【摘要】：背景:胃癌是全球常見(jiàn)的惡性腫瘤之一,其發(fā)病率和死亡率高,嚴(yán)重威脅人類(lèi)健康。近年來(lái),胃癌的診斷和治療已經(jīng)得到了較大提高;然而,胃癌患者的預(yù)后仍然非常差,癌細(xì)胞的浸潤(rùn)轉(zhuǎn)移是導(dǎo)致其不良預(yù)后的重要原因。胃癌的發(fā)生發(fā)展是多基因參與的復(fù)雜過(guò)程,尋找并探索癌變過(guò)程中的分子機(jī)制成為胃癌研究的重要內(nèi)容,有助于為胃癌的靶向治療提供理論依據(jù)。前期我們課題組通過(guò)送檢芯片并進(jìn)行高通量篩選,發(fā)現(xiàn)多聚胞嘧啶結(jié)合蛋白3(poly C binding protein 3,PCBP3)在發(fā)生淋巴結(jié)轉(zhuǎn)移的胃癌組織中表達(dá)量顯著高于未發(fā)生淋巴結(jié)轉(zhuǎn)移的胃癌組織。多聚胞嘧啶結(jié)合蛋白是一類(lèi)RNA結(jié)合蛋白,能夠特異性結(jié)合RNA的多聚胞嘧啶區(qū)域。該家族分成兩大類(lèi):即hnRNPK/J和PCBP1-4。這些蛋白主要參與轉(zhuǎn)錄水平調(diào)控、mRNA穩(wěn)定性、翻譯增強(qiáng)或沉默。目前,愈來(lái)愈多研究表明,該家族在人類(lèi)多種腫瘤異常表達(dá),影響腫瘤的發(fā)生發(fā)展。PCBP3作為該家族成員之一,參與mu阿片受體啟動(dòng)子區(qū)的活性調(diào)節(jié),但其與腫瘤的關(guān)系尚無(wú)文獻(xiàn)報(bào)道。本課題通過(guò)檢測(cè)PCBP3在人胃癌組織中的表達(dá),以及對(duì)胃癌細(xì)胞生物學(xué)行為的影響和異常表達(dá)機(jī)制的研究,初步揭示了PCBP3對(duì)胃癌浸潤(rùn)、轉(zhuǎn)移的潛在作用及相關(guān)機(jī)制。方法:收集47例新鮮的胃癌組織標(biāo)本,整理相關(guān)臨床病理資料。提取胃癌組織中RNA進(jìn)行逆轉(zhuǎn)錄,隨后采用RT-qPCR技術(shù)檢測(cè)組織中PCBP3的mRNA表達(dá)情況。采用免疫組織化學(xué)技術(shù)檢測(cè)34例非腫瘤性胃黏膜石蠟標(biāo)本及97例胃癌石蠟標(biāo)本中PCBP3的蛋白表達(dá)情況,并分析PCBP3的表達(dá)與胃癌患者臨床病理參數(shù)之間的關(guān)系。應(yīng)用PCBP3的干擾RNA和過(guò)表達(dá)載體瞬時(shí)轉(zhuǎn)染胃癌細(xì)胞系MKN45和BGC823,通過(guò)MTS、EdU、Transwell實(shí)驗(yàn)及流式細(xì)胞術(shù),檢測(cè)胃癌細(xì)胞增殖、遷移、侵襲及凋亡能力的改變。通過(guò)生物信息學(xué)軟件預(yù)測(cè)與PCBP3的3' UTR結(jié)合的microRNA,并采用雙熒光素酶報(bào)告基因和Western blot對(duì)其進(jìn)行驗(yàn)證。結(jié)果:1.RT-qPCR檢測(cè)顯示:與未發(fā)生淋巴結(jié)轉(zhuǎn)移的胃癌組織相比,PCBP3在發(fā)生淋巴結(jié)轉(zhuǎn)移的胃癌組織中表達(dá)水平明顯升高。2.免疫組化檢測(cè)顯示:PCBP3的蛋白表達(dá)水平在非腫瘤性胃黏膜組織、無(wú)淋巴結(jié)轉(zhuǎn)移的胃癌組織和淋巴結(jié)轉(zhuǎn)移性胃癌組織中呈現(xiàn)逐級(jí)升高趨勢(shì),且其表達(dá)量與淋巴結(jié)轉(zhuǎn)移、TNM分期等臨床病理參數(shù)顯著相關(guān)。3.細(xì)胞功能學(xué)實(shí)驗(yàn):干擾PCBP3,其表達(dá)水平明顯降低。隨后Transwell實(shí)驗(yàn)表明,干擾PCBP3,胃癌細(xì)胞的遷移、浸潤(rùn)能力較對(duì)照組明顯降低。相反,過(guò)表達(dá)PCBP3,其表達(dá)水平明顯升高,胃癌細(xì)胞的遷移、浸潤(rùn)能力也明顯升高。而EdU、MTS及流式細(xì)胞術(shù)結(jié)果顯示,干擾或過(guò)表達(dá)PCBP3,胃癌細(xì)胞的增殖和凋亡能力均沒(méi)有明顯變化。以上結(jié)果提示我們,PCBP3可以促進(jìn)胃癌細(xì)胞的遷移、侵襲,而對(duì)增殖、凋亡沒(méi)有明顯影響。4.PCBP3表達(dá)的上游調(diào)控研究:通過(guò)microRNA、TargetScan和miRDB等生物學(xué)軟件預(yù)測(cè)能與PCBP3的3' UTR結(jié)合的microRNA,并結(jié)合相關(guān)文獻(xiàn),選定4個(gè)microRNA。經(jīng)過(guò)雙熒光素酶報(bào)告基因和Western blot進(jìn)一步驗(yàn)證,證實(shí)miR-141和miR-200a與PCBP3的3' UTR結(jié)合,負(fù)向調(diào)控PCBP3的表達(dá)。結(jié)論:本研究發(fā)現(xiàn),PCBP3在胃癌組織中高表達(dá),具有促進(jìn)胃癌細(xì)胞遷移、浸潤(rùn)的能力。miR-141和miR-200a參與PCBP3表達(dá)的上游調(diào)控。PCBP3的表達(dá)及相關(guān)機(jī)制的探索,可能為研究胃癌進(jìn)展提供重要線索。
[Abstract]:BACKGROUND: Gastric cancer is one of the most common malignant tumors in the world with high morbidity and mortality, which seriously threatens human health. In recent years, the diagnosis and treatment of gastric cancer have been greatly improved. However, the prognosis of patients with gastric cancer is still very poor, and the invasion and metastasis of cancer cells are the important reasons leading to poor prognosis. To find and explore the molecular mechanism of carcinogenesis is an important part of gastric cancer research, which is helpful to provide theoretical basis for targeted treatment of gastric cancer. Polycytidine-binding proteins are a class of RNA-binding proteins that specifically bind to the polycytidine domain of RNA. The family is divided into two groups: hnRNPK/J and PCBP1-4. These proteins are mainly involved in transcriptional regulation and mRNA stabilization. At present, more and more studies have shown that PCBP3 is abnormally expressed in many human tumors and affects the development of tumors. As a member of this family, PCBP3 participates in the regulation of mu opioid receptor promoter activity, but the relationship between PCBP3 and tumors has not been reported. Methods: 47 fresh gastric cancer tissues were collected and the clinical and pathological data were collected. RNA was extracted from gastric cancer tissues for reverse transcription, and then RT-qPCR was used. The expression of PCBP3 mRNA in tissues was detected by immunohistochemistry. The expression of PCBP3 protein in 34 paraffin specimens of non-tumor gastric mucosa and 97 paraffin specimens of gastric cancer was detected. The relationship between the expression of PCBP3 and clinicopathological parameters of gastric cancer was analyzed. Gastric cancer cell lines MKN45 and BGC823 were stained by MTS, EdU, Transwell assay and flow cytometry to detect the proliferation, migration, invasion and apoptosis of gastric cancer cells. The expression of PCBP3 in gastric cancer tissues with lymph node metastasis was significantly higher than that in gastric cancer tissues without lymph node metastasis. Cytological experiment: interfering with PCBP3, the expression level of PCBP3 was significantly decreased. Subsequently Transwell experiment showed that interfering with PCBP3, the migration and infiltration ability of gastric cancer cells were significantly lower than that of the control group. The results of EdU, MTS and flow cytometry showed that the proliferation and apoptosis of gastric cancer cells were not significantly changed by interfering with or overexpressing PCBP3. These results suggest that PCBP3 can promote the migration and invasion of gastric cancer cells, but has no significant effect on proliferation and apoptosis. Upstream regulation studies: MicroRNA, TargetScan and microRNADB were used to predict the 3'UTR-binding microRNA of PCBP 3. Four microRNAs were selected according to the relevant literature. Further verification by double luciferase reporter gene and Western blot confirmed that microRNA-141 and microRNA-200a bind to the 3'UTR of PCBP 3 and negatively regulate the expression of PCBP 3. Conclusion: PCBP3 is highly expressed in gastric cancer tissues and has the ability to promote the migration and infiltration of gastric cancer cells.
【學(xué)位授予單位】：山東大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類(lèi)號(hào)】：R735.2

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