本文選題：結(jié)直腸癌 + 微小RNA�。� 參考：《南方醫(yī)科大學(xué)》2015年碩士論文

【摘要】：背景大腸癌(colorectal carcinoma, CRC)根據(jù)腫瘤發(fā)生的部位,可分為直腸癌和結(jié)腸癌,是最常見消化道惡性腫瘤之一。在我國,隨著生活水平的提高,飲食結(jié)構(gòu)及生活習(xí)慣的改變,其發(fā)病率呈逐年上升趨勢(shì)。近年來,盡管結(jié)直腸癌的診治水平不斷提高,但是其5年生存率并未明顯提高,究其原因,在于結(jié)直腸癌發(fā)生發(fā)展的確切分子機(jī)制仍未完全闡明。因此進(jìn)一步闡明結(jié)直腸癌發(fā)生發(fā)展的確切分子機(jī)制是目前的研究熱點(diǎn)。微小RNAs (microRNAs, miRNA)是一類長18-25個(gè)核苷酸的內(nèi)源性單鏈非編碼小RNA,是在人類分子腫瘤學(xué)領(lǐng)域里又一個(gè)新的研究熱點(diǎn),主要是通過與靶mRNA上的3’非編碼區(qū)域(Untranslated region, UTR)的特異序列結(jié)合,于轉(zhuǎn)錄后水平促進(jìn)靶mRNA的降解,和(或)抑制翻譯過程而參與多種腫瘤的發(fā)生和發(fā)展的。近來研究發(fā)現(xiàn),很多miRNA在結(jié)直腸癌中存在異常高表達(dá)或者低表達(dá),并通過調(diào)節(jié)多種靶基因的表達(dá),在結(jié)直腸癌的發(fā)生發(fā)展等過程中發(fā)揮抑癌或促癌作用。星形膠質(zhì)細(xì)胞升高基因-1(Astrocyte elevated gene-1, AEG-1),亦被稱為轉(zhuǎn)移粘附分子(Metadherin, MTDH),是人免疫缺陷病毒(Human immunodeficiency virus-1, HIV-1)感染或腫瘤壞死因子α(Tumor necrosis factor-α, TNF-α)誘導(dǎo)胚胎星形細(xì)胞后導(dǎo)致表達(dá)升高的一種基因,最初是Su等通過快速消減雜交的方法克隆出來的,位于人染色體8q22,此位點(diǎn)是與多種惡性腫瘤的發(fā)生高度相關(guān)的地帶。AEG-1在多種惡性腫瘤中高表達(dá),我們的前期研究發(fā)現(xiàn)AEG-1與結(jié)直腸癌的形成和發(fā)展密切相關(guān),可促進(jìn)大腸癌的增殖、遷移、侵襲能力,誘導(dǎo)腫瘤新生血管的形成和化療耐藥等。miR-375是microRNAs家族中的一員,研究發(fā)現(xiàn)miR-375可通過靶向調(diào)節(jié)AEG-1基因的表達(dá),從而參與肝癌和頭頸部鱗狀細(xì)胞癌等的發(fā)生發(fā)展過程,但miR-375與AEG-1的關(guān)系尚未在結(jié)直腸癌中得到證實(shí)。本研究使用化學(xué)合成的miR-375模擬物轉(zhuǎn)染結(jié)腸癌HCT116細(xì)胞,上調(diào)結(jié)腸癌HCT116細(xì)胞中miR-375的表達(dá),通過研究miR-375對(duì)結(jié)腸癌細(xì)胞株HCT116生物學(xué)行為的影響,旨在為結(jié)腸癌的發(fā)病機(jī)制提供新的理論依據(jù)。目的脂質(zhì)體轉(zhuǎn)染法將miR-375 mimics轉(zhuǎn)入HCT116細(xì)胞上調(diào)miR-375的表達(dá),用實(shí)時(shí)定量-PCR法檢測(cè)miR-375和AEG-1 mRNA的表達(dá)情況；MTT法檢測(cè)細(xì)胞活力的改變情況；流式細(xì)胞技術(shù)檢測(cè)miR-375對(duì)細(xì)胞凋亡及細(xì)胞周期的影響；我們通過研究miR-375對(duì)結(jié)腸癌細(xì)胞株HCT116生物學(xué)行為的影響,旨在為結(jié)腸癌的發(fā)病機(jī)制提供新的理論依據(jù)。材料和方法1.材料：人結(jié)直腸癌細(xì)胞株Caco2、HCT116、SW480、SW620均購自CTCC(中國模式培養(yǎng)物集存庫Chinese Type Culture Collection,中國北京)；DMEM培養(yǎng)基、RPMI-1640培養(yǎng)基和胎牛血清均購自美國Gibco公司；0.25%胰蛋白酶購自杭州吉諾生物有限公司；miR-375 mimics和negative control轉(zhuǎn)染試劑盒購自廣州銳博生物科技有限公司；Lipofectmanine TM 2000及TRIzol試劑均購自美國Invitrogen公司；噻唑藍(lán)(MTT)購自廣州威佳公司；二甲基亞砜(DMSO)購自美國Sigma公司；細(xì)胞周期及凋亡檢測(cè)試劑盒購于杭州聯(lián)科生物技術(shù)股份有限公司；RevertAid First Strand cDNA Synthesis Kit購自Fermentas公司；SYBR Premix Ex Taq Ⅱ(Tli RNaseH Plus)為TaKaRa公司產(chǎn)品。2.細(xì)胞培養(yǎng)：HCT116用含10%胎牛血清的DMEM培養(yǎng)基進(jìn)行培養(yǎng),SW480、SW620用含10%胎牛血清的RPMI-1640培養(yǎng)基進(jìn)行培養(yǎng)。培養(yǎng)條件：置于37℃、CO2體積分?jǐn)?shù)為5%的培養(yǎng)箱中培養(yǎng)。根據(jù)細(xì)胞生長情況每1～2 d換液培養(yǎng)1次,當(dāng)細(xì)胞覆蓋瓶底壁大部分表面時(shí),進(jìn)行細(xì)胞傳代或收集細(xì)胞。3.實(shí)時(shí)定量-PCR檢測(cè)不同結(jié)直腸癌細(xì)胞中miR-375的表達(dá)情況：收集細(xì)胞,按照TRIzol試劑說明書抽提結(jié)直腸癌細(xì)胞株Caco2、HCT116、SW620和SW480中的總RNA,-80℃凍存?zhèn)溆�。采用�?shí)時(shí)定量-PCR檢測(cè)結(jié)直腸癌細(xì)胞中miR-375的表達(dá),以U6作為內(nèi)參,引物均購自廣州銳博公司。結(jié)果采用2-△Ct法計(jì)算目的基因的相對(duì)表達(dá)量,△Ct=目的基因平均Ct值-內(nèi)參基因平均Ct值。4.細(xì)胞轉(zhuǎn)染：轉(zhuǎn)染前1 d,將適量(約4×104～5×104)生長狀態(tài)穩(wěn)定的HCT116細(xì)胞接種在24孔細(xì)胞培養(yǎng)板上,每孔加入不含抗生素的細(xì)胞培養(yǎng)液400μL。密切觀察細(xì)胞生長情況,當(dāng)細(xì)胞密度達(dá)到30%-50%時(shí),開始進(jìn)行實(shí)驗(yàn)組(miR-375 mimics)和對(duì)照組(negative control,NC)的轉(zhuǎn)染。用50μL不含血清的培養(yǎng)基Opti-MEM對(duì)5μL濃度為20μmol/L的miR-375 mimics和negative control進(jìn)行稀釋,輕輕振蕩混勻,室溫下溫育5 min。用50μL的不含血清培養(yǎng)基Opti-MEM對(duì)1.5μL Lipofectmanine TM 2000進(jìn)行稀釋,輕輕振蕩混勻,室溫下溫育5 min。將稀釋好的miR-375 mimics和negative control與LipofectmanineTM 2000混合,輕柔混勻,室溫下溫育20 min,以形成混合物。100μL混合物加到培養(yǎng)板的孔中,輕輕搖晃細(xì)胞培養(yǎng)板使其與培養(yǎng)液混勻。將細(xì)胞培養(yǎng)板置于CO2體積分?jǐn)?shù)為5%的培養(yǎng)箱中,在37℃下培養(yǎng)6h后,移除每孔中含有混合物的培養(yǎng)液,更換新的培養(yǎng)液。繼續(xù)在37℃、CO2體積分?jǐn)?shù)為5%的條件下培養(yǎng)24 h后,進(jìn)行轉(zhuǎn)染后的其他實(shí)驗(yàn)檢測(cè)。5.實(shí)時(shí)定量-PCR檢測(cè)各轉(zhuǎn)染組細(xì)胞中miR-375和AEG-1 mRNA的表達(dá)情況：轉(zhuǎn)染48 h后收集細(xì)胞,按照TRIzol說明書抽提細(xì)胞中的總RNA,-80℃凍存?zhèn)溆�。采用�?shí)時(shí)熒光定量-PCR檢測(cè)結(jié)腸癌細(xì)胞中miR-375的表達(dá),以U6作為內(nèi)參照,引物購自廣州銳博公司。實(shí)時(shí)熒光定量-PCR檢測(cè)細(xì)胞中AEG-1mRNA的表達(dá)情況,以GAPDH作為內(nèi)參,引物購自TaKaRa公司。結(jié)果采用2-ΔΔCt法計(jì)算目的基因的相對(duì)表達(dá)量,ACt=目的基因平均Ct值-內(nèi)參基因平均Ct值。6.MTT法檢測(cè)細(xì)胞的生長情況：將轉(zhuǎn)染后的實(shí)驗(yàn)組和陰性對(duì)照組細(xì)胞分別接種于96孔培養(yǎng)板中,每孔接種細(xì)胞約3000個(gè)(每孔加培養(yǎng)液100μL),置于37℃、CO2體積分?jǐn)?shù)為5%的培養(yǎng)箱中進(jìn)行培養(yǎng)。分別于培養(yǎng)4h、24 h、48 h和72 h后加入MTT工作液,每孔加入0.5% MTT 20μL,繼續(xù)培養(yǎng)4h后,輕輕吸出孔內(nèi)液體,然后在每孔中加入二甲基亞砜150μL,應(yīng)用全自動(dòng)酶標(biāo)儀,檢測(cè)波長490 nm下的吸光度(OD)值。以時(shí)間作為橫坐標(biāo),計(jì)算所得OD值的均數(shù)為縱坐標(biāo),繪制細(xì)胞生長曲線。7.流式細(xì)胞術(shù)檢測(cè)細(xì)胞周期：轉(zhuǎn)染48 h后消化收集細(xì)胞,制成單細(xì)胞懸液,然后1200 r/min離心5 min,棄上清。預(yù)冷PBS洗滌細(xì)胞2次,PI細(xì)胞染色,即加入1000 μL staining buffer (A)及10 μL reagent B染色,混勻后室溫避光孵育30 min,立即上流式細(xì)胞儀進(jìn)行檢測(cè)。8.流式細(xì)胞術(shù)檢測(cè)細(xì)胞凋亡：轉(zhuǎn)染48 h后消化收集細(xì)胞,制成單細(xì)胞懸液,然后1200 r/min離心5 min,棄上清。預(yù)冷PBS洗滌細(xì)胞2次,用500μL1×binding buffer重懸細(xì)胞,加入5 μL FITC標(biāo)記的annexin-V,加入10μL的P1�；靹蚝笫覝乇芄夥跤�5 min,立即上流式細(xì)胞儀檢測(cè)。9.應(yīng)用SPSS 20.0統(tǒng)計(jì)學(xué)軟件對(duì)實(shí)驗(yàn)數(shù)據(jù)進(jìn)行統(tǒng)計(jì)學(xué)分析。所有的細(xì)胞學(xué)實(shí)驗(yàn)均重復(fù)3次,計(jì)量數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(mean±SD)表示,符合正態(tài)分布且方差齊者采用t檢驗(yàn)或者ANOVA檢驗(yàn),方差不齊采用Dunnett T3校正,以P0.05為有統(tǒng)計(jì)學(xué)意義。結(jié)果1.miR-375在結(jié)腸癌細(xì)胞株中的表達(dá)情況：提取結(jié)直腸癌細(xì)胞株(Caco2、 HCT116、SW620、SW480)中的總RNA,以U6作為內(nèi)參,運(yùn)用實(shí)時(shí)定量-PCR法檢測(cè)miR-375的相對(duì)表達(dá)量。結(jié)果采用2-△Ct法計(jì)算miR-375的相對(duì)表達(dá)量。其中以HCT116細(xì)胞中miR-375的表達(dá)水平最低,因此選取HCT116細(xì)胞株作為我們進(jìn)一步研究的對(duì)象。2. miR-375 mimics對(duì)HCT 116細(xì)胞中miR-375和AEG-1 mRNA表達(dá)的影響：miR-375 mimics瞬時(shí)轉(zhuǎn)染HCT116細(xì)胞48 h后,采用實(shí)時(shí)定量-PCR檢測(cè)轉(zhuǎn)染后實(shí)驗(yàn)組及陰性對(duì)照組HCT116細(xì)胞中miR-375和AEG-1 mRNA的表達(dá)水平。結(jié)果顯示,轉(zhuǎn)染miR-375 mimic后miR-375的表達(dá)水平明顯上調(diào),其表達(dá)水平約是陰性對(duì)照組的2000倍(P0.01),同時(shí)miR-375的高表達(dá)導(dǎo)致AEG-1mRNA表達(dá)下調(diào)(P0.05)。以上結(jié)果提示,miR-375 mimics可上調(diào)miR-375的表達(dá),同時(shí)過表達(dá)miR-375可抑制AEG-1 mRNA的表達(dá)。3.miR-375過表達(dá)對(duì)HCT116細(xì)胞生長的影響：MTT法檢測(cè)結(jié)果顯示,miR-375 mimics組細(xì)胞轉(zhuǎn)染24 h、48 h、72 h時(shí)的OD值均低于陰性對(duì)照組,差異有統(tǒng)計(jì)學(xué)意義(P0.01)。miR-375過表達(dá)對(duì)HCT116細(xì)胞的活力具有明顯抑制作用。4.miR-375過表達(dá)對(duì)HCT116細(xì)胞周期的影響：流式細(xì)胞儀檢測(cè)轉(zhuǎn)染48h后各組細(xì)胞的周期分布,結(jié)果顯示,miR-375 mimics組與陰性對(duì)照組在G1期、S期和G2期細(xì)胞所占比例分別為(68.323±2.975)%vs(54.973±3.056)%、(16.443±0.422)% vs (27.767±3.636)%, (14.200±0.943)% vs (17.26±2.268)%,2組間經(jīng)比較,miR-375 mimics組G1期細(xì)胞所占比例明顯增多(P0.01),S期細(xì)胞所占比例明顯減少(P0.05)。5.miR-375過表達(dá)對(duì)HCT116細(xì)胞凋亡率的影響：流式細(xì)胞儀檢測(cè)轉(zhuǎn)染48 h后各組細(xì)胞的凋亡率,結(jié)果顯示,miR-375 mimics組和陰性對(duì)照組的細(xì)胞凋亡率分別為(8.468±1.546)%和(6.252±1.201)%,陰性對(duì)照組的細(xì)胞凋亡率明顯低于miR-375 mimics組,差異有統(tǒng)計(jì)學(xué)意義(P0.05)。結(jié)論1. miR-375mimics組中miR-375表達(dá)量較對(duì)照組明顯上調(diào)；miR-375抑制結(jié)腸癌細(xì)胞HCT116的細(xì)胞活性,介導(dǎo)細(xì)胞周期阻滯并促進(jìn)其凋亡2.miR-375高表達(dá)可以顯著抑制AEG-1 mRNA的表達(dá)水平,miR-375作為一種抑癌因子,在結(jié)腸癌中可能通過抑制癌基因AEG-1的表達(dá)發(fā)揮抑癌作用。
[Abstract]:Background colorectal carcinoma (CRC) is one of the most common malignant tumors of the digestive tract based on the site of the tumor. It is one of the most common digestive malignant tumors. In China, with the improvement of the living standard and the change of diet structure and living habits, the incidence of the cancer is increasing year by year. In recent years, although the diagnosis and treatment of colorectal cancer has continued, the level of the diagnosis and treatment of colorectal cancer has continued. However, the 5 year survival rate has not been improved significantly. The reason is that the exact molecular mechanism of the development of colorectal cancer is still not fully elucidated. Therefore, further clarification of the exact molecular mechanism of the development of colorectal cancer is the current research focus. The micro RNAs (microRNAs, miRNA) is a class of endogenous single chains of a class of 18-25 nucleotides. The non coding small RNA is another new research hotspot in the field of human molecular oncology, mainly by combining the specific sequence of the 3 'non coding region (Untranslated region, UTR) on the target mRNA, promoting the degradation of target mRNA at the post transcriptional level, and (or) inhibiting the translation process to participate in the occurrence and development of a variety of tumors. It is found that many miRNA have abnormal high expression or low expression in colorectal cancer, and play the role of inhibiting cancer or promoting cancer in the process of the development of colorectal cancer by regulating the expression of a variety of target genes. The astrocyte elevated gene -1 (Astrocyte elevated gene-1, AEG-1) is also known as the transfer adhesion molecule (Metadherin, MTD). H) is a gene that is induced by human immunodeficiency virus (Human immunodeficiency virus-1, HIV-1) infection or tumor necrosis factor alpha (Tumor necrosis factor- a, TNF- a) induced embryonic astrocytes, which was first cloned by rapid subtractive hybridization, located in the human chromosome 8q22, and this site is a variety of genes. .AEG-1 is highly expressed in a variety of malignant tumors. Our previous study found that AEG-1 is closely related to the formation and development of colorectal cancer. It can promote the proliferation, migration, invasion, formation of neovascularization and chemotherapeutic resistance of cancer, and.MiR-375 is a member of the microRNAs family. The study found that miR-375 can regulate the expression of AEG-1 gene by targeting to participate in the development of hepatocellular carcinoma and squamous cell carcinoma of the head and neck, but the relationship between miR-375 and AEG-1 has not been confirmed in colorectal cancer. This study used the miR-375 analog of chemical synthesis to transfect colon cancer HCT116 cells to up regulate the HCT116 cells of colon cancer. The expression of miR-375, through the study of the effect of miR-375 on the biological behavior of colon cancer cell line HCT116, aims to provide a new theoretical basis for the pathogenesis of colon cancer. Objective liposome transfection was used to transfer miR-375 mimics into HCT116 cells to up regulate the expression of miR-375, and to detect the expression of miR-375 and AEG-1 mRNA by real-time quantitative -PCR. The effect of miR-375 on cell apoptosis and cell cycle was detected by flow cytometry; the effect of miR-375 on the biological behavior of HCT116 in colon cancer cell line was investigated by flow cytometry. The purpose of this study was to provide a new theoretical basis for the pathogenesis of colon cancer. Material and methods 1.: human colorectal cancer cell line Ca CO2, HCT116, SW480, and SW620 were purchased from CTCC (China model culture storage storage Chinese Type Culture Collection, Beijing, China); DMEM medium, RPMI-1640 medium and fetal bovine serum were purchased from the American Gibco company; 0.25% trypsin was purchased from the Hangzhou Kino biological Limited company; Guangzhou sharp biological science and Technology Co., Ltd., Lipofectmanine TM 2000 and TRIzol reagents are purchased from American Invitrogen company; thiazolyl blue (MTT) purchased from Guangzhou company; two methyl sulfoxide (DMSO) purchased from American Sigma company; cell cycle and apoptosis detection kit purchased in Hangzhou biologic biotechnology Limited by Share Ltd; RevertAid F IRST Strand cDNA Synthesis Kit purchased from Fermentas company; SYBR Premix Ex Taq II (Tli RNaseH) culture: culture medium containing 10% fetal bovine serum, culture medium with 10% fetal bovine serum. Culture conditions: at 37, volume fraction 5% culture in the incubator. 1 times per 1~2 D for cell growth, and when cells covered most of the surface of the bottom wall of the bottle, the expression of miR-375 in different colorectal cancer cells was detected by cell passage or collection of cell.3. real-time quantitative -PCR: collect the cells and extract the colorectal cancer cell line Caco according to the TRIzol reagent instruction manual 2, the total RNA in HCT116, SW620 and SW480 was stored in -80. The expression of miR-375 in colorectal cancer cells was detected by real-time quantitative -PCR, and U6 was used as the internal parameter. The primers were purchased from Guangzhou sharp company. The result was calculated by 2- Delta Ct method. : 1 d before transfection, appropriate (about 4 x 104~5 * 104) growth stable HCT116 cells were inoculated on the 24 cell culture plate. The cell growth was observed closely with the cell culture solution containing no antibiotics in each hole. When the cell density reached 30%-50%, the test group (miR-375 mimics) and the control group (negative control, NC) were started. Transfection. MiR-375 mimics and negative control with 5 mu L concentration of 20 mu mol/L were diluted with a serum free medium of 50 micron Opti-MEM, and miR-375 mimics and negative control were diluted with a gentle oscillation. At room temperature, 5 min. was incubated at room temperature with 50 micron L without the medium Opti-MEM on 1.5 micron L 2000. The light light oscillation was mixed and the temperature 5 would be diluted well at room temperature. MiR-375 mimics and negative control are mixed with LipofectmanineTM 2000, gently and well mixed, and incubated at room temperature for 20 min. The mixture of.100 mu L mixture is added to the hole of the culture plate, and the cell culture plate is gently swayed to mix it with the culture medium. The cell culture plate is placed in a incubator with CO2 volume fraction of 5%, and after 6h at 37. In addition to the culture solution containing the mixture in each hole, the new culture fluid was replaced. After 37 centigrade, the volume fraction of CO2 was 5%, the other experiments were carried out to detect the expression of miR-375 and AEG-1 mRNA in the cells of each transfected group after transfection. The expression of miR-375 and AEG-1 mRNA in the cells of each transfected group was detected by.5.. After transfecting 48 h, the cells were collected and extracted in accordance with the TRIzol specification. The expression of miR-375 in colon cancer cells was detected by real time fluorescence quantitative -PCR and U6 was used as internal reference. The primers were purchased from Guangzhou Rubo company. The expression of AEG-1mRNA in the cells was detected by real-time fluorescent quantitative -PCR. GAPDH was used as the internal parameter and the primers were purchased from TaKaRa company. The results were calculated by 2- Delta Delta Ct method. The relative expression of the gene, the average Ct value of the ACt= target gene and the average Ct value of the internal reference gene were detected by the.6.MTT method. The transfected experimental group and the negative control group were inoculated in the 96 hole culture plate respectively. The cells were inoculated 3000 cells per pore (100 mu L per pore), and were placed in a incubator with 37 and CO2 volume fraction of 5%. After culture 4h, 24 h, 48 h and 72 h, MTT working fluid was added, 0.5% MTT 20 mu L was added to each hole, after 4H was added, the liquid in the hole was gently sucked out, and two methyl sulfoxide 150 micron per hole was added to each hole, and the full automatic enzyme labeling instrument was used to detect the absorbance (OD) value under the wavelength 490 nm. The cell growth curve.7. flow cytometry was used to detect cell cycle. After 48 h transfection, the cells were digested and collected to form a single cell suspension, then 1200 r/min centrifuged 5 min, abandoned supernatant. The pre cooled PBS washing cells were 2 times and PI cells were stained, that was, 1000 mu L staining buffer (A) and 10 mu L reagent were stained, and then incubated at room temperature after mixing. At 30 min,.8. flow cytometry was used to detect the apoptosis of cells. After transfection, the cells were collected and collected to form a single cell suspension after transfection of 48 h. Then 1200 r/min centrifuged 5 min and abandoned the supernatant. The pre cooled PBS washing cells were used for 2 times, and 500 mu L1 x buffer suspended cells were added to 5 mu L FITC annexin-V, and 10 micron mixed mixtures were added. At room temperature, 5 min was incubated at room temperature, and the experimental data were statistically analyzed by.9. SPSS 20 statistical software. All the cytological experiments were repeated 3 times, and the measured data were expressed with mean + standard deviation (mean + SD). The normal distribution was in conformity with the normal distribution with the t test or ANOVA test, and the variance was not homogeneous. The Dunnett T3 correction was statistically significant. Results the expression of 1.miR-375 in the colon cancer cell line: the total RNA in the colorectal cancer cell line (Caco2, HCT116, SW620, SW480) was extracted, and U6 was used as the internal parameter. The relative expression of miR-375 was detected by real time quantitative -PCR method. The expression level of miR-375 in HCT116 cells is the lowest, so the HCT116 cell line is selected as the object of our further study,.2. miR-375 mimics on the expression of miR-375 and AEG-1 mRNA in HCT 116 cells: miR-375 mimics transient transfection of the cell 48 after the transient quantitative detection of the transfected experimental group and negative control The expression level of miR-375 and AEG-1 mRNA in HCT116 cells showed that the expression level of miR-375 after transfection of miR-375 mimic was obviously up up, and the expression level was about 2000 times of that of the negative control group (P0.01), and the high expression of miR-375 resulted in the downregulation of AEG-1mRNA (P0.05). At the same time, overexpression of miR-375 could inhibit the effect of AEG-1 mRNA expression on the growth of HCT116 cells: MTT assay results showed that miR-375 mimics group cells transfected 24 h, 48 h, 72 h were lower than the negative control group, and the difference was statistically significant (P0.01) obviously inhibited the vitality of the cells. The effect of.4.miR-375 overexpression on HCT116 cell cycle: flow cytometry detected the cell cycle distribution after 48h transfection. The results showed that miR-375 mimics group and negative control group were in G1 phase, and the proportion of S and G2 phase cells were (68.323 + 2.975)%vs (54.973 + 3.056)%, (16.443 + 0.422)% vs (27.767 + 3.636)%, (14.200 + 0.943). )% vs (17.26 + 2.268)%, compared with the 2 groups, the proportion of G1 cells in the miR-375 mimics group increased significantly (P0.01). The proportion of S phase cells significantly decreased (P0.05).5.miR-375 overexpression on the apoptosis rate of HCT116 cells: the flow cytometry was used to detect the apoptosis rate of each group after transfection of 48 h. The results showed that miR-375 mimics and negative controls were shown. The apoptosis rate of the group was (8.468 + 1.546)% and (6.252 + 1.201)% respectively. The apoptosis rate of the negative control group was significantly lower than that of the miR-375 mimics group, and the difference was statistically significant (P0.05). Conclusion the expression of miR-375 in the 1. miR-375mimics group was significantly higher than that in the control group; miR-375 inhibited the cell activity of HCT116 in the colon cancer cells and mediated the cell cycle. Blocking and promoting the high expression of apoptotic 2.miR-375 can significantly inhibit the expression of AEG-1 mRNA. As a tumor suppressor factor, miR-375 may inhibit cancer by inhibiting the expression of oncogene AEG-1 in colon cancer.
【學(xué)位授予單位】：南方醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2015
【分類號(hào)】：R735.35

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