LncRNA UCA1通过下调miR-27b表达增强胃癌多药耐药性的研究
发布时间：2018-07-24 19:48
【摘要】：第一部分LncRNA UCA1在胃癌的发生发展中的作用研究目的:LncRNAUCA1在胃癌的发生发展中的作用研究方法:诊断为原发性胃癌且未进行化疗或放疗的56位患者行胃切除术,取胃癌组织和癌旁正常组织切除后立即用液氮进行冰冻,然后保存于-80℃以备用。首先把由4%多聚甲醛浸泡了 48小时以后的由手术切除的胃粘膜组织,用石蜡进行包埋,再经4μm连续切片以后的用石蜡包埋好的组织再行HE染色,染色以后再由病理学专业的专家对上述切片给予分析和评价。再经免疫组化染色后提取胃癌样本总RNA,抽提细胞总RNA并进行变性琼脂糖电泳检测以及RNA逆转录反应。在进行反转录反应之后,应用Real-time PCR技术对lnc RNA、mRNA的表达丰度分别进行检测。结果:1 HE及免疫组化染色胃癌组织和癌旁正常组织的石蜡标本经HE染色,病理学复查示诊断和实验前诊断一致;免疫组织化学染色表明,胃癌组织标本呈现出高度度表达,而癌旁正常组织基本上不作出表达。2 胃癌组织中的lnc RNA表达水平明显上调采用Real-time PCR方法对病理采集(癌组织和癌旁胃组织)中,lnc RNA UCA1的表达进行检测,采集标本共56对。lnc RNA UCA1在癌旁组织中的相对含量(1.29 ± 0.26)低于癌组组织(2.85±0.74),Wilcoxon matched pairs test 有显著统计学意义(p0.01)。表明lnc RNA UCA1极有可能是一种存在于胃癌组织中的促癌基基因。3 lnc RNA UCA1在胃癌和7癌旁胃正常组织中相对含量的比值同胃癌的临床病理特征、临床预后相关进一步研究。分析在56例胃癌以及胃癌旁组织内的lnc RNA表达出的相对含量的C/P比值与性别、年龄、癌肿大小、分期、浸润程度、分化情况、淋巴结侵袭之间的关系。结果发现:C/P比值与肿瘤大小、肿瘤的分化程度有明显关联;其于低分化的胃癌病人中的表达有增加趋势,分析差异具有统计学意义;与性别、年龄无明显相关,分析差异没有统计学意义,但其和癌肿的浸润程度、淋巴结转移、TNM分期及远处转移亦无明显相关,分析差异没有统汁学意义,怀疑此结果可能与样本量偏少有关。在此过程中我们把56例标本平均分成两组,分别为C/PC/P平均值组和C/PC/P平均值组,对术后生存时间(hour)进行统计学分析。发现C/PC/P平均值组患者术后生存时间长于C/PC/P平均值组,且差异没有统计学意义,究其原因可能受随访时间过短及胃癌术后5年生存率较高等因素影响。结论:lncRNAUCA1与胃癌的发生、发展及分化程度相关,在胃癌中起到促癌基因的作用。第二部分LncRNA UCA1通过下调miR-27b表达增强胃癌多药耐药性研究目的:探索UCA1和miR-27b在胃癌的发生发展中的联系,并进一步研究其在胃癌的多药耐药性中的作用。探讨lncRNAUCA1通过下调miR-27b的表达强度和胃癌的多药耐药性的关系。筛选和验证胃癌耐药相关lnc RNA分子。通过实验验证lnc RNA UCA1在胃癌的多药耐药性发生中所起的作用;并验证lnc RNA UCA1能够调节胃癌多药耐药性的分子机理。方法:异常表达的lncRNA的基因芯片数据来源于GEO数据库。采用实时荧光定量 PCR的方法对28组癌症和癌旁的正常组织样本给予分析以评估lncRNAUCA1和miR27b的表达情况。采用人类胃胃癌细胞系SGC-7901细胞和SGC-7901衍生的具有阿霉素抗性的SGC-7901/ADR细胞,顺铂耐药的SGC-7901/DDP细胞和5-氟尿嘧啶耐药的SGC-7901/FU细胞作为体外细胞模型来评估UCA1和miR-27b在胃癌多药耐药性中的作用。诊断为原发性胃癌且未进行化疗或放疗的28位患者行胃切除术,取胃癌组织和癌旁正常组织切除后立即用液氮进行冰冻,然后保存于-80℃以备用。细胞培养和转染癌症细胞培养在RPMI-1640培养基内,并加入10%FBS,100μg/mL青霉素,及100U/mL链霉素。通过采用Ribobio试剂盒采用75 nM UCA1 siRNA或混合阴性对照(Ribobio)对 SGC-7901/ADR,SGC-7901/DDP 和 SGC-7901/FU 细胞进行转染,而采用 100 nMmiR-27b模拟物或阴性对照(Ribobio)对SGC-7901/ADR细胞进行转染,使用的试剂盒为Lipofectamine 2000 48小时后,收集siRNA转染的细胞,并用qRT-PCR测定其抑制的程度。对与UCA1基因互补的DNA进行扩增,并插入到pcDNA3.1的BamhI/XhoI位点之间。采用pcDNA3.1-UCA1表达载体或50 nM miR-27b抑制剂(Ribobio)SGC-7901细胞进行转染。胃癌组织和相应的癌旁正常组织的lncRNA表达谱的基因芯片数据调采用DIANA工其对UCA1和miR-27b的结合位点进行预测。采用TRIzol试剂提取组织及细胞样品的总RNA。进行反转录以获得一链cDNA。采用TaqMan MicroRNA Assay Kit进行实时荧光定量PCR分析。结果采用2-AACT法进行计算。采用线性回归分析对UCA1表达的水平和miR-27b表达的水平的相关性进行评估。采用Cy3标记的抗生物素抗体在37℃反应30min时检测信号。采用DAPI来复染细胞核,然后用FV1000荧光显微镜检测免疫荧光。采用UCA1 siRNA或miR-27b模拟物对SGC-7901/ADR细胞进行转染,而采用UCA1表达载体或miR-27b抑制剂对SGC-7901细胞进行转染。转染24小时后,在96孔板上接种细胞。24小时后,用不同浓度的ADR,DDP或5-FU对细胞进行处理48小时。采用传统MTT芯片来对细胞活性进行测量。采用酶标仪来记录490 nm的吸光度情况。通过绘制剂量-反应曲线计算 IC50 值。采用 Annexin V-FITC Apoptosis Detection Kit 来检测细胞的凋亡情况,采用流式细胞仪计算凋亡率。将含有30μg总蛋白的样品被置于每一条带上,然后用10%SDS-PAGE进行分离。之后,电泳使蛋白样品转移至硝酸纤维膜上。对硝酸纤维膜进行封闭、洗涤并用抗BCL-2一抗(ab32124,Abeam)和裂解的半胱天冬蛋门酶-3(#9661.Cell Signaling.Danvers,MA,USA)以及β-肌动蛋白(ab8227,Abcam)孵育过夜。洗涤之后,用HRP共轭的二抗对硝酸纤维膜进行孵育。采用化学发光超敏显色试剂盒super ECL detection reagent对蛋白条带进行视觉化。采用GraphPad Prism 6.0进行数据分析。采用非配对双侧t检验方法来进行组间差异评估。p0.05表示差异具有统计显著性。结果:1胃癌中UCA1和miR-27b呈负相关在本部分中,我们首先通过提取GEO数据库的基因芯片数据研究了胃癌组织中异常表达的lncRNA。有一项近期的研究基于6个癌症组织样品和6个配对的癌旁组织样品对lncRNA的表达谱进行了分析。通过回顾他们的基因芯片数据(accession No.GSE53137),我们观察到UCA1是胃癌组织样品中被最大程度上调表达的lncRNA之一。为了进一步验证这种异常表达情况,我们进一步基于28对癌症组织和癌旁正常组织样品进行了实时荧光定量PCR分析(qRT-PCR)。结果表明,UCA1在癌症组织中被显著上调(图2-1B)。与此同时,我们的qRT-PCR结果表明miR-27b,一种对胃癌细胞具有抑制多药耐药性的miRNA,在癌症组织中显著下降。通过线性回归分析,我们进一步确认了 UCA1和miR-27b的负相关关系(R2=0.23.p=0.01)。然后,我们决定进一步调查它们之间是否存在什么关联,以及它们在胃癌细胞多药耐药性形成中的影响。2抑制UCA1可恢复MDR 胃癌细胞中miR-27b的表达水平通过qRT-PCR,我们发现MDR胃癌细胞,包括SGC-7901/ADR,SGC-7901/DDP和SGC-7901/5-FU细胞有进一步增加UCA1表达和降低miR-27b表达。很明显,生物信息分析表明UCA1有两个与miR-27b结合的可能位点。因此,我们决定调查看看UCA1是否对miR-27b的表达具有调控作用。FISH分析结果表明,UCA1和miR-27b在SGC7901和SGC-7901/ADR细胞的细胞核和细胞质中都有分布。UCA1在SGC-7901/ADR细胞中的表达量明显要更高,而miR-27b表达量则在SGC7901细胞中更高(图22-D)。接着,用UCA siRNA对 SGC-7901/ADR,SGC-7901/DDP 和 SGC-7901/5-FU 细胞进行转染。具有较高抑制作用的Si-UCAl-1被用于后续研究。QRT-PCR结果表明,对UCA1的抑制能够显著恢复miR-27b在MDR 胃癌细胞内的表达。3 UCA1抑制和miR-27b过量表达使MDR 胃癌细胞对化疗试剂变得更加敏感为了进一步调查UCA1和miR-27b在胃癌细胞MDR形成中的作用,我们采用MTT芯片对UCAlsiRNA或miR-27b模拟物转染后的SGC-7901/ADR细胞中的ADR.DDP和5-FU的IC50进行检测。结果表明,UCA1抑制和miR-27b过量表达都会降低SGC-7901/ADR细胞中的ADR,DDP和5-FU的IC50。接着,我们采用流式细胞仪检验用20μg/ml ADR处理后的SGC-7901/ADR细胞中ADR诱导的细胞凋亡。结果表明,UCA1抑制和miR-27b的增强表达的细胞能够显著增加ADR处理后的细胞凋亡。因为想要进一步验证这些细胞中由ADR诱导的细胞凋亡,本课题组运用免疫印迹分析以检测抗凋亡蛋白BCL-2和凋亡蛋白裂解的半胱天冬蛋白酶-3的变化情况。结果表明,UCA1抑制和miR-27b过量表达在20μg/mlADR处理后的SGC-7901/ADR细胞内显著降低了 BCL-2农达并增加了半胱天冬蛋白酶-3的表达。上述结果显示UCA1抑制和miR-27b过量表达使MDR胃癌细胞对化疗试剂变得更敏感。4 UCA1过量表达和miR-27b抑制使胃癌细胞产生了 MDR为了进一步调查UCA1和miR-27b对胃癌细胞的多药耐药性的调控作用,首先采用UCA1表达载体或miR-27b抑制剂对SGC-7901细胞进行转染。MTT芯片结果证实UCA1过量表达和miR-27b抑制增加了SGC-7901细胞的ADR,DDP和5-FU的IC50。后续的流式细胞仪分析表明UCA1过量表达和miR-27b抑制显著降低了 ADR(10μ/ml)诱导的细胞凋亡。LncRNAUCA1是胃癌细胞中的促癌因子；LncRNAUCA1能够下调miR-27b表达从而增强胃癌多药耐药性,本论文通过竞争内源性 RNA理论为基础,从而把胃癌细胞多药耐药性相关的lnc RNA表达谱和mi RNA农达谱的数据进行统一合并分析,在ncRNA相互作用的层面表明了UCA1-mi RNA通路在胃癌MDR中的调节作用,结论:在胃癌中,UCA1与miR-27b表达呈负相关关系。抑制UCA1可恢复miR-27b在癌症细胞中的表达。UCA1-miR-27b通路参与了癌症细胞的化学敏感性调节。
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2
本文编号：2142468
[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.
【学位授予单位】：山东大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.2
【参考文献】
相关期刊论文 前3条
1 陈凛;李涛;;胃癌综合治疗现状与进展[J];世界华人消化杂志;2008年06期
2 KatherineDCrew;AlfredINeugut;;Epidemiology of gastric cancer[J];World Journal of Gastroenterology;2006年03期
3 ;Expression of NF-кB and human telomerase reverse transcriptase in gastric cancer and precancerous lesions[J];World Journal of Gastroenterology;2004年02期
，本文编号：2142468
本文链接：https://www.wllwen.com/shoufeilunwen/yxlbs/2142468.html
教材专著
