肝癌差异miRNA分子的鉴定及其功能机制研究
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摘要
【背景】
肝细胞癌（HCC，以下简称肝癌）是最常见的恶性肿瘤之一，但其发生发展的分子机制尚未完全清楚。MicroRNA（miRNA）是近年来在生物体内发现的一大类长度约22核苷酸左右的内源性非编码小RNA。它通过转录后抑制或靶mRNA降解参与细胞增殖、分化、凋亡等生物学过程，目前已鉴定出千余个。miRNA呈现组织特异性表达，可以有多个靶基因。研究发现，miRNA与细胞的癌变有着极为密切的关系。最近的研究表明，miRNA在人类恶性肿瘤中发挥促癌或抑癌的作用。对于miRNA表达调控的研究将是针对人类恶性肿瘤预防的一种新策略。
由于miRNA具有“一对多”的作用方式，可同时调控多个蛋白质编码基因，参与多条与肿瘤发生发展相关的分子通路，所以靶向miRNA的干预能更有效地控制甚至逆转涉及多基因改变的肿瘤恶性表型。然而，miRNA是如何参与肝癌的发生发展？有哪些miRNA参与？它们的作用机制如何？靶向miRNA的干预治疗是否能逆转肝癌细胞的恶性表型？上述问题还远未阐明，miRNA在肝癌发生发展中的作用及作用机制仍需进一步的研究和探索。
【目的】
筛选、鉴定肝癌中差异表达的miRNA，研究miRNA分子在肝癌发生发展过程中的作用及其机制，进一步从新的角度理解肝癌的发生，为肝癌的早期预防、早期诊断以及选择合适的靶点进行干预、治疗提供新的理论依据。
【方法】
1.根据文献报道的芯片筛选结果，选取了miR-199a-1、miR-125a、miR-125b、miR-373、miR34a五个miRNAs分子，对18例肝癌、癌旁及部分转移灶的冰冻和石蜡包埋组织标本，通过实时荧光定量PCR（qRT-PCR）检测、比较肝癌组织和癌旁组织的表达水平，鉴定差异最明显的miRNA分子。
2.补足40例肝癌、癌旁及部分转移灶的冰冻和石蜡包埋组织标本，通过qRT-PCR检测肝癌组织及癌旁组织标本中miR-199a-1的表达水平，分析miR-199a-1的表达水平与肝癌临床病理参数的关系。
3.构建、扩增miR-199a-1的慢病毒表达载体及其阴性对照载体，分别感染miR-199a-1低表达的肝癌细胞系HepG2，建立稳定上调miR-199a-1的肝癌细胞系，用MTT、平板克隆、流式细胞术及裸鼠成瘤等实验，在体外和体内观察miR-199a-1对肝癌细胞生长、增殖的影响。
4.利用生物信息学软件（PicTar、MiRanda、TargetScan等）预测miR-199a-1可能调控的靶基因；通过荧光素酶报告基因实验、半定量PCR和Western blot对候选靶基因进行验证，明确miR-199a-1对其靶基因FZD7的调控作用及两者在肝癌细胞及临床肝癌组织中的表达相关性；Westernblot检测miR-199a-1稳转细胞系中miR-199a-1上调对FZD7通路的影响。
【结果】
1.在选取的五个肝癌差异表达miRNA分子中，miR-199a-1的差异表达最为显著，肝癌组织中的表达水平明显低于癌旁组织，提示miR-199a-1可能是参与肝癌发生发展过程中的关键分子之一。
2. miR-199a-1的表达水平与肝癌临床病理参数的分析结果表明，其表达降低的程度与肝癌的临床分期、淋巴结转移以及肝癌患者的预后密切相关。
3. miR-199a-1高表达慢病毒感染肝细胞，建立稳转细胞系。MTT结果显示miR-199a-1上调表达能显著抑制肝癌细胞的增殖能力；平板克隆形成实验显示miR-199a-1上调表达的肝癌细胞集落生长能力显著降低；流式细胞术检测发现阴性对照组和亲本HepG2细胞系增殖指数（PI）分别为60.7和63.9，明显高于对照组miR-199a-1高表达的HepG2细胞系的PI值40.8。实验结果提示miR-199a-1高表达细胞系发生细胞周期G1期阻滞；裸鼠体内成瘤发现稳定高表达miR-199a-1的肝癌细胞系皮下接种BALB/c裸鼠40天后，肿瘤生长缓慢，实验组瘤体显著小于对照组。体内外功能研究表明miR-199a-1可以抑制肝癌的增殖。
4.生物信息学预测提示，FZD7可能是miR-199a-1的靶基因之一。上调miR-199a-1的肝癌细胞系与其阴性对照细胞系相比，FZD7表达明显降低，与miR-199a-1的表达成负相关性。在肝癌组织标本中，FZD7在肝癌中表达明显高于癌旁组织。Western blot检测经典Wnt信号通路的靶基因β-catenin，Jun, Cyclin D1和Myc均有不同程度的降低。
【结论】
1. miR199a-1在肝癌细胞和肝癌组织中表达显著降低，其表达降低的程度与肝癌的临床分期、淋巴结转移以及肝癌患者的预后密切相关。
2. miR-199a-1可以通过调节细胞周期G1阻滞来抑制体内、外肝癌细胞增殖能力。
3. miR-199a-1通过负性调控细胞膜受体FZD7表达，影响经典的Wnt通路发挥抑癌作用。
【Background】
Hepatocellular carcinoma (HCC) is one of the most common humanmalignancies worldwide, while the underlying mechanisms of HCCcarcinogenesis and progression are still unclear. MicroRNAs (miRNAs) are aclass of single-strand evolutionarily conserved small non-coding RNAs with19–22nucleotides in length. The specificity of miRNA targeting is based onWatson–Crick complementarities with the3’ untranslated region (UTR) of theirtarget mRNAs, which can down-regulate the expression of various target genesinvolved in the malignant process of cancer. For one thing, when the miRNAbinding shows perfect complementarities, the RISC induces mRNA degradation.For another, once an imperfect miRNA–mRNA target pairing occurs, translationinto a protein is blocked. Overall, miRNAs serve as expression regulators infundamental cell progression, including growth, proliferation, apoptosis,differentiation and metastasis. An increasing number of evidence shows that the aberrant expression of certain miRNAs in cancers plays significant roles in thetumorigenesis and progression.Because of miRNAs’ property of ‘one for all’, they are able to targetmultiple genes involved in cancer pathways so that cancer phenotypes can bemodified by targeting gene expression. However, targets of miRNAs intumorigenesis, progression and therapeutic intervention of HCC remains unclear.The corresponding mechanisms require more concentration and commitment oftargeting miRNAs in therapeutic strategies for HCC in future studies.
【Aims】
To screen and identify the aberrantly expressed miRNAs in HCC comparedwith matched non-cancerous tissues, and to investigate the effects involved inHCC carcinogenesis and progression, for better understanding the mechanisms ofHCC from a fresh perspective and providing a new theoretical basis for earlydiagnosis and intervention of HCC.
【Methods】
1. Based on our former experimental data and references, we selected5miRNAs: miR-199a-1, miR-125a、miR-125b、miR-373、miR34a for furtherstudy. The miRNAs levels in18pairs of HCC and corresponding non-tumortissues were detected by quantitative real time-PCR (qRT–PCR), andanalyzed the difference expression of each in HCC and non-tumor tissues.
2. A complementary of22more paires of HCC and corresponding non-tumortissues was carried out with the same treatment as described above. Theexpression levels of miR-199a-1in these22HCC tissues and correspondingnon-tumor mucosa were detected by qRT–PCR. Correlations between themiR-199a-1expression level and clinicopathologic characteristics of HCCwere also analyzed.
3. The expression of miR-199a-1was also detected in HCC cell lines HepG2and SMMC-7721, and normal liver cell line Chang liver. MiR-199a-1wasfound to be lowly expressed in HepG2cells, and over-expression ofmiR-199a-1in HepG2cells using a miR-199a-1expression vector(pGenesil-1-miR-1) was used for further study. The effect of miR-199a-1expression on cell growth and proliferation in vitro and tumorigenicity invivo were determined by MTT assay, flow cytometry, flat cloningexperiments and nude mice.
4. To determine the underlying mechanisms that miR-199a-1contributes to themigration and metastasis of HCC, computative predicting tools includingmiRanda, Pictar, and TargetScan were used for the prediction of potentialregulatory targets of miR-199a-1. FZD7was confirmed to be one of themost inmortant target genes by luciferase, qRT–PCR and western blot assays.The downstream genes of FZD7, including β-catenin, Jun, CyclinD1andMyc were also inverstigated by western blot analysas.
【Results】
1. MiR-199a-1was distinctly over-expressed among all the five selectedmiRNAs, indicating that miR-199a-1might serve as a key regulator in HCCcarcinogenesis.
2. The results of qRT-PCR verified that the miR-199a-1expression level wassignificantly lower in HCC comapred with mathced non-neoplastic tissues.Further analysis showed the levels of miR-199a-1were significantlycorrelated with the patients’ clinical parameters including TNM stage,metastasis and poor prognosis of patients with HCC.
3. Up-regulated miR-199a-1cell line was successfully established in HepG2cells. MTT assays revealed that miR-199a-1could obviously repress HCCcell proliferation. The flat cloning experimental data indicated that miR-199a-1transfected HCC cells had a significantly decrease in theircolony forming ability. Flow cytometry data demonstrated that theproliferation index (PI) of both negative control group and the miR-199a-1hypoexpressd HepG2parental one were60.7and63.9respectively, whichwere dramatically higher than miR-199a-1hyper-expressed HepG2cells,indicating that over-expression of miR-199a-1could obtain a G1arrest.Furthermore, in vivo experiments revealed that the tumor growth withinHCC BALB/c nude mice had sharply slowed down after40days of tumorcell injection. Above all, these data demonstrated that miR-199a-1couldsuccessfully inhibit HCC growth and proliferation in vivo and vitro.
4. Bioinformatics predicted that FZD7may be a target for miR-199a-1. Tofurther test the hypothesis, we analyzed the influence of miR-199a-1onFZD7expression in HCC cell line HepG2. The results showed thatover-expression of miR-199a-1could significantly down-regulate expressionof FZD7, and its downstream genes including β-catenin, Jun, CyclinD1andMyc, indicating that miR-199a-1repressed the development of HCC partlythrough inhibiting FZD7passageway.
【Conclusions】
1. MiR-199a-1expression is significantly down-regulated in HCC, anddecreased expression of miR-199a-1was significantly correlated with themalignant progression of HCC and poor prognosis.
2. Up-regulation of MiR-199a-1expression in HCC cells can significantlysuppress HCC growth and proliferation in vitro and in vivo through G1arrest.
3. MiR-199a-1suppreses HCC progression partly through inhibiting the FZD7pathway.
肝细胞癌（HCC，以下简称肝癌）是最常见的恶性肿瘤之一，但其发生发展的分子机制尚未完全清楚。MicroRNA（miRNA）是近年来在生物体内发现的一大类长度约22核苷酸左右的内源性非编码小RNA。它通过转录后抑制或靶mRNA降解参与细胞增殖、分化、凋亡等生物学过程，目前已鉴定出千余个。miRNA呈现组织特异性表达，可以有多个靶基因。研究发现，miRNA与细胞的癌变有着极为密切的关系。最近的研究表明，miRNA在人类恶性肿瘤中发挥促癌或抑癌的作用。对于miRNA表达调控的研究将是针对人类恶性肿瘤预防的一种新策略。
由于miRNA具有“一对多”的作用方式，可同时调控多个蛋白质编码基因，参与多条与肿瘤发生发展相关的分子通路，所以靶向miRNA的干预能更有效地控制甚至逆转涉及多基因改变的肿瘤恶性表型。然而，miRNA是如何参与肝癌的发生发展？有哪些miRNA参与？它们的作用机制如何？靶向miRNA的干预治疗是否能逆转肝癌细胞的恶性表型？上述问题还远未阐明，miRNA在肝癌发生发展中的作用及作用机制仍需进一步的研究和探索。
【目的】
筛选、鉴定肝癌中差异表达的miRNA，研究miRNA分子在肝癌发生发展过程中的作用及其机制，进一步从新的角度理解肝癌的发生，为肝癌的早期预防、早期诊断以及选择合适的靶点进行干预、治疗提供新的理论依据。
【方法】
1.根据文献报道的芯片筛选结果，选取了miR-199a-1、miR-125a、miR-125b、miR-373、miR34a五个miRNAs分子，对18例肝癌、癌旁及部分转移灶的冰冻和石蜡包埋组织标本，通过实时荧光定量PCR（qRT-PCR）检测、比较肝癌组织和癌旁组织的表达水平，鉴定差异最明显的miRNA分子。
2.补足40例肝癌、癌旁及部分转移灶的冰冻和石蜡包埋组织标本，通过qRT-PCR检测肝癌组织及癌旁组织标本中miR-199a-1的表达水平，分析miR-199a-1的表达水平与肝癌临床病理参数的关系。
3.构建、扩增miR-199a-1的慢病毒表达载体及其阴性对照载体，分别感染miR-199a-1低表达的肝癌细胞系HepG2，建立稳定上调miR-199a-1的肝癌细胞系，用MTT、平板克隆、流式细胞术及裸鼠成瘤等实验，在体外和体内观察miR-199a-1对肝癌细胞生长、增殖的影响。
4.利用生物信息学软件（PicTar、MiRanda、TargetScan等）预测miR-199a-1可能调控的靶基因；通过荧光素酶报告基因实验、半定量PCR和Western blot对候选靶基因进行验证，明确miR-199a-1对其靶基因FZD7的调控作用及两者在肝癌细胞及临床肝癌组织中的表达相关性；Westernblot检测miR-199a-1稳转细胞系中miR-199a-1上调对FZD7通路的影响。
【结果】
1.在选取的五个肝癌差异表达miRNA分子中，miR-199a-1的差异表达最为显著，肝癌组织中的表达水平明显低于癌旁组织，提示miR-199a-1可能是参与肝癌发生发展过程中的关键分子之一。
2. miR-199a-1的表达水平与肝癌临床病理参数的分析结果表明，其表达降低的程度与肝癌的临床分期、淋巴结转移以及肝癌患者的预后密切相关。
3. miR-199a-1高表达慢病毒感染肝细胞，建立稳转细胞系。MTT结果显示miR-199a-1上调表达能显著抑制肝癌细胞的增殖能力；平板克隆形成实验显示miR-199a-1上调表达的肝癌细胞集落生长能力显著降低；流式细胞术检测发现阴性对照组和亲本HepG2细胞系增殖指数（PI）分别为60.7和63.9，明显高于对照组miR-199a-1高表达的HepG2细胞系的PI值40.8。实验结果提示miR-199a-1高表达细胞系发生细胞周期G1期阻滞；裸鼠体内成瘤发现稳定高表达miR-199a-1的肝癌细胞系皮下接种BALB/c裸鼠40天后，肿瘤生长缓慢，实验组瘤体显著小于对照组。体内外功能研究表明miR-199a-1可以抑制肝癌的增殖。
4.生物信息学预测提示，FZD7可能是miR-199a-1的靶基因之一。上调miR-199a-1的肝癌细胞系与其阴性对照细胞系相比，FZD7表达明显降低，与miR-199a-1的表达成负相关性。在肝癌组织标本中，FZD7在肝癌中表达明显高于癌旁组织。Western blot检测经典Wnt信号通路的靶基因β-catenin，Jun, Cyclin D1和Myc均有不同程度的降低。
【结论】
1. miR199a-1在肝癌细胞和肝癌组织中表达显著降低，其表达降低的程度与肝癌的临床分期、淋巴结转移以及肝癌患者的预后密切相关。
2. miR-199a-1可以通过调节细胞周期G1阻滞来抑制体内、外肝癌细胞增殖能力。
3. miR-199a-1通过负性调控细胞膜受体FZD7表达，影响经典的Wnt通路发挥抑癌作用。
【Background】
Hepatocellular carcinoma (HCC) is one of the most common humanmalignancies worldwide, while the underlying mechanisms of HCCcarcinogenesis and progression are still unclear. MicroRNAs (miRNAs) are aclass of single-strand evolutionarily conserved small non-coding RNAs with19–22nucleotides in length. The specificity of miRNA targeting is based onWatson–Crick complementarities with the3’ untranslated region (UTR) of theirtarget mRNAs, which can down-regulate the expression of various target genesinvolved in the malignant process of cancer. For one thing, when the miRNAbinding shows perfect complementarities, the RISC induces mRNA degradation.For another, once an imperfect miRNA–mRNA target pairing occurs, translationinto a protein is blocked. Overall, miRNAs serve as expression regulators infundamental cell progression, including growth, proliferation, apoptosis,differentiation and metastasis. An increasing number of evidence shows that the aberrant expression of certain miRNAs in cancers plays significant roles in thetumorigenesis and progression.Because of miRNAs’ property of ‘one for all’, they are able to targetmultiple genes involved in cancer pathways so that cancer phenotypes can bemodified by targeting gene expression. However, targets of miRNAs intumorigenesis, progression and therapeutic intervention of HCC remains unclear.The corresponding mechanisms require more concentration and commitment oftargeting miRNAs in therapeutic strategies for HCC in future studies.
【Aims】
To screen and identify the aberrantly expressed miRNAs in HCC comparedwith matched non-cancerous tissues, and to investigate the effects involved inHCC carcinogenesis and progression, for better understanding the mechanisms ofHCC from a fresh perspective and providing a new theoretical basis for earlydiagnosis and intervention of HCC.
【Methods】
1. Based on our former experimental data and references, we selected5miRNAs: miR-199a-1, miR-125a、miR-125b、miR-373、miR34a for furtherstudy. The miRNAs levels in18pairs of HCC and corresponding non-tumortissues were detected by quantitative real time-PCR (qRT–PCR), andanalyzed the difference expression of each in HCC and non-tumor tissues.
2. A complementary of22more paires of HCC and corresponding non-tumortissues was carried out with the same treatment as described above. Theexpression levels of miR-199a-1in these22HCC tissues and correspondingnon-tumor mucosa were detected by qRT–PCR. Correlations between themiR-199a-1expression level and clinicopathologic characteristics of HCCwere also analyzed.
3. The expression of miR-199a-1was also detected in HCC cell lines HepG2and SMMC-7721, and normal liver cell line Chang liver. MiR-199a-1wasfound to be lowly expressed in HepG2cells, and over-expression ofmiR-199a-1in HepG2cells using a miR-199a-1expression vector(pGenesil-1-miR-1) was used for further study. The effect of miR-199a-1expression on cell growth and proliferation in vitro and tumorigenicity invivo were determined by MTT assay, flow cytometry, flat cloningexperiments and nude mice.
4. To determine the underlying mechanisms that miR-199a-1contributes to themigration and metastasis of HCC, computative predicting tools includingmiRanda, Pictar, and TargetScan were used for the prediction of potentialregulatory targets of miR-199a-1. FZD7was confirmed to be one of themost inmortant target genes by luciferase, qRT–PCR and western blot assays.The downstream genes of FZD7, including β-catenin, Jun, CyclinD1andMyc were also inverstigated by western blot analysas.
【Results】
1. MiR-199a-1was distinctly over-expressed among all the five selectedmiRNAs, indicating that miR-199a-1might serve as a key regulator in HCCcarcinogenesis.
2. The results of qRT-PCR verified that the miR-199a-1expression level wassignificantly lower in HCC comapred with mathced non-neoplastic tissues.Further analysis showed the levels of miR-199a-1were significantlycorrelated with the patients’ clinical parameters including TNM stage,metastasis and poor prognosis of patients with HCC.
3. Up-regulated miR-199a-1cell line was successfully established in HepG2cells. MTT assays revealed that miR-199a-1could obviously repress HCCcell proliferation. The flat cloning experimental data indicated that miR-199a-1transfected HCC cells had a significantly decrease in theircolony forming ability. Flow cytometry data demonstrated that theproliferation index (PI) of both negative control group and the miR-199a-1hypoexpressd HepG2parental one were60.7and63.9respectively, whichwere dramatically higher than miR-199a-1hyper-expressed HepG2cells,indicating that over-expression of miR-199a-1could obtain a G1arrest.Furthermore, in vivo experiments revealed that the tumor growth withinHCC BALB/c nude mice had sharply slowed down after40days of tumorcell injection. Above all, these data demonstrated that miR-199a-1couldsuccessfully inhibit HCC growth and proliferation in vivo and vitro.
4. Bioinformatics predicted that FZD7may be a target for miR-199a-1. Tofurther test the hypothesis, we analyzed the influence of miR-199a-1onFZD7expression in HCC cell line HepG2. The results showed thatover-expression of miR-199a-1could significantly down-regulate expressionof FZD7, and its downstream genes including β-catenin, Jun, CyclinD1andMyc, indicating that miR-199a-1repressed the development of HCC partlythrough inhibiting FZD7passageway.
【Conclusions】
1. MiR-199a-1expression is significantly down-regulated in HCC, anddecreased expression of miR-199a-1was significantly correlated with themalignant progression of HCC and poor prognosis.
2. Up-regulation of MiR-199a-1expression in HCC cells can significantlysuppress HCC growth and proliferation in vitro and in vivo through G1arrest.
3. MiR-199a-1suppreses HCC progression partly through inhibiting the FZD7pathway.
引文
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