鼻咽癌细胞系5-8F与6-10B总蛋白质与分泌蛋白质的差异蛋白质组学研究
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摘要
鼻咽癌(Nasopharyngeal carcinoma,NPC)是一种好发于东南亚地区及我国南方各省的常见恶性肿瘤。早期转移是NPC的显著特征之一和患者死亡的主要原因,而且NPC放疗后局部残灶复发、远处转移亦是制约其疗效和预后的瓶颈。因此,研究NPC的转移机制、寻找预测转移的分子标志物对NPC疗效和预后起关键作用,具有重要的理论和临床应用价值。
本研究以一对来自同一亲本、具有不同转移潜能的NPC细胞系5-8F和6-10B为转移模型,应用双向凝胶电泳(2-DE)技术分离5-8F和6-10B细胞的总蛋白和分泌蛋白质;利用PDQuest图像分析软件比较两株细胞总蛋白和分泌蛋白质2-DE图谱的异同;应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和电喷雾-四极杆-飞行时间串联质谱(ESI-Q-TOF MS/MS)鉴定差异表达的蛋白质,并利用生物信息学资源对所鉴定蛋白质的功能和亚细胞定位进行分析。然后采用Western blot验证部分差异蛋白质在5-8F和6-10B细胞中的表达水平,采用免疫组化染色检测差异蛋白质nm23-H1在NPC组织和颈淋巴结转移NPC中的表达。主要结果如下:
1.建立了高转移NPC细胞系5-8F和无转移潜能的NPC细胞系6.10B细胞总蛋白和分泌蛋白质的2-DE图谱,识别了123个差异表达的蛋白质点,鉴定出29个非冗余的差异表达的蛋白质,并对这些蛋白质进行了功能分类和亚细胞定位;
2.Western blot分析结果显示:差异蛋白质AnnexinA1、Stratifin、Keratin-8和nm23-H1在5-8F和6-10B细胞中的差异表达水平与比较蛋白质组学研究结果一致;
3.nm23-H1在高转移的5-8F NPC细胞总蛋白中的表达水平高于无转移潜能的6-10B细胞,在颈淋巴结转移NPC中的表达水平亦高于原发NPC;
4、差异蛋白质肽基脯氨酰顺反异构酶A(PPIA)存在乙酰化并瓜氨酸化或者苯乳酸化修饰。
本研究鉴定了29个可能与NPC转移相关的蛋白质,发现nm23-H1可能具有促进NPC转移的作用以及PPIA存在可能影响蛋白质功能的翻译后修饰,研究结果为阐明NPC转移机制以及筛选NPC转移分子标志物提供了实验依据和新线索。
Nasopharyngeal carcinoma (NPC) is a high-incidence malignancy in southern China and Southeast Asia. Early metastasis is one of distinctive characteristics of NPC and the main reason of death in NPC patients. Furthermore, local recurrence after radiotherapy and distant metastasis are the bottlenecks which restrain therapeutic effect and prognosis for NPC. Therefore, elucidating NPC metastasis mechanism and screening the molecular biomarkers are crucial for treatment and prognosis, which have important theoretical and clinical value.
A pair of NPC cell lines (5-8F and 6-10B) with different metastatic potential and the same genetic background was used as metastatic model of NPC in this study. Two-dimensional gel electrophoresis (2-DE) was used to separate the total and secretory proteins from 5-8F and 6-10B. PDQuest software was applied to analyze 2-DE maps of the total and secretory proteins in above two cell lines. Differentially expressed proteins between 5-8F and 6-10B were identified by MALDI-TOF-MS(matrix-assisted laser desorption/ionization time-of-flight mass Spectrometry) and ESI-Q-TOF MS/MS(electrospray ionization-quadrupole time-of-flight MS/MS). The biological functions and subcellular locations of identified proteins were further analyzed using bioinformatic resources. The expression levels of partial identified proteins were validated by Western blot analysis. The expression levels of differential protein nm23-H1 between NPC tissue and cervical lymph-node metastasis were detected by immunohistochemical staining. The results were as following: (1) The 2-DE maps of the total and secretory proteins from highly metastatic 5-8F and non-metastatic 6-10B were established respectively. One hundred and twenty-three differential proteins spots were found between 5-8F and 6-10B, and twenty-nine differential expression non-redundant proteins were identified by MS. Furthermore, the identified proteins were categorized into several protein groups according to their functions or subcellular locations; (2) The results of Western blot showed that Annexin A1, Stratifin, Keratin-8 and nm23-H1 were differential expression proteins in 5-8F and 6-10B, which was consistent with the results of our comparative proteomic analysis; (3) The expression level of nm23-H1 in the total proteins was significantly higher in 5-8F than that in 6-10B. And also the expression level of nm23-H1 in cervical lymph-node metastasis was significantly higher than that in primary NPC; (4) The differential protein—peptidyl-prolyl cis-trans isomerase A (PPIA) was found to have posttranslational modifications (PTM) including acetylation(ACET) and citrullination (CITR) or 3-phenyllactic acid(FLAC).
In this study, twenty-nine differential expression proteins between 5-8F and 6-10B were identified, which may be associated with the metastasis of NPC, nm23-H1 may contribute to the metastasis of NPC, and PPIA was found to have PTM which may influence its function. These data will provide experimental evidences and new clues for elucidating the metastatic mechanism of NPC and screening the metastatic molecular biomarkers of NPC.
本研究以一对来自同一亲本、具有不同转移潜能的NPC细胞系5-8F和6-10B为转移模型,应用双向凝胶电泳(2-DE)技术分离5-8F和6-10B细胞的总蛋白和分泌蛋白质;利用PDQuest图像分析软件比较两株细胞总蛋白和分泌蛋白质2-DE图谱的异同;应用基质辅助激光解吸电离飞行时间质谱(MALDI-TOF-MS)和电喷雾-四极杆-飞行时间串联质谱(ESI-Q-TOF MS/MS)鉴定差异表达的蛋白质,并利用生物信息学资源对所鉴定蛋白质的功能和亚细胞定位进行分析。然后采用Western blot验证部分差异蛋白质在5-8F和6-10B细胞中的表达水平,采用免疫组化染色检测差异蛋白质nm23-H1在NPC组织和颈淋巴结转移NPC中的表达。主要结果如下:
1.建立了高转移NPC细胞系5-8F和无转移潜能的NPC细胞系6.10B细胞总蛋白和分泌蛋白质的2-DE图谱,识别了123个差异表达的蛋白质点,鉴定出29个非冗余的差异表达的蛋白质,并对这些蛋白质进行了功能分类和亚细胞定位;
2.Western blot分析结果显示:差异蛋白质AnnexinA1、Stratifin、Keratin-8和nm23-H1在5-8F和6-10B细胞中的差异表达水平与比较蛋白质组学研究结果一致;
3.nm23-H1在高转移的5-8F NPC细胞总蛋白中的表达水平高于无转移潜能的6-10B细胞,在颈淋巴结转移NPC中的表达水平亦高于原发NPC;
4、差异蛋白质肽基脯氨酰顺反异构酶A(PPIA)存在乙酰化并瓜氨酸化或者苯乳酸化修饰。
本研究鉴定了29个可能与NPC转移相关的蛋白质,发现nm23-H1可能具有促进NPC转移的作用以及PPIA存在可能影响蛋白质功能的翻译后修饰,研究结果为阐明NPC转移机制以及筛选NPC转移分子标志物提供了实验依据和新线索。
Nasopharyngeal carcinoma (NPC) is a high-incidence malignancy in southern China and Southeast Asia. Early metastasis is one of distinctive characteristics of NPC and the main reason of death in NPC patients. Furthermore, local recurrence after radiotherapy and distant metastasis are the bottlenecks which restrain therapeutic effect and prognosis for NPC. Therefore, elucidating NPC metastasis mechanism and screening the molecular biomarkers are crucial for treatment and prognosis, which have important theoretical and clinical value.
A pair of NPC cell lines (5-8F and 6-10B) with different metastatic potential and the same genetic background was used as metastatic model of NPC in this study. Two-dimensional gel electrophoresis (2-DE) was used to separate the total and secretory proteins from 5-8F and 6-10B. PDQuest software was applied to analyze 2-DE maps of the total and secretory proteins in above two cell lines. Differentially expressed proteins between 5-8F and 6-10B were identified by MALDI-TOF-MS(matrix-assisted laser desorption/ionization time-of-flight mass Spectrometry) and ESI-Q-TOF MS/MS(electrospray ionization-quadrupole time-of-flight MS/MS). The biological functions and subcellular locations of identified proteins were further analyzed using bioinformatic resources. The expression levels of partial identified proteins were validated by Western blot analysis. The expression levels of differential protein nm23-H1 between NPC tissue and cervical lymph-node metastasis were detected by immunohistochemical staining. The results were as following: (1) The 2-DE maps of the total and secretory proteins from highly metastatic 5-8F and non-metastatic 6-10B were established respectively. One hundred and twenty-three differential proteins spots were found between 5-8F and 6-10B, and twenty-nine differential expression non-redundant proteins were identified by MS. Furthermore, the identified proteins were categorized into several protein groups according to their functions or subcellular locations; (2) The results of Western blot showed that Annexin A1, Stratifin, Keratin-8 and nm23-H1 were differential expression proteins in 5-8F and 6-10B, which was consistent with the results of our comparative proteomic analysis; (3) The expression level of nm23-H1 in the total proteins was significantly higher in 5-8F than that in 6-10B. And also the expression level of nm23-H1 in cervical lymph-node metastasis was significantly higher than that in primary NPC; (4) The differential protein—peptidyl-prolyl cis-trans isomerase A (PPIA) was found to have posttranslational modifications (PTM) including acetylation(ACET) and citrullination (CITR) or 3-phenyllactic acid(FLAC).
In this study, twenty-nine differential expression proteins between 5-8F and 6-10B were identified, which may be associated with the metastasis of NPC, nm23-H1 may contribute to the metastasis of NPC, and PPIA was found to have PTM which may influence its function. These data will provide experimental evidences and new clues for elucidating the metastatic mechanism of NPC and screening the metastatic molecular biomarkers of NPC.
引文
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