microRNA-187-3p在肝细胞癌中的表达及预后意义
发布时间：2018-07-28 15:33
【摘要】：目的:肝细胞癌是一种世界范围内常见的恶性肿瘤,其死亡率占肿瘤相关死亡率的第三位。尽管以手术治疗为主的综合治疗提高了肝细胞癌患者的5年生存率,但肝细胞癌的发病机制十分复杂,缺乏特异性强和灵敏度高的生物学标记物,肝细胞癌患者5年生存率仅为15.0%-45.7%。因此深入研究肝细胞癌的分子生物学机制,寻求特异的诊断治疗靶点,是目前肝细胞癌研究的重点。micro RNA(miRNA)是一类广泛存在于真核生物中的非编码小片段RNA,其通过调节靶基因的表达进而广泛参与机体的各项生理、病理过程中。miR-187-3p是miR-187家族的一员,定位于人染色体18q12.2。已有研究报道在结直肠癌、前列腺癌、非小细胞肺癌、肾细胞癌、食管腺癌中miR-187-3p呈低表达,这些研究表明miR-187-3p在人类肿瘤发生发展中发挥着抑癌基因的作用。然而,也有实验证实miR-187-3p在外周T细胞淋巴瘤中高表达,并且miR-187-3p能增加乳腺癌细胞的侵袭能力,与乳腺癌患者的预后有关。因此,在不同的肿瘤组织中miR-187-3p发挥的作用可能不同。但是miR-187-3p在肝细胞癌组织中的表达状况及作用报道较少。本研究通过miRNA芯片杂交技术筛选新鲜肝细胞癌组织中差异表达的miRNA,然后通过实时荧光定量PCR检测miR-187-3p的表达情况,并结合患者临床资料、生存期资料及病理学特征分析miR-187-3p在肝细胞癌发生发展、临床诊断以及预后评估中的意义,为miR-187-3p在临床中的应用提供理论依据。方法:1研究对象及标本选取2010年4月至2016年8月于河北医科大学第四医院肝胆外科行手术切除的肝细胞癌患者90例,其中男性75例,女性15例,年龄25-78岁。依据手术前一周实验室的指标、Child-Pugh分级标准评估肝功能,同时确定肿瘤标记物水平和肝炎类型,并根据影像学检查和术中情况评估肝硬化严重程度以及肿瘤特征。90例患者中肿瘤直径2-25cm。肝功能Child A级87例,B级3例。临床分期Ⅰ期39例、Ⅱ期29例,Ⅲ期22例。本实验中患者乙肝表面抗原(HBs Ag)阳性74例,阴性16例。所有研究对象在手术前均未进行过放疗、化疗或其它抗肿瘤治疗。研究标本共180例,其中新鲜肝细胞癌组织标本90例及相对应癌旁组织90例。标本提取后立即保存于液氮中,然后储存于-80℃低温冰箱中以备实验使用。所有标本均经过常规石蜡包埋,HE染色,最终由2位病理科医师进行组织学诊断。收集患者肝炎病史、饮酒史、吸烟史以及家族史等资料,记录患者肝功能、AFP值、血常规、凝血功能、甲乙丙肝炎等实验室指标,登记患者肿瘤病理类型、肿瘤大小和数目、门静脉及其分支是否存在瘤栓等病理学资料。通过电话联系或信访等途径对患者进行随访,随访开始时间即为患者手术日,随访截止时间为2016年10月11日。2 miRNA芯片杂交技术从90例肝细胞癌患者中选取5例利用引物Poly(A)Tailing反转录符合标准的总RNA,然后进行miRNA芯片杂交、洗涤、扫描、最终生成数据。最后进行生物信息学分析以及应用互联网上miRNA靶基因预测软件(Targetscan、Mi Randa)在线服务站点,输出表达差异miRNA的预测靶基因,取2个软件预测靶基因的交集(实验由上海其明生物信息有限公司协助完成)。3检测肝细胞癌组织和癌旁组织中miR-187-3p的表达情况利用Trizol法提取组织总RNA,对RNA进行纯度、完整性和浓度的测定,然后将符合标准的RNA反转录为c DNA,再采用SYBR Green实时荧光定量PCR技术检测90对肝细胞癌组织标本及配对的癌旁组织标本中miR-187-3p表达情况。miR-187-3p特异性茎环反转录引物:5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CCGGCTGC-3’;U6特异性茎环反转录引物:5’-AACGCTTCACGAAT TTGCGT-3’。miR-187-3p实时荧光定量PCR上游引物:5’-GTGCAGGGT CCGAGGTATT-3’;下游引物:5’-GCCGCTCGTGTCTTGTGTTGCAGC-3’。U6实时荧光定量PCR上游引物:5’-CTCGCTTCGGCAGCACA-3’;下游引物:5’-AACGCTTCACGAATTTGCGT-3’。4统计学分析统计学分析使用统计软件SPSS 21.0,实验数据正态性分布检验采用Kolmogorov-Smimov法。计数资料采用χ2检验或Fisher精确检验,两组间的配对样本采用Wilcoxon检验。生存分析使用Kaplan-Meier法,用Log-rank检验进行单因素分析,用COX回归模型进行多因素分析。P0.05为差异具有统计学意义。结果:1肝细胞癌组织中miRNA表达谱分析运用miRNA芯片杂交技术,在5对肝细胞癌及相应癌旁组织中筛选出差异表达在2倍以上的miRNA共148个,其中102个miRNAs在肝细胞癌组织较癌旁组织表达下调,46个miRNAs在肝细胞癌组织较癌旁组织表达上调(实验由上海其明生物信息有限公司协助完成)。2肝细胞癌组织及相应癌旁组织中miR-187-3p的相对表达量及比较在90例肝细胞癌患者中有63例(70.0%)癌组织中miR-187-3p相对于癌旁组织表达量下降,且差异具有统计学意义(Z=-2.891,P=0.004);说明miR-187-3p在肝细胞癌组织中的表达显著低于癌旁组织,这与miRNA芯片结果相一致。3肝细胞癌患者中miR-187-3p的表达状态与临床病理特征的关系肝细胞癌患者中miR-187-3p的异常表达与肿瘤大小、临床分期显著相关(P0.05)。在肿瘤直径≥5cm的患者中miR-187-3p表达下调的阳性率为(75.0%),肿瘤直径5cm的患者中miR-187-3p表达下调的阳性率为(59.1%),其差别具有统计学意义(P=0.017);在临床分期Ⅰ期患者中miR-187-3p表达下调的阳性率为(69.2%),Ⅱ期患者中miR-187-3p表达下调的阳性率为(79.3%),Ⅲ期患者中miR-187-3p表达下调的阳性率为(59.1%),差异具有统计学意义(P=0.002);但在不同性别(P=0.119)、年龄(P=0.976)、乙肝表面抗原(P=0.342)、门静脉及其分支瘤栓(P=0.392)、肿瘤个数(P=0.867)、AFP水平(P=0.198)组别之间差异无显著相关性。4 miR-187-3p的表达状态与肝细胞癌患者术后生存期预后的关系Kaplan-Meier生存分析表明miR-187-3p表达下调的肝细胞癌患者的1年、3年、5年生存率为87.8%、56.2%、24.7%,平均生存时间40.470±4.407个月;上调者的1年生存率为90%,平均生存时间64.600±4.996个月,差异有统计学意义(P=0.011)。门静脉及其分支有瘤栓患者的1年生存率为50.9%,平均生存时间17.568±2.326个月,中位生存时间19个月;无瘤栓患者的1年、3年、5年生存率为93.8%、68.4%、59.3%,平均生存时间51.266±4.164个月,中位生存时间70个月,两者间差异有统计学意义(P=0.005)。肿瘤直径5cm患者的1年生存率为82.9%,平均生存时间58.802±5.791个月,中位生存时间70个月;肿瘤直径≥5cm患者的1年、3年、5年生存率为95%、59.9%、37.2%,平均生存时间41.713±4.606个月,中位生存时间26个月,两者之间的差异有统计学意义(P=0.032)。临床分期Ⅰ期患者的1年、3年生存率86.4%、47.8%,平均生存时间58.629±5.203个月;Ⅱ期患者1年生存率为77.6%,平均生存时间30.593±6.290个月;Ⅲ期患者1年生存率为56.8%,平均生存时间20.729±1.914个月,三者之间差异具有统计学意义(P=0.001)。Child-Pugh分级为A级患者的1年、3年、5年生存率为97.5%、71.2%、63.3%,平均生存时间47.109±3.984个月,中位生存时间63个月;Child-Pugh分级为B级患者的1年、3年、5年生存率为0%,平均生存时间19.667±6.006个月,中位生存时间26个月,两者间差异无统计学意义(P=0.437)。年龄60岁的1年、3年生存率分别为88.9%、62.3%,平均生存时间49.916±4.785个月;年龄≥60岁的1年生存率为78.5%,平均生存时间39.836±6.172个月,两者间差异无统计学意义(P=0.441)。进一步分析肝细胞癌患者术后总生存期的独立危险因子,我们将miR-187-3p表达水平、临床分期、肿瘤大小等影响因素进行多因素COX回归模型分析,结果显示miR-187-3p表达水平、肿瘤个数、临床分期是影响肝细胞癌患者术后总生存期的独立危险因子(P均0.05),其余参数P值均大于0.05。结论:1肝细胞癌组织中存在差异表达的miRNAs,其中miR-187-3p在肝细胞癌组织中的表达较相应癌旁组织表达下调,表明miR-187-3p可能参与了肝细胞癌的发生与发展。2 miR-187-3p的差异表达与肝细胞癌患者肿瘤大小、临床分期有关,表明miR-187-3p异常表达一定程度上反应肝细胞癌患者的病情变化。3 miR-187-3p的表达状态、肿瘤个数、临床分期可作为肝细胞癌患者术后总生存期的独立危险因子,可作为肝细胞癌患者判断预后的指标。
[Abstract]:Objective: hepatocellular carcinoma is a common malignant tumor worldwide, and its mortality accounts for third of the tumor related mortality. Although comprehensive treatment based on surgical treatment improves the 5 year survival rate of patients with hepatocellular carcinoma, the pathogenesis of hepatocellular carcinoma is very complex and lacks specific and sensitive biological markers. The 5 year survival rate of the patients with hepatocellular carcinoma is only 15.0%-45.7%., so the molecular biological mechanism of hepatocellular carcinoma is deeply studied and the specific diagnostic target is sought. The focus of.Micro RNA (miRNA) is a class of non coding small fragment RNA, which is widely used in eukaryotes, and is widely used to regulate the expression of target genes. .miR-187-3p is a member of the miR-187 family during the pathological process of the body. The study on human chromosome 18q12.2. has been reported to have reported low expression of miR-187-3p in colorectal cancer, prostate cancer, non small cell lung cancer, renal cell carcinoma, and esophageal adenocarcinoma. These studies show that miR-187-3p plays an inhibitory role in the development of human tumor. The role of oncogenes. However, there are also experiments that confirm the high expression of miR-187-3p in peripheral T cell lymphoma, and miR-187-3p can increase the invasive ability of breast cancer cells, which is related to the prognosis of breast cancer patients. Therefore, the role of miR-187-3p in different tumor tissues may be different. But miR-187-3p is in the tissue of hepatocellular carcinoma. This study screened the differentially expressed miRNA in the tissues of fresh hepatocellular carcinoma by miRNA chip hybridization, and then detected the expression of miR-187-3p by real-time fluorescence quantitative PCR, combined with the clinical data of the patients, the survival time data and the pathological characteristics analysis of miR-187-3p in the development of hepatocellular carcinoma. The significance of bed diagnosis and prognostic evaluation provided a theoretical basis for the application of miR-187-3p in clinical practice. Methods: 1 subjects and specimens were selected from April 2010 to August 2016 in the Department of hepatobiliary surgery, Fourth Hospital of Hebei Medical University, 90 cases of surgical resection of hepatocellular carcinoma, including 75 male, 15 female, 25-78 years old. The previous week's laboratory index, Child-Pugh grading standard assessed liver function, determined the level of tumor markers and hepatitis type, and evaluated the severity of liver cirrhosis with imaging and intraoperative conditions and the tumor characteristics in.90 patients, 87 cases of 2-25cm. liver function Child A, 3 cases of B grade. Clinical stage I 39 cases, stage II 29. There were 74 cases of hepatitis B surface antigen (HBs Ag) positive and 16 cases negative in this experiment. All the subjects were not treated with radiotherapy, chemotherapy or other antitumor treatment before operation. There were 180 cases of study specimens, of which 90 cases of fresh hepatocellular carcinoma tissue and 90 cases of relative cancerous tissue were preserved in liquid nitrogen immediately after extraction. And then stored in -80 C cryogenic refrigerator for experimental use. All specimens were covered with conventional paraffin embedded, HE staining, and finally by 2 pathologists. The history of hepatitis, the history of drinking, the history of smoking and family history were collected, and the liver function, blood routine, blood clotting function, hepatitis C, etc. were recorded. The pathological type of the patient's tumor, the size and number of the tumor, the size and number of the tumor, the presence of the tumor embolus in the portal vein and its branches. The patients were followed up by telephone or letter visits. The follow-up time was the operation day of the patients. The follow-up time was the.2 miRNA chip in October 11, 2016 from 90 cases of liver. In the patients with cell carcinoma, 5 cases were selected by using primer Poly (A) Tailing reverse transcription of the total RNA, and then miRNA chip hybridization, washing, scanning, and finally generating data. Finally, bioinformatics analysis and the application of miRNA target gene prediction software on the Internet (Targetscan, Mi Randa) online service site were carried out, and the output differential miRNA was expressed. To predict the target gene, take the intersection of 2 software prediction target genes (the experiment was completed by Shanghai Ming biologic information Co., Ltd.) to detect the expression of miR-187-3p in the tissues of hepatocellular carcinoma and the adjacent tissues by.3. The total RNA was extracted by Trizol method, the purity, integrity and concentration of RNA were measured, and the standard RNA reverse transcription was followed. For C DNA, SYBR Green real-time fluorescent quantitative PCR was used to detect the miR-187-3p expression in 90 pairs of hepatocellular carcinoma tissue specimens and paired paracancerous tissue specimens,.MiR-187-3p specific stem ring reverse transcriptional primers: 5 '-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CCGGCTGC-3'; U6 specific stem ring reverse transcription primers: 5 '-AACGCTTCAC GAAT TTGCGT-3 '.MiR-187-3p real-time fluorescence quantitative PCR upstream primers: 5' -GTGCAGGGT CCGAGGTATT-3; downstream primers: 5 '-GCCGCTCGTGTCTTGTGTTGCAGC-3'.U6 real-time fluorescent quantitative PCR upstream primers: 5 '-CTCGCTTCGGCAGCACA-3'; downstream primers: 5 '-AACGCTTCACGAATTTGCGT-3'.4 statistical analysis statistical analysis using statistical software SS 21, Kolmogorov-Smimov method was used to test the normal distribution of the experimental data. The count data were tested by chi 2 or Fisher, and the paired samples between the two groups were tested by Wilcoxon test. The survival analysis used the Kaplan-Meier method, the single factor analysis was carried out by Log-rank test, and the COX regression model was used to analyze the multiple factor analysis.P0.05 as the difference system. Results: 1 the expression profiles of miRNA in hepatocellular carcinoma tissue were analyzed by miRNA chip hybridization, and there were 148 miRNA more than 2 times of differential expression in 5 hepatocellular carcinoma and corresponding para cancerous tissues, of which 102 miRNAs were downregulated in the hepatocellular carcinoma tissue than in the para cancerous tissue, and 46 miRNAs in the hepatocellular carcinoma tissue than in the para cancer group. The relative expression of miR-187-3p in.2 hepatocellular carcinoma tissues and corresponding para cancerous tissues in.2 and the corresponding para cancerous tissues in the 90 cases of hepatocellular carcinoma (70%), the relative expression of miR-187-3p in the tissues and adjacent tissues of the carcinoma was decreased, and the difference was statistically significant (Z=-2.891, P). =0.004): the expression of miR-187-3p in hepatocellular carcinoma was significantly lower than that of para cancerous tissue, which was consistent with the results of miRNA microarray. The expression of miR-187-3p in the patients with.3 hepatocellular carcinoma was closely related to the size of the tumor in the patients with hepatocellular carcinoma (miR-187-3p) in the patients with hepatocellular carcinoma (P0.05). The positive rate of down regulation of miR-187-3p expression in patients with diameter more than 5cm was (75%). The positive rate of down regulation of miR-187-3p expression in patients with tumor diameter 5cm was (59.1%), and the difference was statistically significant (P=0.017). The positive rate of down-regulation of miR-187-3p expression in stage I stage patients was (69.2%), and the positive expression of miR-187-3p expression in stage II patients was positive. The positive rate of miR-187-3p expression in stage III patients was (59.1%), and the difference was statistically significant (P=0.002), but there was no significant correlation between different sex (P=0.119), age (P=0.976), hepatitis B surface antigen (P=0.342), portal vein and its branch tumor suppository (P= 0.392), the number of tumor (P=0.867), and AFP level (P=0.198) group (.4). The relationship between the state of expression of miR-187-3p and the prognosis of postoperative survival of hepatocellular carcinoma patients Kaplan-Meier survival analysis showed that the 5 year survival rate of miR-187-3p expression was 87.8%, 56.2%, 24.7%, and the average survival time was 40.470 + 4.407 months, and the survival rate of the up - regulator was 90%, and the average survival time was 64.600 + 4.996. The difference was statistically significant (P=0.011). The 1 year survival rate of the portal and its branches with tumor embolus was 50.9%, the average survival time was 17.568 + 2.326 months and the median survival time was 19 months. The 5 year survival rate was 93.8%, 68.4%, 59.3%, and average survival time 51.266 + 4.164 months, median survival time spent within 17.568 months. The difference was statistically significant (P=0.005). The 1 year survival rate of the tumor diameter 5cm patients was 82.9%, the average survival time was 58.802 + 5.791 months and the median survival time was 70 months; the tumor diameter more than 5cm patients was 1 years and 3 years. The 5 year survival rate was 95%, 59.9%, 37.2%, average survival time between 41.713 + 4.606 months, median survival time between the two months, both between the two months. The difference was statistically significant (P=0.032). The 3 year survival rate was 86.4%, 47.8%, and the average survival time was 58.629 + 5.203 months in the clinical stage I stage patients. The 1 year survival rate in stage II patients was 77.6%, the average survival time was 30.593 + 6.290 months, the average survival rate of the patients in stage III was 56.8%, the average survival time was 20.729 for 58.629 months. There was a statistically significant (P=0.001).Child-Pugh classification of 1 years and 3 years with a 5 year survival rate of 97.5%, 71.2%, 63.3%, the average survival time was 47.109 + 3.984 months, and the median survival time was 63 months; the Child-Pugh classification was 1 years and 3 years, 5 survival rate was 0%, the average survival time was 5 months and median survival time was months. There was no statistical significance (P=0.437). The 3 year survival rate was 88.9%, 62.3%, and the average survival time was 49.916 + 4.785 months for the age of 60 years. The survival rate of 1 years for the age of 60 years was 78.5% and the average survival time was 39.836 + 6.172 months. The difference was not statistically significant (P=0.441). Further analysis of the total survival of the patients with hepatocellular carcinoma after operation. The miR-187-3p expression level, clinical stage, tumor size and other influencing factors were analyzed by multiple factor COX regression model. The results showed that the level of miR-187-3p expression, the number of tumor and the clinical stage were independent risk factors (all P 0.05) affecting the total survival of the patients with hepatocellular carcinoma (all P 0.05), and the rest of the parameters were more than 0 5. conclusion: 1 the differential expression of miRNAs in the tissues of hepatocellular carcinoma, and the expression of miR-187-3p in the hepatocellular carcinoma tissue is lower than that of the corresponding para cancerous tissue, indicating that miR-187-3p may be involved in the occurrence and development of hepatocellular carcinoma and the differential expression of.2 miR-187-3p is related to the size of the cancer of the hepatocellular carcinoma, and the clinical stage is related to the expression of miR-187-3. The abnormal expression of P, to some extent, reacts to the state of.3 miR-187-3p expression in patients with hepatocellular carcinoma, the number of tumors and clinical stages can be used as an independent risk factor for the total survival of the patients with hepatocellular carcinoma, which can be used as a prognostic indicator for patients with hepatocellular carcinoma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
本文编号：2150727
[Abstract]:Objective: hepatocellular carcinoma is a common malignant tumor worldwide, and its mortality accounts for third of the tumor related mortality. Although comprehensive treatment based on surgical treatment improves the 5 year survival rate of patients with hepatocellular carcinoma, the pathogenesis of hepatocellular carcinoma is very complex and lacks specific and sensitive biological markers. The 5 year survival rate of the patients with hepatocellular carcinoma is only 15.0%-45.7%., so the molecular biological mechanism of hepatocellular carcinoma is deeply studied and the specific diagnostic target is sought. The focus of.Micro RNA (miRNA) is a class of non coding small fragment RNA, which is widely used in eukaryotes, and is widely used to regulate the expression of target genes. .miR-187-3p is a member of the miR-187 family during the pathological process of the body. The study on human chromosome 18q12.2. has been reported to have reported low expression of miR-187-3p in colorectal cancer, prostate cancer, non small cell lung cancer, renal cell carcinoma, and esophageal adenocarcinoma. These studies show that miR-187-3p plays an inhibitory role in the development of human tumor. The role of oncogenes. However, there are also experiments that confirm the high expression of miR-187-3p in peripheral T cell lymphoma, and miR-187-3p can increase the invasive ability of breast cancer cells, which is related to the prognosis of breast cancer patients. Therefore, the role of miR-187-3p in different tumor tissues may be different. But miR-187-3p is in the tissue of hepatocellular carcinoma. This study screened the differentially expressed miRNA in the tissues of fresh hepatocellular carcinoma by miRNA chip hybridization, and then detected the expression of miR-187-3p by real-time fluorescence quantitative PCR, combined with the clinical data of the patients, the survival time data and the pathological characteristics analysis of miR-187-3p in the development of hepatocellular carcinoma. The significance of bed diagnosis and prognostic evaluation provided a theoretical basis for the application of miR-187-3p in clinical practice. Methods: 1 subjects and specimens were selected from April 2010 to August 2016 in the Department of hepatobiliary surgery, Fourth Hospital of Hebei Medical University, 90 cases of surgical resection of hepatocellular carcinoma, including 75 male, 15 female, 25-78 years old. The previous week's laboratory index, Child-Pugh grading standard assessed liver function, determined the level of tumor markers and hepatitis type, and evaluated the severity of liver cirrhosis with imaging and intraoperative conditions and the tumor characteristics in.90 patients, 87 cases of 2-25cm. liver function Child A, 3 cases of B grade. Clinical stage I 39 cases, stage II 29. There were 74 cases of hepatitis B surface antigen (HBs Ag) positive and 16 cases negative in this experiment. All the subjects were not treated with radiotherapy, chemotherapy or other antitumor treatment before operation. There were 180 cases of study specimens, of which 90 cases of fresh hepatocellular carcinoma tissue and 90 cases of relative cancerous tissue were preserved in liquid nitrogen immediately after extraction. And then stored in -80 C cryogenic refrigerator for experimental use. All specimens were covered with conventional paraffin embedded, HE staining, and finally by 2 pathologists. The history of hepatitis, the history of drinking, the history of smoking and family history were collected, and the liver function, blood routine, blood clotting function, hepatitis C, etc. were recorded. The pathological type of the patient's tumor, the size and number of the tumor, the size and number of the tumor, the presence of the tumor embolus in the portal vein and its branches. The patients were followed up by telephone or letter visits. The follow-up time was the operation day of the patients. The follow-up time was the.2 miRNA chip in October 11, 2016 from 90 cases of liver. In the patients with cell carcinoma, 5 cases were selected by using primer Poly (A) Tailing reverse transcription of the total RNA, and then miRNA chip hybridization, washing, scanning, and finally generating data. Finally, bioinformatics analysis and the application of miRNA target gene prediction software on the Internet (Targetscan, Mi Randa) online service site were carried out, and the output differential miRNA was expressed. To predict the target gene, take the intersection of 2 software prediction target genes (the experiment was completed by Shanghai Ming biologic information Co., Ltd.) to detect the expression of miR-187-3p in the tissues of hepatocellular carcinoma and the adjacent tissues by.3. The total RNA was extracted by Trizol method, the purity, integrity and concentration of RNA were measured, and the standard RNA reverse transcription was followed. For C DNA, SYBR Green real-time fluorescent quantitative PCR was used to detect the miR-187-3p expression in 90 pairs of hepatocellular carcinoma tissue specimens and paired paracancerous tissue specimens,.MiR-187-3p specific stem ring reverse transcriptional primers: 5 '-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CCGGCTGC-3'; U6 specific stem ring reverse transcription primers: 5 '-AACGCTTCAC GAAT TTGCGT-3 '.MiR-187-3p real-time fluorescence quantitative PCR upstream primers: 5' -GTGCAGGGT CCGAGGTATT-3; downstream primers: 5 '-GCCGCTCGTGTCTTGTGTTGCAGC-3'.U6 real-time fluorescent quantitative PCR upstream primers: 5 '-CTCGCTTCGGCAGCACA-3'; downstream primers: 5 '-AACGCTTCACGAATTTGCGT-3'.4 statistical analysis statistical analysis using statistical software SS 21, Kolmogorov-Smimov method was used to test the normal distribution of the experimental data. The count data were tested by chi 2 or Fisher, and the paired samples between the two groups were tested by Wilcoxon test. The survival analysis used the Kaplan-Meier method, the single factor analysis was carried out by Log-rank test, and the COX regression model was used to analyze the multiple factor analysis.P0.05 as the difference system. Results: 1 the expression profiles of miRNA in hepatocellular carcinoma tissue were analyzed by miRNA chip hybridization, and there were 148 miRNA more than 2 times of differential expression in 5 hepatocellular carcinoma and corresponding para cancerous tissues, of which 102 miRNAs were downregulated in the hepatocellular carcinoma tissue than in the para cancerous tissue, and 46 miRNAs in the hepatocellular carcinoma tissue than in the para cancer group. The relative expression of miR-187-3p in.2 hepatocellular carcinoma tissues and corresponding para cancerous tissues in.2 and the corresponding para cancerous tissues in the 90 cases of hepatocellular carcinoma (70%), the relative expression of miR-187-3p in the tissues and adjacent tissues of the carcinoma was decreased, and the difference was statistically significant (Z=-2.891, P). =0.004): the expression of miR-187-3p in hepatocellular carcinoma was significantly lower than that of para cancerous tissue, which was consistent with the results of miRNA microarray. The expression of miR-187-3p in the patients with.3 hepatocellular carcinoma was closely related to the size of the tumor in the patients with hepatocellular carcinoma (miR-187-3p) in the patients with hepatocellular carcinoma (P0.05). The positive rate of down regulation of miR-187-3p expression in patients with diameter more than 5cm was (75%). The positive rate of down regulation of miR-187-3p expression in patients with tumor diameter 5cm was (59.1%), and the difference was statistically significant (P=0.017). The positive rate of down-regulation of miR-187-3p expression in stage I stage patients was (69.2%), and the positive expression of miR-187-3p expression in stage II patients was positive. The positive rate of miR-187-3p expression in stage III patients was (59.1%), and the difference was statistically significant (P=0.002), but there was no significant correlation between different sex (P=0.119), age (P=0.976), hepatitis B surface antigen (P=0.342), portal vein and its branch tumor suppository (P= 0.392), the number of tumor (P=0.867), and AFP level (P=0.198) group (.4). The relationship between the state of expression of miR-187-3p and the prognosis of postoperative survival of hepatocellular carcinoma patients Kaplan-Meier survival analysis showed that the 5 year survival rate of miR-187-3p expression was 87.8%, 56.2%, 24.7%, and the average survival time was 40.470 + 4.407 months, and the survival rate of the up - regulator was 90%, and the average survival time was 64.600 + 4.996. The difference was statistically significant (P=0.011). The 1 year survival rate of the portal and its branches with tumor embolus was 50.9%, the average survival time was 17.568 + 2.326 months and the median survival time was 19 months. The 5 year survival rate was 93.8%, 68.4%, 59.3%, and average survival time 51.266 + 4.164 months, median survival time spent within 17.568 months. The difference was statistically significant (P=0.005). The 1 year survival rate of the tumor diameter 5cm patients was 82.9%, the average survival time was 58.802 + 5.791 months and the median survival time was 70 months; the tumor diameter more than 5cm patients was 1 years and 3 years. The 5 year survival rate was 95%, 59.9%, 37.2%, average survival time between 41.713 + 4.606 months, median survival time between the two months, both between the two months. The difference was statistically significant (P=0.032). The 3 year survival rate was 86.4%, 47.8%, and the average survival time was 58.629 + 5.203 months in the clinical stage I stage patients. The 1 year survival rate in stage II patients was 77.6%, the average survival time was 30.593 + 6.290 months, the average survival rate of the patients in stage III was 56.8%, the average survival time was 20.729 for 58.629 months. There was a statistically significant (P=0.001).Child-Pugh classification of 1 years and 3 years with a 5 year survival rate of 97.5%, 71.2%, 63.3%, the average survival time was 47.109 + 3.984 months, and the median survival time was 63 months; the Child-Pugh classification was 1 years and 3 years, 5 survival rate was 0%, the average survival time was 5 months and median survival time was months. There was no statistical significance (P=0.437). The 3 year survival rate was 88.9%, 62.3%, and the average survival time was 49.916 + 4.785 months for the age of 60 years. The survival rate of 1 years for the age of 60 years was 78.5% and the average survival time was 39.836 + 6.172 months. The difference was not statistically significant (P=0.441). Further analysis of the total survival of the patients with hepatocellular carcinoma after operation. The miR-187-3p expression level, clinical stage, tumor size and other influencing factors were analyzed by multiple factor COX regression model. The results showed that the level of miR-187-3p expression, the number of tumor and the clinical stage were independent risk factors (all P 0.05) affecting the total survival of the patients with hepatocellular carcinoma (all P 0.05), and the rest of the parameters were more than 0 5. conclusion: 1 the differential expression of miRNAs in the tissues of hepatocellular carcinoma, and the expression of miR-187-3p in the hepatocellular carcinoma tissue is lower than that of the corresponding para cancerous tissue, indicating that miR-187-3p may be involved in the occurrence and development of hepatocellular carcinoma and the differential expression of.2 miR-187-3p is related to the size of the cancer of the hepatocellular carcinoma, and the clinical stage is related to the expression of miR-187-3. The abnormal expression of P, to some extent, reacts to the state of.3 miR-187-3p expression in patients with hepatocellular carcinoma, the number of tumors and clinical stages can be used as an independent risk factor for the total survival of the patients with hepatocellular carcinoma, which can be used as a prognostic indicator for patients with hepatocellular carcinoma.
【学位授予单位】：河北医科大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.7
【参考文献】
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