microRNA-187-3p在肝細胞癌中的表達及預(yù)后意義
發(fā)布時間：2018-07-28 15:33
【摘要】：目的:肝細胞癌是一種世界范圍內(nèi)常見的惡性腫瘤,其死亡率占腫瘤相關(guān)死亡率的第三位。盡管以手術(shù)治療為主的綜合治療提高了肝細胞癌患者的5年生存率,但肝細胞癌的發(fā)病機制十分復(fù)雜,缺乏特異性強和靈敏度高的生物學標記物,肝細胞癌患者5年生存率僅為15.0%-45.7%。因此深入研究肝細胞癌的分子生物學機制,尋求特異的診斷治療靶點,是目前肝細胞癌研究的重點。micro RNA(miRNA)是一類廣泛存在于真核生物中的非編碼小片段RNA,其通過調(diào)節(jié)靶基因的表達進而廣泛參與機體的各項生理、病理過程中。miR-187-3p是miR-187家族的一員,定位于人染色體18q12.2。已有研究報道在結(jié)直腸癌、前列腺癌、非小細胞肺癌、腎細胞癌、食管腺癌中miR-187-3p呈低表達,這些研究表明miR-187-3p在人類腫瘤發(fā)生發(fā)展中發(fā)揮著抑癌基因的作用。然而,也有實驗證實miR-187-3p在外周T細胞淋巴瘤中高表達,并且miR-187-3p能增加乳腺癌細胞的侵襲能力,與乳腺癌患者的預(yù)后有關(guān)。因此,在不同的腫瘤組織中miR-187-3p發(fā)揮的作用可能不同。但是miR-187-3p在肝細胞癌組織中的表達狀況及作用報道較少。本研究通過miRNA芯片雜交技術(shù)篩選新鮮肝細胞癌組織中差異表達的miRNA,然后通過實時熒光定量PCR檢測miR-187-3p的表達情況,并結(jié)合患者臨床資料、生存期資料及病理學特征分析miR-187-3p在肝細胞癌發(fā)生發(fā)展、臨床診斷以及預(yù)后評估中的意義,為miR-187-3p在臨床中的應(yīng)用提供理論依據(jù)。方法:1研究對象及標本選取2010年4月至2016年8月于河北醫(yī)科大學第四醫(yī)院肝膽外科行手術(shù)切除的肝細胞癌患者90例,其中男性75例,女性15例,年齡25-78歲。依據(jù)手術(shù)前一周實驗室的指標、Child-Pugh分級標準評估肝功能,同時確定腫瘤標記物水平和肝炎類型,并根據(jù)影像學檢查和術(shù)中情況評估肝硬化嚴重程度以及腫瘤特征。90例患者中腫瘤直徑2-25cm。肝功能Child A級87例,B級3例。臨床分期Ⅰ期39例、Ⅱ期29例,Ⅲ期22例。本實驗中患者乙肝表面抗原(HBs Ag)陽性74例,陰性16例。所有研究對象在手術(shù)前均未進行過放療、化療或其它抗腫瘤治療。研究標本共180例,其中新鮮肝細胞癌組織標本90例及相對應(yīng)癌旁組織90例。標本提取后立即保存于液氮中,然后儲存于-80℃低溫冰箱中以備實驗使用。所有標本均經(jīng)過常規(guī)石蠟包埋,HE染色,最終由2位病理科醫(yī)師進行組織學診斷。收集患者肝炎病史、飲酒史、吸煙史以及家族史等資料,記錄患者肝功能、AFP值、血常規(guī)、凝血功能、甲乙丙肝炎等實驗室指標,登記患者腫瘤病理類型、腫瘤大小和數(shù)目、門靜脈及其分支是否存在瘤栓等病理學資料。通過電話聯(lián)系或信訪等途徑對患者進行隨訪,隨訪開始時間即為患者手術(shù)日,隨訪截止時間為2016年10月11日。2 miRNA芯片雜交技術(shù)從90例肝細胞癌患者中選取5例利用引物Poly(A)Tailing反轉(zhuǎn)錄符合標準的總RNA,然后進行miRNA芯片雜交、洗滌、掃描、最終生成數(shù)據(jù)。最后進行生物信息學分析以及應(yīng)用互聯(lián)網(wǎng)上miRNA靶基因預(yù)測軟件(Targetscan、Mi Randa)在線服務(wù)站點,輸出表達差異miRNA的預(yù)測靶基因,取2個軟件預(yù)測靶基因的交集(實驗由上海其明生物信息有限公司協(xié)助完成)。3檢測肝細胞癌組織和癌旁組織中miR-187-3p的表達情況利用Trizol法提取組織總RNA,對RNA進行純度、完整性和濃度的測定,然后將符合標準的RNA反轉(zhuǎn)錄為c DNA,再采用SYBR Green實時熒光定量PCR技術(shù)檢測90對肝細胞癌組織標本及配對的癌旁組織標本中miR-187-3p表達情況。miR-187-3p特異性莖環(huán)反轉(zhuǎn)錄引物:5’-GTCGTATCCAGTGCAGGGTCCGAGGTATTCGCACTGGATACGAC CCGGCTGC-3’;U6特異性莖環(huán)反轉(zhuǎn)錄引物:5’-AACGCTTCACGAAT TTGCGT-3’。miR-187-3p實時熒光定量PCR上游引物:5’-GTGCAGGGT CCGAGGTATT-3’;下游引物:5’-GCCGCTCGTGTCTTGTGTTGCAGC-3’。U6實時熒光定量PCR上游引物:5’-CTCGCTTCGGCAGCACA-3’;下游引物:5’-AACGCTTCACGAATTTGCGT-3’。4統(tǒng)計學分析統(tǒng)計學分析使用統(tǒng)計軟件SPSS 21.0,實驗數(shù)據(jù)正態(tài)性分布檢驗采用Kolmogorov-Smimov法。計數(shù)資料采用χ2檢驗或Fisher精確檢驗,兩組間的配對樣本采用Wilcoxon檢驗。生存分析使用Kaplan-Meier法,用Log-rank檢驗進行單因素分析,用COX回歸模型進行多因素分析。P0.05為差異具有統(tǒng)計學意義。結(jié)果:1肝細胞癌組織中miRNA表達譜分析運用miRNA芯片雜交技術(shù),在5對肝細胞癌及相應(yīng)癌旁組織中篩選出差異表達在2倍以上的miRNA共148個,其中102個miRNAs在肝細胞癌組織較癌旁組織表達下調(diào),46個miRNAs在肝細胞癌組織較癌旁組織表達上調(diào)(實驗由上海其明生物信息有限公司協(xié)助完成)。2肝細胞癌組織及相應(yīng)癌旁組織中miR-187-3p的相對表達量及比較在90例肝細胞癌患者中有63例(70.0%)癌組織中miR-187-3p相對于癌旁組織表達量下降,且差異具有統(tǒng)計學意義(Z=-2.891,P=0.004);說明miR-187-3p在肝細胞癌組織中的表達顯著低于癌旁組織,這與miRNA芯片結(jié)果相一致。3肝細胞癌患者中miR-187-3p的表達狀態(tài)與臨床病理特征的關(guān)系肝細胞癌患者中miR-187-3p的異常表達與腫瘤大小、臨床分期顯著相關(guān)(P0.05)。在腫瘤直徑≥5cm的患者中miR-187-3p表達下調(diào)的陽性率為(75.0%),腫瘤直徑5cm的患者中miR-187-3p表達下調(diào)的陽性率為(59.1%),其差別具有統(tǒng)計學意義(P=0.017);在臨床分期Ⅰ期患者中miR-187-3p表達下調(diào)的陽性率為(69.2%),Ⅱ期患者中miR-187-3p表達下調(diào)的陽性率為(79.3%),Ⅲ期患者中miR-187-3p表達下調(diào)的陽性率為(59.1%),差異具有統(tǒng)計學意義(P=0.002);但在不同性別(P=0.119)、年齡(P=0.976)、乙肝表面抗原(P=0.342)、門靜脈及其分支瘤栓(P=0.392)、腫瘤個數(shù)(P=0.867)、AFP水平(P=0.198)組別之間差異無顯著相關(guān)性。4 miR-187-3p的表達狀態(tài)與肝細胞癌患者術(shù)后生存期預(yù)后的關(guān)系Kaplan-Meier生存分析表明miR-187-3p表達下調(diào)的肝細胞癌患者的1年、3年、5年生存率為87.8%、56.2%、24.7%,平均生存時間40.470±4.407個月;上調(diào)者的1年生存率為90%,平均生存時間64.600±4.996個月,差異有統(tǒng)計學意義(P=0.011)。門靜脈及其分支有瘤栓患者的1年生存率為50.9%,平均生存時間17.568±2.326個月,中位生存時間19個月;無瘤栓患者的1年、3年、5年生存率為93.8%、68.4%、59.3%,平均生存時間51.266±4.164個月,中位生存時間70個月,兩者間差異有統(tǒng)計學意義(P=0.005)。腫瘤直徑5cm患者的1年生存率為82.9%,平均生存時間58.802±5.791個月,中位生存時間70個月;腫瘤直徑≥5cm患者的1年、3年、5年生存率為95%、59.9%、37.2%,平均生存時間41.713±4.606個月,中位生存時間26個月,兩者之間的差異有統(tǒng)計學意義(P=0.032)。臨床分期Ⅰ期患者的1年、3年生存率86.4%、47.8%,平均生存時間58.629±5.203個月;Ⅱ期患者1年生存率為77.6%,平均生存時間30.593±6.290個月;Ⅲ期患者1年生存率為56.8%,平均生存時間20.729±1.914個月,三者之間差異具有統(tǒng)計學意義(P=0.001)。Child-Pugh分級為A級患者的1年、3年、5年生存率為97.5%、71.2%、63.3%,平均生存時間47.109±3.984個月,中位生存時間63個月;Child-Pugh分級為B級患者的1年、3年、5年生存率為0%,平均生存時間19.667±6.006個月,中位生存時間26個月,兩者間差異無統(tǒng)計學意義(P=0.437)。年齡60歲的1年、3年生存率分別為88.9%、62.3%,平均生存時間49.916±4.785個月;年齡≥60歲的1年生存率為78.5%,平均生存時間39.836±6.172個月,兩者間差異無統(tǒng)計學意義(P=0.441)。進一步分析肝細胞癌患者術(shù)后總生存期的獨立危險因子,我們將miR-187-3p表達水平、臨床分期、腫瘤大小等影響因素進行多因素COX回歸模型分析,結(jié)果顯示miR-187-3p表達水平、腫瘤個數(shù)、臨床分期是影響肝細胞癌患者術(shù)后總生存期的獨立危險因子(P均0.05),其余參數(shù)P值均大于0.05。結(jié)論:1肝細胞癌組織中存在差異表達的miRNAs,其中miR-187-3p在肝細胞癌組織中的表達較相應(yīng)癌旁組織表達下調(diào),表明miR-187-3p可能參與了肝細胞癌的發(fā)生與發(fā)展。2 miR-187-3p的差異表達與肝細胞癌患者腫瘤大小、臨床分期有關(guān),表明miR-187-3p異常表達一定程度上反應(yīng)肝細胞癌患者的病情變化。3 miR-187-3p的表達狀態(tài)、腫瘤個數(shù)、臨床分期可作為肝細胞癌患者術(shù)后總生存期的獨立危險因子,可作為肝細胞癌患者判斷預(yù)后的指標。
[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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7
本文編號：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.
【學位授予單位】：河北醫(yī)科大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.7
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