miR-409-3p在結(jié)直腸癌侵襲和轉(zhuǎn)移中的作用及分子機(jī)制
發(fā)布時(shí)間：2018-09-19 10:23
【摘要】：結(jié)直腸癌(Colorectal Cancer, CRC)是世界上第三大常見的癌癥,其發(fā)病率和死亡率均位居腫瘤譜前列。轉(zhuǎn)移是導(dǎo)致CRC患者死亡的主要原因,但其分子機(jī)制尚未完全闡明。miRNAs能夠通過轉(zhuǎn)錄后水平調(diào)控基因的表達(dá)而影響CRC的發(fā)生發(fā)展和轉(zhuǎn)移。因此,明確調(diào)控CRC轉(zhuǎn)移的關(guān)鍵miRNAs并闡明其作用機(jī)制,將有助于全面了解CRC轉(zhuǎn)移的全貌。本課題組前期工作和同行的研究報(bào)道表明,miR-409-3p是一個(gè)腫瘤轉(zhuǎn)移相關(guān)的miRNA,但其在CRC中的生物學(xué)功能及作用機(jī)制尚未明確。為此,本論文以探索闡明CRC轉(zhuǎn)移機(jī)制為研究目標(biāo),分析了miR-409-3p表達(dá)與CRC轉(zhuǎn)移的相關(guān)性,然后系統(tǒng)研究該miRNA在CRC轉(zhuǎn)移中的作用及其分子機(jī)制。 為明確miR-409-3p表達(dá)與CRC發(fā)生發(fā)展及轉(zhuǎn)移的相關(guān)性,本論文首先檢測(cè)了82對(duì)CRC臨床樣本中該miRNA的表達(dá)。結(jié)果顯示,miR-409-3p在CRC組織中的表達(dá)水平明顯低于相對(duì)應(yīng)的癌旁組織。進(jìn)一步的分析顯示,它的表達(dá)下調(diào)和CRC轉(zhuǎn)移發(fā)生具有負(fù)相關(guān)性。對(duì)CRC細(xì)胞系中miR-409-3p的檢測(cè)結(jié)果也顯示,該miRNA在轉(zhuǎn)移部位建株細(xì)胞系中的表達(dá)顯著低于原發(fā)部位建株細(xì)胞系。以上結(jié)果證明,miR-409-3p是一個(gè)CRC轉(zhuǎn)移相關(guān)的miRNA。 進(jìn)一步,本論文通過體、內(nèi)外實(shí)驗(yàn)研究明確miR-409-3p在CRC轉(zhuǎn)移過程中的生物學(xué)功能。細(xì)胞水平的實(shí)驗(yàn)研究顯示,該miRNA能夠抑制CRC細(xì)胞HCT116和RKO的遷移和侵襲,但不影響其增殖和克隆形成；動(dòng)物水平的實(shí)驗(yàn)研究發(fā)現(xiàn),該miRNA能夠抑制HCT116細(xì)胞在小鼠體內(nèi)肺部轉(zhuǎn)移灶的形成,但不影響細(xì)胞移植瘤的形成和生長。因此,我們認(rèn)為miR-409-3p能夠通過抑制CRC細(xì)胞的遷移和侵襲而抑制CRC轉(zhuǎn)移的發(fā)生,即miR-409-3p是一個(gè)特異性抑制CRC轉(zhuǎn)移的miRNA。 為了探索miR-409-3p抑制CRC轉(zhuǎn)移的分子機(jī)制,我們采用“基于特定生物學(xué)事件的靶基因篩選、鑒定和功能研究”策略,全面篩選并鑒定該miRNA調(diào)控的與轉(zhuǎn)移相關(guān)的靶基因。最終,我們篩選到9個(gè)可能與腫瘤轉(zhuǎn)移相關(guān)的miR-409-3p靶基因。通過雙熒光素酶報(bào)告基因分析,證明它在293T/17細(xì)胞中可以直接靶向結(jié)合調(diào)控GAB1、 NR4A、 LMO4的3'UTR區(qū)序列�？紤]到：1)293T/17細(xì)胞不同于CRC細(xì)胞內(nèi)的微環(huán)境可能導(dǎo)致靶基因的誤判；2) miRNA能夠通過結(jié)合非3'UTR區(qū)序列(如5'UTR和編碼區(qū)序列)而調(diào)控基因的表達(dá)。因此,本論文通過Western blot實(shí)驗(yàn)直接檢測(cè)了miR-409-3p對(duì)CRC細(xì)胞中所有候選靶基因以及已報(bào)道的該miRNA靶基因蛋白表達(dá)的影響。雖然由于抗體可獲性方面存在制約,但至少篩選并驗(yàn)證了GAB1是miR-409-3p在CRC細(xì)胞中靶向結(jié)合調(diào)控的靶基因。進(jìn)一步的細(xì)胞遷移和侵襲的功能性實(shí)驗(yàn)分析發(fā)現(xiàn),下調(diào)內(nèi)源性GAB1可以顯著抑制CRC細(xì)胞HCT116遷移和侵襲；同時(shí),靶基因功能回復(fù)實(shí)驗(yàn)結(jié)果顯示,回補(bǔ)GAB1可以將被miR-409-3p抑制的細(xì)胞遷移和侵襲能力回復(fù)到原來80%左右。最后,在八組配對(duì)的新鮮結(jié)直腸癌和癌旁組織中的miR-409-3p和GAB1表達(dá)水平檢測(cè)結(jié)果顯示,相對(duì)于癌旁組織,六例結(jié)直腸癌組織中的miR-409-3p表達(dá)水平顯著降低；而相對(duì)應(yīng)的GAB1的表達(dá)水平均有一定的升高。 綜上,本學(xué)位論文研究顯示,miR-409-3p在CRC中表達(dá)下調(diào),并且與CRC轉(zhuǎn)移發(fā)生具有負(fù)相關(guān)性；該miRNA通過下調(diào)CRC細(xì)胞的遷移和侵襲能力而抑制CRC轉(zhuǎn)移；它對(duì)GAB1的靶向表達(dá)下調(diào)可以部分解釋其抑制CRC細(xì)胞轉(zhuǎn)移的分子機(jī)制。因此,本論文的研究結(jié)果可望為發(fā)展基于miR-409-3p的轉(zhuǎn)移性CRC早期診斷和干預(yù)治療新方案提供新的切入點(diǎn)。
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.34
本文編號(hào)：2249861
[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.
【學(xué)位授予單位】：浙江大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.34
【參考文獻(xiàn)】
相關(guān)期刊論文 前3條
1 Jiao-Jiao Zhou;Shu Zheng;Li-Feng Sun;Lei Zheng;;MicroRNA regulation network in colorectal cancer metastasis[J];World Journal of Biological Chemistry;2014年03期
2 Yong-Bin Zheng;Hai-Ping Luo;Qiang Shi;Zhi-Nan Hao;Yu Ding;Qiu-Shuang Wang;Sheng-Bo Li;Gao-Chun Xiao;Shi-Lun Tong;;miR-132 inhibits colorectal cancer invasion and metastasis via directly targeting ZEB2[J];World Journal of Gastroenterology;2014年21期
3 Verena Stiegelbauer;Samantha Perakis;Alexander Deutsch;Hui Ling;Armin Gerger;Martin Pichler;;MicroRNAs as novel predictive biomarkers and therapeutic targets in colorectal cancer[J];World Journal of Gastroenterology;2014年33期
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