miR-409-3p在结直肠癌侵袭和转移中的作用及分子机制

发布时间：2018-09-19 10:23

【摘要】：结直肠癌(Colorectal Cancer, CRC)是世界上第三大常见的癌症,其发病率和死亡率均位居肿瘤谱前列。转移是导致CRC患者死亡的主要原因,但其分子机制尚未完全阐明。miRNAs能够通过转录后水平调控基因的表达而影响CRC的发生发展和转移。因此,明确调控CRC转移的关键miRNAs并阐明其作用机制,将有助于全面了解CRC转移的全貌。本课题组前期工作和同行的研究报道表明,miR-409-3p是一个肿瘤转移相关的miRNA,但其在CRC中的生物学功能及作用机制尚未明确。为此,本论文以探索阐明CRC转移机制为研究目标,分析了miR-409-3p表达与CRC转移的相关性,然后系统研究该miRNA在CRC转移中的作用及其分子机制。为明确miR-409-3p表达与CRC发生发展及转移的相关性,本论文首先检测了82对CRC临床样本中该miRNA的表达。结果显示,miR-409-3p在CRC组织中的表达水平明显低于相对应的癌旁组织。进一步的分析显示,它的表达下调和CRC转移发生具有负相关性。对CRC细胞系中miR-409-3p的检测结果也显示,该miRNA在转移部位建株细胞系中的表达显著低于原发部位建株细胞系。以上结果证明,miR-409-3p是一个CRC转移相关的miRNA。进一步,本论文通过体、内外实验研究明确miR-409-3p在CRC转移过程中的生物学功能。细胞水平的实验研究显示,该miRNA能够抑制CRC细胞HCT116和RKO的迁移和侵袭,但不影响其增殖和克隆形成；动物水平的实验研究发现,该miRNA能够抑制HCT116细胞在小鼠体内肺部转移灶的形成,但不影响细胞移植瘤的形成和生长。因此,我们认为miR-409-3p能够通过抑制CRC细胞的迁移和侵袭而抑制CRC转移的发生,即miR-409-3p是一个特异性抑制CRC转移的miRNA。为了探索miR-409-3p抑制CRC转移的分子机制,我们采用“基于特定生物学事件的靶基因筛选、鉴定和功能研究”策略,全面筛选并鉴定该miRNA调控的与转移相关的靶基因。最终,我们筛选到9个可能与肿瘤转移相关的miR-409-3p靶基因。通过双荧光素酶报告基因分析,证明它在293T/17细胞中可以直接靶向结合调控GAB1、 NR4A、 LMO4的3'UTR区序列。考虑到：1)293T/17细胞不同于CRC细胞内的微环境可能导致靶基因的误判；2) miRNA能够通过结合非3'UTR区序列(如5'UTR和编码区序列)而调控基因的表达。因此,本论文通过Western blot实验直接检测了miR-409-3p对CRC细胞中所有候选靶基因以及已报道的该miRNA靶基因蛋白表达的影响。虽然由于抗体可获性方面存在制约,但至少筛选并验证了GAB1是miR-409-3p在CRC细胞中靶向结合调控的靶基因。进一步的细胞迁移和侵袭的功能性实验分析发现,下调内源性GAB1可以显著抑制CRC细胞HCT116迁移和侵袭；同时,靶基因功能回复实验结果显示,回补GAB1可以将被miR-409-3p抑制的细胞迁移和侵袭能力回复到原来80%左右。最后,在八组配对的新鲜结直肠癌和癌旁组织中的miR-409-3p和GAB1表达水平检测结果显示,相对于癌旁组织,六例结直肠癌组织中的miR-409-3p表达水平显著降低；而相对应的GAB1的表达水平均有一定的升高。综上,本学位论文研究显示,miR-409-3p在CRC中表达下调,并且与CRC转移发生具有负相关性；该miRNA通过下调CRC细胞的迁移和侵袭能力而抑制CRC转移；它对GAB1的靶向表达下调可以部分解释其抑制CRC细胞转移的分子机制。因此,本论文的研究结果可望为发展基于miR-409-3p的转移性CRC早期诊断和干预治疗新方案提供新的切入点。
[Abstract]:Colorectal Cancer (CRC) is the third most common cancer in the world. Its morbidity and mortality are in the forefront of the cancer spectrum. Metastasis is the main cause of death in CRC patients, but its molecular mechanism has not been fully elucidated. Therefore, to clarify the key microRNAs that regulate CRC metastasis and elucidate their mechanisms will be helpful to understand the overall picture of CRC metastasis. Previous work and peer reports of our group indicate that microRNAs-409-3p are Tumor Metastasis-related microRNAs, but their biological functions and mechanisms in CRC are not yet clear. To clarify the mechanism of CRC metastasis, we analyzed the correlation between the expression of microRNA-409-3p and CRC metastasis, and then studied the role of microRNA in CRC metastasis and its molecular mechanism.
In order to clarify the correlation between the expression of microRNAs-409-3p and the development and metastasis of CRC, the expression of microRNAs in 82 clinical specimens of CRC was detected. The detection of microRNA-409-3p in CRC cell lines also showed that the expression of microRNA in metastatic sites was significantly lower than that in primary sites.
Further, in vivo, in vitro and in vivo studies have demonstrated the biological function of microRNAs-409-3p in CRC metastasis. Cell-level experiments have shown that the microRNAs can inhibit the migration and invasion of HCT116 and RKO in CRC cells, but do not affect their proliferation and cloning formation; animal-level experiments have found that the microRNAs can inhibit the fine HCT116. Therefore, we believe that microRNA-409-3p can inhibit CRC metastasis by inhibiting the migration and invasion of CRC cells, that is, microRNA-409-3p is a microRNA that specifically inhibits CRC metastasis.
To explore the molecular mechanism of microRNAs-409-3p inhibiting CRC metastasis, we used the strategy of "target gene screening, identification and functional research based on specific biological events" to screen and identify the metastasis-related target genes regulated by the microRNAs. Photoenzyme reporter gene analysis showed that it can directly bind and regulate the 3'UTR region sequences of GAB1, NR4A and LMO4 in 293T/17 cells. Therefore, in this study, we directly detected the effects of microRNAs-409-3p on the expression of all candidate target genes in CRC cells and the reported expression of the target gene of microRNAs by Western blot assay. Further functional analysis of cell migration and invasion revealed that down-regulation of endogenous GAB1 significantly inhibited HCT116 migration and invasion in CRC cells. Meanwhile, functional recovery of target genes showed that replenishing GAB1 could restore the migration and invasiveness of cells inhibited by Mi-409-3p to about 80%. The expression levels of microRNA409-3p and GAB1 in the matched fresh colorectal cancer and adjacent tissues showed that the expression levels of microRNA409-3p in six colorectal cancer tissues were significantly lower than those in adjacent tissues, while the corresponding expression levels of GAB1 were increased.
In conclusion, this dissertation shows that the expression of microRNAs-409-3p is down-regulated in CRC and is negatively correlated with CRC metastasis; the microRNAs inhibit CRC metastasis by down-regulating the migration and invasion of CRC cells; and the down-regulation of its targeted expression of GAB1 may partly explain the molecular mechanism of inhibiting CRC metastasis. The results are expected to provide a new entry point for the development of new protocols for early diagnosis and intervention of metastatic CRC based on microarray-409-3p.
【学位授予单位】：浙江大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.34

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