肥胖脂肪组织分泌miR-27a通过靶向FOXO1促进肝癌的发生发展

发布时间：2018-09-13 16:02

【摘要】：背景：大量流行病学研究表明肥胖与各种癌症的发生和发展关系密切，肥胖通常会导致肝脏疾病，如非酒精性脂肪性肝病（NAFLD）和更严重的非酒精性脂肪型肝炎（NASH），进而导致肝脏发生脂肪性炎症，亦可导致肝纤维化，从而诱发肝硬化，而后者是公认的发生肝癌的高危因素之一。有研究报道在肥胖小鼠动物模型中，无论是饮食或遗传性肥胖都是强烈的诱导肝癌发生的因素，大量的研究表明肥胖可能通过诱导促癌细胞因子IL-6和TNF的产生进而促进肝脏炎症，并通过激活致癌基因转录因子STAT等途径促进肝癌发生和发展。microRNA(miRNA)是近年来新发现的一类非编码单链小分子RNA，在转录后水平调控多基因的表达，是基因调控网络中的关键成分，参与调节细胞代谢、增殖、凋亡、分化和发育，其表达异常会导致肿瘤等重大疾病。多项研究证据表明miRNA在肝癌的进展中发挥着重要作用，并且直接参与肝癌的分化、凋亡、侵袭和远处转移的过程。基于此，本论文立足于miR-27a，早先对其的研究发现miR-27a在脂肪细胞的分化中起关键作用，而最近研究表明miR-27a在乳腺癌、肺癌、胰腺癌等多种恶性肿瘤的发生和发展中扮演着癌基因的角色，但是miR-27a在肥胖诱导的肝癌中的作用和机制尚不清楚。本文重点研究miR-27a对肥胖人群肝癌发生发展的作用，并探讨其促进肝癌细胞增殖的分子机制，为肥胖肝癌的诊断和治疗提供新靶点。目的：探讨脂肪组织分泌的miR-27a在促进原发性肝癌发生和介导肥胖相关恶性肿瘤发生的机制，并为肿瘤的生长并干预肿瘤的形成和进展提供新的思路。方法：首先应用Real-time PCR法检测肥胖原发性肝癌和非肥胖原发性肝癌，健康人群对照组血清中miR-27a的表达水平，和肥胖原发性肝癌，非肥胖原发性肝癌患者肝癌组织，癌旁肝组织，以及大网膜组织中miR-27a的表达水平。采用免疫组织化学染色方法测定临床组织样本中FOXO1，P21，P27，CyclinD1的表达情况。通过构建过表达miR-27a的3T3-L1脂肪细胞，并与肝癌细胞株HepG2分组共培养，随后检测各组肝癌细胞增殖活性和细胞周期，，并通过RT-PCR法和western blot法测定FOXO1，P21，P27，CyclinD1的mRNA和蛋白表达情况。结果：所有原发性肝癌患者的肿瘤组织miR-27a水平较癌旁组织明显升高；肥胖肝癌患者的肿瘤组织miR-27a表达水平是非肥胖肝癌患者2倍，这表明miR-27a上调可能可以作为肥胖相关肝癌的生物学诊断标记物；相比非肥胖肝癌患者和健康人群，肥胖肝癌患者血清miR-27a水平明显升高；肥胖肝癌患者体内大网膜脂肪组织中的miR-27a水平，约为非肥胖肝癌患者和健康人群5倍。miR-27a高表达的肝癌组织中FOXO1，P21和P27表达水平显著降低，CyclinD1表达水平显著升高。而miR-27a低表达的癌旁组织中FOXO1，P21和P27表达水平显著升高，而Cyclin D1的表达水平显著降低。3T3-L1脂肪细胞可通过旁分泌miR-27a的降低肝癌细胞FOXO1，P21，P27的mRNA和蛋白表达，并增加Cyclin D1的mRNA和蛋白表达，从而促进细胞周期G1/S转换，诱导肝癌细胞HepG2增殖。结论：脂肪组织分泌的miR-27a通过下调转录因子FOXO1，降低细胞周期抑制蛋白P21和P27的表达，增加细胞周期调节蛋白CyclinD1表达，从而促进G1/S期的细胞周期转换，最终促进肝癌细胞的增殖。
[Abstract]:Background:
A large number of epidemiological studies have shown that obesity is closely related to the occurrence and development of various cancers. Obesity usually leads to liver diseases, such as non-alcoholic fatty liver disease (NAFLD) and more severe non-alcoholic fatty hepatitis (NASH), which in turn leads to fatty inflammation in the liver and liver fibrosis, leading to cirrhosis of the liver. It has been reported that both diet and hereditary obesity are strong factors inducing hepatocarcinogenesis in obese mice. A large number of studies have shown that obesity may promote hepatic inflammation by inducing the production of tumor-promoting cytokines IL-6 and TNF, and by activating carcinogenic bases. MicroRNA (microRNA) is a newly discovered non-coding single-stranded small molecule RNA, which regulates the expression of multiple genes at the post-transcriptional level. It is a key component of gene regulatory network. It is involved in regulating cell metabolism, proliferation, apoptosis, differentiation and development. Abnormal expression of microRNA can lead to cancer. Several studies have shown that microRNAs play an important role in the development of hepatocellular carcinoma and are directly involved in the differentiation, apoptosis, invasion and distant metastasis of hepatocellular carcinoma. 7A plays an oncogene role in the occurrence and development of breast cancer, lung cancer, pancreatic cancer and other malignant tumors, but the role and mechanism of microarray-27a in obesity-induced liver cancer is still unclear. The diagnosis and treatment of fat liver cancer provide a new target.
Objective:
Objective To explore the mechanism of adipose tissue secreted microarray-27a in promoting the occurrence of primary hepatocellular carcinoma (PHC) and mediating the occurrence of obesity-related malignant tumors, and to provide new ideas for tumor growth and intervention of tumor formation and progression.
Firstly, the expression of microRNA-27a in the serum of obese and non-obese primary hepatocellular carcinoma, healthy control group, and obese primary hepatocellular carcinoma, non-obese primary hepatocellular carcinoma tissues, adjacent liver tissues and omentum tissues were detected by Real-time PCR. Immunohistochemical staining was used. Methods The expression of FOXO1, P21, P27 and CyclinD1 in clinical tissue samples was determined. The 3T3-L1 adipocytes overexpressing microarray-27a were constructed and co-cultured with hepatoma cell line HepG2. The proliferation activity and cell cycle of hepatoma cells were detected. The mRNA and eggs of FOXO1, P21, P27, CyclinD1 were detected by RT-PCR and Western blot. White expression.
Result:
The levels of microRNA-27a in tumor tissues of all patients with primary hepatocellular carcinoma were significantly higher than those of adjacent tissues; the levels of microRNA-27a in tumor tissues of obese patients with hepatocellular carcinoma were twice as high as those of non-obese patients with hepatocellular carcinoma, suggesting that the up-regulation of microRNA-27a might serve as a biomarker for biological diagnosis of obesity-related hepatocellular carcinoma; compared with non-obese patients with non-obe The levels of serum microRNA-27a in obese hepatocellular carcinoma patients were significantly higher than those in non-obese hepatocellular carcinoma patients and healthy controls. The levels of FOXO1, P21 and P27 in liver cancer tissues with high expression of microRNA-27a were significantly lower than those in non-obese hepatocellular carcinoma patients and healthy controls. The levels of CyclinD1 were significantly higher in hepatocellular carcinoma tissues with low expression of microRNA-27a. The expression levels of FOXO1, P21 and P27 were significantly increased, while the expression level of Cyclin D1 was significantly decreased. 3T3-L1 adipocytes could decrease the mRNA and protein expression of FOXO1, P21 and P27, and increase the mRNA and protein expression of Cyclin D1, thereby promoting G1/S transition of cell cycle and inducing the proliferation of HepG2.
Conclusion:
Mi-27a secreted by adipose tissue can promote the cell cycle transition in G1/S phase and ultimately promote the proliferation of hepatocellular carcinoma cells by down-regulating the transcription factor FOXO1, decreasing the expression of cyclin-inhibitory proteins P21 and P27, and increasing the expression of cyclin-regulated proteins Cyclin D1.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.7

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