FAM172A在大腸癌轉(zhuǎn)移的作用機(jī)制研究
發(fā)布時間：2019-04-04 08:25
【摘要】：大腸癌(Colorectal Cancer,CRC)作為常見的人類惡性腫瘤,全世界每年大約120萬新發(fā)病例,同時至少60萬患者死亡。轉(zhuǎn)移是影響大腸癌患者預(yù)后的重要原因,大多數(shù)伴有轉(zhuǎn)移的大腸癌患者不適合傳統(tǒng)治療方式。大腸癌轉(zhuǎn)移主要與個體基因突變有關(guān),大腸癌中基因突變不超過50%,故可認(rèn)定大腸癌的轉(zhuǎn)移尚有其它新的抑癌基因或癌基因在起作用。FAM172A是本課題組前期研究發(fā)現(xiàn)的一個新基因,我們最近的研究結(jié)果證實:1)FAM172A在CRC相關(guān)組織中表達(dá)下調(diào),且顯著抑制人大腸癌LoVo細(xì)胞增殖和促進(jìn)凋亡;2)轉(zhuǎn)錄因子STAT1在轉(zhuǎn)錄水平調(diào)控FAM172A表達(dá),增強(qiáng)后者功能。綜上所述,我們推測:FAM172A基因是一個典型的抑癌基因,進(jìn)一步研究其轉(zhuǎn)錄調(diào)控相關(guān)機(jī)制具有重要意義�；虮磉_(dá)調(diào)控除發(fā)生在轉(zhuǎn)錄水平,轉(zhuǎn)錄后調(diào)控也是關(guān)鍵環(huán)節(jié),miRNAs是近年來發(fā)現(xiàn)參與轉(zhuǎn)錄后水平調(diào)控的重要因子。miRNAs是一種由內(nèi)源基因編碼的、長度大概為22個核苷酸的非編碼單鏈小RNA分子。研究證實miRNA異常表達(dá)與大腸癌轉(zhuǎn)移相關(guān),如miR-21在大腸癌細(xì)胞中表達(dá)明顯增高,并通過抑制PDCD4表達(dá)促進(jìn)腫瘤細(xì)胞的侵襲。我們前期工作中利用生物信息學(xué)預(yù)測發(fā)現(xiàn)miR-27a可能靶向結(jié)合FAM172A,故我們推測FAM172A功能可能在轉(zhuǎn)錄后水平受到miR-27a的調(diào)控。本課題擬在前期工作基礎(chǔ)上,在轉(zhuǎn)錄后水平進(jìn)一步闡明FAM172A抑制大腸癌侵襲轉(zhuǎn)移的調(diào)控機(jī)制,為大腸癌的基因治療提供科學(xué)依據(jù)和實驗基礎(chǔ)。目的:1、明確FAM172A對大腸癌細(xì)胞遷移和侵襲能力的影響;2、生物信息學(xué)篩選和驗證調(diào)控FAM172A的重要miRNA;3、明確大腸癌組織及細(xì)胞株中miR-27a表達(dá)水平及其相關(guān)臨床意義;4、探討miR-27a靶向FAM172A對大腸癌細(xì)胞遷移和侵襲的影響及其機(jī)制;方法:1、構(gòu)建FAM172A重組質(zhì)粒,通過細(xì)胞劃痕及Transwell侵襲實驗分別觀察FAM172A對LoVo細(xì)胞遷移和侵襲的影響;2、生物信息學(xué)篩選靶向FAM172A的重要miRNA,Luciferase報告基因技術(shù)驗證兩者結(jié)合關(guān)系;3、RT-qPCR檢測大腸癌細(xì)胞株及配對組織標(biāo)本中miR-27a表達(dá)水平。4、構(gòu)建穩(wěn)定過表達(dá)miR-27a的LoVo細(xì)胞株;細(xì)胞劃痕及Transwell明確miR-27a調(diào)控FAM172A對大腸細(xì)胞遷移和侵襲影響;western blot檢測miR-27a調(diào)控FAM172A對侵襲轉(zhuǎn)移相關(guān)重要信號通路分子的表達(dá)影響;5、數(shù)據(jù)統(tǒng)計采用SPSS 22.0軟件分析,以均數(shù)±標(biāo)準(zhǔn)誤表示。計量資料組間比較采用兩獨(dú)立樣本t檢驗,多組間比較采用方差分析。P0.05為有統(tǒng)計學(xué)差異。所有實驗均獨(dú)立進(jìn)行三次,取平均值進(jìn)行統(tǒng)計分析。結(jié)果:1、過表達(dá)FAM172A減弱了LoVo細(xì)胞運(yùn)動速度,引起LoVo細(xì)胞侵襲數(shù)量的減少,干擾FAM172A表達(dá)則加快了LoVo細(xì)胞運(yùn)動速度,增加了其侵襲數(shù)量;2、FAM172A 3'UTR存在miR-27a結(jié)合位點(diǎn)且雙熒光報告基因證實轉(zhuǎn)染miR-27a能引起熒光素酶活性下降。3、在大腸癌相關(guān)組織中miR-27a含量明顯高于正常癌旁組織,臨床病例資料分析顯示其表達(dá)水平與TNM分期、淋巴結(jié)轉(zhuǎn)移和遠(yuǎn)處轉(zhuǎn)移與否呈正相關(guān);miR-27a在各人大腸癌細(xì)胞株的表達(dá)均高于正常人大腸上皮細(xì)胞。4、轉(zhuǎn)染miR-27a導(dǎo)致LoVo細(xì)胞遷移加快和侵襲數(shù)量明顯增加,干擾miR-27a表達(dá)則引起LoVo細(xì)胞遷移和侵襲數(shù)量減少;穩(wěn)定過表達(dá)miR-27a的LoVo細(xì)胞在導(dǎo)入FAM172A過表達(dá)質(zhì)粒后,LoVo細(xì)胞遷移減慢及侵襲細(xì)胞數(shù)量減少,且轉(zhuǎn)移相關(guān)重要信號通路分子MMP-2、MMP-9和NF-κ B的蛋白表達(dá)降低。結(jié)論:1、FAM172A抑制大腸癌細(xì)胞的遷移和侵襲;2、miR-27a靶向結(jié)合FAM172A;3、miR-27a的表達(dá)變化對診斷和評估CRC患者病情具有一定的價值;4、FAM172A抑制大腸癌細(xì)胞株LoVo細(xì)胞遷移和侵襲活性的功能受到miR-27a負(fù)性調(diào)控,且可能通過下游NF-κ B信號通路而實現(xiàn)。
[Abstract]:Colorectal cancer (CRC), as a common human malignant tumor, is about 1.2 million new cases every year, and at least 600,000 patients die. Transfer is an important cause of the prognosis of patients with colorectal cancer. Most of the patients with large bowel cancer with metastasis are not suitable for the traditional treatment. The metastasis of colorectal cancer is mainly related to the individual gene mutation, and the gene mutation in the large intestine is not more than 50%. FAM172A is a new gene found in the previous study of the research group. Our recent results confirm that 1) the expression of FAM172A in CRC-related tissues is down-regulated, and the proliferation and apoptosis of human colorectal cancer LoVo cells are significantly inhibited; and 2) the transcription factor STAT1 regulates the expression of FAM172A at the transcription level, and enhances the latter function. In conclusion, we have speculated that the FAM172A gene is a typical tumor suppressor gene, and it is of great significance to further study the mechanism of transcription regulation. The regulation of gene expression is not only in the level of transcription, but also in the post-transcriptional regulation, and miRNAs are important factors that have been found to be involved in the post-transcriptional level regulation in recent years. MiRNAs is a non-coding single-stranded small-stranded RNA molecule encoded by an endogenous gene with a length of about 22 nucleotides. The study confirmed that the abnormal expression of miRNA was associated with the metastasis of colorectal cancer, such as the expression of miR-21 in colorectal cancer cells, and the inhibition of the invasion of tumor cells by inhibiting the expression of PDCD4. In our earlier work, using bioinformatics to predict that miR-27a may target the binding of FAAM172A, we assume that the FAM172A function may be regulated by miR-27a at the post-transcriptional level. In this paper, on the basis of the preliminary work, the regulation mechanism of FAM172A to inhibit the invasion and metastasis of large intestine cancer is further elucidated on the basis of the post-transcriptional level, and the scientific basis and the experimental basis for gene therapy of the large intestine cancer are provided. Objective:1. To clarify the effect of FAM172A on the migration and invasion of colorectal cancer cells;2. Bioinformatics to screen and verify the important miRNAs of FAM172A;3. To determine the expression level of miR-27a in colorectal cancer tissues and cell lines and its related clinical significance; The effects of miR-27a on the migration and invasion of colorectal cancer cells and its mechanism were discussed. Methods:1. The recombinant plasmid of FAM172A was constructed, and the effects of FAM172A on the migration and invasion of LoVo cells were observed by cell scratch and Transwell.2. The important miRNAs targeting the FAM172A were screened by bioinformatics. 3. The expression level of miR-27a in colorectal cancer cell lines and paired tissue samples was detected by RT-qPCR, and the LoVo cell strain with stable expression of miR-27a was constructed, and the cell scratch and Transwell's specific miR-27a control the migration and invasion of large intestine cells. Western blot was used to detect the effect of miR-27a on the expression of the important signaling pathway related to the invasion and metastasis. Two independent samples of t-test were used for the comparison between the data groups, and the variance of variance was used for many groups. There was a statistical difference between P0.05. All the experiments were carried out independently for three times, and the average value was taken for statistical analysis. Results:1. Overexpression of FAM172A decreased the movement speed of LoVo cells, resulting in a decrease in the invasion number of LoVo cells. in that presence of the miR-27a binding site in the FAM172A 3 'UTR and the double-fluorescent reporter gene confirmed that the transfection of the miR-27a can cause the luciferase activity to decrease.3, the content of the miR-27a in the colorectal cancer-related tissue is obviously higher than that of the normal adjacent tissue, and the clinical case data analysis shows the expression level and the TNM stage, There was a positive correlation between lymph node metastasis and distant metastasis. The expression of miR-27a in human colorectal cancer cell line was higher than that of normal human colorectal epithelial cell. LoVo cells stably overexpressing miR-27a, after the introduction of the FAM172A overexpression plasmid, decreased the migration of LoVo cells and decreased the number of invasive cells, and the protein expression of the related important signaling pathway molecules, MMP-2, MMP-9 and NF-EMAB, was reduced. Conclusion:1, FAM172A inhibits the migration and invasion of colorectal cancer cells;2, the expression of miR-27a in combination with FAM172A;3, the expression of miR-27a has a certain value in the diagnosis and evaluation of CRC patients; and 4, the function of the FAM172A to inhibit the migration and invasion activity of the LoVo cells of the colorectal cancer cell line is negatively regulated by the miR-27a, And may be implemented by a downstream NF-B signal path.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34
本文編號：2453642
[Abstract]:Colorectal cancer (CRC), as a common human malignant tumor, is about 1.2 million new cases every year, and at least 600,000 patients die. Transfer is an important cause of the prognosis of patients with colorectal cancer. Most of the patients with large bowel cancer with metastasis are not suitable for the traditional treatment. The metastasis of colorectal cancer is mainly related to the individual gene mutation, and the gene mutation in the large intestine is not more than 50%. FAM172A is a new gene found in the previous study of the research group. Our recent results confirm that 1) the expression of FAM172A in CRC-related tissues is down-regulated, and the proliferation and apoptosis of human colorectal cancer LoVo cells are significantly inhibited; and 2) the transcription factor STAT1 regulates the expression of FAM172A at the transcription level, and enhances the latter function. In conclusion, we have speculated that the FAM172A gene is a typical tumor suppressor gene, and it is of great significance to further study the mechanism of transcription regulation. The regulation of gene expression is not only in the level of transcription, but also in the post-transcriptional regulation, and miRNAs are important factors that have been found to be involved in the post-transcriptional level regulation in recent years. MiRNAs is a non-coding single-stranded small-stranded RNA molecule encoded by an endogenous gene with a length of about 22 nucleotides. The study confirmed that the abnormal expression of miRNA was associated with the metastasis of colorectal cancer, such as the expression of miR-21 in colorectal cancer cells, and the inhibition of the invasion of tumor cells by inhibiting the expression of PDCD4. In our earlier work, using bioinformatics to predict that miR-27a may target the binding of FAAM172A, we assume that the FAM172A function may be regulated by miR-27a at the post-transcriptional level. In this paper, on the basis of the preliminary work, the regulation mechanism of FAM172A to inhibit the invasion and metastasis of large intestine cancer is further elucidated on the basis of the post-transcriptional level, and the scientific basis and the experimental basis for gene therapy of the large intestine cancer are provided. Objective:1. To clarify the effect of FAM172A on the migration and invasion of colorectal cancer cells;2. Bioinformatics to screen and verify the important miRNAs of FAM172A;3. To determine the expression level of miR-27a in colorectal cancer tissues and cell lines and its related clinical significance; The effects of miR-27a on the migration and invasion of colorectal cancer cells and its mechanism were discussed. Methods:1. The recombinant plasmid of FAM172A was constructed, and the effects of FAM172A on the migration and invasion of LoVo cells were observed by cell scratch and Transwell.2. The important miRNAs targeting the FAM172A were screened by bioinformatics. 3. The expression level of miR-27a in colorectal cancer cell lines and paired tissue samples was detected by RT-qPCR, and the LoVo cell strain with stable expression of miR-27a was constructed, and the cell scratch and Transwell's specific miR-27a control the migration and invasion of large intestine cells. Western blot was used to detect the effect of miR-27a on the expression of the important signaling pathway related to the invasion and metastasis. Two independent samples of t-test were used for the comparison between the data groups, and the variance of variance was used for many groups. There was a statistical difference between P0.05. All the experiments were carried out independently for three times, and the average value was taken for statistical analysis. Results:1. Overexpression of FAM172A decreased the movement speed of LoVo cells, resulting in a decrease in the invasion number of LoVo cells. in that presence of the miR-27a binding site in the FAM172A 3 'UTR and the double-fluorescent reporter gene confirmed that the transfection of the miR-27a can cause the luciferase activity to decrease.3, the content of the miR-27a in the colorectal cancer-related tissue is obviously higher than that of the normal adjacent tissue, and the clinical case data analysis shows the expression level and the TNM stage, There was a positive correlation between lymph node metastasis and distant metastasis. The expression of miR-27a in human colorectal cancer cell line was higher than that of normal human colorectal epithelial cell. LoVo cells stably overexpressing miR-27a, after the introduction of the FAM172A overexpression plasmid, decreased the migration of LoVo cells and decreased the number of invasive cells, and the protein expression of the related important signaling pathway molecules, MMP-2, MMP-9 and NF-EMAB, was reduced. Conclusion:1, FAM172A inhibits the migration and invasion of colorectal cancer cells;2, the expression of miR-27a in combination with FAM172A;3, the expression of miR-27a has a certain value in the diagnosis and evaluation of CRC patients; and 4, the function of the FAM172A to inhibit the migration and invasion activity of the LoVo cells of the colorectal cancer cell line is negatively regulated by the miR-27a, And may be implemented by a downstream NF-B signal path.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R735.34
【參考文獻(xiàn)】
相關(guān)期刊論文 前4條
1 王鵬;郭琳;王強(qiáng);;PTEN、Fas/FasL和MMP-2在胃癌組織的表達(dá)及其臨床意義[J];中國普外基礎(chǔ)與臨床雜志;2009年12期
2 江州華;俞繼衛(wèi);姜波健;;基質(zhì)金屬蛋白酶對胃癌微環(huán)境調(diào)控作用的研究進(jìn)展[J];中國普外基礎(chǔ)與臨床雜志;2009年09期
3 鄭樹;;結(jié)直腸癌的預(yù)防[J];中華腫瘤防治雜志;2006年01期
4 張凌志,邸菁,王運(yùn)杰,趙昕,張朝東;Transwell小室篩選高侵襲性C6細(xì)胞MMP-2和TIMP-2的表達(dá)[J];腫瘤防治雜志;2005年10期
，本文編號：2453642
本文鏈接：http://sikaile.net/yixuelunwen/zlx/2453642.html
最近更新
教材專著
