MicroRNA-652-3p通过抑制Lgl1的表达促进非小细胞肺癌增殖、侵袭、转移

发布时间：2018-11-16 12:45

【摘要】：肺癌是世界上发病率、死亡率最高的恶性肿瘤,虽然在过去几十年中,肺癌在诊断、治疗、发病机制等方面都取得了较大的进步,但总生存率仍然没有明显改善,目前非小细胞肺癌总体5年生存率为17.1%,而对于那些Ⅳ期患者,5年生存率仅为3.6%。因此,深入探讨肺癌的发病机制,寻找新的治疗靶点,对肺癌疾病的诊疗具有重要意义。microRNA是长度为19-24个核苷酸的单链非编码小RNA,其在生物进化过程中表现出高度保守的特性,其主要作用机制是与靶基因mRNA的3'非翻译区(3'UTR)的碱基互补结合,引起靶基因mRNA降解或抑制靶基因翻译。目前发现microRNA也可以通过与靶基因的启动子元件结合而促进其转录,这种现象被称为RNA活化。MicroRNAs可以作为癌基因或抑癌基因,在多种恶性肿瘤的发生发展过程中起重要作用。在非小细胞肺癌中已被证实有多种microRNAs的表达发生变化,并参与肿瘤的形成与进展。我们的前期研究发现miR-652-3p在非小细胞肺癌患者血清中显著升高,可以作为一个新的肺癌辅助诊断血液分子标志物。本研究中,我们检测并发现,与远癌正常肺组织相比,miR-652-3p在非小细胞肺癌组织及细胞系中表达水平显著升高；而且miR-652-3p的高表达与肺癌患者淋巴结转移及TNM分期显著相关,并且miR-652-3p高表达的肺癌患者术后生存时间显著短于低表达患者。细胞实验证实,转染nim-652-3p上调非小细胞肺癌细胞中miR-652-3p的表达水平,促进了细胞的增殖、迁移和侵袭能力,抑制了细胞凋亡的发生；转染/mti-652降低非小细胞肺癌细胞miR-652-3p的表达水平,则抑制了细胞增殖、迁移和侵袭能力,促进了细胞凋亡。在非小细胞肺癌组织和细胞中,miR-652-3p的水平都与Lgl1蛋白水平呈负相关；并且在非小细胞肺癌细胞中miR-652-3p明显抑制了Lgl1蛋白的表达水平,提示LLGL1是miR-652-3p的下游靶基因之一。进一步的荧光素酶报告基因实验表明,在转染报告基因pMIR-WT(包含野生型LLGL1 mRNA 3'UTR序列)的细胞中,上调或降低miR-652-3p表达能有效抑制或促进荧光素酶的表达和活性；miR-652-3p结合位点缺失突变的3'UTR序列则有效消除了miR-652-3p对荧光素酶表达的调节。这一结果证明,在非小细胞肺癌中,LLGL1是niR-652-3p的下游靶基因之一。进一步在非小细胞肺癌细胞中转染不含3'UTR序列的Lgl1表达载体,回复miR-652-3p过表达细胞中Lgl1蛋白的表达,有效阻碍了niR-652-3p促进细胞迁移和侵袭能力的活性。这一结果进一步表明,miR-652-3p促进非小细胞肺癌细胞迁移和侵袭能力的活性至少一部分是通过对Lgl1表达的调节来实现的。综上所述,本研究结果证实,miR-652-3p在非小细胞肺癌组织和细胞系中存在普遍高表达现象,miR-652-3p通过抑制Lgl1蛋白表达发挥其促进肿瘤生长、侵袭和迁移的作用。因此,miR-652-3p在非小细胞肺癌中发挥癌基因的作用,并有可能成为非小细胞肺癌治疗的新靶点。
[Abstract]:Lung cancer is a malignant tumor with the highest morbidity and mortality in the world. Although lung cancer has made great progress in diagnosis, treatment and pathogenesis in the past few decades, the overall survival rate has not been significantly improved. The overall 5-year survival rate was 17. 1% for NSCLC patients, but only 3. 6% for those patients with stage IV lung cancer. Therefore, it is important for the diagnosis and treatment of lung cancer to explore the pathogenesis of lung cancer and find new therapeutic targets. MicroRNA is a single strand non-coding small RNA, with a length of 19-24 nucleotides. It has a highly conserved nature in the course of biological evolution, and its main mechanism is that it is complementary to the 3 'untranslated region (3'UTR) of target gene mRNA, resulting in the degradation of target gene mRNA or inhibition of target gene translation. At present, it has been found that microRNA can also promote transcription by binding to the promoter element of target gene. This phenomenon is called RNA activation. MicroRNAs can be used as oncogene or tumor suppressor gene, and it plays an important role in the occurrence and development of many kinds of malignant tumors. It has been proved that many kinds of microRNAs expression changes in non-small cell lung cancer and participate in the formation and progression of tumor. Our previous study found that miR-652-3p was significantly elevated in the serum of patients with NSCLC, and could be used as a new molecular marker for the auxiliary diagnosis of lung cancer. In this study, we detected and found that the expression of miR-652-3p in non-small cell lung cancer (NSCLC) tissues and cell lines was significantly higher than that in normal lung tissues of distant carcinoma. Moreover, the high expression of miR-652-3p was significantly correlated with lymph node metastasis and TNM stage of lung cancer patients, and the survival time of lung cancer patients with high expression of miR-652-3p was significantly shorter than that of patients with low expression of miR-652-3p. Cell experiments showed that transfection of nim-652-3p upregulated the expression of miR-652-3p in NSCLC cells, promoted cell proliferation, migration and invasion, and inhibited apoptosis. Transfection / mti-652 decreased the expression of miR-652-3p in NSCLC cells, inhibited cell proliferation, migration and invasion, and promoted cell apoptosis. In non-small cell lung cancer (NSCLC), the level of miR-652-3p was negatively correlated with the level of Lgl1 protein. Moreover, miR-652-3p significantly inhibited the expression of Lgl1 protein in NSCLC cells, suggesting that LLGL1 is one of the downstream target genes of miR-652-3p. Further experiments of luciferase reporter gene showed that up-regulation or down-regulation of miR-652-3p expression could effectively inhibit or promote luciferase expression and activity in cells transfected with reporter gene pMIR-WT (including wild-type LLGL1 mRNA 3'UTR sequence). The 3'UTR sequence with deletion mutation of miR-652-3p binding site effectively eliminated the regulation of luciferase expression by miR-652-3p. These results suggest that LLGL1 is one of the downstream target genes of niR-652-3p in non-small cell lung cancer. Further transfection of Lgl1 expression vector without 3'UTR sequence in non-small cell lung cancer cells and reversion of the expression of Lgl1 protein in miR-652-3p overexpression cells effectively hindered the activity of niR-652-3p in promoting cell migration and invasion. These results further suggest that the activity of miR-652-3p in promoting the migration and invasion of NSCLC cells is at least partly mediated by the regulation of Lgl1 expression. In conclusion, the results of this study confirm that miR-652-3p is generally overexpression in non-small cell lung cancer tissues and cell lines, and miR-652-3p plays a role in promoting tumor growth, invasion and migration by inhibiting the expression of Lgl1 protein. Therefore, miR-652-3p plays the role of oncogene in non-small cell lung cancer and may become a new target for the treatment of non-small cell lung cancer.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R734.2

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