微小RNA-1284在胃癌中作用及机制的研究

发布时间：2018-04-24 14:07

本文选题：miR-1284 + 胃癌　；参考：《广西医科大学》2016年博士论文

【摘要】：1背景与目的胃癌是我国最常见的消化道恶性肿瘤,年患病率和死亡率均为世界平均水平的2倍以上。目前临床上主要利用手术、化疗和放疗等综合手段治疗胃癌,但胃癌总体生存率并没有明显改善,特别是进展期胃癌出现侵袭及转移后不仅加重机体损害而且疾病预后恶化。目前,传统的肿瘤药物治疗一般是指化学治疗,具有不分敌我、毒性较大的特点,分子靶向治疗比传统的化疗特异性强、毒副反应小。以EFGR和ALK基因突变的非小细胞肺癌治疗为例,过去传统的化疗有效率始终徘徊在20%到30%左右,生存率在9~10个月,采用抑制此类基因突变的分子靶向药物,治疗有效率能翻一番,超过50%~60%,生存率可延长至18个月。因此,靶向治疗可能成为未来肿瘤的个体化治疗的关键,探寻细胞调控的关键分子靶点已成为当前肿瘤治疗研究的热点。微小RNA((micro RNA,mi RNA))作为内源性非编码单链小RNA,其具有高效调控癌基因或抑癌基因的生物学特性,与肿瘤细胞的生长、侵袭和凋亡等密切相关,被认为是高效靶向调控细胞途径之一。最新的胃癌mi RNA表达谱芯片筛选研究报道了与胃癌相关的多个mi RNA,其中报道了mi R-1284在胃癌淋巴结转移阳性组织比原胃癌组织表达少,然而,对mi R-1284在胃癌发生发展中的功能和作用机制并未见有深入研究和探讨。为了验证芯片结果的准确性,本研究拟通过荧光定量PCR方法检测mi R-1284在胃正常细胞株、胃癌细胞系、胃癌组织和癌旁正常组织的表达情况,明确其在胃癌中的表达,并分析其与患者年龄、组织学类型、淋巴结转移、远处转移、病理分期等的相关性,明确其在胃癌临床诊断和预后中的意义。同时构建mi R-1284慢病毒载体,观察目的mi R-1284在体内外对胃癌肿瘤细胞的功能作用,利用基因芯片、双荧光素酶报告基因实验、实时定量PCR和Western Blot蛋白印迹实验检测靶基因。明确目的mi R-1284与其靶基因在胃癌中的关系,阐明mi R-1284在胃癌发生发展中的功能和作用机制。2方法2.1应用Trizol试剂提取胃癌组织和细胞的总RNA,应用荧光定量PCR方法检测mi R-1284在胃正常细胞株、胃癌细胞系、胃癌组织和癌旁正常组织的表达情况,并分析其与患者年龄、组织学类型、淋巴结转移、远处转移、病理分期等的相关性。2.2构建包含目的mi R-1284的荧光慢病毒载体,阳性重组克隆进行测序验证,收集重组过表达目的mi R-1284的病毒颗粒;Transwell法鉴定不同胃癌细胞的迁移和侵袭能力;采用目的mi R-1284慢病毒颗粒和阴性对照病毒载体分别转染胃癌细胞MGC-803和SGC-7901,嘌呤霉素筛选过表达mi R-1284胃癌稳定细胞株,荧光定量PCR检测转染细胞株mi R-1284的表达;Transwell法和划痕实验检测mi R-1284对胃癌细胞迁移侵袭能力的影响;CCK-8法检测mi R-1284对胃癌细胞增殖能力的影响;流式细胞术检测mi R-1284对胃癌细胞周期和凋亡的影响;Hoechst染色法和AO-EB双染色法检测mi R-1284对胃癌细胞凋亡的影响。2.3将转染后稳定细胞株的细胞悬液200μl(6×106个/ml)每只,在裸鼠左侧腋窝皮下注射构建人胃癌裸鼠皮下移植瘤模型,隔2天1次,用游标卡尺测量肿瘤体长径(a)和短径(b),计算相对瘤体体积(RTV)并绘制肿瘤生长曲线,21天后脱颈椎法处死裸鼠,完整切取肿瘤组织并用HE和TUNEL染色确认肿瘤组织和检测组织细胞凋亡情况;将转染后稳定细胞株的细胞悬液200μl(4×105个/ml)每只,注射入裸鼠尾静脉构建胃癌裸鼠转移模型,第36天后脱颈椎法处死裸鼠,完整切取裸鼠肺部和肝脏组织,并用HE染色检测胃癌肝肺转移情况。2.4采用全基因表达谱芯片检测mi R-1284过表达慢病毒转染胃癌细胞显著差异表达的基因;生物信息学预测软件预测目的mi R-1284的靶基因,利用双荧光素酶报告基因实验验证靶基因;应用实时定量PCR和Western Blot蛋白印迹实验检测靶基因EIF4A1及其下游基因cMyc、MMP12、JUN、CDH1的m RNA及蛋白表达,应用免疫组化法检测胃癌标本靶基因EIF4A1蛋白表达。3结果3.1 q RT-PCR结果显示,74例胃癌患者中,26例胃癌患者的胃癌组织mi R-1284表达量高于癌旁正常组织,占35.14%;48例胃癌患者的胃癌组织mi R-1284表达量低于癌旁正常组织,占64.86%,mi R-1284在胃癌组织中的表达量明显低于胃癌旁的正常组织(P㩳0.05),mi R-1284在胃癌细胞株AGS、HGC-27、MGC-803和SGC-7901的表达量低于在胃上皮细胞株GES-1的表达量(P㩳0.05)。mi R-1284表达量与胃癌患者的年龄、性别、组织学分级、TNM分期和淋巴结转移的相关无统计学显著意义(P㧐0.05),但mi R-1284表达量与胃癌的肿瘤大小及远处转移的相关有统计学显著意义(P㩳0.05)。3.2阳性重组克隆测序结果显示mi R-1284过表达载体重组序列无误;Transwell实验检测结果显示,与胃癌细胞株AGS、HGC-27相比,胃癌细胞株MGC-803和SGC-7901的迁移和侵袭细胞数较多(P㩳0.05);荧光定量PCR检测结果显示,mi R-1284在胃癌细胞株MGC-803和SGC-7901中实验组的表达量高于阴性对照组的表达量(P㩳0.05);Transwell实验检测结果显示,与阴性对照组相比,mi R-1284过表达的实验组胃癌细胞株MGC-803和SGC-7901的迁移和侵袭细胞数减少(P㩳0.05);划痕实验结果显示,胃癌细胞株SGC-7901在48小时时mi R-1284组细胞迁移间距为(312.42±16.87)μm较NC(阴性对照)组为(123.63±17.69)μm迁移细胞间距大,胃癌细胞株MGC-803在48小时时mi R-1284组细胞迁移间距为(112.46±11.18)μm较NC(阴性对照)组为(28.74±8.62)μm迁移细胞间距大,差异均有统计学显著意义(P㩳0.05);CCK-8实验检测结果显示与阴性对照组相比,mi R-1284过表达的实验组胃癌细胞株MGC-803和SGC-7901的增殖能力降低(P㩳0.05);流式细胞仪检测结果显示,与阴性对照组相比,mi R-1284过表达的实验组胃癌细胞株MGC-803和SGC-7901的细胞周期阻滞在G0/G1期,S期细胞比例减少,mi R-1284过表达的实验组胃癌细胞株MGC-803和SGC-7901的细胞凋亡率增加,差异均有统计学显著意义(P0.05);Hoechst和AO-EB双染检测结果显示,与阴性对照组相比,mi R-1284过表达的实验组胃癌细胞株MGC-803和SGC-7901的细胞凋亡率增加,差异均有统计学显著意义(P0.05)。3.3裸鼠皮下移植瘤模型肿瘤体积测量的结果显示,与阴性对照组相比,mi R-1284过表达的实验组胃癌细胞株SGC-7901、MGC-803的裸鼠皮下移植瘤模型肿瘤体积较小(P㩳0.05),HE染色确认皮下移植瘤组织为恶性胃癌组织,TUNEL检测结果显示mi R-1284过表达的实验组SGC-7901、MGC-803的裸鼠皮下移植瘤凋亡率增加(P㩳0.05);转移瘤模型的肝肺组织病理检查结果显示,mi R-1284过表达的实验组的肺转移率为10.00%,与阴性对照组的肺转移率70.00%相比,mi R-1284过表达的实验组胃癌细胞株MGC-803的细胞肺转移率降低(P㩳0.05)。3.4全基因表达谱芯片检测结果显示,按差异基因的筛选条件设定为log2"#Flod change"#"g1.5且P㩳0.05,共有143个基因为差异性表达,上调基因数97个,下调基因数46个;Mi Randa、Target Scan、micro RNA软件预测mi R-1284的靶基因交集为138个基因;芯片表达差异基因和生物信息学网站数据库预测靶基因的交集为EIF4A1、KLF10、C8orf4和SUMO1基因;双荧光素酶报告实验结果显示:EIF4A1 3'UTR-NC+mi RNA组和EIF4A1 3'UTR+mi RNA组的荧光素酶活性相比,差异有统计学显著意义(P0.05);EIF4A1 3'UTR-NC+mi RNA组和EIF4A1 3'UTR-MU+mi RNA组的荧光素酶活性相比,差异无统计学显著意义(P0.05);EIF4A13'UTR+mi RNA组和EIF4A1 3'UTR-MU+mi RNA组荧光素酶活性相比,EIF4A1 3'UTR-MU+mi RNA组的荧光素酶活性明显增加,差异有统计学显著意义(P0.05);荧光定量PCR和Western blotting实验结果显示:与阴性对照组比较,上调胃癌细胞株MGC-803、SGC-7901的mi R-1284表达后,实验组靶基因EIF4A1和c-Myc、MMP12、JUN的m RNA和蛋白表达降低,CDH1的m RNA和蛋白表达明显增加,差异均有统计学显著意义(P0.05);免疫组化结果显示,人胃癌组织中EIF4A1的蛋白表达量较匹配的癌旁正常组织低,差异有统计学显著意义(P0.05)。4结论4.1 mi R-1284在胃癌细胞株的表达水平低于正常胃上皮细胞株GES-1,在胃癌组织中的表达量明显低于胃癌组织,且mi R-1284的表达量与胃癌的肿瘤大小及远处转移呈负相关。4.2成功构建人mi R-1284过表达慢病毒载体和mi R-1284过表达的胃癌细胞株MGC-803、SGC-7901,mi R-1284过表达抑制胃癌细胞MGC-803、SGC-7901的迁移、侵袭、增殖能力,促进胃癌细胞MGC-803、SGC-7901的凋亡,使MGC-803、SGC-7901的细胞周期阻滞在G0/G1期和S期细胞比例减少。4.3 mi R-1284过表达抑制裸鼠皮下移植瘤模型肿瘤的生长速度,促进肿瘤组织的细胞凋亡,抑制裸鼠转移瘤模型的肿瘤细胞转移能力。4.4 mi R-1284过表达对胃癌细胞基因谱表达有影响,EIF4A1是mi R-1284的直接靶基因,mi R-1284过表达可使胃癌细胞株MGC-803、SGC-7901的EIF4A1和c-Myc、MMP12、JUN基因的m RNA和蛋白表达降低,CDH1的m RNA和蛋白表达增加。4.5在胃癌患者中EIF4A1的蛋白表达与mi R-1284表达量呈负相关,推测mi R-1284过表达极有可能直接通过EIF4A1/CDH1/JUN/MMP12和EIF4A1/c-Myc信号通路抑制胃癌的进展。创新点1体外双荧光素酶报告实验是研究micro RNA和靶基因间相互作用的有效手段,本研究采用了此技术验证mi R-1284和EIF4A1的相互作用,揭示了mi R-1284直接靶向调控EIF4A1的客观存在性。2 micro RNA是机体内调控基因表达的重要分子,与肿瘤的发生发展、侵袭转移密切相关,本课题从mi R-1284在胃癌中的表达入手,通过体外和体内两个方面进行研究,揭示和阐明了mi R-1284靶向调控EIF4A1对胃癌进展的影响其分子机制。
[Abstract]:1 background and objective gastric cancer is the most common malignant tumor in the digestive tract in China. The annual prevalence rate and mortality rate are more than 2 times more than the world average. At present, the main clinical use of surgery, chemotherapy and radiotherapy for the treatment of gastric cancer, but the overall survival rate of gastric cancer is not significantly improved, especially in the progression of gastric cancer after the invasion and metastasis. It not only aggravates the damage of the body but also worsens the prognosis of the disease. At present, the traditional treatment of tumor drugs generally refers to the chemical treatment, which has the characteristics of not dividing the enemy from the enemy, having a large toxicity, and the molecular targeting therapy is stronger than the traditional chemotherapy, and the toxic side effects are small. The traditional chemotherapy has been used as an example with the treatment of EFGR and ALK gene mutations in non small cell lung cancer. The efficiency is always around 20% to 30%, the survival rate is in 9~10 months. The effective rate can be doubled, the survival rate can be more than 50%~60%, and the survival rate can be extended to 18 months. Therefore, the target therapy may be the key to the individualized treatment of the tumor in the future, and the key molecular targets for cell regulation are explored. RNA (micro RNA (MI RNA)), as an endogenous non coding single strand RNA, has a close relationship with the growth, invasion and apoptosis of tumor cells, which is considered to be one of the highly effective targeting cell pathways. The latest gastric cancer mi RNA table is considered as an endogenous non coding single strand RNA. A number of MI RNA related to gastric cancer were reported by the spectrum chip screening. It was reported that the expression of MI R-1284 in gastric cancer lymph node metastasis was less than that of the original gastric carcinoma. However, the function and mechanism of MI R-1284 in the development of gastric cancer had not been further studied and discussed. The expression of MI R-1284 in normal gastric cell line, gastric cancer cell line, gastric cancer tissue and normal tissue were detected by fluorescence quantitative PCR, and its expression in gastric cancer was determined. The correlation with age, histological type, lymph node metastasis, distant metastasis and disease stage was analyzed, and the clinical diagnosis and diagnosis of gastric cancer were also made. The significance of MI R-1284 lentivirus vector was also constructed to observe the function of MI R-1284 on gastric cancer cells in vivo and in vivo and in vivo and in vivo, using gene chip, double luciferase reporter gene test, real-time quantitative PCR and Western Blot blot test to detect target genes. The purpose of this study was to determine the purpose of MI R-1284 and its target gene in gastric cancer. To elucidate the function and mechanism of MI R-1284 in the development of gastric cancer.2 method 2.1 using Trizol reagent to extract the total RNA of gastric cancer tissues and cells, and to detect the expression of MI R-1284 in normal gastric cell lines, gastric cancer cell lines, gastric cancer tissues and normal tissues by Trizol reagent, and to analyze the age and histology of the patients with the gastric cancer cell line, gastric cancer tissue and the carcinoma adjacent to the cancer. Type, lymph node metastasis, distant metastasis, pathological staging, and other correlation.2.2 constructs a fluorescent lentivirus carrier containing target mi R-1284, positive recombinant clones are sequenced and verified, and the recombinant virus particles that express mi R-1284 are collected; the migration and invasion ability of different gastric cancer cells are identified by Transwell method, and the target mi R-1284 lentivirus is used. MGC-803 and SGC-7901 were transfected by granular and negative control virus vectors respectively. The expression of MI R-1284 gastric cancer cell line was screened by purinomycin, the expression of MI R-1284 in the transfected cell line was detected by fluorescence quantitative PCR; the influence of MI R-1284 on the invasion ability of gastric cancer cells was detected by Transwell and scratch test; CCK-8 method was used to detect mi The effect of MI R-1284 on the cell cycle and apoptosis of gastric cancer cells by flow cytometry; the effect of Hoechst staining and AO-EB double staining on the apoptosis of gastric cancer cell line.2.3,.2.3 will be used for 200 mu (6 x 106 /ml) per mouse of stable cell suspension after transfection and subcutaneous injections in the left armpit of nude mice The subcutaneous tumor model of nude mice was built. 2 days 1 times, the tumor body length (a) and short diameter (b) were measured with the vernier caliper. The relative tumor volume (RTV) was calculated and the tumor growth curve was plotted. After 21 days, the nude mice were killed by deactivation of the cervical vertebra. The tumor tissue was stained with HE and TUNEL, and the apoptosis of the tissue cells was confirmed. The cell suspension of the stable cell line was 200 L (4 x 105 /ml) each, and the nude mice were injected into the tail vein of the nude mice to construct the metastatic model of gastric cancer in nude mice. After thirty-sixth days, the nude mice were killed by the deactivation of the cervical vertebra. The lung and liver tissues of the nude mice were completely cut out, and the liver and lung metastasis of the gastric cancer was detected by HE staining. The full gene expression spectrum chip was used to detect the slow expression of MI R-1284. The gene was transfected by the virus, and the target gene of MI R-1284 was predicted by the bioinformatics prediction software, and the target gene was verified by the double luciferase reporter gene experiment. The target gene EIF4A1 and its downstream gene cMyc, MMP12, JUN, CDH1 m RNA and protein were detected by real-time quantitative PCR and Western Blot blotting. The results of the expression of the target gene EIF4A1 protein expression of.3 3.1 Q RT-PCR by immunohistochemistry showed that in 74 cases of gastric cancer, the expression of MI R-1284 in gastric cancer tissues of 26 cases of gastric cancer was higher than that of normal tissue, accounting for 35.14%, and the expression of MI R-1284 in gastric cancer tissues of 48 cases of gastric cancer was lower than that of normal tissue adjacent to cancer, accounting for 64.86%, MI. The expression of R-1284 in gastric cancer tissues was significantly lower than that of normal tissue adjacent to gastric cancer (P? 0.05). The expression of MI R-1284 in gastric cancer cell lines AGS, HGC-27, MGC-803 and SGC-7901 was lower than that in gastric epithelial cell strain GES-1 (P? 0.05).Mi R-1284 expression and age, sex, histological grading, staging and lymph node metastasis of gastric cancer patients. The correlation was not statistically significant (P? 0.05), but the expression of MI R-1284 was significantly correlated with the tumor size and distant metastasis of gastric cancer (P? 0.05).3.2 positive recombinant cloning sequencing results showed that MI R-1284 overexpressed vector recombinant sequence was unmistakable; Transwell experimental results showed that gastric cancer was compared with AGS, HGC-27, gastric cancer. The number of cell migration and invasion of cell lines MGC-803 and SGC-7901 was more (P? 0.05), and the results of fluorescence quantitative PCR detection showed that the expression of MI R-1284 in the gastric cancer cell line MGC-803 and SGC-7901 was higher than that of the negative control group (P? 0.05). The Transwell test results showed that MI R-1284 was over expressed in comparison with the negative control group. The number of migration and invasion cells of gastric cancer cell lines MGC-803 and SGC-7901 decreased (P? 0.05), and the results of scratch test showed that the cell migration interval of the MI R-1284 group was (312.42 + 16.87) mu m at 48 hours and (123.63 + 17.69) mu m migrating cell spacing in the mi R-1284 group, and MGC-803 of the gastric cancer cell line was mi R at 48 hours. The cell migration distance of group -1284 was (112.46 + 11.18) mu m than that of NC (28.74 + 8.62) mu m (28.74 + 8.62). The difference was significant (P? 0.05). The results of CCK-8 test showed that the proliferation ability of gastric cancer cell line MGC-803 and SGC-7901 was lower than that of negative control group (P? 0.05). The results of flow cytometry showed that compared with the negative control group, the cell cycle block of gastric cancer cell line MGC-803 and SGC-7901 in the MI R-1284 overexpressed experimental group was in G0/G1 stage, and the proportion of S phase cells decreased, and the apoptosis rate of MGC-803 and SGC-7901 increased in the experimental group of MI R-1284, and the difference was statistically significant. (P0.05), the results of Hoechst and AO-EB double staining showed that compared with the negative control group, the apoptosis rate of MGC-803 and SGC-7901 in the experimental group of gastric cancer cells with overexpression of MI R-1284 increased, and the difference was statistically significant (P0.05) in.3.3 nude mice, the results of the tumor volume measurement of the subcutaneous transplanted tumor model showed that the MI R-128 compared with the negative control group. 4 over expression of the experimental group of gastric cancer cell line SGC-7901, MGC-803 nude mice subcutaneous tumor model tumor volume smaller (P? 0.05), HE staining confirmed that subcutaneous transplantation tumor tissue was malignant gastric cancer tissue, TUNEL test results showed that MI R-1284 overexpressed experimental group SGC-7901, MGC-803 nude mice subcutaneous transplantation tumor apoptosis rate increased (P? 0.05); metastatic tumor model The pathological examination of liver and lung tissue showed that the lung metastasis rate of the MI R-1284 overexpressed experimental group was 10%, compared with the lung metastasis rate of the negative control group 70%. The lung metastasis rate of the gastric cancer cell line MGC-803 in the MI R-1284 overexpressed group was reduced (P? 0.05).3.4 whole gene expression spectrum chip detection results showed that the differential gene was screened by the differential gene screening. The condition was set as log2 "#Flod change", "g1.5 and P? 0.05, there were 143 genes of differential expression, 97 up-regulated genes and 46 down regulated genes; Mi Randa, Target Scan, micro RNA software predicted the target gene of MI to be 138 genes, and the intersection of chip expression differentially gene and bioinformatics website database predicted target gene. F4A1, KLF10, C8orf4 and SUMO1 genes, and the results of the double luciferase reporter experiment showed that the difference of luciferase activity of EIF4A1 3'UTR-NC+mi RNA group and EIF4A1 3'UTR+mi RNA group was statistically significant (P0.05), and there was no significant difference in the activity of luciferase activity. (P0.05); the luciferase activity of the EIF4A1 3'UTR-MU+mi RNA group was significantly increased in the group of EIF4A13'UTR+mi RNA and the EIF4A1 3'UTR-MU+mi RNA group, and the difference was statistically significant (P0.05). After the expression of R-1284, the target gene EIF4A1 and c-Myc, MMP12, JUN, m RNA and protein expression decreased, the m RNA and protein expression of CDH1 increased significantly, and the difference was statistically significant (P0.05). The immunohistochemical results showed that the expression of EIF4A1 protein in human gastric carcinoma tissue was lower than that of the normal tissue adjacent to the cancer, and the difference was statistically significant. The expression level of P0.05.4 conclusion 4.1 mi R-1284 in gastric cancer cell line is lower than that of normal gastric epithelial cell strain GES-1, and the expression in gastric cancer tissue is significantly lower than that of gastric cancer tissue, and the expression of MI R-1284 is negatively correlated with the size and distant metastasis of gastric cancer, which is a successful construction of the MI R-1284 overexpressed lentivirus carrier and MI R-1284. The expression of MGC-803, SGC-7901, and MI R-1284 overexpressed the migration, invasion, and proliferation of gastric cancer cell MGC-803, SGC-7901, and promoted the apoptosis of MGC-803 and SGC-7901 in gastric cancer cells, so that the cell cycle block in MGC-803 and SGC-7901 decreased in the G0/G1 phase and the S phase cells to inhibit the subcutaneous tumor model in nude mice The growth rate of tumor, promoting the apoptosis of tumor tissue, inhibiting the metastasis ability of tumor cells in the metastatic tumor model of nude mice,.4.4 mi R-1284 overexpression affects gene expression of gastric cancer cells. EIF4A1 is the direct target gene of MI R-1284. Mi R-1284 overexpression can make the gastric cancer cell strain MGC-803, SGC-7901 EIF4A1 and c-Myc The expression of M RNA and protein decreased, CDH1 m RNA and protein expression increased.4.5 expression in gastric cancer patients with MI R-1284 expression in negative correlation. It is presumed that MI R-1284 overexpression may inhibit the progression of gastric cancer directly through EIF4A1/CDH1/JUN/MMP12 and signaling pathway. Innovation point 1 in vitro double Luciferase Report The test is an effective means to study the interaction between micro RNA and target genes. This study uses this technique to verify the interaction between MI R-1284 and EIF4A1, and reveals that the objective existence.2 micro RNA of MI R-1284 targeting EIF4A1 is an important molecule in the regulation of gene expression in the body, which is closely related to the development of the tumor and the invasion and metastasis of the tumor. This topic begins with the expression of MI R-1284 in gastric cancer and through two aspects in vitro and in vivo, reveals and clarifies the molecular mechanism of the effect of MI R-1284 targeting EIF4A1 on the progression of gastric cancer.

【学位授予单位】：广西医科大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R735.2

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