食管癌微环境中PKM2参与细胞恶性表型的调控机制研究

发布时间：2018-01-05 06:36

  本文关键词：食管癌微环境中PKM2参与细胞恶性表型的调控机制研究　出处：《新疆医科大学》2017年博士论文　论文类型：学位论文

  更多相关文章： 食管癌 M2型丙酮酸激酶(PKM2) 恶性表型 调控机制

【摘要】：目的:探讨M2型丙酮酸激酶(Pyruvate Kinase M2,PKM2)与食管鳞状细胞癌(Esophageal Squamous Cell Carcinoma,ESCC)临床恶性表型的相关性;验证微环境中PKM2对食管癌细胞恶性表型的功能影响;初步探讨食管癌微环境中PKM2表达参与的细胞调控通路。方法:(1)在组织水平,通过免疫组化方法检测PKM2在食管鳞癌癌组织及癌旁正常组织中的表达,分析PKM2与食管癌临床病理参数(患者年龄、性别,肿瘤浸润深度、淋巴结转移等)和预后的相关性。此外,运用酶联免疫吸附法(Enzyme Linked Immunosorbent Assay,ELISA)验证PKM2在食管癌患者血清中的表达。(2)通过实时荧光定量PCR(Quantitative Real Time PCR,qRT-PCR)检测不同食管癌细胞系中PKM2的本底表达量差异。设计合成三条不同的PKM2干扰序列,通过siRNA干扰技术敲低食管癌Eca109细胞中PKM2的表达,qRT-PCR技术检测siRNA瞬时转染后PKM2 mRNA表达水平的变化;Westem blot技术检测蛋白水平PKM2的表达改变;采用四甲基偶氮唑盐(MTT)比色法检测细胞增殖变化;流式细胞术(Flow Cytometry,FCM)检测细胞凋亡及周期改变;细胞划痕实验检测对食管癌细胞迁移能力的影响;Transwell实验检测对食管癌细胞侵袭能力的影响。(3)进一步选择干扰效率最高的siRNA序列设计合成携带GFP的干扰PKM2表达的慢病毒包装载体,同时设计合成携带GFP的PKM2过表达慢病毒包装载体,通过转染食管癌Eca109细胞、FCM分选得到稳定转染的干扰以及过表达PKM2的细胞株,MTT法验证细胞增殖变化,细胞划痕实验验证PKM2表达量改变对食管癌细胞迁移能力的影响,Transwell实验验证PKM2表达量改变对食管癌细胞侵袭能力的影响。(4)选取4~5周龄14~17g的雌性BALB/c Nude鼠,构建裸鼠异位移植瘤模型。采用PKM2干扰及过表达慢病毒包装载体稳定转染的食管癌Eca109细胞系,细胞计数后按2×107个细胞/0.2m L/鼠的剂量接种于裸鼠右侧后肢部皮下,接种后每周测量裸鼠体重并观察瘤块的生长情况。瘤块长出后,用游标卡尺测量裸鼠瘤块长径(A)及垂直横径(B),按公式V=A×B2/2计算裸鼠肿瘤体积。组织样本,福尔马林固定后制成石蜡包埋的组织样本,切片后进行HE染色及免疫组化染色,观察移植瘤组织形态并检测移植瘤组织中PKM2的表达。(5)为探讨PKM2调控食管癌恶性表型的机制,将PKM2 siRNA转染食管癌eca109细胞及kyse150细胞,正常培养的eca109细胞及kyse150细胞设为对照组,转染后48h收集细胞并提取总rna,明确pkm2sirna干扰效果后通过mrna表达谱芯片检测,分析基因的改变和信号通路变化。结果:(1)食管癌组织中pkm2阳性表达为在细胞胞浆中呈棕褐色着色,pkm2在食管癌组织中的表达(69/75,92.0%)明显高于其在配对癌旁正常组织中的表达(9/75,12.0%),差异有统计学意义(p0.05)。kaplan-meier生存分析发现,与pkm2低表达的食管癌患者相比较,pkm2高表达的患者显示出生存时间较短的趋势(log-rank检验,p0.05)。此外,检测哈族食管癌临床样本中pkm2的表达,发现:pkm2在食管癌组织中的表达(37/54,68.5%)明显高于其在配对癌旁正常组织中的表达(8/54,14.8%),差异有统计学意义(p0.05)。通过kaplan-meier生存分析,显示:pkm2高表达的哈族食管癌患者其生存期明显短于pkm2低表达的哈族食管癌患者(log-rank检验,p0.05),即pkm2高表达的哈族食管癌患者预后较差。食管癌患者血清中pkm2的表达(78.84ng/ml)显著高于正常对照人群血清中pkm2的表达(13.55ng/ml,p0.05)。(2)在细胞水平检测不同食管癌细胞系中pkm2的本底表达量差异,结果发现在eca109、ec9706、kyse30、kyse150、kyse450及t46种细胞系中,kyse450细胞中pkm2表达量最高,其次是t4、ec9706、eca109、kyse150,kyse30细胞中pkm2表达量最低。为验证微环境中pkm2表达对食管癌细胞恶性表型的功能影响,通过设计、合成并瞬时转染pkm2sirna,结果发现三条pkm2sirna在mrna水平和蛋白水平均能显著敲低食管癌eca109细胞中pkm2的表达,其表达量改变与随机序列对照组(sirna-scramble)以及正常培养对照组(normalcontrol)比较均显著降低,差异有统计学意义(p0.05)。通过pkm2sirna瞬时转染干扰pkm2表达后进行细胞功能学实验,mtt检测发现敲低pkm2的表达后食管癌eca109细胞增殖受到显著抑制,划痕实验和transwell侵袭实验发现eca109迁移能力和侵袭能力均显著降低,fcm检测发现敲低pkm2的表达后细胞凋亡增多,细胞周期被阻滞在g0/g1期。检测细胞功能变化,发现:sirna瞬时转染成功敲低pkm2的表达后,食管癌eca109细胞的增殖受到抑制,细胞凋亡增加,细胞侵袭、迁移能力显著下降,同时细胞周期被阻滞在g0/g1期。(3)为进一步明确pkm2表达对食管癌细胞功能的影响,采用慢病毒稳定转染pkm2基因并在体内验证该结果,本研究选择干扰效果最显著的sirna序列进一步设计合成干扰pkm2稳定表达的慢病毒包装载体,以及pkm2过表达慢病毒包装载体,根据细胞转染的慢病毒种类将实验分5组:干扰pkm2表达实验组(转染sirna-pkm2慢病毒包装载体)、干扰pkm2表达对照组(转染sirna-scramble慢病毒包装载体)、pkm2过表达实验组(转染pkm2过表达慢病毒包装载体)、pkm2过表达对照组(转染pkm2-scramble慢病毒包装载体)、阴性对照组(正常培养的eca109细胞)。通过转染干扰以及过表达pkm2的慢病毒载体改变pkm2的表达后,检测细胞功能变化,细胞增殖实验结果显示,干扰PKM2表达后食管癌Eca109细胞的增殖受到显著抑制,过表达PKM2促进了Eca109细胞的增殖;划痕实验结果发现,干扰PKM2表达后Eca109细胞的相对迁移距离减小,过表达PKM2后Eca109细胞的相对迁移距离增加;Transwell侵袭实验发现,干扰PKM2表达后侵袭细胞数显著减少,过表达PKM2后侵袭细胞数显著增多,即干扰PKM2表达抑制了食管癌Eca109细胞的增殖、迁移和侵袭,过表达PKM2促进食管癌Eca109细胞的增殖、迁移和侵袭。(4)在裸鼠皮下分别注射干扰、过表达PKM2的Eca109稳定转染细胞株一周后成瘤,注射干扰PKM2表达的Eca109细胞成瘤后组织低表达PKM2,注射过表达PKM2的Eca109细胞成瘤后组织高表达PKM2,此外,PKM2过表达后裸鼠皮下移植瘤体积大于对照组,统计分析结果显示两组之间平均瘤体体积差异有统计学意义(P0.05),即过表达PKM2促进了裸鼠皮下移植瘤的生长。以上结果说明在体内PKM2的表达影响食管癌细胞的增殖。(5)mRNA表达谱芯片差异基因筛选常规标准为FC(abs)在2.0倍以上,按此标准,干扰PKM2表达后Eca109细胞中有4592个上调基因,2497个下调基因;干扰PKM2表达后KYSE150细胞中有1796个上调基因,1936个下调基因。干扰PKM2表达后Eca109细胞和KYSE150细胞中mRNA水平表达均上调的基因有298个,其中差异倍数大于5倍的基因共有16个;表达均下调的基因有277个,其中差异倍数大于4倍的基因共有8个。此外,Eca109细胞中干扰PKM2表达后受到影响的细胞功能和信号通路包括p53信号通路、自噬、蛋白质消化和吸收、补体系统以及化学致癌作用;KYSE150细胞中干扰PKM2表达后受到影响的功能和信号通路变化包括核糖体起源、N-聚糖生物合成、凋亡、TNF信号通路、Notch信号通路、DNA复制、细胞周期以及对转录的异常调控。结论:PKM2在食管癌中呈高表达,哈族食管癌患者高表达PKM2其预后差。PKM2高表达能促进食管鳞癌细胞的增殖、迁移以及侵袭,抑制细胞凋亡,并影响细胞的周期分布。PKM2对食管癌细胞恶性表型的影响可能受p53信号通路、TNF信号通路和Notch信号通路等多种因素的调控。
[Abstract]:Objective: To investigate the effect of pyruvate kinase M2 (Pyruvate Kinase M2, PKM2) and esophageal squamous cell carcinoma (Esophageal Squamous Cell Carcinoma, ESCC) the clinical relevance of the malignant phenotype; influence of PKM2 verification in the microenvironment of the malignant phenotype of esophageal cancer cell function; preliminary study of cell regulation pathways involved in the expression of PKM2 esophageal cancer microenvironment. Methods: (1) at the organizational level, expression was detected by immunohistochemistry method PKM2 in esophageal squamous cell carcinoma and normal tissues, analysis of PKM2 and the clinical pathological parameters of esophageal cancer (age, gender, tumor invasion depth, lymph node metastasis) correlation and prognosis. In addition, by ELISA immunoassay (Enzyme Linked Immunosorbent Assay, ELISA) to confirm the expression of PKM2 in esophageal cancer. (2) by real-time fluorescence quantitative PCR (Quantitative Real Time PCR, qRT-PCR) detection of esophageal cancer cells In the end the difference in the expression of PKM2. The design and synthesis of three different PKM2 interference sequence expression by siRNA interference on PKM2 in esophageal cancer Eca109 cells, qRT-PCR detection of siRNA after transient transfection of PKM2 mRNA expression; to detect protein expression level changes PKM2 Westem blot technology; using four methyl thiazolyl tetrazolium (MTT) assay in cell proliferation; flow cytometry (Flow Cytometry, FCM) changes of apoptosis and cell cycle detection; effects of cell scratch assay and migration of esophageal carcinoma cells; effect of Transwell assay on invasion of esophageal carcinoma cells. (3) lentiviral vector packaging further interference PKM2 interference siRNA sequence design of the highest efficiency of synthesis carrying GFP expression, at the same time the design and synthesis of GFP carrying PKM2 overexpression lentivirus packaging carrier by transfection of human esophageal carcinoma cells Eca109 and FCM Isolated interference stable transfection and overexpression of PKM2 cell line, cell proliferation was determined by MTT method, the expression of cell scratch experiments to verify the PKM2 volume change effect on esophageal cancer cell migration and expression of Transwell experiment PKM2 effects on invasion of esophageal carcinoma cells. (4) female BALB/c Nude rats from 4~5 week at the age of 14~17g, construct the heterotopic nude mouse model. Using PKM2 interference and overexpression of Eca109 in esophageal carcinoma cell lines lentiviral vector was successfully transfected into the packaging cell, after counting according to the dose inoculation of 2 * 107 cells in /0.2m L/ rats in nude rat right hindlimb subcutaneous growth of mice weight was measured weekly, and observe the tumor after inoculation of tumor. After the long, measured with vernier caliper tumor block length (A) and vertical diameter (B), calculate the tumor volume according to the formula of V=A * B2/2. Tissue samples were formalin fixed after made of stone Paraffin embedded tissue samples, sections were stained with HE and immunohistochemistry to observe the expression of PKM2 in transplanted tumor tissue morphology were detected in transplanted tumor tissue. (5) to explore the mechanism of regulation of malignant phenotype of PKM2 esophageal carcinoma, PKM2 siRNA transfected esophageal cancer Eca109 cells and kyse150 cells, the normal cultured Eca109 and kyse150 cells as control group, transfected 48h cells were collected and extracted the total RNA, clear pkm2sirna jamming effect by mRNA microarray detection, change and signal pathway of gene. Results: (1) the positive expression of pkm2 in esophageal cancer tissue was brown coloration in the cell cytoplasm, the expression of pkm2 in esophageal carcinoma (69/75,92.0%) was significantly higher than the expression in adjacent normal tissues (9/75,12.0%), the difference was statistically significant (P0.05) and pkm2.Kaplan-meier survival analysis showed that low expression of esophageal cancer patients In comparison, patients with high expression of pkm2 showed a survival in a relatively short time trend (log-rank test, P0.05). In addition, detected expression of Kazakh's esophageal cancer in clinical samples of pkm2: the expression of pkm2 in esophageal carcinoma (37/54,68.5%) was significantly higher than the expression in adjacent normal tissues (8/54,14.8%), the difference was statistically significant (P0.05). The Kaplan-Meier survival analysis showed that the high expression of pkm2 in Kazakh esophageal cancer patients whose survival was significantly shorter than the low expression of pkm2 in Kazakh esophageal cancer patients (log-rank test, P0.05), the high expression of pkm2 in Kazakh esophageal cancer patients with poor prognosis. The expression of pkm2 in serum of patients with esophageal cancer in (78.84ng/ml) pkm2 expression was significantly higher than that of normal control group in serum (13.55ng/ml, P0.05). (2) pkm2 at the cellular level detection of esophageal carcinoma cell line in the bottom of the expression differences were found in the Eca109, EC9706, kyse30, Kyse150, KYSE450 and T46 cell lines, the highest expression of pkm2 in KYSE450 cells, followed by T4, EC9706, Eca109, kyse150, pkm2 was the lowest in kyse30 cells. Pkm2 verification in the micro environment affect the expression, the malignant phenotype of esophageal cancer cell function through the design, synthesis and transient transfection of pkm2sirna results found that the expression of three pkm2sirna were significantly lower on pkm2 in esophageal cancer Eca109 cells at mRNA and protein level, the expression changes and random sequence control group (sirna-scramble) and normal control group (normalcontrol) training is significantly reduced, the difference was statistically significant (P0.05). Experimental study by cell function the expression of pkm2sirna after transient transfection of pkm2 interference, MTT detected that the expression knockdown of pkm2 after esophageal cancer Eca109 cells proliferation was significantly inhibited, cell scratch test and Transwell test showed that Eca109 migration and The invasion ability decreased significantly, the apoptosis that knockdown of pkm2 expression after FCM detection increased, cell cycle arrest in g0/g1 phase. It is found that changes in detection of cell function: siRNA transient transfection knockdown pkm2 protein after Eca109 esophageal cancer cell proliferation inhibition, cell apoptosis, cell invasion and migration ability at the same time significantly decreased, cell cycle arrest in g0/g1 phase. (3) to further clarify the effect of pkm2 expression on esophageal cancer cell function, using lentivirus transfected with pkm2 gene and verified the results in vivo, siRNA sequences in this study disturbance is the most significant effect of the further design package lentivirus vectors for the synthesis of stable expression of pkm2 interference the expression of pkm2 and lentiviral packaging carrier, according to the types of cells transfected with lentivirus were divided into 5 groups: interference of pkm2 expression in the experimental group (sirna-pkm2 transfected with lentiviral packaging carrier), PK interference The expression of M2 in the control group (transfected with sirna-scramble lentiviral vector packaging), over expression of pkm2 in experimental group (transfected with pkm2 overexpression lentivirus vector, overexpression of pkm2 packaging) and control group (transfected with pkm2-scramble lentiviral vector packaging), negative control group (normal Eca109 cells). By transfection of interference and overexpression of Lentivirus Expression Vector the change of pkm2 after pkm2, to detect the changes of cell function, cell proliferation experiments showed that PKM2 knockdown esophageal carcinoma Eca109 cells significantly inhibited the proliferation, overexpression of PKM2 promotes the proliferation of Eca109 cells; scratch experiment results showed that the relative migration in PKM2 knockdown Eca109 cells after overexpression of PKM2 distance decreases. The relative migration distance of Eca109 cells increased; Transwell invasion experiments showed that PKM2 knockdown significantly reduce the number of invasive cells, overexpression of PKM2 cells increased significantly after the invasion, dry PKM2 interference inhibited the expression of Eca109 esophageal cancer cell proliferation, migration and invasion, overexpression of PKM2 promotes Eca109 esophageal cancer cell proliferation, migration and invasion. (4) in nude mice were injected with interference and overexpression of Eca109 stable transfected cell line PKM2 carrying tumor one week, tumor tissue with low expression of PKM2 rejection PKM2 the expression of Eca109 stem cell injection, injection of PKM2 overexpressing Eca109 cells into tumor tissue after high expression of PKM2, in addition, over expression of PKM2 volume after subcutaneous transplantation tumor in nude mice than in the control group, statistical analysis showed that there were statistically significant differences in average tumor volume between the two groups (P0.05), which promotes the over expression of PKM2 in nude mice the growth of xenografts. The above results showed that the effect of the proliferation of esophageal carcinoma cells PKM2 expression in vivo. (5) the difference of gene expression microarray screening standard for FC mRNA (ABS) in more than 2 times, according to this standard, the expression of PKM2 after interference There are 4592 up-regulated genes in Eca109 cells, 2497 down regulated genes; interference PKM2 expression of KYSE150 cells after 1796 up-regulated, 1936 down regulated genes. PKM2 knockdown Eca109 cells and KYSE150 cells in the expression of mRNA were up-regulated and 298 genes, of which more than 5 times the number of times the difference between the total gene 16; the expression of 277 genes were down regulated, which showed over 4 times for a total of 8 genes. In addition, cellular functions and signaling pathways affected by PKM2 expression including p53 signaling pathway, Eca109 interference cell autophagy, protein digestion and absorption, complement system and chemical carcinogenesis; function and signal affected by the expression of PKM2 pathway changes including the ribosome origin interference in KYSE150 cells, N- glycan biosynthesis, apoptosis, TNF signaling pathway, Notch signaling pathway, DNA replication, cell cycle and abnormal regulation of transcription. Conclusion: the high expression of PKM2 in esophageal carcinoma was high expression in patients with esophageal carcinoma PKM2 Kazakh poor prognosis of esophageal squamous cell carcinoma with high expression of.PKM2 can promote cell proliferation, migration and invasion, apoptosis, and cell cycle distribution of.PKM2 on the malignant phenotype of esophageal carcinoma cells may be affected by the impact of the p53 signaling pathway, regulate a variety of the factors of TNF signaling pathway and Notch signaling pathway.

【学位授予单位】：新疆医科大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.1

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