【摘要】：第一部分LncRNA UCA1在胃癌的發(fā)生發(fā)展中的作用研究目的:LncRNAUCA1在胃癌的發(fā)生發(fā)展中的作用研究方法:診斷為原發(fā)性胃癌且未進(jìn)行化療或放療的56位患者行胃切除術(shù),取胃癌組織和癌旁正常組織切除后立即用液氮進(jìn)行冰凍,然后保存于-80℃以備用。首先把由4%多聚甲醛浸泡了 48小時(shí)以后的由手術(shù)切除的胃粘膜組織,用石蠟進(jìn)行包埋,再經(jīng)4μm連續(xù)切片以后的用石蠟包埋好的組織再行HE染色,染色以后再由病理學(xué)專業(yè)的專家對(duì)上述切片給予分析和評(píng)價(jià)。再經(jīng)免疫組化染色后提取胃癌樣本總RNA,抽提細(xì)胞總RNA并進(jìn)行變性瓊脂糖電泳檢測(cè)以及RNA逆轉(zhuǎn)錄反應(yīng)。在進(jìn)行反轉(zhuǎn)錄反應(yīng)之后,應(yīng)用Real-time PCR技術(shù)對(duì)lnc RNA、mRNA的表達(dá)豐度分別進(jìn)行檢測(cè)。結(jié)果:1 HE及免疫組化染色胃癌組織和癌旁正常組織的石蠟標(biāo)本經(jīng)HE染色,病理學(xué)復(fù)查示診斷和實(shí)驗(yàn)前診斷一致;免疫組織化學(xué)染色表明,胃癌組織標(biāo)本呈現(xiàn)出高度度表達(dá),而癌旁正常組織基本上不作出表達(dá)。2 胃癌組織中的lnc RNA表達(dá)水平明顯上調(diào)采用Real-time PCR方法對(duì)病理采集(癌組織和癌旁胃組織)中,lnc RNA UCA1的表達(dá)進(jìn)行檢測(cè),采集標(biāo)本共56對(duì)。lnc RNA UCA1在癌旁組織中的相對(duì)含量(1.29 ± 0.26)低于癌組組織(2.85±0.74),Wilcoxon matched pairs test 有顯著統(tǒng)計(jì)學(xué)意義(p0.01)。表明lnc RNA UCA1極有可能是一種存在于胃癌組織中的促癌基基因。3 lnc RNA UCA1在胃癌和7癌旁胃正常組織中相對(duì)含量的比值同胃癌的臨床病理特征、臨床預(yù)后相關(guān)進(jìn)一步研究。分析在56例胃癌以及胃癌旁組織內(nèi)的lnc RNA表達(dá)出的相對(duì)含量的C/P比值與性別、年齡、癌腫大小、分期、浸潤(rùn)程度、分化情況、淋巴結(jié)侵襲之間的關(guān)系。結(jié)果發(fā)現(xiàn):C/P比值與腫瘤大小、腫瘤的分化程度有明顯關(guān)聯(lián);其于低分化的胃癌病人中的表達(dá)有增加趨勢(shì),分析差異具有統(tǒng)計(jì)學(xué)意義;與性別、年齡無(wú)明顯相關(guān),分析差異沒(méi)有統(tǒng)計(jì)學(xué)意義,但其和癌腫的浸潤(rùn)程度、淋巴結(jié)轉(zhuǎn)移、TNM分期及遠(yuǎn)處轉(zhuǎn)移亦無(wú)明顯相關(guān),分析差異沒(méi)有統(tǒng)汁學(xué)意義,懷疑此結(jié)果可能與樣本量偏少有關(guān)。在此過(guò)程中我們把56例標(biāo)本平均分成兩組,分別為C/PC/P平均值組和C/PC/P平均值組,對(duì)術(shù)后生存時(shí)間(hour)進(jìn)行統(tǒng)計(jì)學(xué)分析。發(fā)現(xiàn)C/PC/P平均值組患者術(shù)后生存時(shí)間長(zhǎng)于C/PC/P平均值組,且差異沒(méi)有統(tǒng)計(jì)學(xué)意義,究其原因可能受隨訪時(shí)間過(guò)短及胃癌術(shù)后5年生存率較高等因素影響。結(jié)論:lncRNAUCA1與胃癌的發(fā)生、發(fā)展及分化程度相關(guān),在胃癌中起到促癌基因的作用。第二部分LncRNA UCA1通過(guò)下調(diào)miR-27b表達(dá)增強(qiáng)胃癌多藥耐藥性研究目的:探索UCA1和miR-27b在胃癌的發(fā)生發(fā)展中的聯(lián)系,并進(jìn)一步研究其在胃癌的多藥耐藥性中的作用。探討lncRNAUCA1通過(guò)下調(diào)miR-27b的表達(dá)強(qiáng)度和胃癌的多藥耐藥性的關(guān)系。篩選和驗(yàn)證胃癌耐藥相關(guān)lnc RNA分子。通過(guò)實(shí)驗(yàn)驗(yàn)證lnc RNA UCA1在胃癌的多藥耐藥性發(fā)生中所起的作用;并驗(yàn)證lnc RNA UCA1能夠調(diào)節(jié)胃癌多藥耐藥性的分子機(jī)理。方法:異常表達(dá)的lncRNA的基因芯片數(shù)據(jù)來(lái)源于GEO數(shù)據(jù)庫(kù)。采用實(shí)時(shí)熒光定量 PCR的方法對(duì)28組癌癥和癌旁的正常組織樣本給予分析以評(píng)估lncRNAUCA1和miR27b的表達(dá)情況。采用人類胃胃癌細(xì)胞系SGC-7901細(xì)胞和SGC-7901衍生的具有阿霉素抗性的SGC-7901/ADR細(xì)胞,順鉑耐藥的SGC-7901/DDP細(xì)胞和5-氟尿嘧啶耐藥的SGC-7901/FU細(xì)胞作為體外細(xì)胞模型來(lái)評(píng)估UCA1和miR-27b在胃癌多藥耐藥性中的作用。診斷為原發(fā)性胃癌且未進(jìn)行化療或放療的28位患者行胃切除術(shù),取胃癌組織和癌旁正常組織切除后立即用液氮進(jìn)行冰凍,然后保存于-80℃以備用。細(xì)胞培養(yǎng)和轉(zhuǎn)染癌癥細(xì)胞培養(yǎng)在RPMI-1640培養(yǎng)基內(nèi),并加入10%FBS,100μg/mL青霉素,及100U/mL鏈霉素。通過(guò)采用Ribobio試劑盒采用75 nM UCA1 siRNA或混合陰性對(duì)照(Ribobio)對(duì) SGC-7901/ADR,SGC-7901/DDP 和 SGC-7901/FU 細(xì)胞進(jìn)行轉(zhuǎn)染,而采用 100 nMmiR-27b模擬物或陰性對(duì)照(Ribobio)對(duì)SGC-7901/ADR細(xì)胞進(jìn)行轉(zhuǎn)染,使用的試劑盒為L(zhǎng)ipofectamine 2000 48小時(shí)后,收集siRNA轉(zhuǎn)染的細(xì)胞,并用qRT-PCR測(cè)定其抑制的程度。對(duì)與UCA1基因互補(bǔ)的DNA進(jìn)行擴(kuò)增,并插入到pcDNA3.1的BamhI/XhoI位點(diǎn)之間。采用pcDNA3.1-UCA1表達(dá)載體或50 nM miR-27b抑制劑(Ribobio)SGC-7901細(xì)胞進(jìn)行轉(zhuǎn)染。胃癌組織和相應(yīng)的癌旁正常組織的lncRNA表達(dá)譜的基因芯片數(shù)據(jù)調(diào)采用DIANA工其對(duì)UCA1和miR-27b的結(jié)合位點(diǎn)進(jìn)行預(yù)測(cè)。采用TRIzol試劑提取組織及細(xì)胞樣品的總RNA。進(jìn)行反轉(zhuǎn)錄以獲得一鏈cDNA。采用TaqMan MicroRNA Assay Kit進(jìn)行實(shí)時(shí)熒光定量PCR分析。結(jié)果采用2-AACT法進(jìn)行計(jì)算。采用線性回歸分析對(duì)UCA1表達(dá)的水平和miR-27b表達(dá)的水平的相關(guān)性進(jìn)行評(píng)估。采用Cy3標(biāo)記的抗生物素抗體在37℃反應(yīng)30min時(shí)檢測(cè)信號(hào)。采用DAPI來(lái)復(fù)染細(xì)胞核,然后用FV1000熒光顯微鏡檢測(cè)免疫熒光。采用UCA1 siRNA或miR-27b模擬物對(duì)SGC-7901/ADR細(xì)胞進(jìn)行轉(zhuǎn)染,而采用UCA1表達(dá)載體或miR-27b抑制劑對(duì)SGC-7901細(xì)胞進(jìn)行轉(zhuǎn)染。轉(zhuǎn)染24小時(shí)后,在96孔板上接種細(xì)胞。24小時(shí)后,用不同濃度的ADR,DDP或5-FU對(duì)細(xì)胞進(jìn)行處理48小時(shí)。采用傳統(tǒng)MTT芯片來(lái)對(duì)細(xì)胞活性進(jìn)行測(cè)量。采用酶標(biāo)儀來(lái)記錄490 nm的吸光度情況。通過(guò)繪制劑量-反應(yīng)曲線計(jì)算 IC50 值。采用 Annexin V-FITC Apoptosis Detection Kit 來(lái)檢測(cè)細(xì)胞的凋亡情況,采用流式細(xì)胞儀計(jì)算凋亡率。將含有30μg總蛋白的樣品被置于每一條帶上,然后用10%SDS-PAGE進(jìn)行分離。之后,電泳使蛋白樣品轉(zhuǎn)移至硝酸纖維膜上。對(duì)硝酸纖維膜進(jìn)行封閉、洗滌并用抗BCL-2一抗(ab32124,Abeam)和裂解的半胱天冬蛋門酶-3(#9661.Cell Signaling.Danvers,MA,USA)以及β-肌動(dòng)蛋白(ab8227,Abcam)孵育過(guò)夜。洗滌之后,用HRP共軛的二抗對(duì)硝酸纖維膜進(jìn)行孵育。采用化學(xué)發(fā)光超敏顯色試劑盒super ECL detection reagent對(duì)蛋白條帶進(jìn)行視覺(jué)化。采用GraphPad Prism 6.0進(jìn)行數(shù)據(jù)分析。采用非配對(duì)雙側(cè)t檢驗(yàn)方法來(lái)進(jìn)行組間差異評(píng)估。p0.05表示差異具有統(tǒng)計(jì)顯著性。結(jié)果:1胃癌中UCA1和miR-27b呈負(fù)相關(guān)在本部分中,我們首先通過(guò)提取GEO數(shù)據(jù)庫(kù)的基因芯片數(shù)據(jù)研究了胃癌組織中異常表達(dá)的lncRNA。有一項(xiàng)近期的研究基于6個(gè)癌癥組織樣品和6個(gè)配對(duì)的癌旁組織樣品對(duì)lncRNA的表達(dá)譜進(jìn)行了分析。通過(guò)回顧他們的基因芯片數(shù)據(jù)(accession No.GSE53137),我們觀察到UCA1是胃癌組織樣品中被最大程度上調(diào)表達(dá)的lncRNA之一。為了進(jìn)一步驗(yàn)證這種異常表達(dá)情況,我們進(jìn)一步基于28對(duì)癌癥組織和癌旁正常組織樣品進(jìn)行了實(shí)時(shí)熒光定量PCR分析(qRT-PCR)。結(jié)果表明,UCA1在癌癥組織中被顯著上調(diào)(圖2-1B)。與此同時(shí),我們的qRT-PCR結(jié)果表明miR-27b,一種對(duì)胃癌細(xì)胞具有抑制多藥耐藥性的miRNA,在癌癥組織中顯著下降。通過(guò)線性回歸分析,我們進(jìn)一步確認(rèn)了 UCA1和miR-27b的負(fù)相關(guān)關(guān)系(R2=0.23.p=0.01)。然后,我們決定進(jìn)一步調(diào)查它們之間是否存在什么關(guān)聯(lián),以及它們?cè)谖赴┘?xì)胞多藥耐藥性形成中的影響。2抑制UCA1可恢復(fù)MDR 胃癌細(xì)胞中miR-27b的表達(dá)水平通過(guò)qRT-PCR,我們發(fā)現(xiàn)MDR胃癌細(xì)胞,包括SGC-7901/ADR,SGC-7901/DDP和SGC-7901/5-FU細(xì)胞有進(jìn)一步增加UCA1表達(dá)和降低miR-27b表達(dá)。很明顯,生物信息分析表明UCA1有兩個(gè)與miR-27b結(jié)合的可能位點(diǎn)。因此,我們決定調(diào)查看看UCA1是否對(duì)miR-27b的表達(dá)具有調(diào)控作用。FISH分析結(jié)果表明,UCA1和miR-27b在SGC7901和SGC-7901/ADR細(xì)胞的細(xì)胞核和細(xì)胞質(zhì)中都有分布。UCA1在SGC-7901/ADR細(xì)胞中的表達(dá)量明顯要更高,而miR-27b表達(dá)量則在SGC7901細(xì)胞中更高(圖22-D)。接著,用UCA siRNA對(duì) SGC-7901/ADR,SGC-7901/DDP 和 SGC-7901/5-FU 細(xì)胞進(jìn)行轉(zhuǎn)染。具有較高抑制作用的Si-UCAl-1被用于后續(xù)研究。QRT-PCR結(jié)果表明,對(duì)UCA1的抑制能夠顯著恢復(fù)miR-27b在MDR 胃癌細(xì)胞內(nèi)的表達(dá)。3 UCA1抑制和miR-27b過(guò)量表達(dá)使MDR 胃癌細(xì)胞對(duì)化療試劑變得更加敏感為了進(jìn)一步調(diào)查UCA1和miR-27b在胃癌細(xì)胞MDR形成中的作用,我們采用MTT芯片對(duì)UCAlsiRNA或miR-27b模擬物轉(zhuǎn)染后的SGC-7901/ADR細(xì)胞中的ADR.DDP和5-FU的IC50進(jìn)行檢測(cè)。結(jié)果表明,UCA1抑制和miR-27b過(guò)量表達(dá)都會(huì)降低SGC-7901/ADR細(xì)胞中的ADR,DDP和5-FU的IC50。接著,我們采用流式細(xì)胞儀檢驗(yàn)用20μg/ml ADR處理后的SGC-7901/ADR細(xì)胞中ADR誘導(dǎo)的細(xì)胞凋亡。結(jié)果表明,UCA1抑制和miR-27b的增強(qiáng)表達(dá)的細(xì)胞能夠顯著增加ADR處理后的細(xì)胞凋亡。因?yàn)橄胍M(jìn)一步驗(yàn)證這些細(xì)胞中由ADR誘導(dǎo)的細(xì)胞凋亡,本課題組運(yùn)用免疫印跡分析以檢測(cè)抗凋亡蛋白BCL-2和凋亡蛋白裂解的半胱天冬蛋白酶-3的變化情況。結(jié)果表明,UCA1抑制和miR-27b過(guò)量表達(dá)在20μg/mlADR處理后的SGC-7901/ADR細(xì)胞內(nèi)顯著降低了 BCL-2農(nóng)達(dá)并增加了半胱天冬蛋白酶-3的表達(dá)。上述結(jié)果顯示UCA1抑制和miR-27b過(guò)量表達(dá)使MDR胃癌細(xì)胞對(duì)化療試劑變得更敏感。4 UCA1過(guò)量表達(dá)和miR-27b抑制使胃癌細(xì)胞產(chǎn)生了 MDR為了進(jìn)一步調(diào)查UCA1和miR-27b對(duì)胃癌細(xì)胞的多藥耐藥性的調(diào)控作用,首先采用UCA1表達(dá)載體或miR-27b抑制劑對(duì)SGC-7901細(xì)胞進(jìn)行轉(zhuǎn)染。MTT芯片結(jié)果證實(shí)UCA1過(guò)量表達(dá)和miR-27b抑制增加了SGC-7901細(xì)胞的ADR,DDP和5-FU的IC50。后續(xù)的流式細(xì)胞儀分析表明UCA1過(guò)量表達(dá)和miR-27b抑制顯著降低了 ADR(10μ/ml)誘導(dǎo)的細(xì)胞凋亡。LncRNAUCA1是胃癌細(xì)胞中的促癌因子；LncRNAUCA1能夠下調(diào)miR-27b表達(dá)從而增強(qiáng)胃癌多藥耐藥性,本論文通過(guò)競(jìng)爭(zhēng)內(nèi)源性 RNA理論為基礎(chǔ),從而把胃癌細(xì)胞多藥耐藥性相關(guān)的lnc RNA表達(dá)譜和mi RNA農(nóng)達(dá)譜的數(shù)據(jù)進(jìn)行統(tǒng)一合并分析,在ncRNA相互作用的層面表明了UCA1-mi RNA通路在胃癌MDR中的調(diào)節(jié)作用,結(jié)論:在胃癌中,UCA1與miR-27b表達(dá)呈負(fù)相關(guān)關(guān)系。抑制UCA1可恢復(fù)miR-27b在癌癥細(xì)胞中的表達(dá)。UCA1-miR-27b通路參與了癌癥細(xì)胞的化學(xué)敏感性調(diào)節(jié)。
[Abstract]:Part I research on the role of LncRNA UCA1 in the development of gastric cancer: the study of the role of LncRNAUCA1 in the development of gastric cancer: 56 patients diagnosed as primary gastric cancer without chemotherapy or radiotherapy were treated with gastrectomy, and the gastric tissue and normal tissue were removed and frozen immediately after the resection of normal tissues and then preserved. At -80 C for reserve. First, the surgical excised gastric mucosa was soaked by 4% polyformaldehyde for 48 hours, and paraffin was embedded with paraffin. After 4 mu m, the paraffin embedded tissues were then stained with HE. After the staining, the pathology professional experts analyzed and evaluated the above sections. After staining, the total RNA of gastric cancer samples was extracted, the total RNA of the cells was extracted and the agarose electrophoresis and RNA reverse transcription were performed. After the reaction, the expression of LNC RNA and mRNA was detected by Real-time PCR. Results: 1 HE and immunohistochemical staining of gastric cancer tissue and paraffin paraffin specimens of the normal tissues adjacent to cancer After HE staining, the pathological examination showed that the diagnosis was consistent with the diagnosis before and before the experiment. The immunohistochemical staining showed that the tissue specimens of gastric cancer showed a high degree of expression, while the normal tissues beside the carcinoma basically did not express the expression of LNC RNA in the.2 gastric carcinoma tissue, and the Real-time PCR method was used for pathological collection (cancer tissue and paracancerous stomach group). In the fabric, the expression of LNC RNA UCA1 was detected. The relative content of 56 pairs of.Lnc RNA UCA1 in the adjacent tissues (1.29 + 0.26) was lower than that of the cancer group (2.85 + 0.74), and the Wilcoxon matched pairs test had significant statistical significance (P0.01). The relative ratio of C RNA UCA1 in gastric carcinoma and 7 paracancerous normal gastric tissues is related to the clinicopathological features of gastric cancer and the clinical prognosis. The ratio of C/P to the relative content of LNC RNA expressed in 56 cases of gastric cancer and paracathal tissue, age, tumor size, stage, infiltration, differentiation, and lymph node invasion are analyzed. The results showed that the ratio of C/P was significantly associated with the size of tumor and the degree of differentiation of the tumor; the expression in the patients with low differentiated gastric cancer had an increasing trend, and the difference was statistically significant; there was no significant correlation with sex and age, and the analysis of differences was not significant, but it was associated with the degree of cancer and lymph node metastasis. There was no significant correlation between TNM staging and distant metastasis. There was no statistical significance in analysis. The results were suspected to be related to less sample size. In this process, we divided 56 specimens into two groups, which were the average value group of C/PC/P and the C/PC/P average group, and the postoperative survival time (hour) was statistically analyzed. The mean value group of C/PC/P was found. The survival time of the patients was longer than the C/PC/P average group, and the difference was not statistically significant. The reason may be influenced by the short follow-up time and the 5 year survival rate of gastric cancer. Conclusion: lncRNAUCA1 is related to the occurrence, development and differentiation of gastric cancer, and the role of the oncogene in gastric cancer. The second part of LncRNA UCA1 The aim of the study of miR-27b expression to enhance the multidrug resistance of gastric cancer: To explore the relationship between UCA1 and miR-27b in the development of gastric cancer and to further study its role in the multidrug resistance of gastric cancer. To explore the relationship between the expression intensity of miR-27b and the multidrug resistance of gastric cancer, and to screen and verify the drug resistance phase of gastric cancer. LNC RNA molecules. The role of LNC RNA UCA1 in the occurrence of multidrug resistance in gastric cancer was tested and the molecular mechanism of LNC RNA UCA1 to regulate the multidrug resistance of gastric cancer. Method: the abnormal expression of lncRNA gene chip data was derived from GEO database. 28 groups of cancer and cancer were used by real time fluorescence quantitative PCR. Analysis of the normal tissue samples was given to evaluate the expression of lncRNAUCA1 and miR27b. SGC-7901/ADR cells with adriamycin resistance derived from human gastric cancer cell line SGC-7901 cells and SGC-7901 were used to evaluate UCA, the cisplatin resistant SGC-7901/DDP cells and the 5- fluorouracil resistant SGC-7901/FU cells were used as an in vitro cell model to evaluate the UCA The role of 1 and miR-27b in the multidrug resistance of gastric cancer. 28 patients who were diagnosed as primary gastric cancer without chemotherapy or radiotherapy were treated with gastrectomy and frozen immediately after the resection of gastric cancer tissues and normal tissues, and then stored at -80 C for reserve. Cell culture and transfected cancer cells were cultured in the RPMI-1640 culture base, 10%FBS, 100 g/mL penicillin, and 100U/mL streptomycin were transfected by Ribobio kit with 75 nM UCA1 siRNA or mixed negative control (Ribobio) for SGC-7901/ADR, SGC-7901/DDP and SGC-7901/FU cells, and transfected with 100 nMmiR-27b simulants or negative controls. After the kit was Lipofectamine 200048 hours, the cells transfected with siRNA were collected and the degree of inhibition was measured by qRT-PCR. The DNA which complemented the UCA1 gene was amplified and inserted between the BamhI/XhoI loci of pcDNA3.1. The pcDNA3.1-UCA1 expression vector or the 50 nM miR-27b inhibitor (Ribobio) SGC-7901 cells were transfected. The gene chip data of the lncRNA expression profiles of the corresponding normal tissues adjacent to the carcinoma were adjusted by DIANA to predict the binding sites of UCA1 and miR-27b. TRIzol reagent was used to extract the total RNA. of tissue and cell samples for reverse transcription in order to obtain a chain cDNA. using TaqMan MicroRNA Assay Kit for real time fluorescence quantitative PCR analysis. The 2-AACT method was used to calculate the correlation between the level of UCA1 expression and the level of miR-27b expression. The Cy3 labeled anti biotin antibody was used to detect the signal at 37 C for 30min. DAPI was used to restain the nucleus, and then the immunofluorescence was detected by the FV1000 fluorescence microscope. UCA1 siRNA or miR-27b modules were used. The SGC-7901/ADR cells were transfected by the pseudo substance, and the SGC-7901 cells were transfected with the UCA1 expression vector or miR-27b inhibitor. After 24 hours transfection, the cells were treated with different concentrations of ADR, DDP or 5-FU for 48 hours after transfection for 48 hours. The cell activity was measured by traditional MTT chip. The absorbance of 490 nm was recorded. The IC50 value was calculated by plotting the dose response curve. The apoptosis of cells was detected by Annexin V-FITC Apoptosis Detection Kit. The apoptosis rate was calculated by flow cytometry. The samples containing 30 mu g protein were placed on each band and then separated with 10%SDS-PAGE. The protein samples were transferred to the nitric acid fiber membrane. The nitric acid fiber membrane was closed, washed and incubated for the night with the anti BCL-2 ab32124 (Abeam) and the lysed caspase -3 (#9661.Cell Signaling.Danvers, MA, USA) and the beta actin (ab8227, Abcam). After washing, the two anti nitric acid fiber membrane was incubated with the HRP conjugate. Super ECL detection reagent, a chemiluminescence hypersensitive reagent box, was used to visualize the protein bands. The data were analyzed with GraphPad Prism 6. The discrepancy was statistically significant by non paired bilateral t test, and the difference between the groups was statistically significant. Results: in 1 gastric cancer, UCA1 and miR-27b were negatively correlated in this department In the division, we first studied the abnormal expression of lncRNA. in gastric cancer by extracting the gene chip data from the GEO database. A recent study was based on the analysis of the expression profiles of lncRNA based on 6 cancer tissue samples and 6 paired paracancerous tissue samples. By reviewing their gene chip data (accession No.GSE53137), I have reviewed their gene chip data (accession No.GSE53137). We observed that UCA1 was one of the most up-regulated lncRNA expressions in gastric cancer tissue samples. To further verify this abnormal expression, we further conducted real-time quantitative PCR analysis (qRT-PCR) based on 28 cancerous tissues and normal tissue samples (qRT-PCR). The results showed that UCA1 was significantly up-regulated in cancer tissues (Figure 2-1B). At the same time, our qRT-PCR results showed that miR-27b, a miRNA with inhibition of multidrug resistance to gastric cancer cells, was significantly reduced in cancer tissue. We further confirmed the negative correlation between UCA1 and miR-27b by linear regression analysis (R2=0.23.p=0.01). Then, we decided to further investigate whether there was anything between them. We found that MDR gastric cancer cells, including SGC-7901/ADR, SGC-7901/DDP and SGC-7901/5-FU cells, can further increase the expression of UCA1 and reduce miR-27b expression in MDR gastric cancer cells, including SGC-7901/ADR, SGC-7901/DDP, and SGC-7901/5-FU cells. It is obvious that the bioinformatics of the MDR gastric cancer cells, including SGC-7901/ADR, SGC-7901/DDP and SGC-7901/5-FU cells, are obvious, biologically,.2. The analysis showed that UCA1 had two possible sites for binding with miR-27b. Therefore, we decided to investigate whether UCA1 had a regulatory effect on the expression of miR-27b..FISH analysis showed that UCA1 and miR-27b were distributed in both SGC7901 and SGC-7901/ADR cells in the nuclei and cytoplasm of the cytoplasm, and the expression of.UCA1 in SGC-7901/ADR cells was significantly more. The expression of miR-27b was higher in SGC7901 cells (Figure 22-D). Then, SGC-7901/ADR, SGC-7901/DDP and SGC-7901/5-FU cells were transfected with UCA siRNA. The high inhibitory Si-UCAl-1 was used for follow-up study of.QRT-PCR. The inhibition of UCA1 could significantly restore the expression of miR-27b in gastric cancer cells. UCA1 inhibition and overexpression of miR-27b make MDR gastric cancer cells more sensitive to chemotherapeutic agents in order to further investigate the role of UCA1 and miR-27b in the formation of MDR in gastric cancer cells. We used MTT chips to detect ADR.DDP and 5-FU in SGC-7901/ADR cells transfected by UCAlsiRNA or miR-27b analogue. The overexpression of ADR, DDP and 5-FU in SGC-7901/ADR cells could decrease the IC50. of ADR, DDP and 5-FU. We used flow cytometry to test the apoptosis induced by ADR in the SGC-7901/ADR cells treated with 20 mu g/ml ADR. The results showed that the cells with UCA1 inhibition and enhanced miR-27b expression could significantly increase the apoptosis after treatment. In order to further verify the apoptosis induced by ADR in these cells, the group used Western blot analysis to detect the changes in caspase -3 of anti apoptotic protein BCL-2 and apoptotic protein lysis. The results showed that UCA1 inhibition and miR-27b overexpression were significantly reduced in SGC-7901/ADR cells treated with 20 uh g /mlADR. The results showed that UCA1 inhibition and excessive expression of miR-27b made MDR gastric cancer cells more sensitive to chemotherapeutic agents,.4 UCA1 overexpression and miR-27b inhibition, so that gastric cancer cells produced MDR in order to further investigate the multidrug resistance of UCA1 and miR-27b on gastric cancer cells, the above results showed that UCA1 inhibition and miR-27b overexpression made the MDR gastric cancer cells more sensitive to.4 UCA1 overexpression and miR-27b inhibition. The UCA1 expression vector or miR-27b inhibitor was first transfected to SGC-7901 cells by.MTT chip. The results of UCA1 overexpression and miR-27b inhibition increased the ADR of SGC-7901 cells. The IC50. follow up flow cytometer analysis of DDP and 5-FU showed that the excess expression of UCA1 and inhibition significantly reduced the cells induced by 10 micron. Apoptotic.LncRNAUCA1 is a cancer promoting factor in gastric cancer cells; LncRNAUCA1 can down regulate the expression of miR-27b to enhance the multidrug resistance of gastric cancer. This paper is based on competitive endogenous RNA theory, so as to combine the LNC RNA expression profiles associated with multidrug resistance of gastric cancer cells and the data of MI RNA agricultural Da spectrum to analyze each other in ncRNA. The functional level indicates the regulatory role of UCA1-mi RNA pathway in gastric cancer MDR. Conclusion: in gastric cancer, the expression of UCA1 and miR-27b is negatively correlated. Inhibition of UCA1 can restore the expression of miR-27b in cancer cells and participate in the chemical sensitivity regulation of cancer cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2017
【分類號(hào)】：R735.2

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