长链非编码RNA UCA1在肝癌中的表达、功能及其调控机制研究

发布时间：2019-07-01 17:11

【摘要】：[背景]肝细胞肝癌(hepatocellular carcinoma, HCC)是我国最常见的恶性肿瘤之一,发病率位居各大恶性肿瘤的第四位,且近年来呈逐年上升的趋势；因其恶性程度高、进展十分迅速、预后极差,死亡率已位居各大恶性肿瘤的第二位。有必要积极探索与HCC相关的基因,为HCC发病机制研究以及临床诊疗提供新思路和新靶标。长链非编码RNA (long non-coding RNAs, IncRNAs)是近年来发现的与肿瘤发生、发展密切相关的一类缺乏蛋白编码能力的RNA分子。新近研究表明,lncRNAs可作为竞争性内源RNA (competing endogenous RNA, ceRNA),通过其微小RNA反应元件(]mniRNA response elements, MREs)竞争结合miRNAs,抑制miRNAs的功能与活性,从而在转录后水平调节miRNAs靶基因mRNAs表达。UCA1 (Urothelial carcinoma associated 1)是首先在膀胱癌中发现的一种新的IncRNA分子,文献报道其在膀胱癌、结直肠癌、乳腺癌等肿瘤中高表达并与肿瘤恶性进展密切相关。但UCA1在HCC中的表达模式、功能角色及潜在作用机制尚未见文献报道。[目的]探讨UCA1在HCC中的表达模式及临床意义；分析RNA干扰UCA1对HCC细胞增殖、克隆形成、细胞周期、侵袭与迁移以及裸鼠移植瘤生长的影响；并深入探讨基于ceRNA模式的UCA1--miRNA--mRNA--pathway调控网络在HCC发生发展中的作用机制。[方法](1)1ncRNA芯片筛选HCC癌组织中的IncRNA分子；(2)实时荧光定量PCR (Quantitative Real-time PCR, qRT-PCR)验证筛选的UCAl;(3)Fisher精确概率法、Kaplan-Meier生存曲线以及Cox回归模型分析UCA1表达与HCC患者临床病理参数及预后的相关性；(4)构建UCA1短发夹RNA干扰载体,转染高表达UCA1的HCC细胞株,检测干扰效率；(5)CCK-8法检测细胞增殖、流式细胞术检测细胞周期、平板法检测细胞克隆形成、Transwell小室法检测细胞侵袭与迁移以及裸鼠皮下成瘤实验对UCA1在HCC中的生物学功能进行分析；(6)生物信息学预测可与UCA1发生作用的miRNA,以及相应miR NA的靶基因；(7)qRT-PCR及免疫组化分析临床HCC标本中UCA1、miRNA以及靶基因三者表达之间的相关性；(8)双荧光素酶试验及RNA结合蛋白免疫沉淀(RNA Binding Protein Immunoprecipitation, RIP)试验验证UCA1可与miRNA作用于RNA诱导沉默复合物(RNA-induced silencing complex, RISC); (9)分析UCA1是否可抑制miRNA的生物学功能；(10)双荧光素酶试验验证miRNA与靶基因之间的关系；(11) qRT-PCR与Western blot分析UCA1、miRNA对其靶基因mRNA及蛋白表达的影响；(12)Western blot分析与靶基因相关的信号通路蛋白及其磷酸化蛋白表达变化。[结果](1)通过IncRNA基因芯片检测并且qRT-PCR证实,UCA1在HCC癌组织中表达明显高于癌旁组织,P0.001。(2)UCA1与HCC患者TNM分期、是否有远距离转移密切相关,P0.001；生存曲线显示高表达UCAl的HCC患者总生存时间明显缩短,P0.001；单因素及多因素回归分析显示UCA1可用于判断HCC患者的不良预后。(3)构建两个针对UCA1的RNA干扰载体,分别转染高表达UCA1的HCC细胞株(SMMC-7721和HepG2细胞),干扰效率分别为siUCA1#1：81%和78%；siUCAl#2:54%和47%; siUCA1#1+siUCAl#2:66%和60%,选用siUCA1#1干扰载体(即siUCA1)进行后继UCA1功能学研究。(4)siUCA1转染SMMC-7721和HepG2细胞后,可体外抑制细胞增殖、克隆形成能力以及侵袭与迁移,并且诱导HCC细胞周期G0/G1期阻滞；同时,siUCA1转染HCC细胞,可体内抑制裸鼠移植瘤的生长。(5)生物信息学预测UCA1序列中存在可与miR-216b、miR-665、miR-326、miR-212-5p、miR-338-3p、 miR-567及 miR-136-3p互补结合的MREs；在siUCA1转染的HCC细胞中,仅miR-216b表达明显升高(2倍)；而其余6个miRNAs的表达无明显变化或有轻微变化(1.5倍)。(6)构建UCA1的荧光素酶报告载体,与miR-216b共转染HCC细胞,双荧光素酶试验证实二者可通过MRE互补结合；RIP试验进一步证实UCA1与miR-216b相互作用于RISC (7) r niR-216b在HCC癌组织中表达与癌旁相比明显降低,P0.01,且HCC癌组织中UCA1与miR-216b的表达呈显著负相关,r=-0.6224, P0.0001。 (8) miR-216b可抑制HCC细胞的增殖与克隆形成、侵袭与转移、裸鼠移植瘤的生长以及诱导细胞周期G0/G1期阻滞；而UCA1可在体、内外逆转miR-216b对HCC细胞生长与转移的抑制效应。(9)生物信息学预测成纤维生长因子受体1 (fibroblast growth factor receptor 1, FGFR1)是miR-216b的靶基因之一。双荧光素酶试证实FGFR1是miR-216b的靶基因,]miR-216b或siUCA1均可下调HCC细胞中FGFR1 mRNA及蛋白的表达；UCA1过表达可上调FGFR1 mRNA及蛋白的表达；而当miR-216b与UCA1同时共转染时可恢复FGFR1 mRNA及蛋白的表达。(10)在HCC组织标本中,FGFR1蛋白与mRNA的表达与UCA1的表达呈显著正相关,r=0.7114与0.6116,P0.0001；而与miR-216b的表达呈显著负相关,r=-0.5040与-0.7094,P0.0001。(11)Western blot分析显示,UCA1不是通过FGFR1-JNK及FGFR1-p38 MAPK信号通路,而是通过FGFR1-ERK1/2信号通路来促进HCC恶性进展。[结论]UCA1在HCC中高表达,且与TNM分期、转移及患者预后相关。UCA1可作为一个癌基因促进HCC细胞增殖、侵袭与迁移以及裸鼠移植瘤的形成。机制研究发现UCA1可作为ceRNA竞争结合miR-216 b,可逆转miR-216b对HCC细胞的生长及转移的抑制作用,并解除miR-216b对其靶基因FGFR1的抑制,FGFR1表达增高,且通过ERK信号(UCA1-miR-216b-FGFR1-ERK pathway)促进HCC发生发展。本研究为HCC发病机制的探索提供了新的思路,也为靶向IncRNAs的HCC诊断与治疗提供了新的发展方向以及新的靶标。
[Abstract]:[BACKGROUND] HEPATOCELLULAR CARCINOMA (HCC) is one of the most common malignant tumors in China. The incidence of HCC is the fourth of the major malignant tumors, and has been increasing year by year in recent years, because of its high degree of malignancy, rapid progress and poor prognosis. The mortality rate has been the second in the major malignancies. It is necessary to actively explore the gene related to HCC, and provide a new thought and a new target for the pathogenesis of HCC and the clinical diagnosis and treatment. Long-chain non-coding RNAs (IncRNAs) is a kind of RNA molecule which is closely related to tumorigenesis and development in recent years. Recent studies have shown that lncRNAs can be used as competitive endogenous RNA (cRNA) to compete with miRNAs through its microRNA response elements (MREs) to inhibit the function and activity of miRNAs, so as to regulate the expression of miRNAs target gene mRNAs at the post-transcriptional level. UCA1 (Urodynamic carcinoma associated 1) is a new kind of IncRNA molecule, which is first found in bladder cancer. It is reported that it is highly expressed in bladder cancer, colorectal cancer, breast cancer and other tumors and is closely related to the malignant progression of the tumor. However, the expression pattern, function and potential mechanism of UCA1 in HCC have not been reported in the literature. [Objective] To study the expression pattern and clinical significance of UCA1 in HCC, and to analyze the effect of RNA interference on proliferation, clone formation, cell cycle, invasion and migration of HCC cells and the growth of transplanted tumor in nude mice. The role of UCA1--miRNA--patway control network in the development of HCC was discussed. [Methods] (1)1 ncRNA chip was used to screen the IncRNA molecule in the tissue of HCC. (2) Real-time fluorescence quantitative PCR (qRT-PCR) was used to verify the screening UCAl; (3) Fisher's exact probability method, the Kaplan-Meier survival curve and the Cox regression model were used to analyze the relationship between the expression of UCA1 and the clinicopathological parameters and prognosis of HCC patients. (4) constructing a UCA short hairpin RNA interference vector, transfecting the HCC cell strain with high expression of UCA1, detecting the interference efficiency, (5) detecting cell proliferation by the CCK-8 method, detecting the cell cycle by flow cytometry, and detecting the cell clone formation by the plate method, The biological function of UCA1 in HCC was analyzed by Transwell's cell method, and the biological function of UCA1 in HCC was analyzed by transwell chamber method. (6) Bioinformatics was used to predict the miRNAs that could interact with UCA1 and the target genes of the corresponding miR-NA; (7) qRT-PCR and immunohistochemistry were used to analyze the UCA1 in the clinical HCC specimens. The correlation between the expression of the miRNA and the target gene; (8) the double-luciferase assay and the RNA Binding Protein Immunopreservation (RIP) test verify that the UCA1 and the miRNA act on the RNA-induced silencing complex (RISC); (9) analyzing whether the UCA1 can inhibit the biological function of the miRNA; (10) the double-luciferase test verifies the relation between the miRNA and the target gene; (11) qRT-PCR and Western blot analysis the influence of the UCA1 and the miRNA on the mRNA and the protein expression of the target gene; (12) Western blot was used to analyze the expression of signal pathway protein and its phosphorylated protein in the target gene. [Results] (1) The expression of UCA1 in the tissues of HCC was significantly higher than that in the adjacent tissues (P 0.01). (2) The TNM staging of UCA1 and HCC was closely related to the long-distance metastasis, and the survival time of the HCC patients with high expression of UCAl was significantly shortened in the survival curve, P 0.001; single factor and multi-factor regression analysis showed that UCA1 could be used to judge the poor prognosis of HCC patients. (3) Two cell lines (SMMC-7721 and HepG2 cells) with high expression of UCA1 were constructed, and the interference efficiency was siUCAl # 1:81% and 78% respectively; siUCAl # 2:54% and 47%; siUCAl # 1 + siUCAl # 2:66% and 60%, respectively. A follow-up UCA1 functional study was performed using the siUCA1 # 1 interference vector (i.e., siUCA1). (4) After the SMMC-7721 and HepG2 cells were transfected with siUCA1, the cell proliferation, the formation of the clone and the invasion and migration can be inhibited in vitro, and the cell cycle G0/ G1 arrest of HCC was induced. (5) Bioinformatics predicts that there are MREs that can be complementary to miR-216b, miR-665, miR-326, miR-212-5p, miR-338-3p, miR-567 and miR-136-3p in the UCA1 sequence; in the HCC cells transfected with siUCA1, only miR-216b expression is significantly increased (2-fold); while the expression of the remaining 6 miRNAs has no significant change or slight change (1.5 times). (6) The luciferase reporter vector of UCA1 was constructed, and the HCC cells were co-transfected with miR-216b. The two-luciferase assay confirmed that both of them could be combined by MRE. The RIP test further confirmed that the expression of UCA1 and miR-216b in RISC (7) r niR-216b was significantly lower than that in the adjacent tissues (P0.01). The expression of UCA1 and miR-216b in HCC was negatively correlated with the expression of miR-216b, r =-0.6224, P.0001. (8) miR-216b can inhibit the proliferation and clone formation, invasion and metastasis of HCC cells, the growth of transplanted tumor in nude mice and the induction of cell cycle G0/ G1 phase, and UCA1 can reverse the inhibitory effect of miR-216b on the growth and metastasis of HCC cells. (9) Bioinformatics predicts that the fibroblast growth factor receptor 1 (FGFR1) is one of the target genes of miR-216b. Double-luciferase assay confirmed that FGFR1 is a target gene of miR-216b, and both miR-216b or siUCA1 can downregulate the expression of FGFR1 mRNA and protein in the HCC cell; the overexpression of UCA1 can upregulate the expression of FGFR1 mRNA and protein; and when miR-216b is co-transfected with UCA1, the expression of FGFR1 mRNA and protein can be recovered. (10) In the specimens of HCC, the expression of FGFR1 protein and mRNA was positively correlated with the expression of UCA1, r = 0.7114 and 0.6116, P.0001; while the expression of FGFR1 and miR-216b was negatively correlated with the expression of miR-216b, r =-0.5040 and-0.7094, P.0001. (11) Western blot analysis showed that UCA1 was not a MAPK signal pathway through FGFR1-JNK and FGFR1-p38, but instead of the FGFR1-ERK1/2 signaling pathway, the malignant progression of HCC was promoted. [Conclusion] UCA1 is highly expressed in HCC and is related to TNM staging, metastasis and prognosis of patients. UCA1 can be used as an oncogene to promote the proliferation, invasion and migration of HCC cells and the formation of transplanted tumor in nude mice. The mechanism study found that UCA1 could be used as ceRNA to bind to miR-216b, which could reverse the inhibition of miR-216b on the growth and metastasis of HCC cells, and release the inhibition of miR-216b on the target gene FGFR1, and the expression of FGFR1 was increased, and the development of HCC was promoted by the ERK signal (UCA1-miR-216b-FGFR1-ERK patway). This study provides a new way for the exploration of the pathogenesis of HCC, and also provides a new development direction and a new target for the diagnosis and treatment of HCC.
【学位授予单位】：南京医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.7

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