HMGCR对食管鳞癌细胞恶性行为的影响及分子机制研究

发布时间：2018-08-28 17:14

【摘要】：食管癌是我国最常见的消化道肿瘤之一。虽然随着外科、麻醉、围手术期处理以及重症监护条件和技术取得较大的提高,但是单纯手术治疗效果仍不理想。其原因在于食管癌发生和发展的分子机制目前仍不清楚,导致食管癌疗效欠佳。肿瘤细胞代谢异常是肿瘤细胞显著区别于正常细胞的显著特征之一。从肿瘤代谢的角度寻找食管癌的治疗靶点很可能为食管癌的治疗带来新的希望。甲羟戊酸(MVA,Mevalonate)途径除了终产物胆固醇外,还能产生许多中间代谢产物,如类异戊二烯,泛醌等。这些物质在细胞重要的生理过程中发挥作用。甲羟戊酸代谢途径在食管鳞癌发生中的功能目前仍不清楚。在本研究中,我们克隆了甲羟戊酸代谢限速酶3-羟基-3-甲基戊二酰辅酶A还原酶(3-Hydroxy-3-methylglutaryl coenzyme A reductase,HMGCR),并对其进行了功能和所涉机制的初步研究。在本研究中,我们收集食管鳞癌组织标本,培养食管鳞癌细胞系,利用荧光定量PCR检测食管鳞癌组织及其配对的正常组织中HMGCR的mRNA水平,利用蛋白印迹检测食管鳞癌组织和食管鳞癌细胞中HMGCR的蛋白水平。在细胞水平上,我们通过脂质体转染食管鳞癌细胞,建立过表达HMGCR的食管鳞癌稳定细胞株;设计RNA干扰序列,下调HMGCR在食管鳞癌细胞中的表达。我们利用MTT实验、Boyden Chamber和软琼脂集落实验分析HMGCR对食管鳞癌细胞生长、迁移和克隆形成的影响;利用GST-pull down和蛋白印迹检测HMGCR对Ras-ERK信号通路的活化作用。最后,我们在食管鳞癌细胞中过表达或下调c-Myc的表达,通过荧光定量PCR和蛋白印迹检测c-Myc的表达对HMGCR mRNA和蛋白水平的影响。我们的研究结果显示,与正常食管粘膜组织相比,HMGCR在食管鳞癌组织中的mRNA水平和蛋白水平均呈现上调趋势。HMGCR在食管鳞癌细胞株中的表达水平高于永生化的食管粘膜上皮细胞。在食管鳞癌细胞Eca109和正常食管粘膜细胞SHEE中过表达HMGCR促进食管鳞癌细胞生长、迁移和在软琼脂上克隆集落;在食管鳞癌细胞Eca109和Caes-17中下调HMGCR的表达抑制食管鳞癌细胞迁移和在软琼脂上克隆集落。在分子机制研究中,我们发现hmgcr过表达促进ras活化,上调erk磷酸化水平。在食管鳞癌细胞中,我们发现代谢的重要调控因子c-myc上调hmgcr的mrna水平和蛋白水平。综上所述,我们的研究结果提供了充分的证据,证明hmgcr在食管鳞癌发生发展过程中的重要功能。同时,我们的研究也提示hmgcr的抑制剂他汀用于食管鳞癌治疗的可能性。本研究可以分为如下三个部分:第一部分hmgcr在食管鳞癌组织和食管鳞癌细胞中表达上调目的:研究hmgcr在食管鳞癌组织及配对的正常组织、食管鳞癌细胞株中的表达模式。方法:首先利用荧光定量pcr(real-timepcr)检测hmgcr在40例食管鳞癌组织及配对的正常组织中的mrna水平;使用蛋白印迹法,检测随机挑选的6例食管鳞癌组织及配对的正常组织中hmgcr的蛋白水平,验证荧光定量pc结果;最后,利用蛋白印迹检测正常食管上皮细胞和食管鳞癌细胞中hmgcr的蛋白水平。结果:mrna水平的检测结果显示,食管鳞癌组织中hmgcr的mrna水平较配对的正常组织显著升高;蛋白印迹实验结果表明,食管鳞癌组织中hmgcr的蛋白水平较配对的正常组织显著上调,这一结果与荧光定量pcr的结果一致;最后,对多种食管鳞癌细胞和正常食管上皮细胞的蛋白印迹结果显示,hmgcr在正常食管上皮细胞中的表达水平较低,在食管鳞癌细胞中的表达水平较高。结论:在食管鳞癌组织和细胞系中,hmgcr的mrna水平和蛋白水平均有较强的表达。与正常组织相比较,食管鳞癌组织中hmgcr的表达水平上升;与正常食管上皮细胞相比,hmgcr在鳞癌细胞中的表达水平升高。这些结果提示hmgcr在食管鳞癌发生发展过程中很可能起着非常重要的作用。第二部分hmgcr对食管鳞癌细胞生长、迁移和克隆集落的影响目的:研究hmgcr对食管鳞癌细胞恶性行为(如生长、迁移、克隆集落等)的影响,揭示hmgcr在食管鳞癌进展中的功能。方法:利用脂质体转染hmgcr的表达载体(myc-hmgcr,带myc标签)至食管鳞癌细胞eca109和正常食管上皮细胞shee中;通过mtt法检测hmgcr对食管鳞癌细胞eca109和正常食管上皮细胞shee生长的影响;通过软琼脂集落法研究hmgcr对食管鳞癌细胞克隆形成能力的影响;借助于细胞迁移模型揭示hmgcr对食管鳞癌细胞eca109和正常食管上皮细胞shee迁移的影响。同时,制备hmgcrrna干扰的病毒载体,在食管鳞癌细胞eca109、caes17中下调hmgcr的表达;利用软琼脂集落法揭示下调hmgcr的表达对食管鳞癌细胞克隆形成的影响;借助细胞迁移模型研究hmgcr表达下调对食管鳞癌细胞迁移能力的影响结果:通过脂质体介导的稳定转染,在食管鳞癌细胞eca109和正常食管上皮细胞shee中建立了稳定表达细胞株,过表达带myc标签的hmgcr。mtt实验结果表明,稳定表达hmgcr促进食管鳞癌细胞eca109、正常食管上皮细胞shee的生长;细胞迁移实验显示,hmgcr在eca109和shee细胞中的表达增加食管鳞癌细胞的运动能力;软琼脂集落形成实验显示,hmgcr在eca109细胞中的表达促进其非锚定性生长。此外,我们构建了hmgcrrna干扰的慢病毒载体,非常有效地干扰hmgcr在食管鳞癌细胞eca109和caes17中的表达。细胞迁移实验表明,hmgcr表达下降削弱了食管鳞癌细胞eca109和caes17的运动能力;克隆集落形成实验显示,干扰hmgcr的表达使食管鳞癌细胞的非锚定性生长的能力受到抑制。结论:hmgcr促进食管鳞癌细胞的生长、迁移、克隆形成能力,干扰hmgcr的表达抑制食管鳞癌细胞非锚定性生长和迁移。hmgcr对食管鳞癌的进展发挥促进作用。第三部分hmgcr在食管鳞癌细胞中激活ras-erk信号通路目的:研究hmgcr调控食管鳞癌细胞恶性行为(如生长、迁移和克隆集落)的分子机制。方法:利用gst-pulldown平台,研究hmgcr的表达ras的活化作用及对erk磷酸化水平的影响;通过荧光定量pcr和蛋白印迹,检测c-myc基因的表达对hmgcr的mrna水平和蛋白水平的影响。结果:GST-pull down分析显示,过表达HMGCR促进Ras的活化,上调ERK的磷酸化水平。干扰HMGCR的表达抑制ERK的磷酸化。荧光定量PCR和蛋白印迹的实验结果显示,过表达c-Myc上调HMGCR的mRNA水平和蛋白水平,干扰cMyc的表达抑制HMGCR的mRNA水平和蛋白水平。结论:HMGCR在食管鳞癌细胞中激活Ras-ERK信号通路,代谢调控的关键分子c-Myc调控HMGCR的表达。
[Abstract]:Esophageal cancer is one of the most common gastrointestinal tumors in China. Although the surgical, anesthesia, perioperative management and intensive care conditions and techniques have been greatly improved, the effect of surgical treatment alone is still unsatisfactory. Abnormal metabolism of tumor cells is one of the significant characteristics that distinguish tumor cells from normal cells. Looking for therapeutic targets for esophageal cancer from the perspective of tumor metabolism may bring new hope for the treatment of esophageal cancer. Diene, ubiquinone and so on. These substances play an important role in the physiological process of cells. The function of mevalonate pathway in esophageal squamous cell carcinogenesis is still unclear. In this study, we cloned the mevalonate metabolic rate-limiting enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (3-Hydroxy-3-methylglutaryl coenzyme A reductase) In this study, we collected esophageal squamous cell carcinoma tissue samples, cultured esophageal squamous cell carcinoma cell lines, detected the mRNA level of HMGCR in esophageal squamous cell carcinoma tissues and their matched normal tissues by fluorescence quantitative PCR, and detected esophageal squamous cell carcinoma tissues and esophageal squamous cell carcinoma fine by Western blot. Protein level of HMGCR in esophageal squamous cell carcinoma cells. At the cellular level, we established a stable cell line of esophageal squamous cell carcinoma overexpressing HMGCR by liposome transfection. RNA interference sequence was designed to down-regulate the expression of HMGCR in esophageal squamous cell carcinoma cells. The effect of HMGCR on Ras-ERK signaling pathway was detected by GST-pull down and Western blotting. Finally, we overexpressed or down-regulated the expression of c-Myc in esophageal squamous cell carcinoma cells, and detected the effect of c-Myc expression on HMGCR mRNA and protein levels by fluorescence quantitative PCR and Western blotting. The results showed that HMGCR was up-regulated in both mRNA and protein levels in esophageal squamous cell carcinoma compared with normal esophageal mucosa. The expression level of HMGCR in esophageal squamous cell carcinoma cell lines was higher than that in immortalized esophageal epithelial cells. Promote the growth, migration and cloning of esophageal squamous cell carcinoma cells on soft agar; down-regulate the expression of HMGCR in esophageal squamous cell carcinoma cells Eca109 and Caes-17, inhibit the migration of esophageal squamous cell carcinoma cells and clone colonies on soft agar. In the study of molecular mechanism, we found that overexpression of HMGCR promoted Ras activation and up-regulated ERK phosphorylation. In conclusion, our findings provide sufficient evidence to support the important role of HMGCR in the development of esophageal squamous cell carcinoma. Our findings also suggest that statin, an inhibitor of hmgcr, may be useful in the treatment of esophageal squamous cell carcinoma. This study can be divided into the following three parts: the first part is the up-regulation of HMGCR expression in esophageal squamous cell carcinoma and esophageal squamous cell carcinoma. The protein levels of HMGCR in 6 randomly selected esophageal squamous cell carcinoma tissues and matched normal tissues were detected by Western blot to verify the results of quantitative fluorescence PC. Finally, the protein levels of HMGCR in normal esophageal epithelial cells and esophageal squamous cell carcinoma cells were detected by Western blot. The results of Western blotting showed that the protein level of HMGCR in esophageal squamous cell carcinoma was significantly higher than that in matched normal tissues, which was consistent with the results of fluorescence quantitative pcr. Western blot analysis showed that the expression of HMGCR was lower in normal esophageal epithelial cells and higher in esophageal squamous cell carcinoma cells. Conclusion: The expression of HMGCR in esophageal squamous cell carcinoma tissue and cell line was higher than that in normal esophageal tissue. These results suggest that the expression of HMGCR may play an important role in the development of esophageal squamous cell carcinoma. Part two: The effects of HMGCR on the growth, migration and cloning of esophageal squamous cell carcinoma cells. The effect of Cr on malignant behavior of esophageal squamous cell carcinoma cells (e.g. growth, migration, colony cloning, etc.) and the function of HMGCR in the progression of esophageal squamous cell carcinoma were revealed. The effects of HMGCR on the growth of esophageal epithelial cells ECA 109 and normal esophageal epithelial cells shee; the effects of HMGCR on the clonal formation of esophageal squamous cell carcinoma cells were studied by soft agar colony assay; the effects of HMGCR on the migration of esophageal squamous cell carcinoma cells ECA 109 and normal esophageal epithelial cells shee were revealed by cell migration model. The expression of HMGCR was down-regulated in esophageal squamous cell carcinoma cells Eca109 and caes17; the effect of down-regulated expression of HMGCR on the formation of esophageal squamous cell carcinoma cell clones was revealed by soft agar colony assay; the effect of down-regulated expression of HMGCR on the migration ability of esophageal squamous cell carcinoma cells was studied by cell migration model; the results showed that stable transfection mediated by liposome could induce fine esophageal squamous cell carcinoma cells. A stable expression cell line was established in Eca109 cells and normal esophageal epithelial cells shee. over-expression of myc-tagged hmgcr. MTT assay showed that stable expression of HMGCR promoted the growth of esophageal squamous cell carcinoma cell Eca109 and normal esophageal epithelial cells shee. cell migration assay showed that the expression of HMGCR in Eca109 and shee cells increased the fineness of esophageal squamous cell carcinoma. In addition, we constructed a lentiviral vector interfered by hmgcrrna, which was very effective in interfering with the expression of HMGCR in esophageal squamous cell carcinoma cells ECA 109 and caes17. Cell migration assay showed that the decrease of HMGCR expression weakened the expression of caes17 and ECA 109 cells. The motility of esophageal squamous cell carcinoma cells Eca109 and caes17 was inhibited by interfering with the expression of hmgcr. conclusion: HMGCR can promote the growth, migration and clonal formation of esophageal squamous cell carcinoma cells, and interfere with the expression of HMGCR can inhibit the Non-anchoring growth and Non-anchoring growth of esophageal squamous cell carcinoma cells. The third part is the activation of ras-erk signaling pathway by HMGCR in esophageal squamous cell carcinoma cells. Objective: To study the molecular mechanism of HMGCR in regulating malignant behavior of esophageal squamous cell carcinoma cells (such as growth, migration and clonal colony). Results: GST-pull down analysis showed that over-expression of HMGCR promoted the activation of Ras and up-regulated the phosphorylation level of ERK. Interference of HMGCR expression inhibited the phosphorylation of ERK. The results of Western blotting showed that overexpression of c-Myc up-regulated the mRNA and protein levels of HMGCR, interfered with the expression of cMyc and inhibited the mRNA and protein levels of HMGCR. Conclusion: HMGCR activates Ras-ERK signaling pathway in esophageal squamous cell carcinoma cells, and c-Myc, the key molecule of metabolic regulation, regulated the expression of HMGCR.
【学位授予单位】：上海交通大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.1

【相似文献】