本文關(guān)鍵詞：miR-29a-3p對胃癌腹膜轉(zhuǎn)移的影響及其機制研究　出處：《寧夏醫(yī)科大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： miR-29a-3p 胃癌 增殖轉(zhuǎn)移 HAS3

【摘要】：背景:胃癌是最常見的惡性腫瘤之一,其發(fā)病率在世界范圍惡性腫瘤排第四位,死亡率居第二位,在我國,每年新發(fā)病例約為30萬,嚴重威脅著國人健康,而轉(zhuǎn)移是胃癌患者最常見的致死原因,其發(fā)生的分子機制一直是腫瘤研究領(lǐng)域的熱點。微小RNA(micro RNAs,mi RNAs)是一大類內(nèi)源性的、在進化過程中高度保守的單鏈非編碼小RNA,參與了人體多種生命活動的調(diào)控,如細胞的增殖、分化、凋亡等。mi RNA的表達失調(diào)可導(dǎo)致多種疾病的發(fā)生,并且與多種惡性腫瘤的發(fā)生、發(fā)展有著極為密切的關(guān)系。近年來許多研究表明,在胃癌中也存在mi RNA表達失調(diào)的現(xiàn)象,說明mi RNA也參與了胃癌的發(fā)生。本實驗將在前期的基礎(chǔ)上,對mi R-29a-3p在胃癌細胞系的表達水平、生物學(xué)功能及可能的作用機制進行初步的研究和探討。目的:探索hsa-mi R-29a-3p(mi R-29a)對胃癌腹膜轉(zhuǎn)移的影響并初步研究其作用機制。方法:1.利用q RT-PCR驗證mi R-29a-3p在三對胃癌高低轉(zhuǎn)移細胞系GC9811-P/GC9811,MKN28-M/MKN-28NM,SGC7901-M/SGC7901-NM中的表達情況。2.構(gòu)建過表達及抑制mi R-29a-3p的慢病毒表達載體,將過表達mi R-29a-3p的慢病毒及其無關(guān)陰性對照轉(zhuǎn)染入胃癌腹膜高轉(zhuǎn)移細胞系GC9811-P中,相反,將抑制mi R-29a-3p表達的慢病毒及其無關(guān)陰性對照轉(zhuǎn)入親本細胞系GC9811中。3.用MTT比色實驗、平板克隆形成實驗、Transwell、劃痕實驗及流式細胞學(xué)技術(shù)分別觀察mi R-29a-3p對胃癌細胞的增殖、轉(zhuǎn)移及凋亡能力的影響。4.采用生物信息學(xué)的方法預(yù)測mi R-29a-3p的靶基因,查閱文獻選出與轉(zhuǎn)移相關(guān)的基因并采用q RT-PCR及western blot驗證與mi R-29a-3p的關(guān)系。5.轉(zhuǎn)染si RNA及空載體沉默基因表達,進一步行功能試驗驗證其功能及是否為mi R-29a-3p的靶基因。結(jié)果:1.mi R-29a-3p在胃癌高轉(zhuǎn)移細胞系(GC9811-P、MKN28-M、SGC7901-M)中高表達。2.成功構(gòu)建高低表達mi R-29a-3p的細胞系,q RT-PCR驗證轉(zhuǎn)染成功。3.功能試驗結(jié)果表明,過表達mi R-29a-3p可以抑制細胞的增殖及轉(zhuǎn)移能力而促進細胞的凋亡能力;相反,抑制mi R-29a-3p的表達可以促進細胞的增殖及轉(zhuǎn)移能力而抑制細胞的凋亡能力。4.生物信息學(xué)預(yù)測提示HAS3可能為mi R-29a-3p的潛在靶基因之一,q RT-PCR結(jié)果顯示,在上調(diào)mi R-29a-3p的細胞系中,HAS3表達有降低的趨勢但無統(tǒng)計學(xué)意義;在下調(diào)mi R-29a-3p的細胞系中,HAS3表達有升高的趨勢但無統(tǒng)計學(xué)意義。Western blot結(jié)果顯示,上調(diào)mi R-29a-3p的表達,HAS3表達下調(diào),下調(diào)mi R-29a-3p的表達,HAS3表達上調(diào)。5.沉默HAS3的表達,胃癌細胞增殖及遷移能力減弱,說明HAS3促進胃癌細胞的增殖與轉(zhuǎn)移。在高表達mi R-29a-3p的細胞系中沉默HAS3的表達后,細胞的增殖及轉(zhuǎn)移能力減弱,與過表達mi R-29a-3p有類似的效果,說明mi R-29a-3p可負性調(diào)控HAS3,進一步證實HAS3可為mi R-29a-3p的潛在靶基因之一。結(jié)論:mi R-29a-3p抑制胃癌細胞的增殖與轉(zhuǎn)移而促進凋亡,并可能通過其下游靶分子之一HAS3發(fā)揮其作用。
[Abstract]:Background: gastric cancer is one of the most common malignant tumors, the incidence of cancer ranked 4th in the world, the death rate ranked second. In China, the annual new cases are about 300,000, which seriously threaten the health of people. Metastasis is the most common cause of death in patients with gastric cancer, and its molecular mechanism has been a hot spot in the field of cancer research. Small RNA(micro RNAs. Mi RNAss are a large class of endogenous, highly conserved, single-stranded, non-coding RNAs that participate in the regulation of a variety of human life activities, such as cell proliferation and differentiation. The expression imbalance of .mi RNA, such as apoptosis, can lead to the occurrence of many diseases, and is closely related to the occurrence and development of many malignant tumors. There is also an imbalance in the expression of mi RNA in gastric cancer, indicating that mi RNA is also involved in the development of gastric cancer. The expression level of mi R-29a-3p in gastric cancer cell line. Biological function and possible mechanism of action were preliminarily studied and discussed. Objective: to explore hsa-mi R-29a-3pmmi R-29a. Effect on peritoneal metastasis of gastric cancer and preliminary study of its mechanism. Methods: 1. Using Q RT-PCR to verify mi. R-29a-3p was detected in three pairs of gastric cancer cell lines GC9811-P/GC9811. MKN28-M/MKN-28NM. The expression of SGC7901-M/SGC7901-NM. 2. Construct the expression vector of lentivirus which overexpressed and inhibited mi R-29a-3p. The overexpression of mi R-29a-3p lentivirus and its unrelated negative control were transfected into the peritoneal high metastasis cell line GC9811-P of gastric cancer. The lentivirus which inhibited the expression of mi R-29a-3p and its unrelated negative control were transferred into the parental cell line GC9811. 3. MTT colorimetric assay and plate clone formation test were used. The proliferation of gastric cancer cells induced by miR-29a-3p was observed by flow cytometry and scratch test. Effects of Metastasis and apoptosis. Bioinformatics was used to predict the target gene of mi R-29a-3p. Search the literature to select genes related to metastasis and verify the relationship with mi R-29a-3p by Q RT-PCR and western blot .5.transfect si. RNA and empty vector silenced gene expression. Further functional tests were performed to verify its function and whether it was the target gene of mi R-29a-3p. Results: 1. Mi R-29a-3p was detected in gastric cancer cell line GC9811-P. High expression of MKN28-MGC7901-M2.The cell line with high and low expression of miR-29a-3p was successfully constructed. The results of functional test showed that overexpression of mi R-29a-3p could inhibit cell proliferation and metastasis and promote cell apoptosis. On the contrary. Inhibition of the expression of mi R-29a-3p could promote cell proliferation and metastasis and inhibit cell apoptosis. 4. Bioinformatics prediction suggested that HAS3 might be mi. One of the potential target genes of R-29a-3p. The results of Q RT-PCR showed that the expression of HAS3 decreased in the cell line of miR-29a-3p, but had no statistical significance. The down-regulation of the expression of HAS3 in the cell line of miR-29a-3p showed a tendency to increase, but no statistical significance. Western blot results showed. Up regulated the expression of miR-29a-3p and down-regulated the expression of miR-29a-3p and up-regulated the expression of HAS3. Silenced the expression of HAS3. The ability of proliferation and migration of gastric cancer cells was decreased, which indicated that HAS3 promoted proliferation and metastasis of gastric cancer cells. After silencing the expression of HAS3 in the cell lines with high expression of miR-29a-3p. The ability of cell proliferation and metastasis was decreased, which was similar to that of overexpression of mi R-29a-3p, indicating that mi R-29a-3p could negatively regulate HAS3. It is further confirmed that HAS3 may be one of the potential target genes of mi R-29a-3p. Conclusion: MiR-29a-3p inhibits the proliferation and metastasis of gastric cancer cells and promotes apoptosis. And it may play its role through one of its downstream target molecules, HAS3.
【學(xué)位授予單位】：寧夏醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：R735.2

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