MiR-375对人结肠癌细胞株HCT116的生物学功能的影响

发布时间：2018-06-24 12:22

本文选题：结直肠癌 + 微小RNA　；参考：《南方医科大学》2015年硕士论文

【摘要】：背景大肠癌(colorectal carcinoma, CRC)根据肿瘤发生的部位,可分为直肠癌和结肠癌,是最常见消化道恶性肿瘤之一。在我国,随着生活水平的提高,饮食结构及生活习惯的改变,其发病率呈逐年上升趋势。近年来,尽管结直肠癌的诊治水平不断提高,但是其5年生存率并未明显提高,究其原因,在于结直肠癌发生发展的确切分子机制仍未完全阐明。因此进一步阐明结直肠癌发生发展的确切分子机制是目前的研究热点。微小RNAs (microRNAs, miRNA)是一类长18-25个核苷酸的内源性单链非编码小RNA,是在人类分子肿瘤学领域里又一个新的研究热点,主要是通过与靶mRNA上的3’非编码区域(Untranslated region, UTR)的特异序列结合,于转录后水平促进靶mRNA的降解,和(或)抑制翻译过程而参与多种肿瘤的发生和发展的。近来研究发现,很多miRNA在结直肠癌中存在异常高表达或者低表达,并通过调节多种靶基因的表达,在结直肠癌的发生发展等过程中发挥抑癌或促癌作用。星形胶质细胞升高基因-1(Astrocyte elevated gene-1, AEG-1),亦被称为转移粘附分子(Metadherin, MTDH),是人免疫缺陷病毒(Human immunodeficiency virus-1, HIV-1)感染或肿瘤坏死因子α(Tumor necrosis factor-α, TNF-α)诱导胚胎星形细胞后导致表达升高的一种基因,最初是Su等通过快速消减杂交的方法克隆出来的,位于人染色体8q22,此位点是与多种恶性肿瘤的发生高度相关的地带。AEG-1在多种恶性肿瘤中高表达,我们的前期研究发现AEG-1与结直肠癌的形成和发展密切相关,可促进大肠癌的增殖、迁移、侵袭能力,诱导肿瘤新生血管的形成和化疗耐药等。miR-375是microRNAs家族中的一员,研究发现miR-375可通过靶向调节AEG-1基因的表达,从而参与肝癌和头颈部鳞状细胞癌等的发生发展过程,但miR-375与AEG-1的关系尚未在结直肠癌中得到证实。本研究使用化学合成的miR-375模拟物转染结肠癌HCT116细胞,上调结肠癌HCT116细胞中miR-375的表达,通过研究miR-375对结肠癌细胞株HCT116生物学行为的影响,旨在为结肠癌的发病机制提供新的理论依据。目的脂质体转染法将miR-375 mimics转入HCT116细胞上调miR-375的表达,用实时定量-PCR法检测miR-375和AEG-1 mRNA的表达情况；MTT法检测细胞活力的改变情况；流式细胞技术检测miR-375对细胞凋亡及细胞周期的影响；我们通过研究miR-375对结肠癌细胞株HCT116生物学行为的影响,旨在为结肠癌的发病机制提供新的理论依据。材料和方法1.材料：人结直肠癌细胞株Caco2、HCT116、SW480、SW620均购自CTCC(中国模式培养物集存库Chinese Type Culture Collection,中国北京)；DMEM培养基、RPMI-1640培养基和胎牛血清均购自美国Gibco公司；0.25%胰蛋白酶购自杭州吉诺生物有限公司；miR-375 mimics和negative control转染试剂盒购自广州锐博生物科技有限公司；Lipofectmanine TM 2000及TRIzol试剂均购自美国Invitrogen公司；噻唑蓝(MTT)购自广州威佳公司；二甲基亚砜(DMSO)购自美国Sigma公司；细胞周期及凋亡检测试剂盒购于杭州联科生物技术股份有限公司；RevertAid First Strand cDNA Synthesis Kit购自Fermentas公司；SYBR Premix Ex Taq Ⅱ(Tli RNaseH Plus)为TaKaRa公司产品。2.细胞培养：HCT116用含10%胎牛血清的DMEM培养基进行培养,SW480、SW620用含10%胎牛血清的RPMI-1640培养基进行培养。培养条件：置于37℃、CO2体积分数为5%的培养箱中培养。根据细胞生长情况每1～2 d换液培养1次,当细胞覆盖瓶底壁大部分表面时,进行细胞传代或收集细胞。3.实时定量-PCR检测不同结直肠癌细胞中miR-375的表达情况：收集细胞,按照TRIzol试剂说明书抽提结直肠癌细胞株Caco2、HCT116、SW620和SW480中的总RNA,-80℃冻存备用。采用实时定量-PCR检测结直肠癌细胞中miR-375的表达,以U6作为内参,引物均购自广州锐博公司。结果采用2-△Ct法计算目的基因的相对表达量,△Ct=目的基因平均Ct值-内参基因平均Ct值。4.细胞转染：转染前1 d,将适量(约4×104～5×104)生长状态稳定的HCT116细胞接种在24孔细胞培养板上,每孔加入不含抗生素的细胞培养液400μL。密切观察细胞生长情况,当细胞密度达到30%-50%时,开始进行实验组(miR-375 mimics)和对照组(negative control,NC)的转染。用50μL不含血清的培养基Opti-MEM对5μL浓度为20μmol/L的miR-375 mimics和negative control进行稀释,轻轻振荡混匀,室温下温育5 min。用50μL的不含血清培养基Opti-MEM对1.5μL Lipofectmanine TM 2000进行稀释,轻轻振荡混匀,室温下温育5 min。将稀释好的miR-375 mimics和negative control与LipofectmanineTM 2000混合,轻柔混匀,室温下温育20 min,以形成混合物。100μL混合物加到培养板的孔中,轻轻摇晃细胞培养板使其与培养液混匀。将细胞培养板置于CO2体积分数为5%的培养箱中,在37℃下培养6h后,移除每孔中含有混合物的培养液,更换新的培养液。继续在37℃、CO2体积分数为5%的条件下培养24 h后,进行转染后的其他实验检测。5.实时定量-PCR检测各转染组细胞中miR-375和AEG-1 mRNA的表达情况：转染48 h后收集细胞,按照TRIzol说明书抽提细胞中的总RNA,-80℃冻存备用。采用实时荧光定量-PCR检测结肠癌细胞中miR-375的表达,以U6作为内参照,引物购自广州锐博公司。实时荧光定量-PCR检测细胞中AEG-1mRNA的表达情况,以GAPDH作为内参,引物购自TaKaRa公司。结果采用2-ΔΔCt法计算目的基因的相对表达量,ACt=目的基因平均Ct值-内参基因平均Ct值。6.MTT法检测细胞的生长情况：将转染后的实验组和阴性对照组细胞分别接种于96孔培养板中,每孔接种细胞约3000个(每孔加培养液100μL),置于37℃、CO2体积分数为5%的培养箱中进行培养。分别于培养4h、24 h、48 h和72 h后加入MTT工作液,每孔加入0.5% MTT 20μL,继续培养4h后,轻轻吸出孔内液体,然后在每孔中加入二甲基亚砜150μL,应用全自动酶标仪,检测波长490 nm下的吸光度(OD)值。以时间作为横坐标,计算所得OD值的均数为纵坐标,绘制细胞生长曲线。7.流式细胞术检测细胞周期：转染48 h后消化收集细胞,制成单细胞悬液,然后1200 r/min离心5 min,弃上清。预冷PBS洗涤细胞2次,PI细胞染色,即加入1000 μL staining buffer (A)及10 μL reagent B染色,混匀后室温避光孵育30 min,立即上流式细胞仪进行检测。8.流式细胞术检测细胞凋亡：转染48 h后消化收集细胞,制成单细胞悬液,然后1200 r/min离心5 min,弃上清。预冷PBS洗涤细胞2次,用500μL1×binding buffer重悬细胞,加入5 μL FITC标记的annexin-V,加入10μL的P1。混匀后室温避光孵育5 min,立即上流式细胞仪检测。9.应用SPSS 20.0统计学软件对实验数据进行统计学分析。所有的细胞学实验均重复3次,计量数据以均数±标准差(mean±SD)表示,符合正态分布且方差齐者采用t检验或者ANOVA检验,方差不齐采用Dunnett T3校正,以P0.05为有统计学意义。结果1.miR-375在结肠癌细胞株中的表达情况：提取结直肠癌细胞株(Caco2、 HCT116、SW620、SW480)中的总RNA,以U6作为内参,运用实时定量-PCR法检测miR-375的相对表达量。结果采用2-△Ct法计算miR-375的相对表达量。其中以HCT116细胞中miR-375的表达水平最低,因此选取HCT116细胞株作为我们进一步研究的对象。2. miR-375 mimics对HCT 116细胞中miR-375和AEG-1 mRNA表达的影响：miR-375 mimics瞬时转染HCT116细胞48 h后,采用实时定量-PCR检测转染后实验组及阴性对照组HCT116细胞中miR-375和AEG-1 mRNA的表达水平。结果显示,转染miR-375 mimic后miR-375的表达水平明显上调,其表达水平约是阴性对照组的2000倍(P0.01),同时miR-375的高表达导致AEG-1mRNA表达下调(P0.05)。以上结果提示,miR-375 mimics可上调miR-375的表达,同时过表达miR-375可抑制AEG-1 mRNA的表达。3.miR-375过表达对HCT116细胞生长的影响：MTT法检测结果显示,miR-375 mimics组细胞转染24 h、48 h、72 h时的OD值均低于阴性对照组,差异有统计学意义(P0.01)。miR-375过表达对HCT116细胞的活力具有明显抑制作用。4.miR-375过表达对HCT116细胞周期的影响：流式细胞仪检测转染48h后各组细胞的周期分布,结果显示,miR-375 mimics组与阴性对照组在G1期、S期和G2期细胞所占比例分别为(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组间经比较,miR-375 mimics组G1期细胞所占比例明显增多(P0.01),S期细胞所占比例明显减少(P0.05)。5.miR-375过表达对HCT116细胞凋亡率的影响：流式细胞仪检测转染48 h后各组细胞的凋亡率,结果显示,miR-375 mimics组和阴性对照组的细胞凋亡率分别为(8.468±1.546)%和(6.252±1.201)%,阴性对照组的细胞凋亡率明显低于miR-375 mimics组,差异有统计学意义(P0.05)。结论1. miR-375mimics组中miR-375表达量较对照组明显上调；miR-375抑制结肠癌细胞HCT116的细胞活性,介导细胞周期阻滞并促进其凋亡2.miR-375高表达可以显著抑制AEG-1 mRNA的表达水平,miR-375作为一种抑癌因子,在结肠癌中可能通过抑制癌基因AEG-1的表达发挥抑癌作用。
[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.
【学位授予单位】：南方医科大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R735.35

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