【摘要】：胃癌(Gastric cancer, GC)不僅僅是世界上最為常見的惡性腫瘤之一,其死亡率亦居惡性腫瘤的前列。盡管人們認識到了胃癌的早期診斷對改善其預后的重要性,但大多數胃癌患者就診時已經為進展期,從而錯失了診治的最佳時機。由于傳統(tǒng)的手術、化療及放療在進展期胃癌患者的治療中效果欠佳,因此新的、更為有效的治療方式,比如免疫治療,受到越來越多的關注。近期的研究表明,長鏈非編碼RNA (long non-coding RNA, IncRNAs) HOTAIR在腫瘤的發(fā)展及轉移方面起著重要作用。同時,胃癌組織中HOTAIR和HLA-G表達水平的相關性分析表明,HOTAIR和HLA-G水平呈正性相關,提示HOTAIR在腫瘤的免疫逃逸方面起著重要作用。然而,HOTAIR在腫瘤免疫逃逸中的可能機制還未明確。在本研究中,我們首先采用實時熒光定量PCR技術對60例胃癌患者癌組織中HOTAIR的表達水平進行了檢測,并且和已經表明與腫瘤的免疫逃逸機制有密切關系的人白細胞抗原-G (HLA-G)做了相關性分析。結果發(fā)現,在胃癌組織中HOTAIR呈顯著高表達,而且HOTAIR表達水平與癌組織中HLA-G mRNA水平及外周血中的HLA-G蛋白水平高度正性相關。然后,我們對HOTAIR進行了獲得性和失活性功能研究,結果表明,HOTAIR能在體外實驗中上調HLA-G的mRNA水平和蛋白水平。另一方面,生物信息學分析表明HOTAIR與miR-152之間存在相互作用關系,提示miR-152可能會參與HOTAIR對HLA-G的表達調節(jié)。我們的組織學研究發(fā)現,在胃癌組織中miR-152的表達亦發(fā)生了改變,而且miR-152的水平與HOTAIR的水平呈負性相關。我們采用報告基因檢測技術在體外細胞實驗中也證實了miR-152對胃癌細胞的HLA-G表達存在負性調節(jié)作用,而突變實驗研究表明HOTAIR的過表達能夠消除miR-152誘導的HLA-G 3'UTR活性降低。因此,我們認為：高表達的HOTAIR有可能通過抑制miR-152來上調HLA-G表達從而參與胃癌的免疫逃逸。不僅如此,本研究也表明了將HOTAIR用于胃癌免疫治療以改善患者的預后、提高患者生存率的潛在可能性。第一部分IncRNAs HOTAIR在胃癌組織中的表達及可能作用機制研究目的研究HOTAIR在胃癌組織中的表達及可能作用機制。研究方法獲取山東大學齊魯醫(yī)院60例胃癌患者癌組織及相應癌旁組織(距腫瘤邊緣5cm)標本,同時采集相應患者術前1天的外周血標本。所有患者術前檢查均未發(fā)現其他臟器腫瘤病變,均接受根治性胃癌切除手術,術前均未接受針對腫瘤的放療、化療及免疫治療,所有腫瘤標本均經術后病理證實。本研究符合《赫爾辛基宣言》標準,通過山東大學齊魯醫(yī)院倫理委員會批準,所有納入患者均在手術前簽署知情同意書。1.胃癌組織及相應癌旁組織中HOTAIR、miR-152及HLA-G的表達檢測常規(guī)胃癌根治術后留取胃癌組織及相應癌旁組織標本,編號后立即放入-80℃液氮罐冷凍備用。應用Trizol法分別提取胃癌組織及相應癌旁組織標本的總RNA。為了檢測HLA-G mRNA和HOTAIR,按產品說明使用SuperScript Ⅲ Reverse Transcriptase將oligo-dT primers標定的RNA反轉錄入cDNA,用磷酸甘油酸脫氫酶(GAPDH)作為內參。為了檢測成熟的miR-152,用mirVana miRNA Isolation kit提取小RNA,用小核RNA U6作為內參。用Takara公司的ABI 7300實時熒光定量試劑盒(SYBR Premix Ex Taq)實施實時熒光定量PCR(qRT-PCR)檢測和收集數據。對每個樣品進行重復檢測3次,并進行溶解曲線檢測。實驗數據采用比較閾值法(2-△△CT法)進行相對定量分析。2.相應外周血標本中可溶性HLA-G (sHLA-G)濃度檢測外周血標本用乙二胺四乙酸(EDTA)做處理留取血漿后存放入-80℃液氮罐冷凍備用。測定前將血漿標本在無血清培養(yǎng)基孵育48小時,然后用sHLA-G試劑盒按說明書使用檢測外周血中可溶性HLA-G的濃度。重復所有實驗3次,然后計算3次實驗結果的平均值,以此作為最后的實驗結果。3.生物信息學分析HOTAIR轉錄和miR-152之間的相互作用用DIANA TOOLS對HOTAIR轉錄和miR-152之間的相互作用進行生物信息學分析。4.統(tǒng)計分析所得數據以均數±標準差(X±SD)表示。用Pearson's correlation法進行相關分析。單因素方差分析或x2檢驗法分析組間的差異。用SPSS 17.0統(tǒng)計學軟件進行統(tǒng)計分析。P0.05被認為差異有顯著統(tǒng)計學意義。研究結果1. HOTAIR在胃癌組織中的表達水平是癌旁組織的1.76倍。2. HOTAIR和HLA-G表達水平之間存在顯著正性相關(R2=0.57),HOTAIR表達和HLA-G濃度之間也存在顯著正性相關(R2=0.582)。3. HOTAIR轉錄中存在miR-152的三個潛在結合域,在這些結合域中常見的程序模塊序列"GCACUG"被"AAGAGA"代替,從而顯露一個Mut-HOTAIR用于接下來的突變研究。4.miR-152在胃癌組織中的表達水平是癌旁組織的64%。HOTAIR和miR-152表達呈顯著負性相關(R2=0.5168)。結論1. 胃癌組織中HOTAIR呈高表達水平。2. HOTAIR水平和HLA-G在胃癌組織中的mRNA水平及外周血中的蛋白水平呈正相關；HOTAIR參與了胃癌組織中HLA-G的表達上調。3.HOTAIR轉錄中存在miR-152的三個潛在結合域,在這些結合域中常見的程序模塊序列"GCACUG"被"AAGAGA"代替,從而顯露一個Mut-HOTAIR用于接下來的突變研究。4.胃癌組織中miR-152呈低表達水平,且與HOTAIR呈顯著負相關。HOTAIR能負性調節(jié)miR-152的表達。第二部分IncRNAs HOTAIR在胃癌細胞免疫逃逸中的作用機制研究目的研究HOTAIR參與胃癌細胞免疫逃逸的可能機制。研究方法兩種人胃癌細胞株SGC7901和MGC-803均為中國科學院提供。1.細胞培養(yǎng)胃癌細胞株SGC7901和MGC-803用含補充10%胎牛血清(fetalbovineserum, FBS)和抗生素(1%鏈霉素／青霉素)的RMPI-1640培養(yǎng)液培養(yǎng),培養(yǎng)在37℃恒溫、含5%CO2的飽和濕度的培養(yǎng)箱中進行。2. HOTAIR高表達質粒的構建和小干擾RNA(siRNAs)用pCDNA3.1載體構建HOTAIR高表達質粒,其引物序列forward: 5'-CCAGTTCTCAGGCGAGAGCC-3'; reverse:5'-TTTATATTCAGGACATGTAA-3'。采用定點誘變法對HOTAIR中miR-152結合位點進行定點突變誘導,從而構建Mut-HOTAIR質粒。所有用于轉染的質粒載體經擴增后由DNA Midiprep kit提取。siRNAs及對照siRNA (si-NC)購買于Invitrogen公司。三個獨立的HOTAIR siRNAs序列為：siHOTAIR-1:GAACGGGAGUACAGAGAGAUU; siHOTAIR-2:CCACAUGAACGCCCAGAGAUU; siHOTAIR-3:UAACAAGACCAGAGAGCUGUU。3.細胞轉染miR-152的高表達質粒和干擾質粒及其對照RNA購買于GenePharma公司。SGC7901和MGC-803細胞(2.5×105)接種于6孔培養(yǎng)板上,孵育24小時后將HOTAIR質粒、siHOTAIR序列、miR-152的高表達質粒以及干擾質粒參照Lipofectamin 2000說明書分別轉染入胃癌細胞中。4.轉染后胃癌細胞株中HOTAIR、miR-152及HLA-G的表達檢測采用Trizol法提取轉染后胃癌細胞中的總RNA。為了檢測HLA-G mRNA和HOTAIR,按產品說明書使用SuperScript Ⅲ Reverse Transcriptase將oligo-dT primers標定的RNA反轉錄入cDNA,用磷酸甘油酸脫氫酶(GAPDH)作為內參。為了檢測成熟的miR-152,用mirVana miRNA Isolation kit提取小RNA,用小核RNA U6作為內參。用Takara公司的ABI 7300實時熒光定量試劑盒(SYBR Premix Ex Taq)進行實時熒光定量PCR (qRT-PCR)檢測和數據收集。每個樣品進行3次重復檢測,并進行溶解曲線檢測。采用比較閾值法對實驗數據進行相對定量分析。5.轉染后胃癌細胞株SGC7901和MGC-803培養(yǎng)液中可溶性HLA-G濃度檢測收集在無血清培養(yǎng)基中孵育48小時后的細胞培養(yǎng)上清液,用sHLA-G試劑盒按說明書檢測培養(yǎng)上清液中可溶性HLA-G的濃度。重復所有實驗3次,然后計算3次實驗結果的平均值,以此作為最后的實驗結果。6.雙熒光素酶檢測用PGL3載體構建含有HLA-G 3'UTR的螢光素酶報告質粒。引物為：HLA-G-3'UTR-forward:5'-GGGGTACCGATGGGTGAGTTCAACGAGA-3'; HLA-G-3'UTR-reverse:5'-CCCTCGAGAGGGTGGGTGTCATCAGG-3'.然后將PGL3-HLA-G和HOTAIR、 siHOTAIR、miR-152高表達質粒及干擾質粒共轉染,進行熒光素酶分析。轉染24小時后,用雙熒光素酶報告1000分析系統(tǒng)檢測螢光素酶活性。7.統(tǒng)計分析所得數據以均數±標準差(X±SD)表示。單因素方差分析或x2檢驗法分析組間的差異。用SPSS 17.0統(tǒng)計學軟件進行統(tǒng)計分析。P0.05被認為差異有顯著統(tǒng)計學意義。研究結果1. HOTAIR在兩組胃癌細胞內的表達水平：用HOTAIR高表達質粒轉染后均增長大約16.1倍,用干擾質粒(si-HOTAIR)轉染后均下降約63%。2.HLA-G在兩組胃癌細胞內的mRNA水平和蛋白水平：用HOTAIR高表達質粒轉染后均增長大約2倍,用干擾質粒(si-HOTAIR)轉染后均下降約50%。3.miR-152在兩組胃癌細胞內的表達水平：用HOTAIR高表達質粒轉染后均下降約52%,用si-HOTAIR轉染后均增長大約1.72倍,而Mut-HOTAIR對miR-152表達沒有顯著影響；用miR-152高表達質粒轉染后均增長約17.3倍,用miR-152干擾質粒轉染后均下降約67%。4. HLA-G在兩組細胞內的mRNA水平和蛋白水平：用miR-152高表達質粒轉染后均下降約52%,用miR-152干擾質粒轉染后均增長大約2.2倍；用HOTAIR高表達質粒轉染后均增長約1.8倍,用siHOTAIR轉染后均下降約47%；而用mut-HOTAIR轉染后無明顯變化。5.兩組胃癌細胞內的HLA-G 3'UTR活性：用miR-152高表達質粒轉染后均下降約51%,用miR-152干擾質粒轉染后均增長約1.75倍；用HOTAIR高表達質粒轉染后均增長約1.53倍,用siHOTAIR轉染后均下降約48%；而用mut-HOTAIR轉染后無明顯變化；用miR-152高表達質粒與HOTAIR質粒共轉染后均下降約1.2%,用miR-152高表達質粒與mut-HOTAIR質粒共轉染后均下降約52%。結論1. HOTAIR能促進胃癌細胞HLA-G的表達。2. 胃癌細胞中,HOTAIR與miR152有直接的交互影響,并負性調節(jié)miR-152的表達。3. HLA-G的相對表達量、濃度可被miR-152下調,被HOTAIR上調。miR-152誘導的HLA-G 3'UTR活性下調能被HOTAIR高表達所逆轉。4. 胃癌細胞中HOTAIR通過干擾miR-152促進HLA-G表達。
[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.
【學位授予單位】：山東大學
【學位級別】：博士
【學位授予年份】：2015
【分類號】：R735.2

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