胃癌中PCBP3异常表达及其调控胃癌浸润转移的研究

发布时间：2018-08-27 06:15

【摘要】：背景:胃癌是全球常见的恶性肿瘤之一,其发病率和死亡率高,严重威胁人类健康。近年来,胃癌的诊断和治疗已经得到了较大提高;然而,胃癌患者的预后仍然非常差,癌细胞的浸润转移是导致其不良预后的重要原因。胃癌的发生发展是多基因参与的复杂过程,寻找并探索癌变过程中的分子机制成为胃癌研究的重要内容,有助于为胃癌的靶向治疗提供理论依据。前期我们课题组通过送检芯片并进行高通量筛选,发现多聚胞嘧啶结合蛋白3(poly C binding protein 3,PCBP3)在发生淋巴结转移的胃癌组织中表达量显著高于未发生淋巴结转移的胃癌组织。多聚胞嘧啶结合蛋白是一类RNA结合蛋白,能够特异性结合RNA的多聚胞嘧啶区域。该家族分成两大类:即hnRNPK/J和PCBP1-4。这些蛋白主要参与转录水平调控、mRNA稳定性、翻译增强或沉默。目前,愈来愈多研究表明,该家族在人类多种肿瘤异常表达,影响肿瘤的发生发展。PCBP3作为该家族成员之一,参与mu阿片受体启动子区的活性调节,但其与肿瘤的关系尚无文献报道。本课题通过检测PCBP3在人胃癌组织中的表达,以及对胃癌细胞生物学行为的影响和异常表达机制的研究,初步揭示了PCBP3对胃癌浸润、转移的潜在作用及相关机制。方法:收集47例新鲜的胃癌组织标本,整理相关临床病理资料。提取胃癌组织中RNA进行逆转录,随后采用RT-qPCR技术检测组织中PCBP3的mRNA表达情况。采用免疫组织化学技术检测34例非肿瘤性胃黏膜石蜡标本及97例胃癌石蜡标本中PCBP3的蛋白表达情况,并分析PCBP3的表达与胃癌患者临床病理参数之间的关系。应用PCBP3的干扰RNA和过表达载体瞬时转染胃癌细胞系MKN45和BGC823,通过MTS、EdU、Transwell实验及流式细胞术,检测胃癌细胞增殖、迁移、侵袭及凋亡能力的改变。通过生物信息学软件预测与PCBP3的3' UTR结合的microRNA,并采用双荧光素酶报告基因和Western blot对其进行验证。结果:1.RT-qPCR检测显示:与未发生淋巴结转移的胃癌组织相比,PCBP3在发生淋巴结转移的胃癌组织中表达水平明显升高。2.免疫组化检测显示:PCBP3的蛋白表达水平在非肿瘤性胃黏膜组织、无淋巴结转移的胃癌组织和淋巴结转移性胃癌组织中呈现逐级升高趋势,且其表达量与淋巴结转移、TNM分期等临床病理参数显著相关。3.细胞功能学实验:干扰PCBP3,其表达水平明显降低。随后Transwell实验表明,干扰PCBP3,胃癌细胞的迁移、浸润能力较对照组明显降低。相反,过表达PCBP3,其表达水平明显升高,胃癌细胞的迁移、浸润能力也明显升高。而EdU、MTS及流式细胞术结果显示,干扰或过表达PCBP3,胃癌细胞的增殖和凋亡能力均没有明显变化。以上结果提示我们,PCBP3可以促进胃癌细胞的迁移、侵袭,而对增殖、凋亡没有明显影响。4.PCBP3表达的上游调控研究:通过microRNA、TargetScan和miRDB等生物学软件预测能与PCBP3的3' UTR结合的microRNA,并结合相关文献,选定4个microRNA。经过双荧光素酶报告基因和Western blot进一步验证,证实miR-141和miR-200a与PCBP3的3' UTR结合,负向调控PCBP3的表达。结论:本研究发现,PCBP3在胃癌组织中高表达,具有促进胃癌细胞迁移、浸润的能力。miR-141和miR-200a参与PCBP3表达的上游调控。PCBP3的表达及相关机制的探索,可能为研究胃癌进展提供重要线索。
[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.
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R735.2

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