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Pheochromocytoma and paraganglioma microRNAs study

Author: ZhangZuo
Tutor: ZengZhengPei
School: Beijing Union Medical College
Course: Internal Medicine
Keywords: Pheochromocytoma Paraganglioma miRNA miR-483-5p miR-183 miR-210 miR-21Pheochromocytoma serum miRNA Succinate dehydrogenase Hypoxia-inducible factor PTEN
CLC: R736.6
Type: PhD thesis
Year: 2012
Downloads: 77
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Abstract


ObjectiveCurrently the diagnosis of malignant pheochromocytoma or paraganglioma can only be made when there is clinical evidence of metastasis or extensive local invasion. The differential diagnosis between malignant and benign pheochromocytomaor paraganglioma is still regarded as difficult issue at present, which is the reason to find out a novel biomarker in purpose of assessing pheochromocytoma or paraganglioma malignant potential essence. MiRNA expression profiling has beenperformed on many types of human cancers and indicates that malignancy involves miRNAdysregulation and that particular miRNA expression signatures could be useful formolecular diagnosis and/or prognosis. In his part, we analyzed four miRNAs expression, which included miR-483-5p, miR-183, miR-210and miR-21in benign and malignant pheochromocytoma or paraganglioma in a bid to evaluate its relationship with benign and malignant tumors along with else clinical features.MethodsTotal RNA of32pheochromocytoma,24paraganglioma and6normal adrenal medulla tissue samples were extracted by Trizol reagent according to the manufacturer’s instructions. Reverse transcription reactions were performed by using first-strand cDNA synthesis Kit with stem-loop primers of miR-483-5p, miR-183, miR-210and miR-21, respectively. PCR products were amplified using specific primers and the SYBR(?) Premix Ex TagTM Kit, and detected using7500Real-TimePCR System. All PCRreactions were run in triplicateand no cDNA template were used for negativecontrols. The expression of U6was used as endogenous control for each sample. The relative gene expression was calculated by comparing cycle times for target PCR using the following equation:relative gene expression=2-(ΔCtsample-ΔCtcontrol). The correlation miRNA expression along with clinical data and tumors’nature was investigated.Results1. The expression of miR-483-5p in PCC and/or PGL.(1).The miR-483-5p in tumor tissue of malignant PCC and PGL obviously exceeded that of benign PCC, benign PGL and normal adrenal medulla(P<0.05).(2). No significant difference was detected with the expression of miR-483-5pin benign PPC or PGL, compared with normal adrenal medulla.(3). No significant difference was found with the expression of miR-483-5pin malignant PCC and PGL, benign PPC and PGL.2. The expression of miR-183in PCC and/or PGL(1).The miR-183level in tumor tissue of malignant PCC and PGL is higher than that of benign PCC and PGLrespectively(P<0.05).(2). No significant difference was studied with the expression of miR-183in malignant PCC and PGL, benign PPC and PGL.3. The expression of miR-21in PCC and/or PGL(1).The miR-21level in tumor tissue of malignant PCC or PGL surpassed that of benign one (P<0.05).(2).The miR-21expression in malignant PCC tumor tissue exceeded normal adrenal medulla (P<0.05), while in malignant PGL with ascending trend (P<0.054). No significant difference was detected with the expression of miR-21in benign PCC or benign PGL, compared with normal adrenal medulla.(3). No significant difference was detected with the expression of miR-21in malignant PCC and PGL, benign PPC and PGL.4. The expression of miR-210in PCC and/or PGL(1).The miR-210level in tumor tissue of malignant PCC or PGL surpassed that of normal adrenal medulla, higher than benign PCC or PGL respectively as well (P<0.05).(2). No significant difference was detected with the expression of miR-210in benign PCC or benign PGL, compared with normal adrenal medulla.(3). No significant difference was detected with the expression of miR-210in malignant PCC and PGL, benign PPC and PGL.5. The miR-483-5p, miR-183, miR-210, miR-21expression in PCC tumor tissue suffered of VHL disease.(1). The miR-483-5p, miR-183, miR-210, miR-21expression in VHL disease patients’PCC tumor tissue was higher than that of benign PCC (P<0.05).(2). The miR-210expression in VHL disease patients’PCC tumor tissue outstriped that of malignant PCC (P<0.05), while the expression of miR-483-5p, miR-183and miR-21in VHL reached the level of malignant PCC.6. The relationship between miR-483-5p, miR-183, miR-210, miR-21expression and PCC/PGL patients’clinical information.(1).The expression of miR-21in patients with multiple tumors was higher than single ones (P<0.05), while no statistical difference was found by miR-483-5p, miR-183, miR-210expression in such tumors.(2). There was no significant miRNA expression differences when samples were compared based on age, gender, tumor site, tumor size and Ki-67level.Conclusion1. The miR-483-5p, miR-183, miR-210, miR-21are highly expressed in malignant PCC/PGL tissue, compared with benign PCC/PGL and normal adrenal medulla. There were no differences between the miR-483-5p, miR-183, miR-210, miR-21expression in benign PCC/PGL and normal adrenal medulla. MiR-483-5p, miR-183, miR-210and miR-21could be diagnosis biomarkers for distinguishing malignant potential. Werecommend a long-term and close follow-up for individuals with higher expression of miR-483-5p, miR-183, miR-210or miR-21.2. The expression level of miR-483-5p, miR-183, miR-210and miR-21in PCC tumor tissue with VHL disease were up-regulated, which indicates that the expression level of miRNA may be correlated with hereditary factors. ObjectiveA handful of studies showed that miRNAs could be released into bloodstream. Because of the high rate of proliferation and cell lysis in tumours, non-specific passive release could also exist in cancer that contribute to the abundance of miRNAs in the bloodstream. Not only reflecting physiological and pathological changes, these circulating miRNAs could be functional and correlated with tumour progression and clinical outcomes. Serum miR-483-5p, miR-21and miR-210expression in patients with pheochromocytoma and paraganglioma was tested in purpose of discussing whether these miRNAs could be detected in serum and whether there was correlation of their expression in between serum and tumor tissues as well as their changing comparison pre and post operation.Methods1.The total RNA was extracted from the serum of9PCC,13PGL patients including pre-operation and post-operation according to manufacturer’s protocols of MaxRecoveryTMBiooPureTMRNA Isolation Reagent. Reverse transcription reactions were performed by using first-strand cDNA synthesis Kit with stem-loop primers of miR-483-5p, miR-183, miR-210and miR-21, respectively. PCR products were amplified using specific primers and the SYBR(?) Premix Ex TagTM Kit, and detected using7500HT Real-TimePCR System. All PCRreactions were run in triplicateand no cDNA template were used for negativecontrols.The expression of miR-16was used as endogenous control for each sample. The relative gene expression was calculated by comparing cycle times for target PCR using the following equation:relative gene expression=2-(ΔCtsample-ΔCtcoinrol).2. The expression of miR-483-5p, miR-210and miR-21among PCC/PGL pre and post operation was compared.3. The miR-483-5p, miR-210and miR-21expression relativity in tumor tissue and blood serum was analyzed. Results1.The miR-483-5p, miR-210and miR-21expression could all be detected in serum of patients with pheochromocytoma and paraganglioma.2. No correlation was confirmed as of the miR-483-5p, miR-210and miR-21level between serum and tumor tissue.3. The serum miR-21and miR-483-5p expression level in PCC/PGL patients both decreased post-operation (p<0.05). Serum miR-210had a tendency to decrease after operation (p=0.082)Conclusion1.The miR-483-5p, miR-210and miR-21expression could all be detected in serum of patients with pheochromocytoma and paraganglioma.2. The serum miR-483-5p, miR-210and miR-21expression level in PCC/PGL were markedly reduced after surgery, which indicated that they might have certain significance on the issue of post-operation monitoring. ObjectivePI3K/PTEN/ARK/HIF signal pathway, upon which HIF activation is taken as a central issue, plays important role in pheochromocytoma and paraganglioma pathogenesis mechanism. Induced by hypoxia, miR-210targets SDHD gene and regulates its expression. The miR-21which is located at upstream pathway of hypoxia activation targets PTEN gene and regulates its expression. In this section, HIF-1α, HIF-2α, SDHD, SDHB and PTEN mRNA level was examined in a bid to explore the correlation between miR-210or miR-21with HIF expression as well as miR-210with SDH, miR-21and PTEN expression.MethodsThe total RNA of32cases of PCC,24PGL tumor tissue along with6normal adrenal medulla, which was reversely transcripted to cDNA by Oligo(dT)18, was extracted by way of Trizol reagent as described in the manuscription. HIF-1α, HIF-2α, SDHD, SDHB and PTEN relative expression level in different tissue was investigated by real-time quantitative PCR.The analysis of each gene was performed in triplicate, and relative mRNA levels were determined using HPRT1as internal control. The relationship between HIF-1α, HIF-2α, SDHD, SDHB and PTEN mRNA expression level in tumor tissue and tumor nature was analyzed. The relativity between HIF-1α, HIF-2α mRNA expression level in tumor tissue and miR-210, miR-21expression was analyzed, as well as that of SDHD, SDHB mRNA expression level and miR-210, PTEN mRNA expression level and miR-21.Results1. The relationship between HIF-1α, HIF-2α, SDHD, SDHB, PTEN mRNA expression level and tumor nature.(1). The HIF-1α mRNA expression in malignant PGL tumor tissue washigher than benign PCC/PGL and normal adrenal medulla in some degree(P value as of0.063,0.055,0.090, respectively). No significant HIF-1a mRNA expression difference was detected between benign PCC/PGL tumor tissueand normal adrenal medulla.(2). The SDHB mRNA expression level in malignant PCC/PGL and benign PGL was less than that of normal adrenal medulla(P<0.05). The SDHD mRNA expression level in malignant PCC and benign PGL was less than that of normal adrenal medulla(P<0.05).(3). No statistical difference of HIF-2α, PTEN mRNA expression was found between benign PCC, malignant PCC, benign PGL, malignant PGL and normal adrenal medulla.2. The relationship of mRNA and miRNA expression(1).The miR-21, miR-210expression level in PCC/PGL tumor tissue was positively correlated with HIF—1α mRNA level, with correlation coefficient as of0.335(P=0.013) and0.466(P<0.001) respectively.(2). No significant correlation was probed from miR-21, miR-210expression level in PCC/PGL tumor tissue to HIF-2α mRNA level, with lvalue as of0.116and0.076respectively.(3). No significant correlation was found between miR-210expression level in PCC/PGL tumor tissue and SDHB, SDHD mRNA level, with lvalue as of0.400and0.184respectively.(4). No significant correlation was detected between miR-21expression level in PCC/PGL tumor tissue and PTEN mRNA level (P=0.141).Conclusion1. The miR-21, miR-210expression level was positively correlated with HIF-1a mRNA level, indicating that miR-21, miR-210expression in PCC/PGL was related with HIF-1α.2. HIF-1α mRNA expression in malignant PGL tumor tissue surpassed benign PGL and PCC, implying that HIF-1α might play a certain role in malignant PGL pathogenesis mechanism.3. The SDHB mRNA expression descended in PCC/PGL, which led to the speculation of SDHB downregulation in PCC/PGL.

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