【作者】 房锋；

【导师】 杨连粤；

【作者基本信息】 中南大学 ， 外科学， 2011， 博士

【摘要】 [背景和目的]肝细胞癌(hepatocellular carcinoma, HCC)是世界上最常见的恶性肿瘤之一,在亚洲东部和非洲南部的发病率相对较高。HCC恶性程度较高,全球每年HCC死亡病例数高达600,000,高居全球癌症死因的第三位。虽然目前HCC的治疗手段已取得显著进步,但其5年生存率仍长期徘徊于30%-40%。究其原因在于,目前依然对晚期肝癌和肝癌的复发转移缺乏有效的治疗手段。累积的研究发现,HCC的发生和发展是一多步骤的复杂过程,其中涉及到众多的生长因子、癌基因和抑癌基因的异常表达或激活。因而,阐明HCC发生发展过程中的分子机制对HCC的诊断、预防和治疗就显得尤为重要。在过去的几十年间,国内外学者在该领域开展了大量研究,并已发现p53、Rb、PI3K/AKT/mTOR、Ras/MAPK及Egfl7等一系列与肝癌发生和发展密切相关的基因或信号通路。尽管如此,HCC发生发展的具体分子机制仍然大部分未知。微小FRNA (microRNA, miRNA)是一类长度为17-25个核苷酸的非编码调控RNA。MiRNAs作为转录后调控因子,可与其靶基因信使RNAs (messenger RNAs, mRNAs)的3’非读码区(3’untranslated region,3’-UTR)特异性结合,诱导靶基因mRNAs(?)勺降解或抑制其翻译,负性调控其靶基因的表达。自1993年Ambros和Ruvkun等发现并报道第一个miRNA以来,大量的(?)miRNAs及其相关文献被鉴定和发表,miRNAs研究已成为当今生命科学研究领域的一大热点。迄今在人类基因组中检测并证实的miRNAs已超过1000个。研究发现,miRNAs参与了生命过程中的一系列重要进程,包括肿瘤的发生和发展。最近的多项研究证实,miRNAs在HCC的发生发展中具有重要作用,可调控HCC细胞的生长、凋亡、侵袭转移和DNA甲基化等众多病理进程。尽管,miRNAs在肝癌发生发展调控中的重要性业已吸引了国内外众多学者的关注,绝大多数1niRNAs在肝癌中的作用及其作用机制依然未知,迫切需要进一步研究。此前,我们在长期的临床实践中发现并命名了一特殊类型的肝癌—孤立性大肝癌(Solitary large hepatocellular carcinoma, SLHCC),系指病灶呈单个、直径大于5cm、呈膨胀性生长、常有包膜或假包膜的一类大肝癌。据此,我们将肝癌按照大体类型分为三类：SLHCC、结节性肝癌(结节数≥2个；Nodular HCC, NHCC)和小肝癌(肿瘤直径≤5cm；Small HCC, SHCC)。进一步研究证实,SLHCC具有相对独特的临床病理特征,且其复发转移潜能与SHCC目近,均显著低于NHCC。根治性肝切除术后,SLHCC的总生存时间和无瘤生存时间与SHCC相比无显著性差异,但显著性优于NHCC。为深入了解SLHCC的分子病理特点,我们采用基因芯片技术比较了三种类型肝癌之间的基因表达差异,结果发现,7.89%(668/8464)的基因在SLHCC、SHCC和NHCC之间存在差异表达。以上结果提示上述三种类型肝癌具备各自独特的临床病理和分子病理特点。基于mRNAs可调控人类基因组中30%的基因表达这一事实,我们推测,SLHCC、SHCC和NHCC亦可能具有各自独特的niRNAs表达谱。为验证这一假说,我们在本研究中首先利用miRNAs芯片技术比较了SLHCC、SHCC和NHCC的(?)niRNAs表达谱,以筛选三者之间的差异表达(?)niRNAs。我们选取在三者中存在差异表达的miR-188-5p作为研究对象,深入研究了其在肝癌中的表达、生物学功能及其作用的分子机制。我们的研究结果证实,miR-188-5p在肝癌内表达显著性下调,并与肝癌患者的预后显著性相关,且过表达miR-188-5p可在体内外抑制肝癌细胞的增殖和侵袭转移；而抗凋亡克隆11(Antiapoptosis clone 11, AAC11)基因系miR-188-5p的直接功能性靶基因。我们的研究结果表明,miR-188-5p可作为肝癌预后监测的新标志物和治疗的新型靶点。[方法和结果]1.SLHCC、SHCC和NHCC中差异表达miRNAs的筛选：利用miRNA芯片分析,我们检测了SLHCC、SHCC和NHCC中的miRNAs表达谱,我们发现,1miR-188-5p的表达水平在SLHCC和SHCC中无显著性差异,但均显著高于NHCC。这种表达差异与上述三种类型肝癌之间临床病理特征的差异具有一致性,提示miR-188-5p可能参与了肝癌发生发展的调控。因而,我们选取miR-188-5p作为进一步深入研究的对象。2. miR-188-5p在肝癌内的表达水平显著性下降：我们采用实时定量RT-PCR技术,检测了72例肝癌组织及其对应的邻近非癌肝组织(adjacent nontumorous liver tissue, ANLT)内miR-188-5p的表达水平,结果显示,与ANLTs相比,miR-188-5p在HCC内的表达水平显著性下调,平均下调倍数为3.75倍。且与芯片结果相一致的是,miR-188-5p在SLHCC中的表达水平与SHCC相近(0.52vs.0.65,P=0.017),但显著性高于NHCC (0.52 vs.0.21, P<0.001)。此外,我们进一步分析了miR-188-5p与肝癌侵袭转移的关系,通过检测其在ANLT、HCC和静脉癌栓(vein cancer embolus of HCC, HCC-M)中的表达水平,我们发现,HCC-M中miR-188-5p的表达水平最低(相对表达水平分别为1.0、0.57和0.12),提示miR-188-5p的表达水平与肝癌的侵袭转移潜能呈反比。3. miR-188-5p低表达与HCC临床病理特征及预后密切相关：我们根据qRT-PCR结果,将72例肝癌分为miR-188-5p高表达组(n27)和低表达组(n=45),并分析了其表达水平与肝癌临床病理特征和预后的关系。结果显示,miR-188-5p表达水平与HCC的肿瘤结节数目(P=0.010)和有无静脉浸润(P=0.002)密切相关。此外,miR-188-5p高表达HCC患者的总体生存时间及无瘤生存时间均明显高于miR-188-5p低表达的HCC患者(中位生存时间,35个月vs.16个月,P=0.044；中位无瘤生存时间,23个月vs.14个月,P0.048)4. miR-188-5p抑制肝癌的生长和侵袭转移：我们首先检测了miR-188-5p在2种正常肝细胞系(L02和CCL13)和3种具有不同复发转移潜能的肝癌细胞系(HepG2、MHCC97-L和HCCLM3)内的表达水平,结果显示,miR-188-5p在肝癌细胞系内的表达水平显著低于正常肝细胞系,且在具备最高复发转移潜能的HCCLM3细胞系内的表达水平最低(相对表达水平依次为：1.0、0.62、0.45、0.41和0.06)。我们选取HCCLM3作为研究对象,采用慢病毒介导的过表达技术,建立了miR-188-5p稳定过表达HCCLM3肝癌细胞系和相应的对照细胞系—HCCLM3miR-188-5p和HCCLM3control细胞系,进了检测了两者之间细胞生物学行为的差异。结果显示,HCCLM3miR-188-5p细胞的生长速度显著慢于HCCLM3control细胞(P<0.005)；集落形成实验证实,相比于HCCLM3control细胞,HCCLM3miR-188-5p细胞的集落数目减少了60%以上(200±30 vs.75±18,P<0.001)；划痕愈合实验证实,HCCLM3miR-188-5p细胞的划痕愈合能力显著慢于HCCLM3control细胞(94% vs.48%,P<0.01)；基质胶Transwell侵袭小室实验的结果亦显示,HCCLM3miR-188-5p细胞迁移运动能力较HCCLM3control细胞明显下降(50±9 vs.125±15,尸<0.001)。5.AAC11是miR-188-5p的直接靶基因：采用生物信息学软件miRanda(microrna.org和miRbase)和TargetScan搜寻miR-188-5p的潜在靶基因,发现AAC11在其3’-UTR存在两个miR-188-5p的结合位点。其中,一结合位点在黑猩猩、恒河猴、狗、猫、马和人等不同物种间存在高度保守性,而另一结合位点仅在恒河猴和人之间存在保守性。为进一步证实miR-188-5p能否直接与AAC11的3’-UTR结合,我们构建了包含/AAC11 3’-UTR全长序列、结合位点1序列(野生型和突变型)或结合位点2序列(野生型和突变型)的荧光素酶报告基因载体。荧光素酶报告基因分析证实,miR-188-5p可与AAC11的全长3’-UTR结合,并显著抑制其荧光素酶的信号强度(约70%),而突变型则不影响其信号强度。Western blot分析亦证实,miR-188-5p的过表达可显著抑制HCCLM3细胞的内源性AAC11的蛋白表达水平。以上结果提示,AAC11是miR-188-5p的直接下游靶基因。6.AAC11表达水平的恢复可消除(?)niR-188-5p过表达诱导的肝癌细胞生长和侵袭转移抑制：为进一步证实miR-188-5p调控细胞生长和侵袭转移的能力是否通过AAC11,我们构建了AAC11的慢病毒过表达载体,并将其转入HCCLM3miR-188-5p细胞中,以恢复AAC11的表达水平。生长曲线分析、集落形成实验、划痕愈合实验及基质胶Transwell侵袭小室实验的结果均显示,AAC11表达水平恢复后,HCCLM3miR-188-5p细胞的细胞生长和侵袭转移能力亦得到恢复,提示AAC11是miR-188-5p的功能性靶基因。7.miR-188-5p过表达可在体内抑制肝癌细胞的生长和转移：为在活体内观察miR-188-5p过表达对HCC生长和侵袭转移能力的影响,我们构建了裸鼠原位肝癌转移模型。结果显示,HCCLM3miR-188-5p细胞形成的肝脏原位肿瘤体积明显小于HCCLM3control细胞(P0.05),而HCCLM3miR-188-5p+AAC11细胞形成的肝脏原位瘤体积明显大于HCCLM3miR-188-5p细胞(P<0.05),但与HCCLM3control细胞相比明显差异(P>0.05)。裸鼠肝和肺脏的连续切片观察结果显示,HCCLM3miR-188-5p肿瘤无肝内和远处肺内转移,而HCCLM3control肿瘤的肝内和肺内转移率分别为100%和80%,两者相比具有显著性差异(P<0.05)。此外,恢复AAC11表达后,HCCLM3的侵袭转移能力已得到了大部分恢复,其肝内和肺内转移率分别为80%和60%。我们的这些结果证明,体内过表达miR-188-5p可通过靶向AAC11抑制HCC的生长、肝内转移及肺转移。[主要创新点]1、本研究首次发现miR-188-5p在HCC中低表达,且其表达水平与HCC临床病理特征及预后密切相关,提示(?)niR-188-5p是一个新的HCC预后标志物,这首次将(?)niR-188-5p引入了HCC及恶性肿瘤研究领域。2、本研究首次证实了miR-188-5p在HCC生长和侵袭转移调控中具有重要作用,并阐明了miR-188-5p通过靶向AAC11抑制肝癌细胞生长和迁移运动的分子机制,有助于加深对肝癌发生发展分子机制的了解。3、本研究通过动物实验证明,miR-188-5p的过表达可在动物活体内显著抑制HCC的生长、肝内转移和肺转移,提示miR-188-5p可作为是一个新的HCC干预治疗的靶点,在HCC分子干预研究中具有重要价值。[结论]本研究首次发现miR-188-5p在HCC中呈低表达并与肝癌的临床病理特征和预后显著相关,进而阐明了miR-188-5p通过靶向AAC11抑制肝癌细胞生长、侵袭和转移的分子机制,揭示了miR-188-5p在HCC发生和发展中的关键性生物学作用,提示miR-188-5p是一个新的HCC预后标志物和一个新的HCC干预治疗靶点。更多还原

【Abstract】 【Background and Aims】Hepatocellular carcinoma (HCC) is one of the most common human malignancies worldwide, especially in East Asia and South Africa. Duing to it’s highly malignant potential, HCC ranks as the third leading cause of cancer death in the world, resulting in almost 600,000 deaths each year. Despite great improvement in the treatment options, the long-time survival of patients with HCC remains unsatisfactory, with a 5-year survival rate of 20% to 30% reported in the literature, because of the challenging problems for advanced and metastatic HCC treatment. Carcinogenesis and progression of HCC are multistage process that involve lots of growth factors, oncogenes and tumor suppressor genes. Making clear the molecular events underlied the tumorgenesis of HCC are important for its screening, prevention, and treatment. In past decades, many factors involved in the pathogenesis of HCC have been identified, including p53, Rb, PI3K/AKT/mTOR, Ras/MAPK, Egfl7 and so on. However, the molecular mechanisms of HCC are still largely unknown.MicroRNAs (miRNAs) are highly conserved small non-coding regulatory RNAs with sizes of 17-25 nucleotides. As posttranscriptional regulators, miRNAs can negatively regulate gene expression by binding directly to the 3’untranslated region (3’-UTR) of corresponding target messenger RNAs (mRNAs) in a sequence-specific manner, which induces mRNA degradation or protein translation repression. Since the first miRNA was reported by Ambros and Ruvkun in 1993, the number of miRNAs as well as related publications have expanded enormously. Currently, more than 1000 human miRNAs have been identified, and aberrant expression of these miRNAs is involved in various physiological and pathological processes, including human carcinogenesis. Recently, emerging evidence links the biological function of miRNAs to the initiation, promotion, and progression of HCC, and studies have confirmed that miRNAs played multiple roles in pathological processes of HCC, such as cell proliferation, cell death, metastasis and DNA methylation, etc. Although, the importance of miRNAs in hepatocarcino-genesis has attracted much attention in recent years, the pathological relevance and significance of the majority of miRNAs in HCC remain unclear.Previously, we have found a specific subtype of HCCs, which were >5 cm in diameter, had just single lession, and always grows expansively within an intact capsule or pseudocapsule, and we categorized them as solitary large hepatocellular carcinoma (SLHCC). Accordingly, we routinely classified HCCs into 3 different subtypes:SLHCC, nodular HCC (NHCC, node number≥2) and small HCC (SHCC, diameter<5 cm). Further study confirmed that SLHCC had unique clinical and pathological characteristics, and its metastastic potential was comparable with SHCC, but significantly lower than NHCC. After hepatic resection, SLHCC exhibited a similar long-time overall and disease-free survival with SHCC, but much better than NHCC. To gain a better understanding of the molecular biologic characteristics of SLHCC, we analyzed the gene expression pattern of SLHCC by using cDNA microarray and we noticed that 7.89%(668 of 8464) human genes had significantly different expression levels between SLHCC, SHCC and NHCC. These findings together with the fact that miRNAs can posttranscriptionally regulate almost 30% of human genes, lend support to the hypothesis that different types of HCCs may also have individual miRNAs expression profiles, which contribute to their specific clinical, pathological and molecular characteristics.Therefore, in the present study, we first compare the miRNAs expression profiles of SLHCC, SHCC and NHCC by miRNA array analysis. We primarily focused on miR-188-5p for further study, and comprehensively investigate its expression, biological functions and molecular mechanism in carcinogenesis of HCC. We found that miR-188-5p was significantly decreased in HCC tissues, and enforced expression of miR-188-5p could inhibit HCC cell proliferation and metastasis in vitro and in vivo. In addition, Antiapoptosis clone 11 (AAC11) was confirmed as a direct and functional target of miR-188-5p. These findings indicate miR-188-5p could serve as a potential therapeutic target for HCC.【Methods and Results】1. Identification of miRNAs Differentially Expressed in SLHCC, SHCC and NHCC:By using miRNA array analysis, we compared the miRNAs expression profiles displayed by SLHCC, SHCC and NHCC. We found that the expression level of miR-188-5p in SLHCC and SHCC was comparable, and both significantly high than that in NHCC. The expression pattern of miR-188-5p was similar with differences of the clinicalopathological characteristics of these three HCC subtypes, suggesting miR-188-5p involved in the carcinogenesis and development of HCC. So, we focused miR-188-5p for further study.2. miR-188-5p Is Frequently Down-regulated in HCC:To explore the expression of miR-188-5p in HCC,72 cases of HCC tissues and the corresponding ANLTs was measured by qRT-PCR. The results showed that the expression of miR-188-5p was remarkably down-regulated in HCC tissues, and the median decrease fold was 3.75 times. Consistent with the miRNA array data, the median miR-188-5p expression level in SLHCC was similar with that of SHCC (0.52 vs.0.65, P=0.017), but significantly higher than that of NHCC (0.52 vs.0.21, P<0.001). Furthermore, the relationship between the expression of miR-188-5p and the metastatic status of HCC was also analyzed. Our results indicated that compared with ANLT and primary lession of HCC, the vein cancer embolus of HCC (HCC-M) had the lowest expression level of miR-188-5p (relative expression=1.0,0.57 and 0.12; respevtively).3. Correlations of miR-188-5p Expression with Clinicopathologic Characteristics and Prognosis of HCC:According to the qRT-PCR results, the expression of miR-188-5p in HCC tissues was divided into low expression group (n=45) and high expression group (n=27), and the correlations of miR-188-5p expression with clinicopathologic characteristics and prognosis of HCC were analyzed. The miR-188-5p expression levels were found to be significantly higher in HCCs with multiple nodules or with vein invasion (P=0.010 and P=0.002, respectively). Additionally, HCC patients with high miR-188-5p expression had better overall survival (median survival time,35 months vs.16 months, P=0.044) and disease-free survival (median disease-free survival time,23 months vs.14 months, P=0.048) than those with the low expression.4. miR-188-5p Inhibits HCC Cell Proliferation and Metastasis in Vitro:We firstly confirmed the expression of miR-188-5p in two normal liver cell lines (LO2 and CCL13) and three HCC cell lines (HepG2, MHCC97-L, and HCCLM3). With LO2 cell as control, the expression of miR-188-5p in these five cell lines was 1.0,0.62,0.45,0.41 and 0.06, separately, which showed that HCC cells had lower miR-188-5p expression than normal liver cells. Furthermore, for the three HCC cells with different metastatic potential, HCCLM3 cell had the lowest miR-188-5p expression level, implicating a potential role for miR-188-5p in HCC metastasis.Among these five cells, HCCLM3 was selected to establish miR-188-5p stably expressing cell (HCCLM3miR-188-5p). Our results showed that the cell proliferation of HCCLM3miR-188-5p cells were significantly slower than that HCCLM3control cells (cells transfected with empty vector). Moreover, colony formation assay indicated that enforced expression of miR-188-5p resulted in more than 60% decrease in colony numbers when compared with the vector control (200±30 vs.75±18, P <0.001). The wound healing assay showed that the closure of HCCLM3miR-188-5p cells was significantly slower than that of HCCLM3control cells (94% vs.48%, P<0.01). Transwell assays with Matrigel demonstrated that miR-188-5p could dramatically inhibit the invasive capacities of HCCLM3 cell compared with vector control (125±15 vs.50±9, P<0.001). These results suggested that miR-188-5p can significantly inhibit HCC cell migration and invasion in vitro.5. AAC11 Was a Driect Downstream Target of miR-188-5p:The potential miR-188-5p target genes were searched with miRanda (microrna.org and miRbase) and TargetScan. We found that AAC11 contained two miR-188-5p binding site on its 3’-UTR, and one sequnence of the binding site is highly conserved across different species (chimpanzee, rhesus, dog, cat, horse and human), but another sequnence of the binding site is only conserved in rhesus and human. To confirm whether miR-188-5p could direct binding to the 3’-UTR of AAC11, vectors containing a series of 3’-UTR fragments, including fulllength 3’-UTR, binding site 1 (wild-type and mutant), and binding site 2 (wild-type and mutant), were by inserting the sequences into the region immediately downstream of the luciferase reporter gene. The luciferase activity assays indicated that the relative luciferase activity, normalized by luciferase activity of Renilla, was remarkably reduced (almost 70%) by miR-188-5p when the full-length wild-type 3’-UTR of AAC11 was present, whereas the reduction with mutant AAC11 3’-UTR was not as sharp as that observed in the wild-type counterpart. Additionally, our results also showed that mutation of the two binding sites separately could restore the luciferase activity, suggesting both of the binding sites of miR-188-5p were involved in AAC11 posttranscriptionally down-regulation. Western blot analysis further demonstrated that the high expression of miR-188-5p dramatically suppressed the endogenous protein level of AAC11 in HCCLM3 cell. Taken together, these results indicated that AAC11 was a direct downstream target for miR-188-5p in HCC cells.6. Restoration of AAC11 Abrogated miR-188-5p Induced Cell Proliferation and Metastasis Suppression:We constructed a lentiviral expression vector of AAC11 without the 3’-UTR and reintroduced AAC11 into HCCLM3 cell stably expressing miR-188-5p. Western-blot results confirmed that the expression of AAC11 was recovered. And the reintroduction of AAC11 enhanced the proliferation ability of miR-188-5p expression cell. Moreover, colony formation inhibition of miR-188-5p was also antagonized by enforced expression of AAC11 Furthermore, the restoration of AAC11 significantly abrogated miR-188-5p mediated HCC cell migration and invasion suppression. These findings suggested that AAC11 was a functional target of miR-188-5p.7. miR-188-5p Inhibits HCC Cell Proliferation and Metastasis by Targeting AAC11 in Vivo:Consistant with the in vitro results, miR-188-5p inhibited tumor growth in vivo, and the size of liver local tumors of HCCLM3 cell stably expressing miR-188-5p was dramatically smaller than that of control vector group. In addition, based on the fact that miR-188-5p overexpression could decrease the expression of AAC11 and restoration of of AAC11 abrogated the growth inhibition effect of miR-188-5p, we can draw the conclusion that miR-188-5p inhibited HCC growth through down-regulation of AAC11 in vivo. Moreover, in light of the in vitro results implicating a role for miR-188-5p in HCC cell migration and invasion, we examined the mice for liver and lung metastasis of the carcinoma cells. The results showed that the intrahepatic and lung metastasis rate of control group was 100% and 80% respectively, wherase no metastasis was found in miR-188-5p group. However, the reintroduction of AAC11 enhanced the metastasis ability of miR-188-5p expression cell, with 80% intrahepatic metastasis and 60% lung metastasis. Together, these data support an important role for miR-188-5p in HCC growth and metastasis in vivo.【Main Innovative Points】1. For the first time, the present study documented that miR-188-5p was down-regulated in HCC and correlated significantly to clinicopathologic characteristics as well as prognosis of HCC, which implicated miR-188-5p as a potential prognostic marker of HCC and extend the research of miR-188-5p to HCC.2. The present study has showed the critical roles of miR-188-5p in suppressing cell proliferation and metastasis of HCC by driectly targeting AAC11, which help understanding the molecular mechanism of carcinogenesis and development of HCC.3. Through HCC mouse model, the present study documented that miR-188-5p overexpression could suppress the proliferation, intrahepatic and pulmonary metastasis of HCC in vivo, implicating miR-188-5p as a novel potential therapeutic target for HCC 【Conclusions】The present study firstly confirmed that miR-188-5p was down-regulated in HCC and correlated significantly to clinicopathologic characteristics as well as prognosis of HCC, and demonstrated the critical role of miR-188-5p in suppressing cell proliferation and metastasis of HCC by driectly targeting AAC11, which suggested miR-188-5p as a novel prognostic marker and a candidate therapeutic target for HCC.更多还原