MiCroRNA-23a对胰腺癌细胞上皮间质转化影响及机制研究

发布时间：2018-07-27 21:37

【摘要】：目的:1研究MiCroRNA-23a在胰腺癌组织中的表达情况及其与临床病理资料的关系。2研究MiCroRNA-23a在不同胰腺癌细胞株(Aspc-1,Bxpc-3,Cfpac-1,Panc-1)的表达规律。3研究MiCroRNA-23a体内体外促进胰腺癌细胞侵袭转移。4研究MiCroRNA-23a通过靶向抑制上皮剪接调节蛋白1(epithelial splicing regulator protein 1,ESRP1)改变CD44亚型表达,促进胰腺癌细胞发生上皮间质转化(epithelial mesenchymal transition,EMT),促进胰腺癌细胞侵袭转移。方法:1.收集西南医院肝胆外科2013年1月~2013年12月手术切除的胰腺癌标本及患者临床病理资料共计47例,记录患者FOS及OS。采用RT-PCR方法检测MiCro RNA-23a在胰腺癌原发灶,转移淋巴结组织以及癌旁正常胰腺组织中的表达情况,比较MiCroRNA-23a在上述三种组织中的表达差异。计算癌组织中MiCro RNA-23a的相对平均表达量,并根据该值将所有标本分为MiCroRNA-23a高表达组和低表达组,分析MiCroRNA-23a表达与临床病理资料的关系。采用Kaplan-Meier软件进行生存分析,研究不同表达水平的MiCro RNA-23a对胰腺癌患者预后的影响。2.qRT-PCR和Western blot检测EMT标志物E-钙黏蛋白(E-cad),N-钙黏蛋白(N-cad),波形蛋白(VIM)的表达,观察细胞形态,TGF-β1诱导EMT处理,区分胰腺癌细胞系Aspc-1,Bxpc-3,Cfpac-1,Panc-1的上皮、间质表型。qRT-PCR检测正常胰腺导管上皮细胞(PDC)和胰腺癌细胞(Aspc-1,Aspc-1+TGF-β1,Bxpc-3,Bxpc-3+TGF-β1,Cfpac-1,Panc-1)mi RNA-23a的表达,观察其表达与胰腺癌细胞EMT表型的关系。干扰mi R-23a,观察对TGF-β1诱导EMT的影响。3.miR-23a mimic和mi R-23a inhibitor分别转染Aspc-1和Panc-1,观察其侵袭迁移能力的变化;构建mi R-23a sponges慢病毒表达载体,转染Panc-1细胞,建立稳定细胞株,裸鼠体内实验观察mi R-23a促进胰腺癌转移。4.基因芯片分析和生物信息学网站(TargetScan.com)搜索结果交叉比较,双荧光素酶报告质粒实验验证ESRP1是mi R-23a的直接靶基因。5.研究干扰miR-23a对ESRP1表达的影响,qRT-PCR检测胰腺癌组织ESRP1的表达,与miR-23a的表达进行相关性分析;干扰或过表达ESRP1,观察对胰腺癌细胞侵袭转移能力的影响;ESRP1回复实验,观察对mi R-23a影响胰腺癌细胞EMT的逆转作用。6.Western blot、qRT-PCR分别检测mi R-23a对ESRP1及其下游CD44s/CD44v、FGFR2 IIIb/IIIc剪接亚型表达的影响。ESRP1回复实验观察干扰mi R-23a表达对下游剪接分子表达的影响。结果:1.胰腺癌转移淋巴结组织中mi R-23a的表达显著高于胰腺癌原发灶和癌旁正常组织,胰腺癌MiCroRNA-23a表达与肿瘤组织分化程度、肿瘤浸润深度(T分期)以及胰腺癌淋巴结转移(N分期)具有显著相关性,而其表达与年龄、性别,肿瘤发生部位及远处转移(M分期)等无相关性。生存分析发现,MiCroRNA-23a表达与胰腺癌肿瘤患者预后具有相关性,高表达组预后差,生存期均低于低表达组,两者相比较差异具有统计学意义。2.1 Aspc-1、Bxpc-3细胞呈上皮表型,Cfpac-1、Panc-1为间质表型,TGF-β1诱导Aspc-1、Bxpc-3后,发生EMT。miR-23a在间质表型的胰腺癌细胞中表达明显增高。结合mi R-23a在胰腺癌转移淋巴结组织中表达增高,推测miR-23a可能促进胰腺癌细胞上皮间质转化及侵袭转移。干扰mi R-23a,抑制了TGF-β1诱导上皮表型胰腺癌细胞EMT的发生。3.体外实验证明,mi R-23a mimic转染Aspc-1导致其侵袭迁移能力增强,mi R-23a inhibitor转染Panc-1导致其侵袭迁移能力减弱;体内实验证明,mi R-23a sponges慢病毒表达载体转染Panc-1细胞,可以抑制miR-23a的功能,从而抑制胰腺癌细胞生长及转移。4.基因芯片分析及生物信息学检索,证实ESRP1是miR-23a的直接的下游靶基因,荧光素酶报告质粒实验证实ESRP1是mi R-23a的直接靶基因。5.ESRP1维持胰腺癌细胞的上皮表型,干扰mi R-23a,ESRP1的表达发生变化;胰腺癌组织中,ESRP1的表达与miR-23a的表达呈显著负相关。ESRP1si RNA转染Aspc-1后导致其侵袭迁移能力增强;ESRP1过表达质粒转染Panc-1后导致其侵袭迁移能力减弱。ESRP1回复实验,可以部分逆转干扰mi R-23a表达后对胰腺癌细胞EMT的影响。6.qRT-PCR、WB检测证实mi R-23a靶向抑制ESRP1的表达,从而导致下游剪接分子CD44s/CD44v、FGFR2 IIIb/IIIc的表达变化,ESRP1回复实验可以部分逆转干扰mi R-23a表达对下游剪接分子表达的影响。结论:1.MiCroRNA-23a在胰腺癌淋巴结转移灶中的表达明显高于胰腺癌原发灶及癌旁正常胰腺组织,MiCroRNA-23a表达与肿瘤分化程度、肿瘤浸润深度以及胰腺癌淋巴结转移呈显著正相关,MiCroRNA-23a表达与胰腺癌预后具有显著相关性。2.上皮表型胰腺癌细胞低表达MiCro RNA-23a,间皮表型胰腺癌细胞高表达MiCroRNA-23a,当上皮表型的胰腺癌细胞发生EMT时,MiCroRNA-23a的表达显著增高,因此MiCroRNA-23a可能促进了胰腺癌细胞EMT。3.过表达MiCroRNA-23a能够促进胰腺癌细胞由上皮表型向间质表型转化;抑制MiCroRNA-23a表达能够促进胰腺癌细胞由间质表型向上皮表型转化。4.MiCroRNA-23a能够增强胰腺癌细胞体外培养细胞以及裸鼠体内的侵袭转移能力。5.MiCroRNA-23a通过靶向抑制ESRP1的表达对胰腺癌细胞上皮间质转化发挥调控作用,从而促进胰腺癌细胞侵袭转移。6.ESRP1通过调控CD44s/CD44v选择性剪接以及FGFR2IIIb/IIIc分子的表达变化影响胰腺癌细胞上皮间质转化。
[Abstract]:Objective: 1 to study the expression of MiCroRNA-23a in pancreatic carcinoma and its relationship with clinicopathological data.2 study on the expression of MiCroRNA-23a in different pancreatic cancer cell lines (Aspc-1, Bxpc-3, Cfpac-1, Panc-1).3 research MiCroRNA-23a in vivo and in vitro promoting the invasion and metastasis of pancreatic cancer cells in vitro,.4 MiCroRNA-23a through targeted inhibition of epithelium Splicing regulation protein 1 (epithelial splicing regulator protein 1, ESRP1) changes the expression of CD44 subtype and promotes epithelial mesenchymal transformation of pancreatic cancer cells (epithelial mesenchymal transition, EMT), and promotes invasion and metastasis of pancreatic cancer cells. Methods: 1. collection of pancreatic cancer markers in Department of hepatobiliary surgery, Southwest Hospital, January 2013 ~2013 year December. This and the patient's clinicopathological data were 47 cases. The FOS and OS. were recorded by RT-PCR method to detect the expression of MiCro RNA-23a in the primary pancreatic cancer, the metastasis lymph node tissue and the normal pancreatic tissue adjacent to the carcinoma. The difference of the expression of MiCroRNA-23a in the above three tissues was compared. The relative average of MiCro RNA-23a in the cancer tissue was calculated. According to this value, all the specimens were divided into MiCroRNA-23a high expression group and low expression group. The relationship between MiCroRNA-23a expression and clinicopathological data was analyzed. Kaplan-Meier software was used to carry out survival analysis, and the effects of MiCro RNA-23a on the prognosis of pancreatic cancer patients with different expression levels were detected by.2.qRT-PCR and Western blot to detect EMT marker E-. The expression of calcium mucin (E-cad), N- calcium mucin (N-cad) and vimentin (VIM), observed cell morphology, TGF- beta 1 induced EMT treatment, differentiating the epithelium of pancreatic cancer cell lines Aspc-1, Bxpc-3, Cfpac-1, Panc-1,.QRT-PCR detection of normal pancreatic duct epithelial cells (PDC) and pancreatic cancer cells (PDC) and pancreatic cancer cells. PAC-1, Panc-1) mi RNA-23a expression and the relationship between the expression of EMT and the EMT phenotype of pancreatic cancer cells. Interfering mi R-23a, and observing the effect of TGF- beta 1 on EMT induced by.3.miR-23a mimic and MI. To establish a stable cell line, the experimental observation of MI R-23a in nude mice to promote pancreatic cancer metastasis.4. gene chip analysis and bioinformatics website (TargetScan.com) search results cross comparison, double luciferase reporter plasmid experiment verifying that ESRP1 is the direct target gene.5. of MI R-23a to study the effect of dry disturbance miR-23a on ESRP1 expression, qRT-PCR detection of the pancreas The expression of ESRP1 in the carcinoma tissue was correlated with the expression of miR-23a, and the effect of ESRP1 on the invasion and metastasis of pancreatic cancer cells was observed, and ESRP1 recovery experiment was used to observe the reverse effect of MI R-23a on the EMT in pancreatic cancer cells.6.Western blot, qRT-PCR division and MI R-23a. The effect of the expression of MI R-23a on the expression of the downstream splicing molecules was disturbed by the expression of b/IIIc splice subtype. Results: 1. the expression of MI R-23a in the metastatic lymph nodes of pancreatic cancer was significantly higher than that of the primary pancreatic cancer and the normal tissue adjacent to the carcinoma. The MiCroRNA-23a table of the pancreatic cancer was associated with the degree of differentiation of the tumor tissue and the depth of the tumor (the depth of the tumor). There was a significant correlation between T staging and lymph node metastasis (N staging) of pancreatic cancer. The expression was not related to age, sex, tumor site and distant metastasis (M staging). Survival analysis showed that the expression of MiCroRNA-23a was related to the prognosis of pancreatic cancer patients. The prognosis of the high expression group was poor and the survival time was lower than that of the low expression group. The difference was statistically significant.2.1 Aspc-1, Bxpc-3 cells were epithelial phenotype, Cfpac-1, Panc-1 were interstitial phenotypes, TGF- beta 1 induced Aspc-1, Bxpc-3, and EMT.miR-23a expressed significantly in the pancreatic cancer cells with interstitial phenotypes. Mi R-23a in pancreatic cancer metastatic lymph node tissues increased, presumably that miR-23a may promote pancreas Epithelial mesenchymal transformation and invasion and metastasis of adenocarcinoma cells interfere with MI R-23a and inhibit the occurrence of EMT in epithelial pancreatic cancer cells induced by TGF- beta 1 in vitro..3. in vitro experiments show that MI R-23a mimic transfected Aspc-1 leads to the enhancement of its invasion and migration ability, MI R-23a inhibitor, which leads to the weakening of its invasion and migration ability. The transfection of NGEs Lentivirus Expression Vector to Panc-1 cells inhibits the function of miR-23a and inhibits the growth and metastasis of pancreatic cancer cells and.4. gene chip analysis and bioinformatics retrieval. It is proved that ESRP1 is a direct downstream target gene of miR-23a, and the luciferase reporter plasmid experiment confirms that ESRP1 is the direct target gene of MI R-23a to maintain the pancreas. The epithelial phenotype of adenocarcinoma cells interfered with MI R-23a and the expression of ESRP1. In pancreatic cancer tissue, the expression of ESRP1 and miR-23a expressed a significant negative correlation with.ESRP1si RNA transfection to Aspc-1, resulting in enhanced invasion and migration ability; ESRP1 overexpression plasmid transfected to Panc-1 led to its invasion and migration ability to weaken.ESRP1 response experiment. The effect of MI R-23a expression on the EMT of pancreatic cancer cells.6.qRT-PCR. WB detection confirmed that MI R-23a target inhibited the expression of ESRP1, which led to the expression of CD44s/CD44v and FGFR2 IIIb/IIIc in the downstream splicing molecules, and the ESRP1 response experiment could partly reverse the influence of the expression of the downstream splice molecules. The expression of roRNA-23a in the lymph node metastasis of pancreatic cancer was significantly higher than that of the primary pancreatic cancer and the normal pancreatic tissue near the carcinoma. The expression of MiCroRNA-23a was significantly correlated with the degree of tumor differentiation, the depth of tumor invasion and the lymph node metastasis of pancreatic cancer. The expression of MiCroRNA-23a was significantly correlated with the.2. epithelial phenotype of pancreatic cancer. The cells expressed low expression of MiCro RNA-23a, and the mesothelial phenotype of pancreatic cancer cells expressed MiCroRNA-23a. When the epithelial phenotype of pancreatic cancer cells was EMT, the expression of MiCroRNA-23a was significantly increased. Therefore, MiCroRNA-23a may promote the EMT.3. overexpression of pancreatic cancer cells to promote the transformation of pancreatic cancer cells from the epithelial phenotype to the interstitial phenotype. Inhibition of MiCroRNA-23a expression can promote the transformation of pancreatic cancer cells from the interstitial phenotype to the epithelial phenotype..4.MiCroRNA-23a can enhance the invasion and metastasis ability of pancreatic cancer cells in vitro and in nude mice..5.MiCroRNA-23a can play a regulatory role in the transformation of pancreatic carcinoma cells through the target inhibition of ESRP1 expression. The invasion and metastasis of pancreatic cancer cells.6.ESRP1 affects the epithelial mesenchymal transition of pancreatic cancer cells by regulating the selective splicing of CD44s/CD44v and the expression of FGFR2IIIb/IIIc molecules.
【学位授予单位】：第三军医大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.9

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