IncRNAs HOTAIR参与胃癌免疫逃逸的机制研究
发布时间：2018-09-19 10:01
【摘要】：胃癌(Gastric cancer, GC)不仅仅是世界上最为常见的恶性肿瘤之一,其死亡率亦居恶性肿瘤的前列。尽管人们认识到了胃癌的早期诊断对改善其预后的重要性,但大多数胃癌患者就诊时已经为进展期,从而错失了诊治的最佳时机。由于传统的手术、化疗及放疗在进展期胃癌患者的治疗中效果欠佳,因此新的、更为有效的治疗方式,比如免疫治疗,受到越来越多的关注。近期的研究表明,长链非编码RNA (long non-coding RNA, IncRNAs) HOTAIR在肿瘤的发展及转移方面起着重要作用。同时,胃癌组织中HOTAIR和HLA-G表达水平的相关性分析表明,HOTAIR和HLA-G水平呈正性相关,提示HOTAIR在肿瘤的免疫逃逸方面起着重要作用。然而,HOTAIR在肿瘤免疫逃逸中的可能机制还未明确。在本研究中,我们首先采用实时荧光定量PCR技术对60例胃癌患者癌组织中HOTAIR的表达水平进行了检测,并且和已经表明与肿瘤的免疫逃逸机制有密切关系的人白细胞抗原-G (HLA-G)做了相关性分析。结果发现,在胃癌组织中HOTAIR呈显著高表达,而且HOTAIR表达水平与癌组织中HLA-G mRNA水平及外周血中的HLA-G蛋白水平高度正性相关。然后,我们对HOTAIR进行了获得性和失活性功能研究,结果表明,HOTAIR能在体外实验中上调HLA-G的mRNA水平和蛋白水平。另一方面,生物信息学分析表明HOTAIR与miR-152之间存在相互作用关系,提示miR-152可能会参与HOTAIR对HLA-G的表达调节。我们的组织学研究发现,在胃癌组织中miR-152的表达亦发生了改变,而且miR-152的水平与HOTAIR的水平呈负性相关。我们采用报告基因检测技术在体外细胞实验中也证实了miR-152对胃癌细胞的HLA-G表达存在负性调节作用,而突变实验研究表明HOTAIR的过表达能够消除miR-152诱导的HLA-G 3'UTR活性降低。因此,我们认为：高表达的HOTAIR有可能通过抑制miR-152来上调HLA-G表达从而参与胃癌的免疫逃逸。不仅如此,本研究也表明了将HOTAIR用于胃癌免疫治疗以改善患者的预后、提高患者生存率的潜在可能性。第一部分IncRNAs HOTAIR在胃癌组织中的表达及可能作用机制研究目的研究HOTAIR在胃癌组织中的表达及可能作用机制。研究方法获取山东大学齐鲁医院60例胃癌患者癌组织及相应癌旁组织(距肿瘤边缘5cm)标本,同时采集相应患者术前1天的外周血标本。所有患者术前检查均未发现其他脏器肿瘤病变,均接受根治性胃癌切除手术,术前均未接受针对肿瘤的放疗、化疗及免疫治疗,所有肿瘤标本均经术后病理证实。本研究符合《赫尔辛基宣言》标准,通过山东大学齐鲁医院伦理委员会批准,所有纳入患者均在手术前签署知情同意书。1.胃癌组织及相应癌旁组织中HOTAIR、miR-152及HLA-G的表达检测常规胃癌根治术后留取胃癌组织及相应癌旁组织标本,编号后立即放入-80℃液氮罐冷冻备用。应用Trizol法分别提取胃癌组织及相应癌旁组织标本的总RNA。为了检测HLA-G mRNA和HOTAIR,按产品说明使用SuperScript Ⅲ Reverse Transcriptase将oligo-dT primers标定的RNA反转录入cDNA,用磷酸甘油酸脱氢酶(GAPDH)作为内参。为了检测成熟的miR-152,用mirVana miRNA Isolation kit提取小RNA,用小核RNA U6作为内参。用Takara公司的ABI 7300实时荧光定量试剂盒(SYBR Premix Ex Taq)实施实时荧光定量PCR(qRT-PCR)检测和收集数据。对每个样品进行重复检测3次,并进行溶解曲线检测。实验数据采用比较阈值法(2-△△CT法)进行相对定量分析。2.相应外周血标本中可溶性HLA-G (sHLA-G)浓度检测外周血标本用乙二胺四乙酸(EDTA)做处理留取血浆后存放入-80℃液氮罐冷冻备用。测定前将血浆标本在无血清培养基孵育48小时,然后用sHLA-G试剂盒按说明书使用检测外周血中可溶性HLA-G的浓度。重复所有实验3次,然后计算3次实验结果的平均值,以此作为最后的实验结果。3.生物信息学分析HOTAIR转录和miR-152之间的相互作用用DIANA TOOLS对HOTAIR转录和miR-152之间的相互作用进行生物信息学分析。4.统计分析所得数据以均数±标准差(X±SD)表示。用Pearson's correlation法进行相关分析。单因素方差分析或x2检验法分析组间的差异。用SPSS 17.0统计学软件进行统计分析。P0.05被认为差异有显著统计学意义。研究结果1. HOTAIR在胃癌组织中的表达水平是癌旁组织的1.76倍。2. HOTAIR和HLA-G表达水平之间存在显著正性相关(R2=0.57),HOTAIR表达和HLA-G浓度之间也存在显著正性相关(R2=0.582)。3. HOTAIR转录中存在miR-152的三个潜在结合域,在这些结合域中常见的程序模块序列"GCACUG"被"AAGAGA"代替,从而显露一个Mut-HOTAIR用于接下来的突变研究。4.miR-152在胃癌组织中的表达水平是癌旁组织的64%。HOTAIR和miR-152表达呈显著负性相关(R2=0.5168)。结论1. 胃癌组织中HOTAIR呈高表达水平。2. HOTAIR水平和HLA-G在胃癌组织中的mRNA水平及外周血中的蛋白水平呈正相关；HOTAIR参与了胃癌组织中HLA-G的表达上调。3.HOTAIR转录中存在miR-152的三个潜在结合域,在这些结合域中常见的程序模块序列"GCACUG"被"AAGAGA"代替,从而显露一个Mut-HOTAIR用于接下来的突变研究。4.胃癌组织中miR-152呈低表达水平,且与HOTAIR呈显著负相关。HOTAIR能负性调节miR-152的表达。第二部分IncRNAs HOTAIR在胃癌细胞免疫逃逸中的作用机制研究目的研究HOTAIR参与胃癌细胞免疫逃逸的可能机制。研究方法两种人胃癌细胞株SGC7901和MGC-803均为中国科学院提供。1.细胞培养胃癌细胞株SGC7901和MGC-803用含补充10%胎牛血清(fetalbovineserum, FBS)和抗生素(1%链霉素／青霉素)的RMPI-1640培养液培养,培养在37℃恒温、含5%CO2的饱和湿度的培养箱中进行。2. HOTAIR高表达质粒的构建和小干扰RNA(siRNAs)用pCDNA3.1载体构建HOTAIR高表达质粒,其引物序列forward: 5'-CCAGTTCTCAGGCGAGAGCC-3'; reverse:5'-TTTATATTCAGGACATGTAA-3'。采用定点诱变法对HOTAIR中miR-152结合位点进行定点突变诱导,从而构建Mut-HOTAIR质粒。所有用于转染的质粒载体经扩增后由DNA Midiprep kit提取。siRNAs及对照siRNA (si-NC)购买于Invitrogen公司。三个独立的HOTAIR siRNAs序列为：siHOTAIR-1:GAACGGGAGUACAGAGAGAUU; siHOTAIR-2:CCACAUGAACGCCCAGAGAUU; siHOTAIR-3:UAACAAGACCAGAGAGCUGUU。3.细胞转染miR-152的高表达质粒和干扰质粒及其对照RNA购买于GenePharma公司。SGC7901和MGC-803细胞(2.5×105)接种于6孔培养板上,孵育24小时后将HOTAIR质粒、siHOTAIR序列、miR-152的高表达质粒以及干扰质粒参照Lipofectamin 2000说明书分别转染入胃癌细胞中。4.转染后胃癌细胞株中HOTAIR、miR-152及HLA-G的表达检测采用Trizol法提取转染后胃癌细胞中的总RNA。为了检测HLA-G mRNA和HOTAIR,按产品说明书使用SuperScript Ⅲ Reverse Transcriptase将oligo-dT primers标定的RNA反转录入cDNA,用磷酸甘油酸脱氢酶(GAPDH)作为内参。为了检测成熟的miR-152,用mirVana miRNA Isolation kit提取小RNA,用小核RNA U6作为内参。用Takara公司的ABI 7300实时荧光定量试剂盒(SYBR Premix Ex Taq)进行实时荧光定量PCR (qRT-PCR)检测和数据收集。每个样品进行3次重复检测,并进行溶解曲线检测。采用比较阈值法对实验数据进行相对定量分析。5.转染后胃癌细胞株SGC7901和MGC-803培养液中可溶性HLA-G浓度检测收集在无血清培养基中孵育48小时后的细胞培养上清液,用sHLA-G试剂盒按说明书检测培养上清液中可溶性HLA-G的浓度。重复所有实验3次,然后计算3次实验结果的平均值,以此作为最后的实验结果。6.双荧光素酶检测用PGL3载体构建含有HLA-G 3'UTR的萤光素酶报告质粒。引物为：HLA-G-3'UTR-forward:5'-GGGGTACCGATGGGTGAGTTCAACGAGA-3'; HLA-G-3'UTR-reverse:5'-CCCTCGAGAGGGTGGGTGTCATCAGG-3'.然后将PGL3-HLA-G和HOTAIR、 siHOTAIR、miR-152高表达质粒及干扰质粒共转染,进行荧光素酶分析。转染24小时后,用双荧光素酶报告1000分析系统检测萤光素酶活性。7.统计分析所得数据以均数±标准差(X±SD)表示。单因素方差分析或x2检验法分析组间的差异。用SPSS 17.0统计学软件进行统计分析。P0.05被认为差异有显著统计学意义。研究结果1. HOTAIR在两组胃癌细胞内的表达水平：用HOTAIR高表达质粒转染后均增长大约16.1倍,用干扰质粒(si-HOTAIR)转染后均下降约63%。2.HLA-G在两组胃癌细胞内的mRNA水平和蛋白水平：用HOTAIR高表达质粒转染后均增长大约2倍,用干扰质粒(si-HOTAIR)转染后均下降约50%。3.miR-152在两组胃癌细胞内的表达水平：用HOTAIR高表达质粒转染后均下降约52%,用si-HOTAIR转染后均增长大约1.72倍,而Mut-HOTAIR对miR-152表达没有显著影响；用miR-152高表达质粒转染后均增长约17.3倍,用miR-152干扰质粒转染后均下降约67%。4. HLA-G在两组细胞内的mRNA水平和蛋白水平：用miR-152高表达质粒转染后均下降约52%,用miR-152干扰质粒转染后均增长大约2.2倍；用HOTAIR高表达质粒转染后均增长约1.8倍,用siHOTAIR转染后均下降约47%；而用mut-HOTAIR转染后无明显变化。5.两组胃癌细胞内的HLA-G 3'UTR活性：用miR-152高表达质粒转染后均下降约51%,用miR-152干扰质粒转染后均增长约1.75倍；用HOTAIR高表达质粒转染后均增长约1.53倍,用siHOTAIR转染后均下降约48%；而用mut-HOTAIR转染后无明显变化；用miR-152高表达质粒与HOTAIR质粒共转染后均下降约1.2%,用miR-152高表达质粒与mut-HOTAIR质粒共转染后均下降约52%。结论1. HOTAIR能促进胃癌细胞HLA-G的表达。2. 胃癌细胞中,HOTAIR与miR152有直接的交互影响,并负性调节miR-152的表达。3. HLA-G的相对表达量、浓度可被miR-152下调,被HOTAIR上调。miR-152诱导的HLA-G 3'UTR活性下调能被HOTAIR高表达所逆转。4. 胃癌细胞中HOTAIR通过干扰miR-152促进HLA-G表达。
[Abstract]:Gastric cancer (GC) is not only one of the most common malignancies in the world, but also one of the leading causes of mortality. Although the importance of early diagnosis of gastric cancer to improve prognosis has been recognized, most patients with gastric cancer are at an advanced stage, thus missing the best opportunity for diagnosis and treatment. Recent studies have shown that long non-coding RNA (IncRNAs) HOTAIR plays an important role in tumor development and metastasis. Correlation analysis of HOTAIR and HLA-G expression levels in gastric cancer tissues showed that there was a positive correlation between HOTAIR and HLA-G levels, suggesting that HOTAIR plays an important role in tumor immune escape. However, the possible mechanism of HOTAIR in tumor immune escape has not been clarified. In this study, we first used real-time fluorescent quantitative PCR in 60 cases. The expression of HOTAIR in gastric cancer tissues was detected and correlated with human leukocyte antigen G (HLA-G), which has been shown to be closely related to the mechanism of tumor immune escape. The acquired and inactivated functions of HOTAIR were studied. The results showed that HOTAIR could up-regulate the mRNA and protein levels of HLA-G in vitro. On the other hand, bioinformatics analysis showed that there was an interaction between HOTAIR and microRNA152, suggesting that microRNA152 might be involved. Our histological study found that the expression of microRNAs-152 was also changed in gastric cancer tissues, and the level of microRNAs-152 was negatively correlated with the level of HOTAIR. We also confirmed that microRNAs-152 had negative HLA-G expression in gastric cancer cells in vitro by reporter gene assay. It is suggested that the overexpression of HOTAIR may play an important role in the immune escape of gastric cancer by inhibiting the expression of HLA-G by inhibiting the expression of miR-152. Objective To study the expression and possible mechanism of IncRNAs HOTAIR in gastric cancer tissues. Methods 60 gastric cancer tissues from Qilu Hospital of Shandong University and their adjacent cancer groups were obtained. Tissue (5 cm from the edge of the tumor) specimens were taken and peripheral blood samples were collected from the corresponding patients one day before operation. All patients underwent radical gastrectomy without other organ tumor lesions. No radiotherapy, chemotherapy and immunotherapy were performed before operation. All tumor specimens were confirmed by postoperative pathology. Following the Helsinki Declaration and approved by the Ethics Committee of Qilu Hospital of Shandong University, all patients were informed consent before operation. 1. The expression of HOTAIR, miR-152 and HLA-G in gastric cancer tissues and corresponding adjacent tissues were detected after routine radical gastrectomy. The specimens of gastric cancer tissues and corresponding adjacent tissues were taken immediately after numbering. To detect HLA-G mRNA and HOTAIR, oligo-dT primers were reverse transcribed into DNA using SuperScript III Reverse Transcriptase and phosphoglycerol dehydrogenase (GAPDH) as internal reference according to product instructions. MirVana microRNA Isolation kit was used to extract small RNA, and micronucleus RNA U6 was used as internal reference. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect and collect data with Takara ABI 7300 Real-time Fluorescence Quantitative Kit (SYBR Premix Ex Taq). Relative quantitative analysis was performed by comparative threshold method (2-delta CT). 2. Soluble HLA-G (sHLA-G) concentration in peripheral blood samples was detected. The peripheral blood samples were treated with EDTA and stored in liquid nitrogen tank at - 80 C for refrigeration. The plasma samples were incubated in serum-free medium for 48 hours before determination. Then the sHLA-G kit was used to detect the concentration of soluble HLA-G in peripheral blood according to the instructions. All the experiments were repeated three times, and then the average value of the three experiments was calculated as the final experimental result. 3. Bioinformatics analysis of the interaction between HOTAIR transcription and microRNA152. Interaction between HOTAIR transcription and microRNA152 using DIANA TOOLS Bioinformatics analysis. 4. Statistical data were expressed as mean [SD]. Correlation analysis was performed using Pearson's correlation method. Differences between groups were analyzed by one-way ANOVA or x2 test. Statistical analysis was performed using SPSS 17.0 statistical software. P 0.05 was considered to be statistically significant. The expression level of HOTAIR in gastric cancer tissue was 1.76 times as high as that in adjacent tissues. 2. There was a significant positive correlation between the expression level of HOTAIR and HLA-G (R2 = 0.57), and a significant positive correlation between the expression of HOTAIR and HLA-G concentration (R2 = 0.582). 3. There were three potential binding domains in the transcription of HOTAIR, which were common in these binding domains. Sequence module sequence "GCACUG" was replaced by "AAGAGA" to reveal a Mut-HOTAIR for subsequent mutation studies. 4. The expression level of Mi-152 in gastric cancer tissues was 64% of that in adjacent tissues. There was a significant negative correlation between the expression of HOTAIR and Mi-152 (R2 = 0.5168). Conclusion 1. HOTAIR was highly expressed in gastric cancer tissues. There are three potential binding domains in the transcription of HOTAIR, in which the program module sequence GCACUG is replaced by AAGAGA, thus revealing a Mut-HOTAIR for subsequent prominence. 4. The expression of microRNA-152 in gastric cancer tissues was low and negatively correlated with HOTAIR. HOTAIR could negatively regulate the expression of microRNA-152. Part 2: The mechanism of IncRNAs HOTAIR in gastric cancer cell immune escape. Objective To investigate the possible mechanism of HOTAIR in gastric cancer cell immune escape. SGC7901 and MGC-803 were both provided by the Chinese Academy of Sciences. 1. Gastric cancer cell lines SGC7901 and MGC-803 were cultured in RMPI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (1% streptomycin / penicillin), and cultured in a constant temperature of 37 C and saturated humidity incubator containing 5% CO 2. 2. HOTAIR high expression plasmid Construction and small interfering RNA (siRNAs) construction of high expression plasmid of HOTAIR with pCDNA3.1 vector, its primer sequence forward: 5'-CCAGTTCTCAGCGAGAGCC-3'; reverse: 5'-TTTATTCAGGACATGTAA-3'. Using site-directed mutagenesis method to induce site-directed mutagenesis of MI-152 binding site in HOTAIR, the mut-HOTAIR plasmid was constructed. Three independent HOTAIR siRNAs were sequenced as follows: siHOTAIR-1: GAACGGAGUACAGAGAUU; siHOTAIR-2: CCACAUGAACGCCCAGAUU; siHOTAIR-3: UAACAAGAGAGAGAGAGAGCUGU.3. Cells transfected with high expression plasmids and interfering plasmids of RNA-152 According to RNA purchased from GenePharma. SGC7901 and MGC-803 cells (2.5 *105) were inoculated on a 6-well culture plate. After incubation for 24 hours, HOTAIR plasmid, siHOTAIR sequence, highly expressed plasmid of microRNA-152 and interfering plasmid were transfected into gastric cancer cells according to Lipofectamin 2000 instructions. 4. After transfection, HOTAIR, microRNA-152 and HLA-G were transfected into gastric cancer cells respectively. In order to detect HLA-G mRNA and HOTAIR, oligo-dT primers were reverse transcribed into DNA using SuperScript III Reverse Transcriptase according to the product specification, and phosphoglyceride dehydrogenase (GAPDH) was used as internal reference. MirVana microRNA was used to detect mature microRNAs-152. A Isolation kit was used to extract small RNA, and micronucleus RNA U6 was used as internal reference. Real-time fluorescence quantitative PCR (qRT-PCR) and data collection were performed with Takara ABI 7300 Real-time Fluorescence Quantitative Kit (SYBR Premix Ex Taq). Each sample was tested three times and the dissolution curves were detected. The experimental data were compared by comparison threshold method. Quantitative analysis.5.Soluble HLA-G concentration in the supernatant of gastric cancer cell lines SGC7901 and MGC-803 after transfection was detected and collected after incubation in serum-free medium for 48 hours. Soluble HLA-G concentration in the supernatant was detected by sHLA-G kit according to the instructions. All the experiments were repeated three times and the results of three experiments were calculated. The average was used as the final experimental result. 6. Double luciferase assay constructed a luciferase reporter plasmid containing HLA-G 3'UTR with PGL3 vector. The primers were HLA-G-3'UTR-forward: 5'-GGGGGTACCGATGGGGTGAGTTCAACGAGA-3'; HLA-G-3'UTR-reverse: 5'-CCCTCGAGGTGGTGATCAGG-3'. Then PGL3-HLA-GHOR and TAIR-HOR, SITAI-152'. Luciferase analysis was performed by co-transfection of high-expression plasmids and interfering plasmids. After 24 hours of transfection, the activity of luciferase was detected by double Luciferase Report 1000 analysis system. 7. The statistical data were expressed by mean (+ SD). Differences between groups were analyzed by one-way ANOVA or x2 test. SPSS 17.0 statistical software was used. Statistical analysis. P 0.05 was considered to have significant statistical significance. Results 1. HOTAIR expression in two groups of gastric cancer cells: after transfection with high-expression plasmid of HOTAIR, both increased by about 16.1 times, and after transfection with interfering plasmid (si-HOTAIR), both decreased by 63%. 2. HLA-G mRNA and protein levels in two groups of gastric cancer cells: after transfection with HOTAIR, the expression of HOTAIR increased by about 63%. The expression level of Mi-152 in gastric cancer cells of both groups decreased by about 52% after transfection with high-expression plasmid of HOTAIR, and increased by about 1.72 times after transfection with si-HOTAIR. However, the expression of Mi-152 was not significantly affected by Mut-HOTAIR. The mRNA and protein levels of HLA-G in both groups of cells were decreased by 52% after transfection with high-expression plasmids of microRNAs-152, and increased by about 2.2 times after transfection with high-expression plasmids of microRNAs-152, and increased by about 1.2 times after transfection with high-expression plasmids of HOTAIR. The activity of HLA-G 3'UTR in gastric cancer cells of both groups decreased by about 51% after transfection with highly expressed plasmid of Mi-152, increased by about 1.75 times after transfection with interfering plasmid of Mi-152, and increased by about 1.53 times after transfection with highly expressed plasmid of HOTAIR. After transfection, the expression of HLA-G in gastric cancer cells was decreased by 48%. However, there was no significant change after transfection with mut-HOTAIR. After transfection with high expression plasmid of Mi-152 and plasmid of HOTAIR, the expression of HOTAIR and plasmid of Mi-152 decreased by 1.2%. After transfection with plasmid of Mi-152 and plasmid of mut-HOTAIR, the expression of HLA-G in gastric cancer cells decreased by 52%. Conclusion 1. HOTAIR can promote the expression of HLA-G in gastric cancer cells. Direct interaction and negative regulation of the expression of microRNAs-152.3. The relative expression of HLA-G can be down-regulated by microRNAs-152 and up-regulated by HOTAIR. The down-regulation of HLA-G 3'UTR activity induced by microRNAs-152 can be reversed by the high expression of HOTAIR. 4. HOTAIR promotes the expression of HLA-G in gastric cancer cells by interfering with microRNAs-152.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2
本文编号：2249807
[Abstract]:Gastric cancer (GC) is not only one of the most common malignancies in the world, but also one of the leading causes of mortality. Although the importance of early diagnosis of gastric cancer to improve prognosis has been recognized, most patients with gastric cancer are at an advanced stage, thus missing the best opportunity for diagnosis and treatment. Recent studies have shown that long non-coding RNA (IncRNAs) HOTAIR plays an important role in tumor development and metastasis. Correlation analysis of HOTAIR and HLA-G expression levels in gastric cancer tissues showed that there was a positive correlation between HOTAIR and HLA-G levels, suggesting that HOTAIR plays an important role in tumor immune escape. However, the possible mechanism of HOTAIR in tumor immune escape has not been clarified. In this study, we first used real-time fluorescent quantitative PCR in 60 cases. The expression of HOTAIR in gastric cancer tissues was detected and correlated with human leukocyte antigen G (HLA-G), which has been shown to be closely related to the mechanism of tumor immune escape. The acquired and inactivated functions of HOTAIR were studied. The results showed that HOTAIR could up-regulate the mRNA and protein levels of HLA-G in vitro. On the other hand, bioinformatics analysis showed that there was an interaction between HOTAIR and microRNA152, suggesting that microRNA152 might be involved. Our histological study found that the expression of microRNAs-152 was also changed in gastric cancer tissues, and the level of microRNAs-152 was negatively correlated with the level of HOTAIR. We also confirmed that microRNAs-152 had negative HLA-G expression in gastric cancer cells in vitro by reporter gene assay. It is suggested that the overexpression of HOTAIR may play an important role in the immune escape of gastric cancer by inhibiting the expression of HLA-G by inhibiting the expression of miR-152. Objective To study the expression and possible mechanism of IncRNAs HOTAIR in gastric cancer tissues. Methods 60 gastric cancer tissues from Qilu Hospital of Shandong University and their adjacent cancer groups were obtained. Tissue (5 cm from the edge of the tumor) specimens were taken and peripheral blood samples were collected from the corresponding patients one day before operation. All patients underwent radical gastrectomy without other organ tumor lesions. No radiotherapy, chemotherapy and immunotherapy were performed before operation. All tumor specimens were confirmed by postoperative pathology. Following the Helsinki Declaration and approved by the Ethics Committee of Qilu Hospital of Shandong University, all patients were informed consent before operation. 1. The expression of HOTAIR, miR-152 and HLA-G in gastric cancer tissues and corresponding adjacent tissues were detected after routine radical gastrectomy. The specimens of gastric cancer tissues and corresponding adjacent tissues were taken immediately after numbering. To detect HLA-G mRNA and HOTAIR, oligo-dT primers were reverse transcribed into DNA using SuperScript III Reverse Transcriptase and phosphoglycerol dehydrogenase (GAPDH) as internal reference according to product instructions. MirVana microRNA Isolation kit was used to extract small RNA, and micronucleus RNA U6 was used as internal reference. Real-time fluorescence quantitative PCR (qRT-PCR) was used to detect and collect data with Takara ABI 7300 Real-time Fluorescence Quantitative Kit (SYBR Premix Ex Taq). Relative quantitative analysis was performed by comparative threshold method (2-delta CT). 2. Soluble HLA-G (sHLA-G) concentration in peripheral blood samples was detected. The peripheral blood samples were treated with EDTA and stored in liquid nitrogen tank at - 80 C for refrigeration. The plasma samples were incubated in serum-free medium for 48 hours before determination. Then the sHLA-G kit was used to detect the concentration of soluble HLA-G in peripheral blood according to the instructions. All the experiments were repeated three times, and then the average value of the three experiments was calculated as the final experimental result. 3. Bioinformatics analysis of the interaction between HOTAIR transcription and microRNA152. Interaction between HOTAIR transcription and microRNA152 using DIANA TOOLS Bioinformatics analysis. 4. Statistical data were expressed as mean [SD]. Correlation analysis was performed using Pearson's correlation method. Differences between groups were analyzed by one-way ANOVA or x2 test. Statistical analysis was performed using SPSS 17.0 statistical software. P 0.05 was considered to be statistically significant. The expression level of HOTAIR in gastric cancer tissue was 1.76 times as high as that in adjacent tissues. 2. There was a significant positive correlation between the expression level of HOTAIR and HLA-G (R2 = 0.57), and a significant positive correlation between the expression of HOTAIR and HLA-G concentration (R2 = 0.582). 3. There were three potential binding domains in the transcription of HOTAIR, which were common in these binding domains. Sequence module sequence "GCACUG" was replaced by "AAGAGA" to reveal a Mut-HOTAIR for subsequent mutation studies. 4. The expression level of Mi-152 in gastric cancer tissues was 64% of that in adjacent tissues. There was a significant negative correlation between the expression of HOTAIR and Mi-152 (R2 = 0.5168). Conclusion 1. HOTAIR was highly expressed in gastric cancer tissues. There are three potential binding domains in the transcription of HOTAIR, in which the program module sequence GCACUG is replaced by AAGAGA, thus revealing a Mut-HOTAIR for subsequent prominence. 4. The expression of microRNA-152 in gastric cancer tissues was low and negatively correlated with HOTAIR. HOTAIR could negatively regulate the expression of microRNA-152. Part 2: The mechanism of IncRNAs HOTAIR in gastric cancer cell immune escape. Objective To investigate the possible mechanism of HOTAIR in gastric cancer cell immune escape. SGC7901 and MGC-803 were both provided by the Chinese Academy of Sciences. 1. Gastric cancer cell lines SGC7901 and MGC-803 were cultured in RMPI-1640 medium supplemented with 10% fetal bovine serum (FBS) and antibiotics (1% streptomycin / penicillin), and cultured in a constant temperature of 37 C and saturated humidity incubator containing 5% CO 2. 2. HOTAIR high expression plasmid Construction and small interfering RNA (siRNAs) construction of high expression plasmid of HOTAIR with pCDNA3.1 vector, its primer sequence forward: 5'-CCAGTTCTCAGCGAGAGCC-3'; reverse: 5'-TTTATTCAGGACATGTAA-3'. Using site-directed mutagenesis method to induce site-directed mutagenesis of MI-152 binding site in HOTAIR, the mut-HOTAIR plasmid was constructed. Three independent HOTAIR siRNAs were sequenced as follows: siHOTAIR-1: GAACGGAGUACAGAGAUU; siHOTAIR-2: CCACAUGAACGCCCAGAUU; siHOTAIR-3: UAACAAGAGAGAGAGAGAGCUGU.3. Cells transfected with high expression plasmids and interfering plasmids of RNA-152 According to RNA purchased from GenePharma. SGC7901 and MGC-803 cells (2.5 *105) were inoculated on a 6-well culture plate. After incubation for 24 hours, HOTAIR plasmid, siHOTAIR sequence, highly expressed plasmid of microRNA-152 and interfering plasmid were transfected into gastric cancer cells according to Lipofectamin 2000 instructions. 4. After transfection, HOTAIR, microRNA-152 and HLA-G were transfected into gastric cancer cells respectively. In order to detect HLA-G mRNA and HOTAIR, oligo-dT primers were reverse transcribed into DNA using SuperScript III Reverse Transcriptase according to the product specification, and phosphoglyceride dehydrogenase (GAPDH) was used as internal reference. MirVana microRNA was used to detect mature microRNAs-152. A Isolation kit was used to extract small RNA, and micronucleus RNA U6 was used as internal reference. Real-time fluorescence quantitative PCR (qRT-PCR) and data collection were performed with Takara ABI 7300 Real-time Fluorescence Quantitative Kit (SYBR Premix Ex Taq). Each sample was tested three times and the dissolution curves were detected. The experimental data were compared by comparison threshold method. Quantitative analysis.5.Soluble HLA-G concentration in the supernatant of gastric cancer cell lines SGC7901 and MGC-803 after transfection was detected and collected after incubation in serum-free medium for 48 hours. Soluble HLA-G concentration in the supernatant was detected by sHLA-G kit according to the instructions. All the experiments were repeated three times and the results of three experiments were calculated. The average was used as the final experimental result. 6. Double luciferase assay constructed a luciferase reporter plasmid containing HLA-G 3'UTR with PGL3 vector. The primers were HLA-G-3'UTR-forward: 5'-GGGGGTACCGATGGGGTGAGTTCAACGAGA-3'; HLA-G-3'UTR-reverse: 5'-CCCTCGAGGTGGTGATCAGG-3'. Then PGL3-HLA-GHOR and TAIR-HOR, SITAI-152'. Luciferase analysis was performed by co-transfection of high-expression plasmids and interfering plasmids. After 24 hours of transfection, the activity of luciferase was detected by double Luciferase Report 1000 analysis system. 7. The statistical data were expressed by mean (+ SD). Differences between groups were analyzed by one-way ANOVA or x2 test. SPSS 17.0 statistical software was used. Statistical analysis. P 0.05 was considered to have significant statistical significance. Results 1. HOTAIR expression in two groups of gastric cancer cells: after transfection with high-expression plasmid of HOTAIR, both increased by about 16.1 times, and after transfection with interfering plasmid (si-HOTAIR), both decreased by 63%. 2. HLA-G mRNA and protein levels in two groups of gastric cancer cells: after transfection with HOTAIR, the expression of HOTAIR increased by about 63%. The expression level of Mi-152 in gastric cancer cells of both groups decreased by about 52% after transfection with high-expression plasmid of HOTAIR, and increased by about 1.72 times after transfection with si-HOTAIR. However, the expression of Mi-152 was not significantly affected by Mut-HOTAIR. The mRNA and protein levels of HLA-G in both groups of cells were decreased by 52% after transfection with high-expression plasmids of microRNAs-152, and increased by about 2.2 times after transfection with high-expression plasmids of microRNAs-152, and increased by about 1.2 times after transfection with high-expression plasmids of HOTAIR. The activity of HLA-G 3'UTR in gastric cancer cells of both groups decreased by about 51% after transfection with highly expressed plasmid of Mi-152, increased by about 1.75 times after transfection with interfering plasmid of Mi-152, and increased by about 1.53 times after transfection with highly expressed plasmid of HOTAIR. After transfection, the expression of HLA-G in gastric cancer cells was decreased by 48%. However, there was no significant change after transfection with mut-HOTAIR. After transfection with high expression plasmid of Mi-152 and plasmid of HOTAIR, the expression of HOTAIR and plasmid of Mi-152 decreased by 1.2%. After transfection with plasmid of Mi-152 and plasmid of mut-HOTAIR, the expression of HLA-G in gastric cancer cells decreased by 52%. Conclusion 1. HOTAIR can promote the expression of HLA-G in gastric cancer cells. Direct interaction and negative regulation of the expression of microRNAs-152.3. The relative expression of HLA-G can be down-regulated by microRNAs-152 and up-regulated by HOTAIR. The down-regulation of HLA-G 3'UTR activity induced by microRNAs-152 can be reversed by the high expression of HOTAIR. 4. HOTAIR promotes the expression of HLA-G in gastric cancer cells by interfering with microRNAs-152.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R735.2
【共引文献】
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