發(fā)布時間：2018-08-27 06:15

【摘要】：背景:胃癌是全球常見的惡性腫瘤之一,其發(fā)病率和死亡率高,嚴重威脅人類健康。近年來,胃癌的診斷和治療已經(jīng)得到了較大提高;然而,胃癌患者的預后仍然非常差,癌細胞的浸潤轉(zhuǎn)移是導致其不良預后的重要原因。胃癌的發(fā)生發(fā)展是多基因參與的復雜過程,尋找并探索癌變過程中的分子機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送檢芯片并進行高通量篩選,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: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、翻譯增強或沉默。目前,愈來愈多研究表明,該家族在人類多種腫瘤異常表達,影響腫瘤的發(fā)生發(fā)展。PCBP3作為該家族成員之一,參與mu阿片受體啟動子區(qū)的活性調(diào)節(jié),但其與腫瘤的關(guān)系尚無文獻報道。本課題通過檢測PCBP3在人胃癌組織中的表達,以及對胃癌細胞生物學行為的影響和異常表達機制的研究,初步揭示了PCBP3對胃癌浸潤、轉(zhuǎn)移的潛在作用及相關(guān)機制。方法:收集47例新鮮的胃癌組織標本,整理相關(guān)臨床病理資料。提取胃癌組織中RNA進行逆轉(zhuǎn)錄,隨后采用RT-qPCR技術(shù)檢測組織中PCBP3的mRNA表達情況。采用免疫組織化學技術(shù)檢測34例非腫瘤性胃黏膜石蠟標本及97例胃癌石蠟標本中PCBP3的蛋白表達情況,并分析PCBP3的表達與胃癌患者臨床病理參數(shù)之間的關(guān)系。應用PCBP3的干擾RNA和過表達載體瞬時轉(zhuǎn)染胃癌細胞系MKN45和BGC823,通過MTS、EdU、Transwell實驗及流式細胞術(shù),檢測胃癌細胞增殖、遷移、侵襲及凋亡能力的改變。通過生物信息學軟件預測與PCBP3的3' UTR結(jié)合的microRNA,并采用雙熒光素酶報告基因和Western blot對其進行驗證。結(jié)果:1.RT-qPCR檢測顯示: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水平明顯升高。2.免疫組化檢測顯示:PCBP3的蛋白表達水平在非腫瘤性胃黏膜組織、無淋巴結(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)逐級升高趨勢,且其表達量與淋巴結(jié)轉(zhuǎn)移、TNM分期等臨床病理參數(shù)顯著相關(guān)。3.細胞功能學實驗:干擾PCBP3,其表達水平明顯降低。隨后Transwell實驗表明,干擾PCBP3,胃癌細胞的遷移、浸潤能力較對照組明顯降低。相反,過表達PCBP3,其表達水平明顯升高,胃癌細胞的遷移、浸潤能力也明顯升高。而EdU、MTS及流式細胞術(shù)結(jié)果顯示,干擾或過表達PCBP3,胃癌細胞的增殖和凋亡能力均沒有明顯變化。以上結(jié)果提示我們,PCBP3可以促進胃癌細胞的遷移、侵襲,而對增殖、凋亡沒有明顯影響。4.PCBP3表達的上游調(diào)控研究:通過microRNA、TargetScan和miRDB等生物學軟件預測能與PCBP3的3' UTR結(jié)合的microRNA,并結(jié)合相關(guān)文獻,選定4個microRNA。經(jīng)過雙熒光素酶報告基因和Western blot進一步驗證,證實miR-141和miR-200a與PCBP3的3' UTR結(jié)合,負向調(diào)控PCBP3的表達。結(jié)論:本研究發(fā)現(xiàn),PCBP3在胃癌組織中高表達,具有促進胃癌細胞遷移、浸潤的能力。miR-141和miR-200a參與PCBP3表達的上游調(diào)控。PCBP3的表達及相關(guān)機制的探索,可能為研究胃癌進展提供重要線索。
[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.
【學位授予單位】：山東大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R735.2

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