62%；总凋亡率为18.19±×1.21%；与未转染组和阴性对照组相比,转染组的凋亡细胞所占比例明显增加(P<0.05)。相反,与未转染组和阴性对照组相比,转染组的活细胞比例明显降低(P<0.05)。
结论：以EN2的特异性siRNA转染膀胱尿路上皮癌细胞,可明显下调膀胱尿路上皮癌细胞中EN2的表达,从而明显抑制膀胱尿路上皮癌细胞的增殖和侵袭能力,并促进细胞的凋亡。因此,EN2有望成为膀胱癌治疗的新靶点,尤其为将来膀胱癌的基因治疗提供了一条新的研究途径。
Bladder cancer (BC) is the first most common cancer in urinary system in China, which is a serious threat to human health. With respect to the pathological type, most of BC belongs to bladder urothelial carcinoma (BUC). The recurrence and invasion of BC is closely related to the pathological grade which is very important to evaluate the prognosis of BC. According to the degree of pathological grade, BC can be divided into papillary urothelial neoplasms of low malignant potential (PUNLMP), low grade urothelial carcinoma and high grade urothelial carcinoma. Pathological stage of BC refers to infiltration depth of the tumor and the metastatic status, which is not only the most valuable parameter judging the prognosis of BC but also the important basis for the BC treatment. According to the seventh edition of TNM staging principle made by union for international cancer control (UICC), BC can be divided into nonmuscle-invasive bladder cancer (NMIBC) including Tis, Ta and T1stage of BC and muscle-invasive bladder cancer (MIBC) including equal to or greater than the T2stage of BC.
To date, transurethral resection of bladder tumor (TURBT) and postoperative adjuvant intravesical chemotherapy is still the main treatment for NMIBC, whereas the main treatment for NMIBC is radical cystectomy and pelvic lymphadencetomy with urinary diversion. However, because bladder cancer cell population present complex heterogeneity with various biological behavior, approximately80%of patients with NMIBC suffer from recurrence within1to2years and about8%will progress to MIBC although most of BC are characterized by NMIBC. For patients with MIBC, despite radical cystectomy and systemic therapy,50%of patients will die from metastasis. Therefore, to further explore the mechanism of the occurrence and development of BC and develop new effective treatments is still an important subject in the future.
RNA interference (RNAi) is a phenomenon which is induced by small double-stranded RNA which can combine with mRNA with complementary sequence and degrade the mRNA. RNAi can silence specific gene with high efficiency, which is a kind of self-protective mechanism intrinsic to the cells against the invasion of exogenous gene. Using RNAi technology, the siRNA imported into tumor cell can lead to mRNA with homologous sequence to degrade, block specific gene expression with high efficiency, make cell present phenotype of specific gene deletion, and then achieve the goal of treatment for some diseases. So, RNAi may be widely used in gene therapy of malignant tumor. Using RNAi technology to block expression of genes or factors closely related to the occurrence and development of BC will change the present status of treatment for BC with more efficient and specific characteristics.
Homeobox (HOX) genes are highly conserved clusters of DNA in evolution. The proteins coded by HOX genes are acted as transcriptional regulation factors in process of animal embryo development. Following maternal genes, fertilized egg genes and segmentation genes, these proteins present as expression before various specific types of cells in each parasegment differentiate and regulate the gene expression of the constituted structures in anterior-and posterior-axis of the body, thus forming expression pattern with strict space-time specificity, which play a key role in the development of embryonic somite and organs and the position of their corresponding location. In addition to regulate the differentiation and proliferation of embryonic cells, HOX genes play an important role in the regulation of the differentiation and development of tissues in adult. Recent studies indicated that the HOX genes may play an important role in occurrence and progression of some cancers. The changed expression of HOX genes makes the development of the individual and the formation of tissues and organs during the embryonal stages abnormal and induces malignant proliferation of cells and carcinogenesis in adult.
Engrailed (EN) is one of HOX genes and widely exists in various phyla including annelids, mollusca, insects, echinoderm, chordate and vertebrata. Due to different functions in vertebrata, EN is divided into EN1and EN2which are functionally complementary in development.
EN2is a highly conserved HOX gene and widely exists in various kinds of animals, which plays a key role in early embryonic development, particularly in nervous system development process. Additionally, EN2gene may be involved in establishment and maintenance of space integrity in the specific region of developing tissues and organs. Recent studies suggested that EN2play an important role in occurrence and progression of some cancers, such as breast cancer, prostate cancer, lung cancer and ovarian cancer, et al, which indicate that it may have the function of the proto-oncogene.
However, to date, there were rare studies about the expression of EN2and its significance in BC. In this study, we used immunohistochemistry (IHC), Western blot analysis, reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time polymerase chain reaction (qRT-PCR) to investigate the expression of EN2in BUC tissues and cells. Moreover, RNAi technology was used to inhibit the expression of EN2gene, thus investigating the effect of EN2on cell proliferation, apoptosis and invasive ability of BUC in order to explore the role of EN2in occurrence and progression of BC and provide the treatment for bladder cancer with a new target.
Part1Expression of EN2in bladder urothelial cancer tissues
Objects:EN2is a member of HOX genes family, which is found to be over-expressed in a number of cancers and may have the function of the proto-oncogene. Nevertheless, there were rare studies about the expression of EN2and its significance in BC. This study aims to investigate the expression of EN2protein and gene in BUC tissues and normal bladder mucous membrane tissues in order to preliminarily identify its function in occurrence and progression of BUC.
Mthods:The tumor samples as experimental group (n=60) were collected from hospitalized patients with BUC between January2012and December2012while normal bladder mucous membrane tissues as control group (n=10) came from patients with chronic bladder mucosal inflammation. The harvested tissues were divided into two parts:one for IHC and another for western blot and RT-PCR.
Results:EN2protein was found to be present in both cytoplasm and nucleus, but mainly in cytoplasm by IHC. Compared with normal bladder urothelial tissues, the expression intensity of EN2protein in BUC tissues significantly increased (P<0.05). Moreover, there existed in positive correlation between the expression intensity of EN2protein and the pathological grade and stage of BUC. However, there was no correlation between patients'gender and the expression intensity of EN2protein in BUC tissues with different pathological grade and stage (P>0.05). Interestingly, there existed in positive correlation between the expression intensity of EN2protein in BUC tissues with T2stage and patients'age by Spearman rank correlation analysis, that is, the older patients'age was, the higher the expression intensity of EN2protein in BUC tissues with T2stage was (P<0.05). For patients with Ta and T1stage of BC, there was no correlation between patients'age and the expression intensity of EN2protein in BUC tissues (P>0.05). Additionally, the expression of EN2protein and mRNA detected by Western blot and RT-PCR were consistent with the expression of EN2protein detected by IHC (P<0.05).
Conclusions:EN2protein was mainly present in cytoplasm of urothelial cell with few in nucleus. Compared with normal bladder urothelial tissues, the expression intensity of EN2protein and gene in BUC tissues significantly increased. Furthermore, there existed in positive correlation between the expression intensity of EN2protein and the pathological grade and stage of BC without significant correlation with patients'gender and age with Ta and T1stage.
Part2Expression of EN2in various human bladder urothelial cancer cell lines
Objects:Based on preliminarily clarifying the expression of EN2in BUC tissues, the expression of EN2protein and gene in three kinds of human BUC cell lines and one kind of human normal bladder urothelial cell line was investigated and the cell line with the highest expression level of EN2was selected.
Methods:Three kinds of human BUC cell lines (EJ, T24and RT4, respectively) and one kind of human normal bladder urothelial cell line (SV-HUC-1) were cultured in vitro. The expression of EN2protien and gene in various cell lines was determined with ELISA, Western blot and qRT-PCR technology, respectively.
Results:Compared with normal bladder urothelial cell line, the expression of EN2protein in BUC cell lines significantly increased with ELISA and Western blot (P<0.05). In three kinds of human BUC cell lines, the expression of EN2protein in EJ cell line is the highest, followed by T24and RT4cell line whereas there was a little expression of EN2protein in normal bladder urothelial cell line (P<0.05). Additionally, the expression of EN2protein was also detected in the supernatant of culture medium for three kinds of BUC cell lines, the level of which presented the same trend as that in cell lines. Similarly, there was also a little expression of EN2protein in normal bladder urothelial cell line (P<0.05). The expression of EN2mRNA determined by RT-PCR was consistent with the expression of EN2protein detected by ELISA and Western blot.
Conclusions:Compared with normal bladder urothelial cell line, the expression of EN2protein and gene in various BUC cell lines significantly increased. However, the expression of EN2protein and gene was significantly different between BUC cell lines with different malignant degrees, that is, compared with BUC cell line with lower malignant degree the expression of EN2protein and gene in BUC cell line with higher malignant degree more significantly increased. Moreover, EN2protein may enter into the extracellular environment with secretion, leakage or other ways, thus further into the urine.
Part3Effects of interference of EN2gene expression by RNAi on biological behaviors of human BUC cell
Objects:Based on clarifying the expression of EN2proten and gene in BUC cell lines, the EN2gene was repressed by chemosynthetic siRNA against EN2with RNAi technology, and then the proliferation, invasion and apoptosis of human BUC cell were detected and the function of EN2in occurrence and progression of BUC was further investigated.
Methods:The BUC cell line (EJ cell line) with the highest expression level of EN2identified by part2experiment was selected. The siRNA against EN2with high specificity was chemically synthesized and then successfully transfected into EJ cell. Transfection efficiency was observed under the inverted flurescence microscopy to identify the best transfection concentration. The expression of EN2mRNA in EJ cell with successfully transfected-siRNAs was detected by qRT-PCR to identify the siRNA with the best inhibition efficiency. The expression of EN2protein was observed with the confocal laser scanning microscopy after the siRNA was transfected. The expression of EN2protein and gene was determined with Western blot and qRT-PCR after the siRNA was transfected, respectively. The apoptosis of EJ was detected with flow cytometry while the proliferation and invasion of EJ cell was determined MTS method and matrigel invasion assay, respectively.
Results:At the48th hour after transfection, the EN2protein was mainly present in the cytoplasm, rarely in the nuclear, under the confocal laser scanning microscopy, indicating that the cytoplasm and/or nuclear in the positive cells was dyed green whereas the cells in the blank group were not dyed. Furthermore, compared with the non-interfered group, the fluorescence intensity in the interfered group was significantly lower, indicationg that the expression of EN2protein was significantly downreulated with the inhibition rate of50.99±0.01%. At the24th and48th hour after the siRNA was successfully transfected into EJ cell, compared with the blank and non-interfered group, the expression of EN2protein and gene in EJ cells in the interfered group significantly decreased with qRT-PCR and Western blot, respectively (P<0.05). Compared with non-interference group and negative control group, the OD value in EJ cell in the transfected group had no significant difference (P>0.05) at the24th h after the transfection of siRNA, which indicated that the proliferation of EJ cell in the transfected group was not significantly affected at this moment (P>0.05). At the48th h after the transfection of siRNA, the OD value in EJ cell in the transfected group significantly decreased in comparison with non-interference group and negative control group, indicating that the inhibition rate rapidly increased to27.85%, and further increasing to43.02%at the72nd hour, which suggested that the proliferation of EJ cell in the transfected group was significantly inhibited (P<0.05). Matrigel invasion assay indicated that the invasive cell number of EJ cell had no difference between non-interference group and negative control group (29.27±4.08and29.47±3.18, respectively)(P>0.05) whereas the invasive cell number of EJ cell in the transfected group significantly decreased in comparison with non-interference group and negative control group (only5.13±1.68)(P<0.05), which suggested that invasive ability to EJ cell in transfected group was significantly inhibited. Flow cytometry analysis indicated that the apoptosis rate of EJ cell in transfected group significantly increased in comparison with non-interference group and negative control group at the48th h after the transfection of siRNA (P<0.05). For EJ cell in transfected group, the early, late and total apoptosis rate were9.72±0.58%,8.46±0.62%and18.19±1.21%, respectively. Compared with non-interference group and negative control group, the proportion of apoptotic cells in the transfected group significantly increased (P<0.05) whereas the proportion of living cells in the transfected group significantly decreased (P<0.05).
Conclusions:The siRNA against EN2with high specificity may significantly downregulate the expression of EN2in BUC cell, thus significantly inhibit the proliferation and invasion of BUC cell while promoting the apoptosis of BUC cell. Therefore, EN2is expected to become the new target for treatment of BC, particularly providing a new pathway with gene therapy of BC in the future.
引文
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