【摘要】：胃癌(Gastric Cancer, GC)是較為常見的消化道系統(tǒng)惡性腫瘤。近年來,在歐美等發(fā)達(dá)國家,GC的發(fā)病率逐年下降,但在東亞地區(qū)尤其是中國,GC的發(fā)病率并未見顯著下降,同時(shí),GC的死亡率也居高不下。GC患者的5年生存率(5-year survival rate)徘徊在20%～25%,中位生存時(shí)間(median survival duration)僅為24個(gè)月。導(dǎo)致胃癌患者生存率低的主要原因是早診率低、轉(zhuǎn)移率高、無敏感有效的治療方法及研究基礎(chǔ)薄弱等。深入研究胃癌的發(fā)生發(fā)展機(jī)理,尋找能夠早期發(fā)現(xiàn)、監(jiān)測疾病進(jìn)展和預(yù)測療效的分子標(biāo)志物,有助于提高胃癌患者的生活質(zhì)量及延長生存期。MicroRNA(MiRNA)是一種調(diào)控癌基因和抑癌基因表達(dá)非編碼單鏈小分子RNA,研究顯示,其進(jìn)化保守,長度約22個(gè)核苷酸,在腫瘤增殖(proliferation)、侵襲(invasion)和轉(zhuǎn)移(metastasis)過程起到重要作用。研究表明miRNAs在胃癌的診斷、療效監(jiān)測和預(yù)后評估中具有重要價(jià)值,但是miRNA在胃癌發(fā)生發(fā)展中的具體作用機(jī)制及其臨床意義還有待于進(jìn)一步研究,因此,本研究將篩選與胃癌發(fā)生相關(guān)的miRNAs表達(dá)譜,并對其中的部分關(guān)鍵的miRNA進(jìn)行功能學(xué)和分子生物學(xué)研究。第一部分 胃癌發(fā)生相關(guān)的microRNA表達(dá)譜系篩選目的：篩選與人胃癌發(fā)生高度相關(guān)的miRNAs,期望為胃癌的診斷提供新的工具和方法。方法：選取胃癌癌旁非腫瘤粘膜上皮組織66例和原發(fā)灶胃癌組織162例。利用RT-PCR檢測兩者之間1920個(gè)miRNAs差異,利用生物信息學(xué)技術(shù)分析找出能區(qū)分胃癌和癌旁非腫瘤上皮組織中的miRNAs表達(dá)模型。結(jié)果：篩選出12個(gè)與胃癌的發(fā)生高度相關(guān)的miRNAs (hsa-miR-196a, has-miR-196b, hsa-miR-204, hsa-miR-133b, hsa-miR-148a, hsa-miR-375, hsa-miR-551b, hsa-miR-31#, hsa-miR-224, hsa-miR-31, hsa-miR-378d和hsa-miR-37),其中2個(gè)上調(diào),10個(gè)下調(diào),能區(qū)分胃癌與正常粘膜,其有效性95.08%,特異性97.56%(P0.005)。結(jié)論：胃癌發(fā)生過程中伴有niRNAs的表達(dá)異常,我們篩選出與胃癌發(fā)生高度相關(guān)的miRNA表達(dá)譜能夠較好的區(qū)分胃癌和非癌組織,為胃癌的診斷提供了新的工具。第二部分：hsa-miR-196a在胃癌細(xì)胞系中功能學(xué)及作用靶點(diǎn)的研究目的：探討hsa-miR-196a促進(jìn)胃癌細(xì)胞增殖、侵襲和轉(zhuǎn)移的功能學(xué)研究,以及潛在的分子生物學(xué)機(jī)制。方法：構(gòu)建hsa-miR-196a-shRNA下調(diào)胃癌細(xì)胞SGC7901和BGC823中miR-196a的表達(dá),利用增殖實(shí)驗(yàn)(MTT)、克隆形成(Colony formation)、劃痕實(shí)驗(yàn)(Wound healing)、侵襲轉(zhuǎn)移實(shí)驗(yàn)(Transwell)檢測miR-196a表達(dá)下調(diào)后胃癌細(xì)胞的功能學(xué)改變,并利用RT-PCR技術(shù)檢測miR-196a以及可能的作用靶點(diǎn)HOXA7、HOXC8、SLC9A6、ZMYND11和CCDC47的mRNA的表達(dá)。結(jié)果:①RT-PCR結(jié)果顯示hsa-miR-196a-shRNA能夠成功干擾SGC7901和BGC823中miR-196a的表達(dá)：②SGC7901和BGC823中miR-196a的表達(dá)下調(diào)后,其細(xì)胞的增殖能力、克隆形成能力、爬行及侵襲能力均明顯下降(P0.05)；③SGC7901和BGC823中miR-196a的表達(dá)下調(diào)后,HOXA7、HOXC8和CCDC47表達(dá)在上述兩種細(xì)胞中均明顯下調(diào),然而SLC9A6、ZMYND11在SGC7901實(shí)驗(yàn)組中表達(dá)上調(diào),在BGC823實(shí)驗(yàn)組中表達(dá)下調(diào)。結(jié)論：miR-196a可能通過調(diào)控HOXA7、HOXC8和CCDC47等基因mRNA水平的改變,從而促進(jìn)胃癌細(xì)胞的增殖、侵襲和轉(zhuǎn)移。第三部分：探討HOXA7和HOXC8在人胃癌中的表達(dá)及其臨床價(jià)值目的：研究HOXA7和HOXC8在人胃癌組織中的表達(dá)及臨床意義、預(yù)后價(jià)值。方法：利用免疫組化(IHC)和組織芯片(TMA)檢測HOXA7和HOXC8在人胃癌組織標(biāo)本中的表達(dá),并統(tǒng)計(jì)分析其表達(dá)與胃癌生物學(xué)行為及預(yù)后的關(guān)系。結(jié)果：①胃癌組織中HOXA7和HOXC8的表達(dá)明顯高于癌旁正常粘膜,兩者的表達(dá)率分別為：55.6%(134/241)和60.6%(146/241)。② HOXA7高表達(dá)與浸潤深度(tumor invasion)呈正相關(guān)(P=0.02), HOXC8高表達(dá)與腫瘤大小(tumor size)、浸潤深度(tumor invasion)、淋巴結(jié)轉(zhuǎn)移(regional lymph node metastasis)、TNM (TNM stage)有相關(guān)性(P0.05),③胃癌組織中HOXA7和HOXC8的表達(dá)與預(yù)后的相關(guān)性：單因素生存分析(Kaplan-Meier analysis)顯示HOXA7過度表達(dá)的GC患者總生存期(overall survival)為56個(gè)月,明顯低于HOXA7低表達(dá)的GC患者[75.6月(P=0.003)]。HOXC8過度表達(dá)的GC患者預(yù)后也明顯較HOXC8低表達(dá)的GC患者差[中位生存時(shí)間(median survival duration):24.9月V.S 90.0月, P0.001], COX多因素回歸分析(Cox regression model)顯示HOXC8過度表達(dá)是判斷GC患者重要的獨(dú)立預(yù)后因素之一。結(jié)論：HOXA7和HOXC8的過度表達(dá)在GC的發(fā)生和發(fā)展過程中起到一定的促進(jìn)作用,有成為潛在的胃癌分子治療靶點(diǎn)和評估GC患者預(yù)后的分子標(biāo)志物(tumor biomarkers)的可能。
[Abstract]:Gastric cancer (GC) is a common malignant tumor of the digestive tract. In recent years, the incidence of GC has declined year by year in developed countries such as Europe and the United States, but in East Asia, especially in China, the incidence of GC has not decreased significantly. At the same time, the mortality rate of GC is still high. The 5-year survival rate of GC patients hovers at 20%. The main reasons for the low survival rate of gastric cancer patients are low early diagnosis rate, high metastasis rate, insensitive and effective treatment methods and weak research foundation. MicroRNA is a small non-coding single-stranded RNA that regulates the expression of oncogenes and tumor suppressor genes. Studies have shown that it is evolutionarily conserved and has a length of about 22 nucleotides, and plays an important role in tumor proliferation, invasion and metastasis. Studies have shown that microRNAs play an important role in the diagnosis, efficacy monitoring and prognosis evaluation of gastric cancer. However, the specific mechanism and clinical significance of microRNAs in the development of gastric cancer need to be further studied. Therefore, this study will screen the expression profiles of microRNAs associated with gastric cancer and some of the key microRNAs. Methods: 66 cases of non-tumor mucosal epithelium adjacent to gastric cancer and 162 cases of primary gastric cancer were selected. Results: Twelve highly correlated microRNAs (hsa-microRNAs-196a, has-microRNAs-196b, hsa-microRNAs-204, hsa-microRNAs-133b, hsa-microRNAs-148a, hsa-microRNAs-148a, hsa-microRNAs-microRNAs) were screened for gastric cancer. - 375, hsa-Mi-551b, hsa-Mi-31#, hsa-Mi-224, hsa-Mi-Mi-31, hsa-Mi-378d and hsa-Mi-37, two of which were up-regulated and 10 down-regulated, were able to distinguish gastric cancer from normal mucosa. The efficacy was 95.08% and specificity was 97.56% (P 0.005). Conclusion: Abnormal expression of niRNAs was associated with gastric cancer. We screened the expression of microRNA highly associated with gastric cancer. Duplex spectroscopy provides a new tool for the diagnosis of gastric cancer. Part II: Functional and functional targets of hsa-microRNA-196a in gastric cancer cell lines. Objective: To investigate the role of hsa-microRNA-196a in promoting the proliferation, invasion and metastasis of gastric cancer cells and its potential molecular biological mechanisms. HSa-microRNA-196a-shRNA was constructed to down-regulate the expression of microRNA-196a in gastric cancer cells SGC7901 and BGC823. Colony formation, Wound healing, invasion and metastasis were used to detect the functional changes of gastric cancer cells after down-regulated expression of microRNA-196a. RT-PCR was used to detect the expression of microRNA-196a and microRNA-196a. Results: The results of RT-PCR showed that hsa-miR-196a-shRNA could successfully interfere with the expression of Mi-196a in SGC7901 and BGC823. After down-regulation of the expression of Mi-196a in SGC7901 and BGC823, the proliferation, cloning, crawling and invasiveness of SGC7901 and BGC823 cells were significantly decreased. The expression of HOXA7, HOXC8 and CCC47 was significantly down-regulated in SGC7901 and BGC823, but the expression of SLC9A6 and ZMYND11 was up-regulated in SGC7901 and down-regulated in BGC823. Conclusion: Mi-196a may regulate the mRNA levels of HOXA7, HOXC8 and CCC47. Objective: To investigate the expression and clinical value of HOXA7 and HOXC8 in human gastric cancer. To study the expression and prognostic value of HOXA7 and HOXC8 in human gastric cancer. Methods: Immunohistochemistry (IHC) and tissue microarray (TMA) were used to detect HOXA7 and HOXC8 in human stomach. Results: The expression of HOXA7 and HOXC8 in gastric cancer tissues was significantly higher than that in adjacent normal mucosa. The expression rates of HOXA7 and HOXC8 were 55.6% (134/241) and 60.6% (146/241), respectively. The high expression of HOXC8 was correlated with tumor size, tumor invasion, regional lymph node metastasis, and TNM stage (P 0.05). The correlation between the expression of HOXA7 and HOXC8 and prognosis in gastric cancer was analyzed by Kaplan-Meier analysis. The overall survival was 56 months, significantly lower than that of GC patients with low HOXA7 expression [75.6 months (P = 0.003)]. The prognosis of GC patients with overexpression of HOXC8 was also significantly worse than that of GC patients with low HOXC8 expression [median survival time: 24.9 months V.S 90.10 months, P.001], COX multivariate regression model CONCLUSION: Overexpression of HOXC8 and HOXA7 may play an important role in the occurrence and development of GC, and may be a potential therapeutic target for gastric cancer and a molecular marker for evaluating the prognosis of GC patients.
【學(xué)位授予單位】：第二軍醫(yī)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2015
【分類號】：R735.2

【參考文獻(xiàn)】

相關(guān)期刊論文 前1條

1 Vivian Yvonne Shin;Kent-Man Chu;;MiRNA as potential biomarkers and therapeutic targets for gastric cancer[J];World Journal of Gastroenterology;2014年30期

，

本文編號：2239482