【作者】 刘洋；

【导师】 王春友；

【作者基本信息】 华中科技大学 ， 外科学， 2013， 博士

【摘要】 研究目的：检测miR-494在胰腺癌组织和细胞系中的表达,分析miR-494异常表达与胰腺癌临床病理特性的临床相关性；以胰腺癌细胞系AsPC-1, PANC-1为研究对象,证实miR-494对胰腺癌细胞增殖,周期,凋亡,侵袭转移,化疗敏感性等生物学特性的影响；进行miR-494与c-Myc和SIRT1相关性分析,明确miR-494靶向调控c-Myc和SIRT1;应用小片段干扰RNA (siRNA)技术抑制c-Myc与SIRT1基因表达,通过研究小片段干扰后PANC-1细胞增殖,周期,凋亡,侵袭转移等生物学特性的影响,进一步明确miR-494的作用机制是通过调控c-Myc和SIRT1实现。研究方法：实时荧光定量聚合酶链式反应(RT-PCR)检测胰腺组(86例胰腺癌组织和41例正常胰腺组织)和胰腺癌细胞系(AsPC-1,Bxpc-3, PANC-1, SW1990, Miapaca-2)中miR-494的表达,分析miR-494的表达与胰腺癌临床病理学特征及其预后的相关性；以胰腺癌细胞系AsPC-1和PANC-1为研究对象,分别转染miR-494类似物(miR-494mimics)和抑制剂(miR-494inhibitor),实时定量PCR检测转染后miR-494的表达,以确定转染效果； MTT(二苯基溴化四氮唑蓝)和平板克隆观察上调miR-494后对胰腺癌细胞增殖的影响；蛋白免疫印迹(western blot)检测上调miR-494后细胞周期与凋亡相关基因P21,CyclinDl,BAX, Bc1-2,P53蛋白的表达；流式细胞术检测细胞周期变化和凋亡的变化；β-半乳糖苷酶染色(β-Galactosidase staining, β-Gal)测定细胞衰老；Transwell检测各组细胞迁移(Migration)和侵袭(Invasion)的影响,并用western blot检测迁移和侵袭相关蛋白基质金属蛋白酶-2(MMP-2)和基质金属蛋白酶-9(-P-9)的表达变化；采用MTT法检测AsPC-1和PANC-1细胞经上调miR-494后对5-氟尿嘧啶(5-FU)和吉西他滨(Gemcitabine, GEM)化疗敏感性的变化；免疫组织化学染色法和实时定量PCR检测c-Myc和SIRT1在胰腺癌组织中的表达,用Graphpad软件对miR-494与c-Myc和SIRT1基因表达进行相关性分析；western blot检测转染miR-494mimics和miR-494inhibitor后c-Myc和SIRT1蛋白水平的变换,并对miR-494同c-Myc和SIRT1蛋白表达进行相关性分析；利用生物信息学预测软件(miRanda, miRDB and TargetScan)寻找miR-494与c-Myc和SIRT1的3’-非编码区结合(3’-UTR)结合位点,构建c-Myc和SIRT1双荧光报告基因质粒,通过双荧光报告初步证实miR-494直接靶向c-Myc和SIRT1;为了进一步明确miR-494的作用机制,以PANC-1为研究对象,小片段干扰RNA(siRNA)技术抑制c-Myc为siRNA-c-Myc,小片段干扰RNA(siRNA)同时抑制c-Myc与SIRT1为siRNA-c-Myc+siRNA-SIRT1(co-transfected,共转染组),对照为siRNA-NC(normal control), western blot检测转染后c-Myc和SIRT1蛋白的变化,以确定转染效果； MTT(二苯基溴化四氮唑蓝)观察各组对PANC-1细胞增殖的影响；蛋白免疫印迹(western blot)检测处理后细胞周期与凋亡相关基因P21, CyclinDl,BAX, Bcl-2,P53蛋白的表达；流式细胞术检测细胞周期变化和凋亡的变化；β-半乳糖苷酶染色(β-Galactosidase staining,β-Gal)测定细胞衰老；Transwell检测各组细胞侵袭(Invasion)的影响；BALB/c裸鼠成瘤实验鉴定上调miR-494和干扰c-Myc后PANC-1细胞的成瘤能力,western blot检测瘤体c-Myc和SIRT1的表达变化。实验结果：miR-494在胰腺癌组织的表达水平明显低于正常胰腺组织(0.567±0.049vs1.000±0.104,P<0.001)；同正常组织相比,miR-494在胰腺癌细胞系AsPC-1,Bxpc-3, PANC-1和SW1990中显著下降,在AsPC-1(0.171±0.023vs1.060±0.131, p=0.0026)和PANC-1细胞(0.270±0.024vs1.060±0.131,p=0.004)中下降最为明显；miR-494高表达患者的生存时间明显长于miR-494低表达患者(P=0.0366)。miR-494表达水平与胰腺癌患者年龄(p=0.0438)、肿瘤大小(p=0.0041)、TNM分期(p=0.0003)、淋巴侵袭(p=0.0010)、远处转移(p=0.0362)密切相关,而与肿瘤组织分化程度、患者性别、肿瘤部位、血管浸润、神经侵犯无相关性(p>0.05)。由于胰腺癌细胞系AsPC-1和PANC-1的miR-494表达水平下降最为明显,因此选取AsPC-1和PANC-1作为实验细胞系,转染miR-494mimics后,miR-494表显著上调(P<0.01),转染miR-494inhibitor后miR-494下调(P<0.05),证实转染有效。同各自的对照相比,转染miR-494mimics后AsPC-1(45.13±5.38％,P<0.01)和PANC-1(41.42±3.95％,P<0.01)细胞增殖受到显著抑制,转染miR-494inhibitor后AsPC-1(23.15±2.18％,P<0.05)和PANC-1(16.59±3.23％,P<0.05)细胞增殖增强。平板克隆结果显示,转染miR-494mimics后AsPC-1和PANC-1克隆数显著降低。流式细胞检测结果显示上调miR-494后,同各自对照相比,AsPC-1(12.3±0.5%vs19.5±0.6%,P<0.05)和PANC-1细胞(8.9±0.8%vs17.1±0.3%,P<0.05)细胞凋亡增强,AsPC-(63.3.1％vs73.7±2.5%,P<0.05)和PANC-1细胞(40.5.6±4.2%vs58.6±3.7%,P<0.01)细胞周期G1期细胞比率显著增加。同各自对照相比,上调miR-494后AsPC-1(17.5±2.1%vs40.7±1.7%,P<0.01)和PANC-1细胞(14.6±3.3%vs60.9±3.8%,P<0.01)衰老显著增加。Western blot检测显示经转染miR-494mimics后Bcl-2和CyclinDl显著下调,乙酰化p53.BAX和p21显著上调。Transwell检测各组细胞侵袭和迁移能力发现,上调miR-494后,AsPC-1和PANC-1细胞侵袭和迁移能力显著减弱,侵袭相关蛋白MMP-2和MMP-9显著下降。药敏结果显示,同各自的对照相比,在转染miR-494后,AsPC-1细胞对5-FU(7.93±1.05vs27.56±2.89ug/mL,P<0.01)和GEM(17.5±1.39vs82.56±4.27ug/mL,P<0.01)IC50值显著下降,PANC-1细胞对5-FU(9.41±1.63vs42.62±3.16ug/mL,P<0.01)和GEM(25.33±1.75vs63.25±5.36ug/mL, P<0.01)的IC50也显著下降,即上调miR-494后,AsPC-1和PANC-1对5-Fu和GEM的药物敏感性明显增强。免疫组织化学结果显示,胰腺癌组织中c-Myc(52.74±1.534％vs26.49±1.56％,P<0.001)和SIRT1(48.35±1.51％vs20.51±1.34％,P<0.001)阳性细胞数比率明显高于正常组织。实时定量PCR显示,同正常组织相比,胰腺癌组织中c-Myc(1.379±0.086vs0.940±0.070,P=0.0017)和SIRT1(1.583±0.0.110vs1-044±0.092,P=0.0022)基因高表达。胰腺组织中miR-494同c-Myc(r=-0.6799,P<0.001)和SIRTl(r=-0.5293,P<0.001)基因表达负相关,提示miR-494可能调控c-Myc和SIRT1.生物信息学预测软件(miRanda, miRDB and TargetScan)显示,miR-494与c-Myc和SIRT1的3’-UTR有共同的结合位点,提示miR-494可能靶向c-Myc和SIRT1.转染miR-494mimics后c-Myc和SIRT1基因和蛋白表达均显著下调,转染miR-494inhibitor后c-Myc和SIRT1基因和蛋白表达均上调。对miR-494同c-Myc和SIRT1基因表达的相关性分析提示：miR-494同c-Myc (r=-0.6374,P=0.0142)和SIRT1(r=-0.5973, P=0.0239)负相关。miR-494mimics与包含野生型c-Myc或SIRT1的3’-UTR质粒共转染的荧光活性明显低于对照组,证实miR-494直接靶向c-Myc和SIRT1.转染c-Myc干扰小片段后,c-Myc和SIRT1显著下降(P<0.01),细胞增殖受抑制,细胞G1期阻滞,细胞衰老增强,侵袭能力减弱(P<0.05)。然而siRNA-c-Myc组对细胞凋亡无明显影响(P>0.05)。提示miR-494的作用不单纯依赖c-Myc。共转染c-Myc和SIRT1干扰小片段后,同单独干扰c-Myc组相比,对增殖和侵袭抑制以及周期阻滞更为明显(P<0.01)。另外,共转染组组PANC-1细胞凋亡率增加(12.65±0.55%vs.16.85±0.75%,P=0.0457),凋亡和周期相关蛋白的结果同上调miR-494结果一致。提示miR-494的作用是通过同时靶向c-Myc和SIRT1实现。BALB/c裸鼠成瘤实验结果显示上调miR-494后PANC-1细胞的成瘤能力下降(0.83±0.18vs0.32±0.07g,P<0.05)。Western blot检测瘤体c-Myc和SIRT1的表达下调。实验结论：1.miR-494在胰腺癌组织中和胰腺癌细胞系的低表达,低表达miR-494与患者年龄,肿瘤大小,胰腺癌侵袭转移以及预后密切相关,检测miR-494的表达有助于判断胰腺癌恶性程度及预后。2.上调miR-494在胰腺癌细胞AsPC-1和PANC-1细胞中的表达,可显著抑制细胞增殖,增加凋亡和衰老细胞比例,发生G1期阻滞,减弱细胞侵袭和迁移能力,增强细胞对5-Fu和GEM的药物敏感性。提示miR-494可能作为胰腺癌基因治疗靶点。3.c-Myc和SIRT1在胰腺癌组织中高表达,并与miR-494负相关。在PANC-1细胞中,上调miR-494后,c-Myc和SIRT1表达下调；下调miR-494后,c-Myc和SIRT1上调；miR-494与c-Myc和SIRT1负相关。结合双荧光结果,证实miR-494直接靶向c-Myc和SIRT1。4.同时下调c-Myc和SIRT1在PANC-1细胞中的表达后,生物学效应及效应蛋白表达改变同上调miR-494一致；单一下调c-Myc后,凋亡未见明显改变,且对细胞增殖抑制和周期阻滞较下调c-Myc和SIRT1弱。提示miR-494可能通过靶向c-Myc和SIRT1发挥生物学效应。上调miR-494抑制胰腺癌细胞的体外成瘤能力。更多还原

【Abstract】 Purpose:MicroRNAs are essential for the regulation of cell proliferation and had been implicated in tumorigenesis. However, the roles and molecular mechanisms of aberrant microRNAs in pancreatic cancer remain unclear. In this study,we investigate the expression levels of miR-494in pancreatic cancer tissues and pancreatic cancer cells, and significance of SIRT1in human pancreatic carcinoma and their association with the clinical pathologic characters, and evaluate the relationship between miR-494expression levels and clinic characteristics. After restoration of miR-494, biological behaviours of Aspc-1and PANC-1cells were investigated such as cellular growth, senescence, cycle progression, apoptosis and chemosensitivity etc. We also explore the mechanism of the antitumor effects of miR-494. To confirm the potential targets of miR-494, we prove the direct interaction between miR-494and its targets c-Myc and SIRT1. To elucidate the antitumor mechanisms of miR-494, we silenced c-Myc/SIRT1expression through the introduction of siRNA-c-Myc/into PANC-1cells. After inhibition of miR-494, biological behaviours were investigated. We hypothesized that miR-494might be involved in pancreatic cancer progression through the modulation of c-Myc and SIRT1.Experimental Design:The expression levels of miR-494were detected in of pancreatic cancer tissues and pancreatic cacer cells(AsPC-1, BxPC-3, PANC-1, SW1990and Miapaca-2) using qRT-PCR. We analyze the correlations among the variety expression levels of SIRT1and pancreatic cancer clinical pathologic characters. To explore the roles of miR-494in pancreatic cancer progression, AsPC-1and PANC-1cells were transfected with miR-494mimics, miR-494inhibitor and their respective negative controls (NC). After transfection, the expression of miR-494in AsPC-1and PANC-1cells was detected by qRT-PCR. MTT was used to measure the proliferation of AsPC-1and PANC-1cells after transfection with miR-494mimics/inhibitor. To further investigate the mechanisms of miR-494in proliferation inhibition, apoptosis and cell cycle were analyzed using flow cytometry while cell senescence was analyzed using β-Galactosidase (β-Gal) staining. Moreover, several associated proteins, including BAX, Bcl-2, acetylated p53, p21and CyclinDl were detected by western blot analysis. The effects of miR-494on the migration and invasive ability of pancreatic cancer cells were investigated using Transwell analysis after transfection with miR-494. We also observed whether miR-494could sensitize pancreatic cancer cells to5-FU and GEM. Immunohistochemistry (IHC)was used to detect the expression of c-Myc and SIRT1in86cancer tissues (PC) and41normal pancreatic tissues (NP). Next, we determined the expression levels of c-Myc and SIRT1mRNAs in PC and NP tissues, and analysis the correlation between miR-494levels and c-Myc and SIRT1mRNA expression in pancreatic cancer tissues. The putative targets of miR-494were revealed by bioinformatic analyses, includingmiRanda, miRDB and TargetScan algorithms. The expression of c-Myc and SIRT1was detected qRT-PCR and western blot results after transfection of miR-494mimics/inhibitor. After that, the correlation between miR-494levels and c-Myc and SIRT1mRNA expression was analysised. Furthermore, we cloned3’-UTR sequences that contained the predicted target sites (wild type, WT) or mutated sequences (mutant type, MUT) of c-Myc and SIRT1into the pGL3vector. A luciferase reporter assay was then used to prove the direct interaction between miR-494and its targets. To confirm the antitumor mechanisms of miR-494, we silenced c-Myc/SIRTl expression through the introduction of siRNA-c-Myc/SIRT1into PANC-1cells. Cell biology behaviors were investigated after inhibition of c-Myc/SIRT1. Further, restoration of miR-494was evaluated in nude mice to confirm whether our findings shown above in vitro would be reproducible in vivo. Results:The mean expression level of miR-494in PC tissues was significantly lower than that of NP tissues (0.567±0.049vs1.000±0.104, P<0.001). We also found that miR-494was significantly down-regulated in four different pancreatic cancer cell lines (AsPC-1, BxPC-3, PANC-1and SW1990) compared with NP tissues, especially in AsPC-1(0.171±0.023vs1.060±0.131, p=0.0026) and PANC-1cells (0.270±0.024vs1.060±0.131, p=0.004). Low miR-494expression was found to be significantly correlated with age (p=0.0438), tumor size (p=0.0041), TNM stage (p=0.0003), lymphatic invasion (p=0.0010) and distant metastasis (p=0.0362). However, there was no obvious correlation between miR-494levels and other clinic features. Kaplan-Meier survival curves showed that patients with high miR-494expression levels had significantly longer survival time than patients with low miR-494levels (P=0.0366). After restoration of miR-494, the proliferation of AsPC-1and PANC-1cells was significantly inhibited by45.13±5.38%and41.42±3.95%, respectively (F<0.01). While after transfection with miR-494inhibitor, the proliferation rate increased by23.15±2.18%and16.59±3.23%(P<0.05). Moreover, AsPC-1and PANC-1cells transfected with miR-494mimics displayed fewer and smaller colonies than negative control. The number of apoptotic cells were markedly increased in AsPC-1(12.3±0.5%vs19.5±0.6%, P<0.05) and PANC-1cells (8.9±0.8%vs17.1±0.3%, P<0.05) transfected with miR-494. Furthermore, overexpression of miR-494notably increased the proportion of cells in the G1phase in AsPC-1(63.3±3.1%vs73.7±2.5%, P<0.05) and PANC-1cells (40.5.6±4.2%vs58.6±3.7%, P<0.01). Analysis of β-Gal staining indicated that miR-494significantly induced senescence of AsPC-1(17.5±2.1%vs40.7±1.7%, P<0.01) and PANC-1cells (14.6±3.3%vs60.9±3.8%, P<0.01). Bcl-2and CyclinD1were significantly down-regulated, whereas acetylated p53, BAX and p21were obviously up-regulated. The migration and invasive ability of pancreatic cancer cells were significantly decreased after transfection with miR-494. After restoration of miR-494, the IC50of AsPC-1cells exposed to5-FU (7.93±1.05vs27.56±2.89ug/mL, P<0.01) and GEM (17.5±1.39vs82.56±4.27ug/mL， P<0.01) were significantly decreased. Moreover, the IC50of PANC-1cells exposed to5-FU (9.41±1vs1.63vs42.62±3.16ug/mL, P<0.01) and GEM (25.33±1.75vs63.25±5.36ug/mL, P<0.01) were also decreased. Compared to the NP group, the percentage of cells that were positive for c-Myc (52.74±1.534%vs26.49±1.56%, P<0.001)and SIRT1(48.35±1.51%vs20.51±1.34%, P<0.001) were significantly increased in the PC group. The expression levels of c-Myc (1.379±0.086vs0.940±0.070, P=0.0017) and SIRT1(1.583±0.0.110vs1.044±0.092, P=0.0022) mRNAs were also significantly up-regulated in the PC group. A significant inverse correlation between miR-494levels and c-Myc(r=-0.6799, P<0.001) and SIRT1(r=-0.5293, P<0.001) mRNA expression was observed in pancreatic cancer tissues. The qRT-PCR and western blot results confirmed that miR-494restoration led to the down-regulation of c-Myc and SIRT1expression, whereas the inhibition of miR-494expression increased c-Myc and SIRT1expression. In addition, we found that miR-494expression was negatively correlated with c-Myc (r=-0.6374, P=0.0142) and SIRTl(r=-0.5973, P=0.0239) expression. Luciferase reporter assay proved that miR-494targeted c-Myc and SIRT1. Specific knockdown of c-Myc by siRNA significantly inhibited the proliferation and invasion of PANC-1cells and induced cell cycle arrest and senescence. The down-regulation of c-Myc did not lead to obvious apoptosis, which implied that the effects of miR-494on pancreatic cancer do not rely exclusively on targeting c-Myc. After co-transfection of PANC-1cells with c-Myc-RNAi and SIRTl-RNAi further inhibited proliferation, invasion and cell cycle arrest. Moreover, the co-transfection induced obvious apoptosis in PANC-1cells (12.65±0.55%vs.16.85±0.75%, P=0.0457), which was similar to the effect of miR-494induction. After co-transfection, the associated proteins BAX, Bcl-2, acetylated p53, p21and CyclinDl expression levels were consistent with induction of miR-494. Restoration of miR-494inhibited the growth of pancreatic cancer cells in vivo (0.83±0.18vs0.32±0.07g, P<0.05). Western blot analysis demonstrated that the expression levels of c-Myc and SIRT1appeared to be significantly down-regulated in the pancreatic cancer xenografts.Conclusions:1. MiR-494was down-regulated in pancreatic cancer tissues and cell lines. Low miR-494expression was significantly correlated with age, tumor proliferation, and could participate in invasion and metastasis of pancreatic cancer. The expression of miR-494may be viewed as a biomarker of malignancy degree and prognosis of pancreatic cacer.2. Overexpression of miR-494in pancreatic cancer cells could significantly inhibit cell proliferation by inducing senescence, G1phase arrest and apoptosis. Moreover, restoration of miR-494expression also inhibited migration and the invasive ability of pancreatic cancer cells. The induction of miR-494enhanced the chemosensitivity of pancreatic cancer cells to5-FU and Gemcitabine. These results indicated that miR-494might be involved in the carcinogenesis of pancreatic cancer and that its low expression levels could be considered as a novel diagnostic and prognostic indicator.3. MiR-494down-regulated c-Myc and SIRT1expression by directly binding to the3’-UTR of c-Myc and SIRT1mRNA. Inhibition of miR-494increased expression of c-Myc and SIRT1. Furthermore, the inverse correlation between miR-494levels and c-Myc or SIRT1expression was observed in pancreatic cancer tissues as well as cell lines. Therefore, these results confirmed that miR-494directly targeted c-Myc and SIRT1.4. After co-transfection of PANC-1cells with c-Myc-RNAi and SIRT1-RNAi, the biological behaviours were similar to the effect of miR-494induction. Howover, the down-regulation of c-Myc did not lead to obvious apoptosis, which implied that the effects of miR-494on pancreatic cancer do not rely exclusively on targeting c-Myc.更多还原