Effects of FEN1 Overexpression on Biological Behaviors of Hepatocellular Carcinoma Cells and Prognosis of Patients
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摘要:目的
探讨FEN1在肝细胞癌中的表达及其在肝细胞癌发生发展中的作用。
方法应用RT-qPCR及免疫组织化学方法检测FEN1基因在52例肝细胞癌组织及相应癌旁组织中的表达。FEN1基因特异性siRNA转染肝癌细胞株HepG2并用Western blot检测转染效率。用MTT及流式细胞技术检测干扰FEN1后HepG2增殖及凋亡情况。Transwell实验检测HepG2迁移、侵袭能力改变。
结果RT-qPCR结果显示与癌旁正常组织相比较,FEN1高表达于肝细胞癌组织(P < 0.01)。免疫组织化学与临床资料统计分析进一步证实FEN1的高表达与肿瘤分化程度(P=0.017)、肝内转移(P=0.046)、临床TNM分期(P=0.020)相关,而与患者性别、年龄、术前AFP值及肿瘤直径无相关性(P > 0.05)。通过siRNA干扰肝癌细胞株HepG2中FEN1基因表达,MTT及流式细胞术检测表明干扰FEN1表达后肝癌细胞株HepG2增殖受到明显抑制(P < 0.05),凋亡率明显增加(P < 0.05)。Transwell实验发现干扰FEN1后可明显抑制HepG2迁移、侵袭能力(P < 0.05)。
结论FEN1在肝细胞癌组织中高表达,高FEN1表达提示肿瘤分化程度低,易转移及预后差,干扰FEN1后抑制肝癌细胞增殖,诱导其凋亡,并降低肝癌细胞体外迁移和侵袭能力。FEN1基因可成为肝细胞癌诊断及治疗的一个新生物靶点。
Abstract:ObjectiveTo investigate the expression level of FEN1 in hepatocellular carcinoma (HCC) cells and its role in the progression of HCC.
MethodsThe expressions of FEN1 in 52 matched pairs of HCC tissues and corresponding normal tissues were measured by RT-qPCR and immunohistochemical staining. Specific FEN1 siRNA was transfected in HepG2 cells and the transfection efficiency was detected by Western blot. Moreover, cell proliferation was analyzed by MTT assay and cells apoptosis was determined by flow cytometry. Then, cell migration and invasion were detected by Transwell assays.
ResultsFEN1 was overexpressed in HCC tissues in comparison with the corresponding normal tissues (P < 0.01). FEN1 expression had positive correlations with differentiation degree (P=0.017), intrahepatic metastasis (P=0.046) and TNM stage (P=0.020) of HCC tissues. However, as regards gender (P=0.731), age (P=0.754), level of AFP (P=0.076) and tumor size (P=0.100) no significant differences were observed between the groups. FEN1 expression in HepG2 cells could be downregulated by siRNA effectively. Moreover, the inhibition of FEN1 expression suppressed the proliferation and induced the apoptosis of HepG2 cells(P < 0.05). Transwell migration assay suggested that the migration and invasion abilities of HepG2 cells were significantly inhibited when FEN1 was interfered (P < 0.05).
ConclusionFEN1 is abundantly expressed in HCC tissues, and abnormal FEN1 expression may associate with low differentiation grade, intrahepatic metastasis and poor patient prognosis. Downregulation of FEN1 expression reduced HCC cells proliferation and induced cells apoptosis, as well as, reduced HCC cells migration and invasion ability in vitro. FEN1 might be a novel target for hepatocellular carcinoma diagnosis and therapy.
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Key words:
- FEN1 /
- Hepatocellular carcinoma /
- Cell proliferation /
- Cell apoptosis /
- Migration /
- Invasion
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0 引言
肝细胞癌是最常见的恶性肿瘤之一,具有起病隐匿、进展迅速、预后差、死亡率高等特点[1-3]。因此早期诊断是影响临床治疗和预后的关键因素,然而肝细胞癌的发生、发展的潜在机制尚未完全清楚,需要进一步研究。
皮瓣状核酸内切酶(FEN1)为具有5’特殊分叉结构的核酸酶[4],其在修复DNA复制叉以及凋亡DNA降解过程中发挥着重要作用,使FEN1在维持基因组的保真性及防止细胞癌变中成为一个关键基因[5-7]。近年来,许多研究表明异常FEN1表达与肿瘤发生发展密切相关[8]。与相应的正常组织相比,FEN1基因在前列腺癌[9]、胰腺癌组织[10]、肺癌组织[11]、乳腺癌及卵巢癌组织[12]中表达上调。
因此,本研究通过RT-qPCR和免疫组织化学染色来检测肝细胞癌组织中FEN1的表达情况,并分析FEN1表达与肝细胞癌临床病理特征之间的相关性,探讨FEN1成为肝细胞癌潜在生物学标志物的可能性,并揭示FEN1在肝细胞癌发生发展中的潜在机制。
1 资料与方法
1.1 临床标本采集
肝细胞癌及其癌旁组织收集于重庆医科大学附属第一医院肝胆外科2013年9月至2014年12月行肝细胞癌手术切除的52例患者,术中无菌取出标本后,一份放入液氮中保存,一份放入4%多聚甲醛中保存。所有病例均经病理切片确诊为肝细胞癌,且患者术前均未行放疗和化疗。患者年龄26~65岁,平均年龄47岁。男34例、女18例。
1.2 免疫组织化学染色
FEN1单克隆抗体购自美国Santa Cruz公司,稀释比例为1:300。实验按照免疫组织化学SP法常规步骤操作。结果分析采取4级半定量积分法,随机选取5个视野(×400)后,计数该视野下肿瘤细胞总数和染色阳性细胞个数,得出阳性率:阳性细胞数≤10%为0分; > 10%~50%为1分, > 50%~80%为2分, > 80%为3分;染色强度分为:无染色为0分,浅黄色为1分,棕黄色为2分,棕褐色为3分。阳性等级根据阳性率和染色强度乘积判定:0分为阴性,1~3分为弱阳性,4~6分为阳性,7~9分为强阳性。
1.3 RT-qPCR检测FEN1在肝细胞癌组织中表达
称取0.2 g冻存肝细胞癌组织及癌旁正常组织研磨匀浆后用Trizol(TaKaRa公司)法提取高质量的细胞总RNA;按试剂盒操作步骤反转录cDNA,以cDNA作为模板进行实时荧光定量PCR检测;扩增FEN1基因引物的序列为上游:5’-AGCCCGTGTATGTCTTTG-3’,下游:5’-AGTCAGGTGTCGCATTAG-3’。以GAPDH作为内参照,引物序列为:上游:5’-ATCTTCTTTTGCGTCGCCAG -3’,下游:5’-TTCCCCATGGTGTCTGAGC-3’。qPCR反应条件为:94℃预变性2 min;93℃变性1 min;54℃褪火1 min;72℃延伸1 min;36个循环;72℃延伸10 min。结果使用标准曲线和CT值比较。
1.4 细胞培养及腺病毒转染
肝癌细胞株HepG2由重庆医科大学附属第一医院实验研究中心提供,培养于含有10%胎牛血清(美国Gibco Invitrogen公司),1%青链霉素的DMEM培养液中,培养箱条件设置为36.8℃,CO2饱和度5%。干扰表达FEN1基因重组腺病毒载体FEN1-siRNA及对照空载腺病毒NC-siRNA采用pSES-HUS系统构建[13],且均以MOI50转染进行后期实验。FEN1-siRNA序列为:正义链:5’-GGACUUGUAGUCCUGCGAUTT-3’,反义链:5’-AUCGCAGGACUACAAGUCCTT-3’。以无干扰序列的空载腺病毒NC-siRNA作为阴性对照组。主要试剂如下:RPMI 1640培养液购于美国Sigma公司;胎牛血清购于美国Gibco公司。
1.5 MTT法检测细胞增殖
对数生长期的HepG2细胞胰酶消化离心后,DMEM重悬并以2×103/孔接种在96孔板内,孵育8 h后弃上清液分别加入无血清RPMI 1640培养液作为空白对照组;NC-siRNA作为阴性对照组;再加入FEN1-siRNA作为实验组。每组设4个复孔。于24、72、120、168 h加入MTT(5 mg/ml)20 μl,继续培养4 h后弃上清液,加入DMSO 100微升/孔,震荡使晶体充分溶解后在酶标仪下测得各孔570 nm处吸光度值。
1.6 流式细胞术检测凋亡
对数生长期的HepG2细胞胰酶消化离心后,DMEM重悬并以5×105/孔接种在6孔板内,孵育8 h后弃上清液后分别加入NC-siRNA作为空白对照组;FEN1-siRNA作为实验组,每组设3个复孔。48 h后流式细胞技术检测各组细胞凋亡。
1.7 Transwell实验
未转染腺病毒及转染了FEN1-siRNA 48 h后的HepG2用胰酶消化离心后用含2%FBS的DMEM培养液配制成5×105/ml的细胞悬液,在Transwell上室(Transwell 8.0 μm Proe Size购于美国Corning Costar公司)中加入100 μl细胞悬液(即细胞数为5×104个),下室每孔内加入300 μl含10%FBS的DMEM培养液,培养24 h后取出小室,弃去液体,用4%多聚甲醛固定20 min;小心用棉签拭去上室膜上的细胞,将Transwell小室用0.1%结晶紫染色并用PBS洗3次后在显微镜下拍照进行细胞计数;在100倍光学显微镜下随机计数5个视野的肿瘤细胞数,取均值。Transwell侵袭实验在迁移实验的基础上用50 mg/L Matrigel 1:8稀释液稀释后包被Transwell小室膜。
1.8 统计学方法
实验数据结果采用SPSS19.0统计软件进行分析。肝细胞癌组织和癌旁正常组织中FEN1 mRNA及蛋白表达差异分别用配对t检验和Wilcoxon秩和检验进行统计分析。临床病理资料与免疫组织化学的结果使用χ2检验差异的显著性。P < 0.05为差异有统计学意义。
2 结果
2.1 FEN1 mRNA在肝细胞癌及癌旁组织中的表达
RT-qPCR结果显示52例肝细胞癌组织中FEN1 mRNA相对表达量为(1.31±0.23),而癌旁组织中为(1.04±0.28),两者比较差异有统计学意义(P < 0.01)。结果显示与正常组织相比,肝细胞癌组织中FEN1 mRNA呈明显高表达状态,见图 1。
图 1 FEN1 mRNA在52对肝细胞癌组织和癌旁正常肝组织中的表达A: higher expression of FEN1 mRNA was detected in 40 pairs of HCC tissues in comparison with the corresponding normal tissues among the 52 pairs; B: the difference of FEN1 mRNA expression between HCC tissues and the matched normal tissues was statistically significant(P < 0.01)Figure 1 Expression of FEN1 mRNA in 52 pairs of HCC tissues and the corresponding normal tissues2.2 FEN1蛋白在52对肝细胞癌和癌旁正常组织中的表达
免疫组织化学染色结果提示FEN1蛋白主要表达于细胞膜及细胞质中,呈棕黄色,见图 2。统计结果显示52例肝细胞癌组织中有40例(77%)FEN1蛋白表达呈阳性,而对应的52例癌旁正常组织中仅11例(21%)FEN1蛋白呈阳性表达。Wilcoxon秩和检验统计分析这两组之间的差异发现肝细胞癌组织中FEN1蛋白表达高于癌旁正常肝组织(P < 0.01),见表 1。
图 2 FEN1蛋白在肝细胞癌组织及癌旁正常肝组织中的表达A, C: the positive expression of FEN1 protein in HCC tissues, mainly in the cytoplasm and membrane (IHC ×200); B, D: the weakly positive or negative expression of FEN1 protein in matched normal tissues (IHC ×200)Figure 2 Immunohistochemical analysis of FEN1 protein expression in HCC and matched normal tissues表 1 肝细胞癌组织和配对的癌旁正常肝组织中FEN1蛋白表达Table 1 Expression of FEN1 protein in HCC tissues and matched normal tissues2.3 FEN1基因表达与肝细胞癌临床病理特征之间的关系
通过52例肝细胞癌组织免疫组织化学染色结果及患者临床资料统计分析可以发现,FEN1蛋白表达与肿瘤分化程度(P=0.017)、肝内转移(P=0.046)、临床TNM分期(P=0.020)相关,而与患者性别、年龄、术前AFP值及肿瘤直径无相关性(P > 0.05),见表 2。
表 2 FEN1蛋白表达与肝细胞癌患者临床病理特征之间的关系Table 2 Relationship between FEN1 expression and clinicopathological features of HCC patients2.4 siRNA显著抑制FEN1基因表达
转染FEN1-siRNA作为实验组,转染空载NC-siRNA作为阴性对照组。两组腺病毒以MOI50转染人肝癌细胞株HepG2 48 h后,通过Western blot检测FEN1基因干扰效率。结果显示FEN1-siRNA能明显敲降FEN1基因表达,抑制率在80%以上,而转染NC-siRNA的HepG2细胞FEN1未见明显敲降,见图 3。
2.5 MTT法检测细胞增殖情况
在各时间点测量吸光度发现未转染腺病毒组、转染NC-siRNA与转染FEN1-siRNA组的A值相比差异有统计学意义(P < 0.05),与未转染腺病毒及转染NC-siRNA相比,转染FEN1-siRNA干扰FEN1表达后HepG2增殖明显受到抑制,见图 4。
2.6 流式细胞术检测细胞凋亡情况
未转染组、转染NC-siRNA及转染FEN1-siRNA组的凋亡率分别为0.09%、3.38%及45.33%,干扰FEN1后HepG2凋亡较未转染组及转染NC-siRNA组明显增加,见图 5,表明体外干扰FEN1后可促进HepG2凋亡(P < 0.05)。
2.7 Transwell实验结果
Transwell实验结果表明对照组Transwell迁移及侵袭试验穿过小室膜的HepG2数为(172±18)及(113±8);干扰组分别为(81±9)及(58±6),见图 6。表明干扰FEN1基因后HepG2迁移及侵袭能力均明显下降(P < 0.05)。
图 6 干扰FEN1后对HepG2细胞迁移、侵袭能力的影响Cells migration and invasive abilities of HepG2 cell lines were measured using the Corning 8.0 μm Transwell® assay. Images (SP ×100) were taken from three independent experiments after 24h. The cells were seeded into the upper Transwell chamber. The initial number of cells for each line was 5×104Figure 6 Effect of FEN1 interferance on migration and invasion of HepG2 cells3 讨论
肝细胞癌最特异性的肿瘤标志物是AFP,其诊断价值已得到肯定,但由于高分化和低分化的肝细胞癌合成AFP较少,肝癌患者AFP阳性率仅60%~70%,并且对于早期肝细胞癌,AFP假阴性率高达20%,在受其他因素影响时也会产生假阳性可能,仅凭AFP诊断原发性肝癌容易发生漏诊与误诊[14]。因此AFP需要结合其他生物指标联合诊断肝细胞癌,FEN1可能为肝细胞癌的潜在肿瘤标志物。FEN1是一种具有特异结构的多功能核酸酶,具有5'-3'flap核酸内切酶、缺口核酸内切酶和核酸外切酶等功能,其在调控冈崎片段成熟、维持端粒酶活性、长片段碱基切除等DNA代谢维护上有着重要作用[15]。近期许多研究提示FEN1功能突变及异常表达为肿瘤发生、发展的重要环节。FEN1在肺腺癌中高表达提示肿瘤侵袭性强预后较差[16];FEN1异常升高提示乳腺癌对化疗药物不敏感,生存期缩短[17];有学者提出FEN1可作为乳腺癌及卵巢癌的肿瘤标志物[18]。之前的一些研究提示FEN1在肿瘤复制中有着关键作用,高表达FEN1可导致错误DNA复制增多,增加肿瘤患病风险;同时异常高表达的FEN1提示肿瘤基因组不稳定,异型性高。因此FEN1在肿瘤发生、发展中都起到了重要作用,其异常高表达往往提示肿瘤恶性程度高、侵袭性强、预后差,这也为FEN1成为肿瘤诊断及治疗的一个新生物靶点提供了理论依据[11]。
虽然研究表明FEN1在不同肿瘤组织中高表达,然而FEN1与肝细胞癌的临床病理关系尚未有明确报道。因此在本研究中,对52对肝细胞癌组织与相应的癌旁正常肝组织中的FEN1进行检测,从mRNA及蛋白质水平两个层面证明了肝细胞癌组织中FEN1表达显著高于癌旁正常肝组织。结果表明FEN1在肝细胞癌组织中明显高表达,且其表达量随肿瘤的进展而逐渐升高,可作为肝细胞癌诊断的一个潜在生物学指标,并且可以辅助判断肝细胞癌恶性程度及转移风险。为了进一步研究FEN1与肝细胞癌的关系,我们采用siRNA干扰了肝癌细胞株HepG2中FEN1基因表达,MTT及流式细胞术检测表明干扰FEN1表达后肝癌细胞株HepG2增殖能力受到明显抑制,凋亡率显著增加。Transwell实验发现干扰FEN1基因表达后肝细胞癌细胞迁移、侵袭能力均明显减弱。
本实验结果同以往许多实验研究[19]相符合,结果表明FEN1作为DNA代谢过程的多功能核酸酶对肿瘤发生、发展起到了关键作用。FEN1的突变可能导致基因组不稳定从而引发细胞的恶性转化;该基因过度表达赋予肿瘤细胞生长优势,导致肿瘤侵袭性上升,易于转移。干扰FEN1基因表达后发现,肝癌细胞侵袭行为显著降低,细胞增殖受到抑制,凋亡率明显升高,侵袭、迁移能力下降,这提示FEN1可作为肝细胞癌诊断及治疗的一个新生物靶点。利用FEN1-siRNA沉默基因的效应,制备高纯度的腺病毒或将RNAi腺病毒与其他抗肿瘤药物耦联, 这些技术有望提高靶向肿瘤基因治疗效果,为肝癌临床治疗提供可能性。目前, 我们已制备了3种FEN1-siRNA, 分别靶向FEN1基因的不同位点。虽然许多研究表明FEN1高表达与肿瘤的侵袭和预后差相关,然而肿瘤的发生、侵袭和转移是一复杂的多步骤的连续过程, 其具体的发生细节和转移机制尚未充分了解, 尤其是FEN1端粒酶作用分子机制尚未完全明了。我们将在今后的实验中进一步研究探讨FEN1基因端粒酶活性与肝细胞癌的关系及机制。
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表 1 肝细胞癌组织和配对的癌旁正常肝组织中FEN1蛋白表达
Table 1 Expression of FEN1 protein in HCC tissues and matched normal tissues
表 2 FEN1蛋白表达与肝细胞癌患者临床病理特征之间的关系
Table 2 Relationship between FEN1 expression and clinicopathological features of HCC patients
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