【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(rèn)識到了胃癌的早期診斷對改善其預(yù)后的重要性,但大多數(shù)胃癌患者就診時已經(jīng)為進展期,從而錯失了診治的最佳時機。由于傳統(tǒng)的手術(shù)、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(guān)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(zhuǎn)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(guān)性分析表明,HOTAIR和HLA-G水平呈正性相關(guān),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(shù)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(jīng)表明與腫瘤的免疫逃逸機制有密切關(guān)系的人白細(xì)胞抗原-G (HLA-G)做了相關(guān)性分析。結(jié)果發(fā)現(xiàn),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(guān)。然后,我們對HOTAIR進行了獲得性和失活性功能研究,結(jié)果表明,HOTAIR能在體外實驗中上調(diào)HLA-G的mRNA水平和蛋白水平。另一方面,生物信息學(xué)分析表明HOTAIR與miR-152之間存在相互作用關(guān)系,提示miR-152可能會參與HOTAIR對HLA-G的表達調(diào)節(jié)。我們的組織學(xué)研究發(fā)現(xiàn),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(fù)性相關(guān)。我們采用報告基因檢測技術(shù)在體外細(xì)胞實驗中也證實了miR-152對胃癌細(xì)胞的HLA-G表達存在負(fù)性調(diào)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(dǎo)的HLA-G 3'UTR活性降低。因此,我們認(rèn)為：高表達的HOTAIR有可能通過抑制miR-152來上調(diào)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(yù)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(xué)齊魯醫(yī)院60例胃癌患者癌組織及相應(yīng)癌旁組織(距腫瘤邊緣5cm)標(biāo)本,同時采集相應(yīng)患者術(shù)前1天的外周血標(biāo)本。所有患者術(shù)前檢查均未發(fā)現(xiàn)其他臟器腫瘤病變,均接受根治性胃癌切除手術(shù),術(shù)前均未接受針對腫瘤的放療、化療及免疫治療,所有腫瘤標(biāo)本均經(jīng)術(shù)后病理證實。本研究符合《赫爾辛基宣言》標(biāo)準(zhǔn),通過山東大學(xué)齊魯醫(yī)院倫理委員會批準(zhǔn),所有納入患者均在手術(shù)前簽署知情同意書。1.胃癌組織及相應(yīng)癌旁組織中HOTAIR、miR-152及HLA-G的表達檢測常規(guī)胃癌根治術(shù)后留取胃癌組織及相應(yīng)癌旁組織標(biāo)本,編號后立即放入-80℃液氮罐冷凍備用。應(yīng)用Trizol法分別提取胃癌組織及相應(yīng)癌旁組織標(biāo)本的總RNA。為了檢測HLA-G mRNA和HOTAIR,按產(chǎn)品說明使用SuperScript Ⅲ Reverse Transcriptase將oligo-dT primers標(biāo)定的RNA反轉(zhuǎn)錄入cDNA,用磷酸甘油酸脫氫酶(GAPDH)作為內(nèi)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(nèi)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(shù)據(jù)。對每個樣品進行重復(fù)檢測3次,并進行溶解曲線檢測。實驗數(shù)據(jù)采用比較閾值法(2-△△CT法)進行相對定量分析。2.相應(yīng)外周血標(biāo)本中可溶性HLA-G (sHLA-G)濃度檢測外周血標(biāo)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(biāo)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(fù)所有實驗3次,然后計算3次實驗結(jié)果的平均值,以此作為最后的實驗結(jié)果。3.生物信息學(xué)分析HOTAIR轉(zhuǎn)錄和miR-152之間的相互作用用DIANA TOOLS對HOTAIR轉(zhuǎn)錄和miR-152之間的相互作用進行生物信息學(xué)分析。4.統(tǒng)計分析所得數(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)差(X±SD)表示。用Pearson's correlation法進行相關(guān)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(xué)軟件進行統(tǒng)計分析。P0.05被認(rèn)為差異有顯著統(tǒng)計學(xué)意義。研究結(jié)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(guān)(R2=0.57),HOTAIR表達和HLA-G濃度之間也存在顯著正性相關(guān)(R2=0.582)。3. HOTAIR轉(zhuǎn)錄中存在miR-152的三個潛在結(jié)合域,在這些結(jié)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(fù)性相關(guān)(R2=0.5168)。結(jié)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(guān)；HOTAIR參與了胃癌組織中HLA-G的表達上調(diào)。3.HOTAIR轉(zhuǎn)錄中存在miR-152的三個潛在結(jié)合域,在這些結(jié)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(fù)相關(guān)。HOTAIR能負(fù)性調(diào)節(jié)miR-152的表達。第二部分IncRNAs HOTAIR在胃癌細(xì)胞免疫逃逸中的作用機制研究目的研究HOTAIR參與胃癌細(xì)胞免疫逃逸的可能機制。研究方法兩種人胃癌細(xì)胞株SGC7901和MGC-803均為中國科學(xué)院提供。1.細(xì)胞培養(yǎng)胃癌細(xì)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(zhì)粒的構(gòu)建和小干擾RNA(siRNAs)用pCDNA3.1載體構(gòu)建HOTAIR高表達質(zhì)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(jié)合位點進行定點突變誘導(dǎo),從而構(gòu)建Mut-HOTAIR質(zhì)粒。所有用于轉(zhuǎn)染的質(zhì)粒載體經(jīng)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(xì)胞轉(zhuǎn)染miR-152的高表達質(zhì)粒和干擾質(zhì)粒及其對照RNA購買于GenePharma公司。SGC7901和MGC-803細(xì)胞(2.5×105)接種于6孔培養(yǎng)板上,孵育24小時后將HOTAIR質(zhì)粒、siHOTAIR序列、miR-152的高表達質(zhì)粒以及干擾質(zhì)粒參照Lipofectamin 2000說明書分別轉(zhuǎn)染入胃癌細(xì)胞中。4.轉(zhuǎn)染后胃癌細(xì)胞株中HOTAIR、miR-152及HLA-G的表達檢測采用Trizol法提取轉(zhuǎn)染后胃癌細(xì)胞中的總RNA。為了檢測HLA-G mRNA和HOTAIR,按產(chǎn)品說明書使用SuperScript Ⅲ Reverse Transcriptase將oligo-dT primers標(biāo)定的RNA反轉(zhuǎn)錄入cDNA,用磷酸甘油酸脫氫酶(GAPDH)作為內(nèi)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(nèi)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(shù)據(jù)收集。每個樣品進行3次重復(fù)檢測,并進行溶解曲線檢測。采用比較閾值法對實驗數(shù)據(jù)進行相對定量分析。5.轉(zhuǎn)染后胃癌細(xì)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(xì)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(fù)所有實驗3次,然后計算3次實驗結(jié)果的平均值,以此作為最后的實驗結(jié)果。6.雙熒光素酶檢測用PGL3載體構(gòu)建含有HLA-G 3'UTR的螢光素酶報告質(zhì)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(zhì)粒及干擾質(zhì)粒共轉(zhuǎn)染,進行熒光素酶分析。轉(zhuǎn)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(shù)據(jù)以均數(shù)±標(biāo)準(zhǔn)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(xué)軟件進行統(tǒng)計分析。P0.05被認(rèn)為差異有顯著統(tǒng)計學(xué)意義。研究結(jié)果1. HOTAIR在兩組胃癌細(xì)胞內(nèi)的表達水平：用HOTAIR高表達質(zhì)粒轉(zhuǎn)染后均增長大約16.1倍,用干擾質(zhì)粒(si-HOTAIR)轉(zhuǎn)染后均下降約63%。2.HLA-G在兩組胃癌細(xì)胞內(nèi)的mRNA水平和蛋白水平：用HOTAIR高表達質(zhì)粒轉(zhuǎn)染后均增長大約2倍,用干擾質(zhì)粒(si-HOTAIR)轉(zhuǎn)染后均下降約50%。3.miR-152在兩組胃癌細(xì)胞內(nèi)的表達水平：用HOTAIR高表達質(zhì)粒轉(zhuǎn)染后均下降約52%,用si-HOTAIR轉(zhuǎn)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(zhì)粒轉(zhuǎn)染后均增長約17.3倍,用miR-152干擾質(zhì)粒轉(zhuǎn)染后均下降約67%。4. HLA-G在兩組細(xì)胞內(nèi)的mRNA水平和蛋白水平：用miR-152高表達質(zhì)粒轉(zhuǎn)染后均下降約52%,用miR-152干擾質(zhì)粒轉(zhuǎn)染后均增長大約2.2倍；用HOTAIR高表達質(zhì)粒轉(zhuǎn)染后均增長約1.8倍,用siHOTAIR轉(zhuǎn)染后均下降約47%；而用mut-HOTAIR轉(zhuǎn)染后無明顯變化。5.兩組胃癌細(xì)胞內(nèi)的HLA-G 3'UTR活性：用miR-152高表達質(zhì)粒轉(zhuǎn)染后均下降約51%,用miR-152干擾質(zhì)粒轉(zhuǎn)染后均增長約1.75倍；用HOTAIR高表達質(zhì)粒轉(zhuǎn)染后均增長約1.53倍,用siHOTAIR轉(zhuǎn)染后均下降約48%；而用mut-HOTAIR轉(zhuǎn)染后無明顯變化；用miR-152高表達質(zhì)粒與HOTAIR質(zhì)粒共轉(zhuǎn)染后均下降約1.2%,用miR-152高表達質(zhì)粒與mut-HOTAIR質(zhì)粒共轉(zhuǎn)染后均下降約52%。結(jié)論1. HOTAIR能促進胃癌細(xì)胞HLA-G的表達。2. 胃癌細(xì)胞中,HOTAIR與miR152有直接的交互影響,并負(fù)性調(diào)節(jié)miR-152的表達。3. HLA-G的相對表達量、濃度可被miR-152下調(diào),被HOTAIR上調(diào)。miR-152誘導(dǎo)的HLA-G 3'UTR活性下調(diào)能被HOTAIR高表達所逆轉(zhuǎn)。4. 胃癌細(xì)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(xué)位授予單位】：山東大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2015
【分類號】：R735.2

【共引文獻】

相關(guān)期刊論文 前10條

1 韓寶惠;鐘華;范小紅;馮光麗;李榕;龔樂羅;儲天晴;張偉;金波;施春雷;趙怡卓;沙惠芳;董強剛;廖美琳;;CEA多肽致敏的樹突狀細(xì)胞(DC)治療晚期非小細(xì)胞肺癌臨床初步研究[J];中國肺癌雜志;2006年04期

2 白澎,張滬生,王毓洲;肺癌免疫治療的進展[J];國外醫(yī)學(xué).呼吸系統(tǒng)分冊;2004年S1期

3 晁紅雷;汪昱;;CXCL4及其受體與結(jié)直腸腫瘤[J];國際腫瘤學(xué)雜志;2013年05期

4 孫衍昶;郭t$t$;賽克;王翦;王潔;陳芙蓉;張宗平;陳忠平;;MicroRNA-181b提高U87細(xì)胞對VM-26的敏感性研究[J];中國神經(jīng)腫瘤雜志;2013年02期

5 汪章勛;周權(quán);孟慧敏;張軍;黃勃;;金龜子綠僵菌MicroRNA在蟬蛻和蠶血淋巴誘導(dǎo)下的差異表達分析[J];安徽科技學(xué)院學(xué)報;2013年05期

6 Wenwen Jia;Wen Chen;Jiuhong Kang;;The Functions of MicroRNAs and Long Non-coding RNAs in Embryonic and Induced Pluripotent Stem Cells[J];Genomics,Proteomics & Bioinformatics;2013年05期

7 安麗娜;董斐斐;王國坤;單冬凱;荊清;;MicroRNA與動脈粥樣硬化[J];國際心血管病雜志;2013年04期

8 馮楠楠;孫品;夏昭林;;凋亡相關(guān)的microRNA分子研究進展[J];工業(yè)衛(wèi)生與職業(yè)病;2013年05期

9 陳娟娟;艾劍剛;黃世峰;曹炬;張莉萍;;HBx介導(dǎo)miR-221上調(diào)-ERα下調(diào)致HepG2細(xì)胞惡性增殖[J];第三軍醫(yī)大學(xué)學(xué)報;2013年20期

10 張世芳;魏彩虹;陸健;張小寧;周鑫磊;張淑珍;王光凱;曹家雪;趙福平;張莉;杜立新;;深度測序鑒定綿羊microRNA轉(zhuǎn)錄組[J];中國畜牧獸醫(yī);2013年09期

相關(guān)會議論文 前9條

1 Ruiwen Fan;Shanshan Yang;Zhanquan Shi;Kaiyuan Ji;Xiaoyun Jia;Jianbo Yao;George W Smith;Changsheng Dong;;Identification of a novel miRNA important for melanogenesis in alpaca(Lama Pacos)[A];2013中國駝業(yè)進展[C];2013年

2 曾清華;周箐;康秀華;;血清microRNA的檢測及在肺癌診斷中應(yīng)用的進展[A];江西省第二次中西醫(yī)結(jié)合呼吸疾病學(xué)術(shù)會議論文集[C];2012年

3 秦燕;張玲云;張德玖;;核糖體展示技術(shù)在非編碼核酸研究中的應(yīng)用[A];生命科學(xué)——專題：RNA研究的新技術(shù)和新方法（第26卷第3期）[C];2014年

4 李文峰;鐘伯雄;蘇松坤;;蜜蜂級型分化機理[A];2014年全國蜂產(chǎn)品市場信息交流會暨中國（哈爾濱）蜂業(yè)博覽會論文集[C];2014年

5 宋毅;謝延風(fēng);張鈴鐺;馮清林;劉明冬;張鵬;范仕兵;杜江峰;;MiRNA在垂體泌乳素腺瘤中表達的初步研究[A];全國高血壓防治知識推廣培訓(xùn)班暨健康血壓中國行海南�？跁撐木C合刊[C];2014年

6 陳軼;陳益耀;蔡曼妮;巫華志;;非酒精性脂肪性肝病患者血清miR-29b水平變化及臨床意義[A];中國轉(zhuǎn)化醫(yī)學(xué)和整合醫(yī)學(xué)研討會論文綜合刊[C];2015年

7 Junli Guo;Shaoping Zheng;Zhihong Weng;Keping Xie;Shaojiang Zheng;;Molecular Biomarkers of Pancreatic Intraepithelial Neoplasias and Implications in Early Diagnosis and Therapeutic Intervention[A];第七屆生命科學(xué)聯(lián)合學(xué)術(shù)大會論文匯編[C];2015年

8 Shaosheng Wang;Xiaohong Zhao;Jie Wang;Yingmei Wen;LinJing Zhang;Dezhu Wang;Huili Chen;Qiuxia Chen;Wei Xiang;;Upregulation of micro RNA-203 is associated with advanced tumor progression and poorprognosis in epithelial ovarian cancer[A];第七屆生命科學(xué)聯(lián)合學(xué)術(shù)大會論文匯編[C];2015年

9 邱小宇;黃勇富;娜日蘇;俄廣鑫;陳李鵬;;山羊卵巢miRNAs的克隆、測序及表達鑒定[A];2015年全國養(yǎng)羊生產(chǎn)與學(xué)術(shù)研討會論文集[C];2015年

相關(guān)博士學(xué)位論文 前10條

1 朱波;腫瘤抗原TRAG-3的免疫識別及其免疫效應(yīng)研究[D];第三軍醫(yī)大學(xué);2003年

2 蔡永國;肝素酶基因修飾的樹突狀細(xì)胞疫苗對胃癌細(xì)胞的免疫效應(yīng)研究[D];第三軍醫(yī)大學(xué);2005年

3 溫華;負(fù)載凋亡的肺腺癌細(xì)胞致樹突狀細(xì)胞成熟并增強自然殺傷細(xì)胞的細(xì)胞毒作用研究[D];中國醫(yī)科大學(xué);2007年

4 李丹;CEA迷你基因串聯(lián)體基因疫苗的構(gòu)建及其免疫學(xué)活性研究[D];吉林大學(xué);2008年

5 楊蕾;癌胚抗原mRNA及SW480細(xì)胞總RNA負(fù)載成熟樹突狀細(xì)胞體外誘導(dǎo)的特異性抗腫瘤作用[D];吉林大學(xué);2009年

6 萬亞峰;肝細(xì)胞癌AFPmRNA轉(zhuǎn)染CD40配體活化的B細(xì)胞誘導(dǎo)抗腫瘤免疫的實驗研究[D];華中科技大學(xué);2010年

7 賈蓓;A型血友病人非整合誘導(dǎo)多能干細(xì)胞建系及其細(xì)胞疾病模型的建立[D];南方醫(yī)科大學(xué);2012年

8 邵耘;MicroRNA-101與環(huán)氧化酶-2在胃癌組織中的表達及意義[D];南京醫(yī)科大學(xué);2013年

9 佘笑梅;三氧化二砷對急性早幼粒細(xì)胞白血病NB4細(xì)胞株Warburg效應(yīng)的影響和機制[D];華中科技大學(xué);2013年

10 陳琦;慢病毒介導(dǎo)的FoxP2 knockdown蝙蝠模型建立與功能研究及長翼蝠小RNA轉(zhuǎn)錄組的分析研究[D];華東師范大學(xué);2013年

相關(guān)碩士學(xué)位論文 前10條

1 王國珍;肝素酶CTL表位多抗原肽（MAP）疫苗的設(shè)計及其抗腫瘤免疫效應(yīng)研究[D];第三軍醫(yī)大學(xué);2011年

2 吳軍;樹突狀細(xì)胞（DC）的培養(yǎng)及CEA-重組痘苗病毒轉(zhuǎn)染DC誘導(dǎo)的特異性T細(xì)胞免疫[D];第一軍醫(yī)大學(xué);2002年

3 賈春實;肺癌血管生成與血管生成抑制物之間相互關(guān)系的研究[D];天津醫(yī)科大學(xué);2004年

4 才志剛;凍融抗原沖擊致敏的樹突狀細(xì)胞誘導(dǎo)產(chǎn)生肺癌特異性免疫應(yīng)答的實驗研究[D];第二軍醫(yī)大學(xué);2004年

5 陳東曉;負(fù)載HPV16E6/18E7基因樹突狀細(xì)胞疫苗的構(gòu)建及其抗食管癌細(xì)胞的體外實驗[D];汕頭大學(xué);2005年

6 陳陵;hTERT基因修飾樹突狀細(xì)胞對腫瘤細(xì)胞的免疫效應(yīng)研究[D];第三軍醫(yī)大學(xué);2006年

7 吳乃剛;MEK作為共刺激因子對DC分化成熟的影響及其疫苗對Lewis肺癌抑瘤作用的研究[D];第四軍醫(yī)大學(xué);2006年

8 楊新靜;凋亡腫瘤細(xì)胞負(fù)載的樹突狀細(xì)胞疫苗抗肺癌的實驗研究[D];蘇州大學(xué);2006年

9 袁欣;腫瘤抗原負(fù)載DC介導(dǎo)特異性免疫應(yīng)答的實驗研究[D];吉林大學(xué);2007年

10 陳婷;肝素酶HLA-A2.1限制性CTL表位的預(yù)測、鑒定及其抗腫瘤的免疫效應(yīng)研究[D];第三軍醫(yī)大學(xué);2007年

，

本文編號：2249807