miR196α基因多态性在原发性肝癌发生发展中的作用及机制研究
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摘要
原发性肝癌(Primary liver cancer, PLC,以下简称肝癌)是常见恶性肿瘤。
由于起病隐匿,早期没有症状或症状不明显,进展迅速,确诊时大多数患者已
经达到局部晚期或发生远处转移,治疗困难,预后很差,如果仅采取支持对症
治疗,自然生存时间很短,严重地威胁人民群众的身体健康和生命安全。我国
是乙肝大国,乙肝相关性肝癌的发病人群更为集中,因此,在我国研究肝癌的
发病机制、生物学行为、诊疗、预后等,显得更为重要和迫切。原发性肝癌主要包括肝细胞癌(HCC)、肝内胆管细胞癌(ICC)和肝细胞癌-
肝内胆管细胞癌混合型等不同病理类型,由于其中HCC占到90%以上,故本文
所指的“肝癌”主要是指HCC。肝癌的发病机制十分复杂,其发生、发展和转移与多种基因的突变、细胞
信号转导通路和新生血管增生异常等密切相关,其中存在着多个关键性环节,
正是进行分子靶向治疗的理论基础和重要的潜在靶点。分子靶向药物治疗在控
制HCC的肿瘤增殖、预防和延缓复发转移以及提高患者的生活质量等方面具有
独特的优势。近年来,应用分子靶向药物治疗HCC已成为新的研究热点,受到
高度的关注和重视。对于编码基因的单核苷酸多态性和肝癌的相关性研究已经
很多,然而,非编码基因miRNA单核苷酸基因多态性和肿瘤的相关性研究刚刚
起步。miRNA被称为微小RNA (microRNAs或miRNAs),是长度为21-23nt的小
型非编码RNA,也是RNA家族的成员,能够识别特定目标mRNA并在转录后
水平通过促进靶mRNA的降解、抑制其翻译过程而发挥负调控基因表达作用。
随着人们对miRNAs研究的深入,逐渐发现miRNAs通过调节细胞增殖,分化
和凋亡的功能,导致组织的增生异常,这些调节作用则大多数是通过调控表达
信号的分子(如细胞因子、转录因子、生长因子、促凋亡和抗凋亡基因)实现
的,并且发现肝癌中也有一些差异表达miRNA,而且miRNA的差异性表达与
HCC增殖、转移等相关。研究发现miR196a(rs11614913),miR-146a(rs2910164C>G),miR-499T>C(rs3746444)在基因变异率大于5%,研究其单核苷酸的多态性和肝癌的关系,可为肝癌的早诊断、分子靶向治疗,提供依据。
miR-196包括miR-196a1,miR-196a2,miR-196b,位于染色体17q21,12q13,和7p15,miR-196a和miR-196b只有一个碱基的差别。miR-196a2(rs11614913)单核苷酸的基因多态性(A single-nucleotide polymorphism,SNP)发生在核苷酸在T和C之间转换。有研究发现,miRl96a在结肠癌,肺癌,乳腺癌等有基因多态性的表达。提示,miR196a可能与肿瘤发生发展有关。
同样,miR-146也包括miR-146a和miR-146b,其在基因的调节、细胞生长周期及免疫系统的调节中都有非常重要的作用。miR-146在大多数肿瘤中是抑癌基因。Hurst等[4]的研究发现,在乳腺癌中乳腺癌转移抑制基(Breastmetastasissuppressor1,BRMS1)蛋白是依赖于miR-146a的蛋白,miR-146a的上调会降低乳腺癌细胞转移的能力。可能也与表皮生长因子受体(EpidermalGrowth FactorReceptor,EGFR)的下调有关.Lin等[5]发现,在胰腺癌中miR-146a的上调参与胰腺细胞的上调修饰机制抑制如异黄酮等物质,导致这些细胞的侵袭能力下降,反过来又导致了EGFR表达的下降。Katakowski等[6]研究报道,在人脑胶质瘤细胞株中,miR-146b-5p抑制EGFR的表达.翻译,并与3'UTR的结合,也降低了脑胶质瘤细胞的侵袭、迁移、和蛋白激酶B(AKT)的磷酸化,MeiJ[7]最近的研究也支持miR-146抑制胶质母细胞瘤。研究也发现miR-146a rs2910164C>G多态性与先天性心脏病,类风湿关节炎,急性及慢性乙型肝炎肝衰竭等多种疾病有相关性。然而,miR-146a与肝癌的研究鲜见报道。
miR-499多被认为是冠心病的保护基因,然目前很多学者在肿瘤等方面的研究也发现,miR-499和细胞衰老,凋亡,免疫应答,肿瘤发生、转移有关,[11,12,13]。近年来也有和肝癌的研究,但尚未定论。有研究报道,miR-196C>T(rs11614913)和miRNA-499A>G(rs3746444)多态性增加乙肝肝癌易感性。miR-146a(rs2910164C>G)和miR-499(rs3746444)多态性可能和病毒有关,直接调节病毒的复制或病毒感染后肝细胞的增殖。然而,Xu等meta分析了miR-146a G>C (rs2910164), miR-196a-2C> T(rs11614913)和miR-499T>C (rs3746444)基因多态性和肝癌的关系,认为miR-146a*C多态性能减少亚洲男性肝癌的发病风险,miR-196a-2*T多态性和肝癌的相关性在高加索人(欧洲、北非等人)群中有统计学意义,miR-499*C多态性和肝癌的无相关性。[14,15,16,17]而目前尚无中国人的数据。
miRNA的单核苷酸多态性可发生在miRNA成熟的任一过程,单核苷酸的基因多态性SNP (A single-nucleotide polymorphism SNP)甚至成为第三代遗传标志,作为人类基因组计划走向应用的重要步骤,影响到药物和疾病的易感性。经基因miRBase查询,miR-146aG>C, miR-196a2C>T在中国人群中变异率处于前三位,不小于5%。综上所述,一些实验说明miR-146突变影响了miRNA的成熟,同时新生成了miR-146序列,miR-146a*C和miR-146a*G,这种序列增加了肝癌的发病率,在中国人群中的分布有多态性的表现。miR196a也同miRl46一样,其CC基因型和C等位基因能显著增加我国男性乙肝患者发生HCC的风险,并有学者也认为T位基因在男性HCC患者中与淋巴结转移有相关性。miR-499基因多态性位于miR499成熟的主要位点,可能使A：U变成G：U,这种变化也影响肿瘤,有报道miR499和肝癌有相关性。然目前此3种基因在我国肝癌的研究中例数较少,有Meta分析因缺乏例数,统计学意义不明显,并且结论不一致。因此,开展对miR146aG>C (rs2910164C>G), miR196a2C>T(rs11614913)和miR-499A>G(rs3746444)单核苷酸基因多态性和肝癌相关性的研究,意义重大。同时,也可为肝癌的早期诊断提供循证医学证据,为临床防治肝癌侵袭和转移寻找分子靶点提供理论依据。
本研究分三部分：
第一部分miRNA146a、miRNA196a、miRNA499单核苷酸基因多态性与原发性肝癌的相关性研究
第二部分miRNA196a的低表达对肝癌细胞系HepG2生物学行为的影响
第三部分miRNA196a对HepG2细胞HOXB8、HOXB9、P53、Caspase-3表达的影响。
第一部分miRNA146a、miRNA196a、miRNA499单核苷酸基因多态性与原发性肝癌的相关性研究
目的：研究miRNA-146a、miRNA-196a、miRNA-499单核苷酸基因多态性与原发性肝癌的相关性研究,为肝癌的诊断、分子靶向治疗提供循证医学的依据。
方法：收集原发性肝癌患者的血285例；健康对照组300例；提取外周血中DNA,利用聚合酶链式反应限制性片段长度多态性方法(RFLP-PCR),检测miR196C>T(rs11614913) miRNA499A>G(rs3746444)miR146aG>C(rs2910164C>G)的多态性,然后利用Hardy-Weinberg平衡方程检验抽样的平衡性,分析基因多态性在原发性肝癌患者人群中的表达情况。
结果：
1、在遗传平衡检验对照组miR196C>T(rs11614913)miRNA499A>G(rs3746444) miR146aG>C (rs2910164C>G)的基因型及其等位基因和基因图谱里比较,(P=0.056,0.051,0.34),此次抽样没有人口分布的不同及样品的偏倚,均符合遗传平衡定律。
2、285个患者中,资料完整,能找到相关配对235(82.46%)被研究,其中169名男性,66名女性。对照组根据资料完整能配对选取281例。利用logistic分析,年龄、性别、吸烟均和肝癌无关。饮酒、家族史、乙肝、乙肝和/或丙肝感染均为原发性肝癌的独立危险因素,95%可信区间表示：数值分别为1.60(1.07-2.39),8.51(1.96-76.88),18.26(10.66-31.61)和64.24(15.51-558.03)。
3、各基因的分布频率及与原发性肝癌相关性分析：
miRNA146aG>C以CC基因为参照,CC、GC比较,3组间无统计学意义(X2=0.99,p=0.61),经性别、年龄、家族史校正后,95%可信区间分别为0.89(0.65-1.24)和0.93(0.48-1.83),无统计学差异(p>0.05)。以C等位基因为参照,与G等位基因组比较,2组间无统计学意义(X2=0.30,p=0.58),经性别、年龄、家族史校正后95%的可信区间1.07(0.82-1.39),P=0.62,无统计学意义。
miRNA196a2C>T以CC基因组为参照,CT、TT比较,3组间有统计学意义(X2=6.60,p=0.037)。经性别、年龄、家族史校正后,CT组95%可信区间0.69(0.45-1.05),P=0.06,TT组95%可信区间0.51(0.28-0.90),P=0.01。C等位基因做参照,T等位基因比较(X2=4.81,p=0.028),经性别、年龄、家族史校正后,等位基因组95%可信区间0.74(0.57-0.95),P=0.02。miR-196a2C>T多态性和原发性肝癌的发病有相关性。
miRNA499A>G以AA基因组为参照,AG、GG比较,3组间无统计学意义(X2=3.74,p=0.154)。经性别、年龄、家族史校正后,95%可信区间分别为1.14(0.72-1.78)和1.91(0.94-3.99),无统计学意义P>0.05。以A等位基因为参照,与G等位基因比较,(X2=3.45,p=0.063),经性别、年龄、家族史校正后95%可信区间分别1.35(0.97-1.87),P=0.06,均无统计学意义。
miRNA196a2C>T的CC基因增加肝癌的风险,经性别、年龄、家族史校正后,OR95%可信区间2.18(1.23-3.80)。miR-196a2C等位基因,病例组和对照组相比增加了1.64倍肝癌风险。4miRNA196a2C>T基因多态性在乙肝、丙肝背景下和肝癌相关性研究
logistic回归分析：miRNA196a2C>T(rs11614913), CC、CT、TT、C等位、T等位,在对照组分别为23.84%,56.94%,19.57%,52.31%,48.4%,在没有病毒感染的病例组27.6%,55.2%,17.2%,55.2%,44.8%,乙肝感染组34.6%,53.4%,12%,61.3%,38.7%,OR95%可信区间1.0(Ref.),0.65(0.39-1.06),0.42(0.20-0.86),1.0(Ref.),0.69(0.51-0.94),丙肝感染组33.3%,52.8%,13.9%,59.7222%,40.2778%,OR95%可信区间1.0(Ref.),0.66(0.29-1.59),0.63(0.16-2.11),1.0(Ref.),0.73(0.43-1.24)。
乙肝感染组miRNA196a2C>T单核苷酸基因多态性和肝癌之间有相关性。在丙肝(无乙肝感染的丙肝组),无病毒感染组,niRNA196a2C>T单核苷酸基因多态性和肝癌之间没有相关性。miRNA196a2TC和CC基因型增加了肝癌的易感性。通过交互作用分析乙肝和miRNA196a2C>T之间交互性P=0.007。
结论：
1本实验发现miRNA196a2C>T的CC基因在肝癌患者中检出率高于对照组,CT、TT低于对照组,单核苷酸基因多态性与肝癌有相关性。而miRNA146aG>C与miRNA499A>G单核苷酸基因多态性和肝癌之间无相关性。
2在乙肝病例组miRMA196a2C>T单核苷酸基因多态性在肝癌的病例中和乙肝有交互作用。
第二部分miRNA196a的低表达对肝癌细胞系HepG2生物学行为的影响
目的：通过构建抑制miRNAl96a2C>T表达的载体,转染HepG2细胞,观察细胞增殖、迁移、凋亡,以探讨miRNA196在肝癌发生.发展中的作用.
方法：针对目标序列miRNA-196a2C>T合成特异性反义序列片段载体,利用海绵法重组干扰miR196a的质粒,利用脂质体技术瞬时转染肝细胞癌HepG2细胞株。本实验设三组分别为niR196a干扰组、阴性对照组(negative control,NC组)和正常对照组(ormal,N组)；qPCR鉴定外表达miR196a基因,用cck8检测细胞增殖率、transwell、室迁移实验检测细胞迁移率,annexinV/PI凋亡实验检测细胞凋亡率。
结果：
1荧光定量PCR验证miRNA196a2C>T的抑制效果
质粒转染48小时后测miRNA196a2mRNA表达量,结果示：干扰组niRl96a mRNA的表达量比NC组,正常对照组表达量均低,以正常对照组为1,干扰组的表达下降了41%。
2CCK8检测各实验组细胞的增殖
CCK8法对各实验组HepG2增殖情况的检测结果显示：正常对照组至24h时的OD值为0.86±0.16,NC组的OD值0.80±0.18,miR196a干扰组0.62±0.25,NC组细胞和正常对照组相比增殖率92.21±1.82%(P<0.01),miR196a干扰组和正常对照组相比72.29±2.51%(P<0.01),可见抑制miR196a表达后可以抑制HepG2的增殖。
3采用Transwell法检测细胞迁移和侵袭能力的改变
迁移实验显示,HepG2细胞在miR196a干扰组每个视野迁移的细胞数(58.4±2.46),正常对照组(101.6±3.77)NC组(99.6±2.62),miR196a干扰组与正常对照组、NC组相比(P<0.01),NC和正常对照组相比无统计学意义(P>0.05)
4AnnexinV-FITC/PI流式细胞术检测各组凋亡
采用Annexin V-FITC及PI(碘化丙啶)双标记流式细胞术检测细胞凋亡率,流式图右上象限为晚期凋亡细胞,右下象限为早期凋亡细胞。检测到HepG2凋亡率为正常组0.35±0.49%；NC组HepG2凋亡率为6.25±0.91%,miR196a干扰组为25.47±1.24%。结果表明miR196a干扰组凋亡明显,三组间比较p<0.05。阴性对照也能导致细胞凋亡,niR196a组与阴性对照组比较和正常组比较均有统计学意义(p<0.05)。
结论：niRNA196a2C>T影响肝癌细胞的生物学行为,miR196a低表达后抑制肝癌细胞增殖,及迁移,促进肝癌细胞的凋亡。
第三部分miRNA196a对HepG2细胞HOXB8、HOXB9、P53、Caspase-3表达的影响
目的：通过研究HOXB8、HOXB9、P53、Caspase-3mRNA和蛋白的表达情况,探讨miRNA-196a影响肝癌细胞增长、凋亡、迁移的机制,为肝癌的诊断及靶点治疗提供依据。
方法：
分组同实验二,分别采用Real-time PCR实时定量聚合酶链反应方法和Western-Blot检测同源基因HoxB8、HoxB9、凋亡基因caspase-3、p53的mRNA和蛋白的表达。
结果：
1Caspase-3mRNA表达水平miR196a干扰组(1.06±0.02),NC组(1.03±0.04),正常对照组(1.02±0.03),miRl96a干扰组显著高于对照组和NC(P<0.05),NC组和对照组无统计学意义(p>0.05)。Western-Blot Caspase-3蛋白表达水平miR196a干扰组(0.68±0.012),NC组(0.46±0.03),正常对照组(0.56±0.03),miR1.96a干扰组显著高于对照组、NC(F=54.13,P<0.05),NC组和对照组无统计学意义(p>0.05)。
2P53mRNA表达水平miR196a干扰组(0.97±0.009),NC组(0.99±0.011),正常对照组(1.00±0.012),miR196a干扰组、NC组、对照组三组之间,miR196a干扰组分别和NC、对照组比较,均无统计学意义(p>0.05)。Western-Blot P53蛋白表达水平miR196a干扰组(0.59±0.03),NC组(0.47±0.01),正常对照组(0.52±0.04),miR196a干扰组高于对照组、NC,但无统计学意义(p>0.05)。
3HoxB8mRNA表达水平miR196a干扰组(1.19±0.01),NC组(0.99±0.09),正常对照组(1.00±0.013),miR196a干扰组显著高于对照组,NC(F=770P<0.05),NC组和对照组无统计学意义(p>0.05)。Western-Blot HoxB8蛋白表达水平miR196a干扰组(0.60±0.05),NC组(0.35±0.05),正常对照组(0.60±0.05),miR196a干扰组显著高于对照组,NC(F=25.257,P<0.05),NC组和对照组无统计学意义(p>0.05)。
4HoxB9mRNA表达水平miR196a干扰组(1.00±0.02),NC组(0.96±0.04),正常对照组(1.00±0.02),miRl96a干扰组、对照组,NC组比较无统计学意义(p>0.05)。
结论：干扰miR-196a表达后导致Caspase-3、HoxB8mRNA和蛋白升高,而HoxB9mRNA及p53mRNA和蛋白表达没有明显变化,证明miRl96a可能通过调节凋亡因子Caspase-3和HoxB8表达对肝癌细胞的增殖、迁移、凋亡起调节作用,此过程和HoxB9mRNA、P53基因无相关性。
HCC is the common malignant tumor. Its high fatality rates, the incidence and mortality rate of HCC are almost equal. The Therapy is very difficult.Its Pathology including HCC,ICC. HCC and ICC et al. we dicuss the relationship miRNA and HCC. It is well known that chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, cirrhosis, aflatoxin B1and excessive alcohol drinking are the main risk factors for HCC. Although many people are exposed to these risk factors of HCC, only about10%of these exposed subjects develop HCC during their lifetime Therefore, genetic factors may play an important role in the development of HCC. microRNAs (miRNAs) are short, non-coding RNAs of approximately23nucleotides that regulate target genes. microRNAs have been demonstrated to have a role in several biochemical pathways in cell differentiation, proliferation, apoptosis and carcinogenesis. In the pathogenesis of HCC, miRNAs may have an important role in progression and directly contribute to cell proliferation, avoidance of apoptotic cell death and metastasis by targeting a large number of critical protein-coding genes.
The molecular mechanisms of HCC is complicated. Angiogenesis and metastasis play important roles in the progression and recurrence of HCC. Molecular therapeutic targets is a new avenue for the development of anti-HCC treatments. It has been well demonstrated that SNPs in protein-coding genes can affect the functions of proteins and in turn influence the individual susceptibility to cancers. In as much as the importance of miRNAs has only been recognized recently, little is known about the biological function of SNPs in the miRNA genes.SNP (A single-nucleotide polymorphism SNP) is the third sign of heredity.Previous epidemiologic studies have shown that genetic variations in miRNAs are associated with various diseases and cancers. SNP of miRNA consists in epigentics, murture of miRNA,mutant of mRNA.SNP effects susceptibility of HCC. Studing SNP make human gene project practice and the potential application of miRNAs as predictive, diagnostic, and prognostic biomarkers of HCC and their potential roles in cancer treatment. By checking mibase, we found the single-nucleotide mutant of miR-196a2C>T and miR-499A>G miR-146aG>C is common.
The gene miR196a are located in the regions of homeobox (HOX) clusters within the genome of vertebrates. One nuclieotide diffence is between miR196aa and miR196ab.
The miR146are consisted of miR146a and miR146b. Breast cancer metastasis suppressor up-regulates miR146, which suppresses breast cancer metastasis.miR-146a suppresses invasion of pancreatic cancer. miR146suppresses EGFR expression and reduces in vitromigration and invasion of gtioma.
miR-499participates in several crucial cancer-related biology processesn such as apoptosis,cell migration,cell senescence, and inflammation.
It is reported that miR-196a2C>T and miR-499A>G polymorphisms are associated with various cancer, such as breast cancer, lung cancer, gastric cancer and esophageal cancer. miR-196aC>T and miR-499A>G are associated with the susceptibility to hepatitis B-virus-related HCC, which suggested that miR-196aC>T and miR-499A>G can serve as repressors for viral infection pathways and play a key regulator in host-virus interaction and regulation of viral replication. Therefore, a recent meta-analysis showed no association between miR-146aG>C and miR-499A>G and risk of HCC. In our study, we investigated the association between polymorphisms in miR-146aG>C, miR-196a2C>T and miR-499A>G and risk of HCC, and its interaction with HBV infection in Chinese population.miR196a are located in HOX cluster.We will study miR196a mechanism in HepG2cell.The discovery of new types or novel functions of miRNAs provides us with more and deeper insights into the molecular mechanism underlying thepathogenesis of HCC.
The project contain three parts as below:
Part1:Associations Between Three Common MicroRNA Polymorphisms and Hepatocellular Carcinoma Risk in Chinese.
Part2:The inhibiting expression of microRNA196on biological behaviors in human hepatic cancer cell line HepG2
Part3:miR196a regulate the expression of HoxB8、HoxB9, caspase-3P53in HepG2cell line
Part1:Associations Between Three Common MicroRNA Polymorphisms and Hepatocellular Carcinoma Risk
Objective:Associations between polymorphisms in miR-146aG>C, miR-196a2C>T and miR-499A>G and risk of HCC, and interaction with HBV infection, were the target of the present research.
Methods:The duplex polymerase-chain-reaction with confronting-two-pair primers (PCR-RFLP) was performed to determine the genotypes of the miR-146aG>C, miR-196a2C>T and miR-499A>G genotypes. Associations of polymorphisms with the risk of HCC were estimated by conditional logistic regression analysis..
Results:Drinking, family history of cancer, HBsAg and HCV were risk factors for HCC. Multivariate regression analyses showed that subjects carrying the miR-196a2CC genotype had significantly increased risk of HCC, with an adjusted OR (95%CI) of2.18(1.23-3.80). In addition, cases carrying the miR-196a2C allele had a1.64-fold increase in the risk for HCC (95%CI=1.03-2.49). The miR-196a2CT
and TT genotypes greatly significantly increased the risk of HCC in subjects with HBV infection, with adjusted ORs (95%CI) of2.02(1.12-3.68) and2.69(1.28-5.71), respectively.
Conclusions:Our results demonstrate that miR-196a2CC genotype and C allele have an important role in HCC risk in Chinese, especially in patients with HBV infection.
Part2:The inhibiting expression of microRNA196on biological behaviors in human hepatic cancer cell line HepG2
Objective:To study the effect of inhibiting miR196a on biological behaviors in human hepatic cancer cell
Methods:The antisense RNA of microRNA-196a was synthesized and cloned into the vector. HepG2cells were infected by inhibiting miRl96a vector. The HepG2cells were devided into miR196a lower expression group, NC group and N group in vitro. The expression of the targets of miR-196a was detected by qPCR. Cell growth was analyzed by cck8assay. The invasion was dectected by transwell method. Apoptosis was detected by annexinV/PI.
Results:.1) The expression level of miR-196a was less than normal (41%).(2) The proliferation of HepG2was also markedly suppressed in inhibiting miR196a at the24h point than normal about72.29±2.51%(P<0.01).(3) The number of cells that migrated through the chamber of miR196a inhibiting group is less than normal and NC (P<0.01).(4) The cell apopotosis in miR196a inhibiting group is more than NC and normal group (p<0.05).
Conclusions:MicroRNA-196a lower expression in HepG2surppresses the proliferation of and migration of HepG2cells, and promotes tumor cell apoptosis.
Part3:miR196a effects expression of Caspase-3P53HoxB8HoxB9in HepG2cell Objective:To explore the role of miR-196a on the regulatory mechanism in Hepatocelluar carcinoma cell line by detecting Caspase-3P53HoxB8HoxB9mRNA and Protein.
Methods:The antisense RNA of microRNA-196a was synthesized and cloned into the vector. HepG2cells were infected by inhibiting miR196a vector. The HepG2cells were devided into miR196a lower expression group, NC group and N group in vitro. The P53,caspase-3, HMGA2,HOXB8mRNA and their Protein was detected by qPCR and Weatern-Blot.
Results:.HOXB8mRNA and protein expression,. in HepG2.cell line miR196a inhibiting group is significantly less than normal,NC (P<0.05). Caspase-3mRNA and Protein expression is maximum in three groups (p<0.05). The P53mRNA and Protein, HOXB9mRNA expression in three groups is no markly difference.
Conclusions:MiR-196a can effect the proliferation, the apoptosis and migration of HepG2cell lines by gene HOXB8,caspase-3regulation. However miR-196a is irrelation with P53, HOXB9.
由于起病隐匿,早期没有症状或症状不明显,进展迅速,确诊时大多数患者已
经达到局部晚期或发生远处转移,治疗困难,预后很差,如果仅采取支持对症
治疗,自然生存时间很短,严重地威胁人民群众的身体健康和生命安全。我国
是乙肝大国,乙肝相关性肝癌的发病人群更为集中,因此,在我国研究肝癌的
发病机制、生物学行为、诊疗、预后等,显得更为重要和迫切。原发性肝癌主要包括肝细胞癌(HCC)、肝内胆管细胞癌(ICC)和肝细胞癌-
肝内胆管细胞癌混合型等不同病理类型,由于其中HCC占到90%以上,故本文
所指的“肝癌”主要是指HCC。肝癌的发病机制十分复杂,其发生、发展和转移与多种基因的突变、细胞
信号转导通路和新生血管增生异常等密切相关,其中存在着多个关键性环节,
正是进行分子靶向治疗的理论基础和重要的潜在靶点。分子靶向药物治疗在控
制HCC的肿瘤增殖、预防和延缓复发转移以及提高患者的生活质量等方面具有
独特的优势。近年来,应用分子靶向药物治疗HCC已成为新的研究热点,受到
高度的关注和重视。对于编码基因的单核苷酸多态性和肝癌的相关性研究已经
很多,然而,非编码基因miRNA单核苷酸基因多态性和肿瘤的相关性研究刚刚
起步。miRNA被称为微小RNA (microRNAs或miRNAs),是长度为21-23nt的小
型非编码RNA,也是RNA家族的成员,能够识别特定目标mRNA并在转录后
水平通过促进靶mRNA的降解、抑制其翻译过程而发挥负调控基因表达作用。
随着人们对miRNAs研究的深入,逐渐发现miRNAs通过调节细胞增殖,分化
和凋亡的功能,导致组织的增生异常,这些调节作用则大多数是通过调控表达
信号的分子(如细胞因子、转录因子、生长因子、促凋亡和抗凋亡基因)实现
的,并且发现肝癌中也有一些差异表达miRNA,而且miRNA的差异性表达与
HCC增殖、转移等相关。研究发现miR196a(rs11614913),miR-146a(rs2910164C>G),miR-499T>C(rs3746444)在基因变异率大于5%,研究其单核苷酸的多态性和肝癌的关系,可为肝癌的早诊断、分子靶向治疗,提供依据。
miR-196包括miR-196a1,miR-196a2,miR-196b,位于染色体17q21,12q13,和7p15,miR-196a和miR-196b只有一个碱基的差别。miR-196a2(rs11614913)单核苷酸的基因多态性(A single-nucleotide polymorphism,SNP)发生在核苷酸在T和C之间转换。有研究发现,miRl96a在结肠癌,肺癌,乳腺癌等有基因多态性的表达。提示,miR196a可能与肿瘤发生发展有关。
同样,miR-146也包括miR-146a和miR-146b,其在基因的调节、细胞生长周期及免疫系统的调节中都有非常重要的作用。miR-146在大多数肿瘤中是抑癌基因。Hurst等[4]的研究发现,在乳腺癌中乳腺癌转移抑制基(Breastmetastasissuppressor1,BRMS1)蛋白是依赖于miR-146a的蛋白,miR-146a的上调会降低乳腺癌细胞转移的能力。可能也与表皮生长因子受体(EpidermalGrowth FactorReceptor,EGFR)的下调有关.Lin等[5]发现,在胰腺癌中miR-146a的上调参与胰腺细胞的上调修饰机制抑制如异黄酮等物质,导致这些细胞的侵袭能力下降,反过来又导致了EGFR表达的下降。Katakowski等[6]研究报道,在人脑胶质瘤细胞株中,miR-146b-5p抑制EGFR的表达.翻译,并与3'UTR的结合,也降低了脑胶质瘤细胞的侵袭、迁移、和蛋白激酶B(AKT)的磷酸化,MeiJ[7]最近的研究也支持miR-146抑制胶质母细胞瘤。研究也发现miR-146a rs2910164C>G多态性与先天性心脏病,类风湿关节炎,急性及慢性乙型肝炎肝衰竭等多种疾病有相关性。然而,miR-146a与肝癌的研究鲜见报道。
miR-499多被认为是冠心病的保护基因,然目前很多学者在肿瘤等方面的研究也发现,miR-499和细胞衰老,凋亡,免疫应答,肿瘤发生、转移有关,[11,12,13]。近年来也有和肝癌的研究,但尚未定论。有研究报道,miR-196C>T(rs11614913)和miRNA-499A>G(rs3746444)多态性增加乙肝肝癌易感性。miR-146a(rs2910164C>G)和miR-499(rs3746444)多态性可能和病毒有关,直接调节病毒的复制或病毒感染后肝细胞的增殖。然而,Xu等meta分析了miR-146a G>C (rs2910164), miR-196a-2C> T(rs11614913)和miR-499T>C (rs3746444)基因多态性和肝癌的关系,认为miR-146a*C多态性能减少亚洲男性肝癌的发病风险,miR-196a-2*T多态性和肝癌的相关性在高加索人(欧洲、北非等人)群中有统计学意义,miR-499*C多态性和肝癌的无相关性。[14,15,16,17]而目前尚无中国人的数据。
miRNA的单核苷酸多态性可发生在miRNA成熟的任一过程,单核苷酸的基因多态性SNP (A single-nucleotide polymorphism SNP)甚至成为第三代遗传标志,作为人类基因组计划走向应用的重要步骤,影响到药物和疾病的易感性。经基因miRBase查询,miR-146aG>C, miR-196a2C>T在中国人群中变异率处于前三位,不小于5%。综上所述,一些实验说明miR-146突变影响了miRNA的成熟,同时新生成了miR-146序列,miR-146a*C和miR-146a*G,这种序列增加了肝癌的发病率,在中国人群中的分布有多态性的表现。miR196a也同miRl46一样,其CC基因型和C等位基因能显著增加我国男性乙肝患者发生HCC的风险,并有学者也认为T位基因在男性HCC患者中与淋巴结转移有相关性。miR-499基因多态性位于miR499成熟的主要位点,可能使A：U变成G：U,这种变化也影响肿瘤,有报道miR499和肝癌有相关性。然目前此3种基因在我国肝癌的研究中例数较少,有Meta分析因缺乏例数,统计学意义不明显,并且结论不一致。因此,开展对miR146aG>C (rs2910164C>G), miR196a2C>T(rs11614913)和miR-499A>G(rs3746444)单核苷酸基因多态性和肝癌相关性的研究,意义重大。同时,也可为肝癌的早期诊断提供循证医学证据,为临床防治肝癌侵袭和转移寻找分子靶点提供理论依据。
本研究分三部分：
第一部分miRNA146a、miRNA196a、miRNA499单核苷酸基因多态性与原发性肝癌的相关性研究
第二部分miRNA196a的低表达对肝癌细胞系HepG2生物学行为的影响
第三部分miRNA196a对HepG2细胞HOXB8、HOXB9、P53、Caspase-3表达的影响。
第一部分miRNA146a、miRNA196a、miRNA499单核苷酸基因多态性与原发性肝癌的相关性研究
目的：研究miRNA-146a、miRNA-196a、miRNA-499单核苷酸基因多态性与原发性肝癌的相关性研究,为肝癌的诊断、分子靶向治疗提供循证医学的依据。
方法：收集原发性肝癌患者的血285例；健康对照组300例；提取外周血中DNA,利用聚合酶链式反应限制性片段长度多态性方法(RFLP-PCR),检测miR196C>T(rs11614913) miRNA499A>G(rs3746444)miR146aG>C(rs2910164C>G)的多态性,然后利用Hardy-Weinberg平衡方程检验抽样的平衡性,分析基因多态性在原发性肝癌患者人群中的表达情况。
结果：
1、在遗传平衡检验对照组miR196C>T(rs11614913)miRNA499A>G(rs3746444) miR146aG>C (rs2910164C>G)的基因型及其等位基因和基因图谱里比较,(P=0.056,0.051,0.34),此次抽样没有人口分布的不同及样品的偏倚,均符合遗传平衡定律。
2、285个患者中,资料完整,能找到相关配对235(82.46%)被研究,其中169名男性,66名女性。对照组根据资料完整能配对选取281例。利用logistic分析,年龄、性别、吸烟均和肝癌无关。饮酒、家族史、乙肝、乙肝和/或丙肝感染均为原发性肝癌的独立危险因素,95%可信区间表示：数值分别为1.60(1.07-2.39),8.51(1.96-76.88),18.26(10.66-31.61)和64.24(15.51-558.03)。
3、各基因的分布频率及与原发性肝癌相关性分析：
miRNA146aG>C以CC基因为参照,CC、GC比较,3组间无统计学意义(X2=0.99,p=0.61),经性别、年龄、家族史校正后,95%可信区间分别为0.89(0.65-1.24)和0.93(0.48-1.83),无统计学差异(p>0.05)。以C等位基因为参照,与G等位基因组比较,2组间无统计学意义(X2=0.30,p=0.58),经性别、年龄、家族史校正后95%的可信区间1.07(0.82-1.39),P=0.62,无统计学意义。
miRNA196a2C>T以CC基因组为参照,CT、TT比较,3组间有统计学意义(X2=6.60,p=0.037)。经性别、年龄、家族史校正后,CT组95%可信区间0.69(0.45-1.05),P=0.06,TT组95%可信区间0.51(0.28-0.90),P=0.01。C等位基因做参照,T等位基因比较(X2=4.81,p=0.028),经性别、年龄、家族史校正后,等位基因组95%可信区间0.74(0.57-0.95),P=0.02。miR-196a2C>T多态性和原发性肝癌的发病有相关性。
miRNA499A>G以AA基因组为参照,AG、GG比较,3组间无统计学意义(X2=3.74,p=0.154)。经性别、年龄、家族史校正后,95%可信区间分别为1.14(0.72-1.78)和1.91(0.94-3.99),无统计学意义P>0.05。以A等位基因为参照,与G等位基因比较,(X2=3.45,p=0.063),经性别、年龄、家族史校正后95%可信区间分别1.35(0.97-1.87),P=0.06,均无统计学意义。
miRNA196a2C>T的CC基因增加肝癌的风险,经性别、年龄、家族史校正后,OR95%可信区间2.18(1.23-3.80)。miR-196a2C等位基因,病例组和对照组相比增加了1.64倍肝癌风险。4miRNA196a2C>T基因多态性在乙肝、丙肝背景下和肝癌相关性研究
logistic回归分析：miRNA196a2C>T(rs11614913), CC、CT、TT、C等位、T等位,在对照组分别为23.84%,56.94%,19.57%,52.31%,48.4%,在没有病毒感染的病例组27.6%,55.2%,17.2%,55.2%,44.8%,乙肝感染组34.6%,53.4%,12%,61.3%,38.7%,OR95%可信区间1.0(Ref.),0.65(0.39-1.06),0.42(0.20-0.86),1.0(Ref.),0.69(0.51-0.94),丙肝感染组33.3%,52.8%,13.9%,59.7222%,40.2778%,OR95%可信区间1.0(Ref.),0.66(0.29-1.59),0.63(0.16-2.11),1.0(Ref.),0.73(0.43-1.24)。
乙肝感染组miRNA196a2C>T单核苷酸基因多态性和肝癌之间有相关性。在丙肝(无乙肝感染的丙肝组),无病毒感染组,niRNA196a2C>T单核苷酸基因多态性和肝癌之间没有相关性。miRNA196a2TC和CC基因型增加了肝癌的易感性。通过交互作用分析乙肝和miRNA196a2C>T之间交互性P=0.007。
结论：
1本实验发现miRNA196a2C>T的CC基因在肝癌患者中检出率高于对照组,CT、TT低于对照组,单核苷酸基因多态性与肝癌有相关性。而miRNA146aG>C与miRNA499A>G单核苷酸基因多态性和肝癌之间无相关性。
2在乙肝病例组miRMA196a2C>T单核苷酸基因多态性在肝癌的病例中和乙肝有交互作用。
第二部分miRNA196a的低表达对肝癌细胞系HepG2生物学行为的影响
目的：通过构建抑制miRNAl96a2C>T表达的载体,转染HepG2细胞,观察细胞增殖、迁移、凋亡,以探讨miRNA196在肝癌发生.发展中的作用.
方法：针对目标序列miRNA-196a2C>T合成特异性反义序列片段载体,利用海绵法重组干扰miR196a的质粒,利用脂质体技术瞬时转染肝细胞癌HepG2细胞株。本实验设三组分别为niR196a干扰组、阴性对照组(negative control,NC组)和正常对照组(ormal,N组)；qPCR鉴定外表达miR196a基因,用cck8检测细胞增殖率、transwell、室迁移实验检测细胞迁移率,annexinV/PI凋亡实验检测细胞凋亡率。
结果：
1荧光定量PCR验证miRNA196a2C>T的抑制效果
质粒转染48小时后测miRNA196a2mRNA表达量,结果示：干扰组niRl96a mRNA的表达量比NC组,正常对照组表达量均低,以正常对照组为1,干扰组的表达下降了41%。
2CCK8检测各实验组细胞的增殖
CCK8法对各实验组HepG2增殖情况的检测结果显示：正常对照组至24h时的OD值为0.86±0.16,NC组的OD值0.80±0.18,miR196a干扰组0.62±0.25,NC组细胞和正常对照组相比增殖率92.21±1.82%(P<0.01),miR196a干扰组和正常对照组相比72.29±2.51%(P<0.01),可见抑制miR196a表达后可以抑制HepG2的增殖。
3采用Transwell法检测细胞迁移和侵袭能力的改变
迁移实验显示,HepG2细胞在miR196a干扰组每个视野迁移的细胞数(58.4±2.46),正常对照组(101.6±3.77)NC组(99.6±2.62),miR196a干扰组与正常对照组、NC组相比(P<0.01),NC和正常对照组相比无统计学意义(P>0.05)
4AnnexinV-FITC/PI流式细胞术检测各组凋亡
采用Annexin V-FITC及PI(碘化丙啶)双标记流式细胞术检测细胞凋亡率,流式图右上象限为晚期凋亡细胞,右下象限为早期凋亡细胞。检测到HepG2凋亡率为正常组0.35±0.49%；NC组HepG2凋亡率为6.25±0.91%,miR196a干扰组为25.47±1.24%。结果表明miR196a干扰组凋亡明显,三组间比较p<0.05。阴性对照也能导致细胞凋亡,niR196a组与阴性对照组比较和正常组比较均有统计学意义(p<0.05)。
结论：niRNA196a2C>T影响肝癌细胞的生物学行为,miR196a低表达后抑制肝癌细胞增殖,及迁移,促进肝癌细胞的凋亡。
第三部分miRNA196a对HepG2细胞HOXB8、HOXB9、P53、Caspase-3表达的影响
目的：通过研究HOXB8、HOXB9、P53、Caspase-3mRNA和蛋白的表达情况,探讨miRNA-196a影响肝癌细胞增长、凋亡、迁移的机制,为肝癌的诊断及靶点治疗提供依据。
方法：
分组同实验二,分别采用Real-time PCR实时定量聚合酶链反应方法和Western-Blot检测同源基因HoxB8、HoxB9、凋亡基因caspase-3、p53的mRNA和蛋白的表达。
结果：
1Caspase-3mRNA表达水平miR196a干扰组(1.06±0.02),NC组(1.03±0.04),正常对照组(1.02±0.03),miRl96a干扰组显著高于对照组和NC(P<0.05),NC组和对照组无统计学意义(p>0.05)。Western-Blot Caspase-3蛋白表达水平miR196a干扰组(0.68±0.012),NC组(0.46±0.03),正常对照组(0.56±0.03),miR1.96a干扰组显著高于对照组、NC(F=54.13,P<0.05),NC组和对照组无统计学意义(p>0.05)。
2P53mRNA表达水平miR196a干扰组(0.97±0.009),NC组(0.99±0.011),正常对照组(1.00±0.012),miR196a干扰组、NC组、对照组三组之间,miR196a干扰组分别和NC、对照组比较,均无统计学意义(p>0.05)。Western-Blot P53蛋白表达水平miR196a干扰组(0.59±0.03),NC组(0.47±0.01),正常对照组(0.52±0.04),miR196a干扰组高于对照组、NC,但无统计学意义(p>0.05)。
3HoxB8mRNA表达水平miR196a干扰组(1.19±0.01),NC组(0.99±0.09),正常对照组(1.00±0.013),miR196a干扰组显著高于对照组,NC(F=770P<0.05),NC组和对照组无统计学意义(p>0.05)。Western-Blot HoxB8蛋白表达水平miR196a干扰组(0.60±0.05),NC组(0.35±0.05),正常对照组(0.60±0.05),miR196a干扰组显著高于对照组,NC(F=25.257,P<0.05),NC组和对照组无统计学意义(p>0.05)。
4HoxB9mRNA表达水平miR196a干扰组(1.00±0.02),NC组(0.96±0.04),正常对照组(1.00±0.02),miRl96a干扰组、对照组,NC组比较无统计学意义(p>0.05)。
结论：干扰miR-196a表达后导致Caspase-3、HoxB8mRNA和蛋白升高,而HoxB9mRNA及p53mRNA和蛋白表达没有明显变化,证明miRl96a可能通过调节凋亡因子Caspase-3和HoxB8表达对肝癌细胞的增殖、迁移、凋亡起调节作用,此过程和HoxB9mRNA、P53基因无相关性。
HCC is the common malignant tumor. Its high fatality rates, the incidence and mortality rate of HCC are almost equal. The Therapy is very difficult.Its Pathology including HCC,ICC. HCC and ICC et al. we dicuss the relationship miRNA and HCC. It is well known that chronic hepatitis B virus (HBV) and hepatitis C virus (HCV) infection, cirrhosis, aflatoxin B1and excessive alcohol drinking are the main risk factors for HCC. Although many people are exposed to these risk factors of HCC, only about10%of these exposed subjects develop HCC during their lifetime Therefore, genetic factors may play an important role in the development of HCC. microRNAs (miRNAs) are short, non-coding RNAs of approximately23nucleotides that regulate target genes. microRNAs have been demonstrated to have a role in several biochemical pathways in cell differentiation, proliferation, apoptosis and carcinogenesis. In the pathogenesis of HCC, miRNAs may have an important role in progression and directly contribute to cell proliferation, avoidance of apoptotic cell death and metastasis by targeting a large number of critical protein-coding genes.
The molecular mechanisms of HCC is complicated. Angiogenesis and metastasis play important roles in the progression and recurrence of HCC. Molecular therapeutic targets is a new avenue for the development of anti-HCC treatments. It has been well demonstrated that SNPs in protein-coding genes can affect the functions of proteins and in turn influence the individual susceptibility to cancers. In as much as the importance of miRNAs has only been recognized recently, little is known about the biological function of SNPs in the miRNA genes.SNP (A single-nucleotide polymorphism SNP) is the third sign of heredity.Previous epidemiologic studies have shown that genetic variations in miRNAs are associated with various diseases and cancers. SNP of miRNA consists in epigentics, murture of miRNA,mutant of mRNA.SNP effects susceptibility of HCC. Studing SNP make human gene project practice and the potential application of miRNAs as predictive, diagnostic, and prognostic biomarkers of HCC and their potential roles in cancer treatment. By checking mibase, we found the single-nucleotide mutant of miR-196a2C>T and miR-499A>G miR-146aG>C is common.
The gene miR196a are located in the regions of homeobox (HOX) clusters within the genome of vertebrates. One nuclieotide diffence is between miR196aa and miR196ab.
The miR146are consisted of miR146a and miR146b. Breast cancer metastasis suppressor up-regulates miR146, which suppresses breast cancer metastasis.miR-146a suppresses invasion of pancreatic cancer. miR146suppresses EGFR expression and reduces in vitromigration and invasion of gtioma.
miR-499participates in several crucial cancer-related biology processesn such as apoptosis,cell migration,cell senescence, and inflammation.
It is reported that miR-196a2C>T and miR-499A>G polymorphisms are associated with various cancer, such as breast cancer, lung cancer, gastric cancer and esophageal cancer. miR-196aC>T and miR-499A>G are associated with the susceptibility to hepatitis B-virus-related HCC, which suggested that miR-196aC>T and miR-499A>G can serve as repressors for viral infection pathways and play a key regulator in host-virus interaction and regulation of viral replication. Therefore, a recent meta-analysis showed no association between miR-146aG>C and miR-499A>G and risk of HCC. In our study, we investigated the association between polymorphisms in miR-146aG>C, miR-196a2C>T and miR-499A>G and risk of HCC, and its interaction with HBV infection in Chinese population.miR196a are located in HOX cluster.We will study miR196a mechanism in HepG2cell.The discovery of new types or novel functions of miRNAs provides us with more and deeper insights into the molecular mechanism underlying thepathogenesis of HCC.
The project contain three parts as below:
Part1:Associations Between Three Common MicroRNA Polymorphisms and Hepatocellular Carcinoma Risk in Chinese.
Part2:The inhibiting expression of microRNA196on biological behaviors in human hepatic cancer cell line HepG2
Part3:miR196a regulate the expression of HoxB8、HoxB9, caspase-3P53in HepG2cell line
Part1:Associations Between Three Common MicroRNA Polymorphisms and Hepatocellular Carcinoma Risk
Objective:Associations between polymorphisms in miR-146aG>C, miR-196a2C>T and miR-499A>G and risk of HCC, and interaction with HBV infection, were the target of the present research.
Methods:The duplex polymerase-chain-reaction with confronting-two-pair primers (PCR-RFLP) was performed to determine the genotypes of the miR-146aG>C, miR-196a2C>T and miR-499A>G genotypes. Associations of polymorphisms with the risk of HCC were estimated by conditional logistic regression analysis..
Results:Drinking, family history of cancer, HBsAg and HCV were risk factors for HCC. Multivariate regression analyses showed that subjects carrying the miR-196a2CC genotype had significantly increased risk of HCC, with an adjusted OR (95%CI) of2.18(1.23-3.80). In addition, cases carrying the miR-196a2C allele had a1.64-fold increase in the risk for HCC (95%CI=1.03-2.49). The miR-196a2CT
and TT genotypes greatly significantly increased the risk of HCC in subjects with HBV infection, with adjusted ORs (95%CI) of2.02(1.12-3.68) and2.69(1.28-5.71), respectively.
Conclusions:Our results demonstrate that miR-196a2CC genotype and C allele have an important role in HCC risk in Chinese, especially in patients with HBV infection.
Part2:The inhibiting expression of microRNA196on biological behaviors in human hepatic cancer cell line HepG2
Objective:To study the effect of inhibiting miR196a on biological behaviors in human hepatic cancer cell
Methods:The antisense RNA of microRNA-196a was synthesized and cloned into the vector. HepG2cells were infected by inhibiting miRl96a vector. The HepG2cells were devided into miR196a lower expression group, NC group and N group in vitro. The expression of the targets of miR-196a was detected by qPCR. Cell growth was analyzed by cck8assay. The invasion was dectected by transwell method. Apoptosis was detected by annexinV/PI.
Results:.1) The expression level of miR-196a was less than normal (41%).(2) The proliferation of HepG2was also markedly suppressed in inhibiting miR196a at the24h point than normal about72.29±2.51%(P<0.01).(3) The number of cells that migrated through the chamber of miR196a inhibiting group is less than normal and NC (P<0.01).(4) The cell apopotosis in miR196a inhibiting group is more than NC and normal group (p<0.05).
Conclusions:MicroRNA-196a lower expression in HepG2surppresses the proliferation of and migration of HepG2cells, and promotes tumor cell apoptosis.
Part3:miR196a effects expression of Caspase-3P53HoxB8HoxB9in HepG2cell Objective:To explore the role of miR-196a on the regulatory mechanism in Hepatocelluar carcinoma cell line by detecting Caspase-3P53HoxB8HoxB9mRNA and Protein.
Methods:The antisense RNA of microRNA-196a was synthesized and cloned into the vector. HepG2cells were infected by inhibiting miR196a vector. The HepG2cells were devided into miR196a lower expression group, NC group and N group in vitro. The P53,caspase-3, HMGA2,HOXB8mRNA and their Protein was detected by qPCR and Weatern-Blot.
Results:.HOXB8mRNA and protein expression,. in HepG2.cell line miR196a inhibiting group is significantly less than normal,NC (P<0.05). Caspase-3mRNA and Protein expression is maximum in three groups (p<0.05). The P53mRNA and Protein, HOXB9mRNA expression in three groups is no markly difference.
Conclusions:MiR-196a can effect the proliferation, the apoptosis and migration of HepG2cell lines by gene HOXB8,caspase-3regulation. However miR-196a is irrelation with P53, HOXB9.
引文
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