目的:探讨DNA甲基化影响TP63的表达及其与子宫颈癌的相关性。方法:收集宫颈癌患者40例(CC)和因子宫良性病变行全子宫切除术的45例患者的宫颈组织。采用全基因组DNA甲基化分析(WGBS)和RNA sequence联合筛查宫颈癌组织和正常宫颈组织(各3...目的:探讨DNA甲基化影响TP63的表达及其与子宫颈癌的相关性。方法:收集宫颈癌患者40例(CC)和因子宫良性病变行全子宫切除术的45例患者的宫颈组织。采用全基因组DNA甲基化分析(WGBS)和RNA sequence联合筛查宫颈癌组织和正常宫颈组织(各3例)的DNA甲基化度和mRNA表达。采用亚硫酸氢盐测序法(BSP)和实时定量PCR(qPCR)法检测TP63基因启动子区DNA甲基化和RUNX2、TP63、survivin的mRNA表达。CHIP法检测RUNX2在宫颈组织中与TP63基因启动子的结合情况,并分析TP63基因DNA甲基化状态与SCC病理参数的关系。结果: WGBS和RNA-seq联合筛查发现,TP63启动子DNA甲基化水平升高与其mRNA表达减少相关。BSP结果证实,与正常宫颈组织比较,宫颈癌组织中TP63基因启动子区-688 CpG位点DNA甲基化率显著升高,差异有统计学意义[77.5%(31/40)vs 31.1%(14/45), P <0.05]。qPCR结果显示,与对照组比较,宫颈癌组织中RUNX2和survivin的mRNA表达上调,TP63 mRNA表达下调( P 均<0.05)。染色质免疫共沉淀技术(ChIP)证实,宫颈癌组织中TP63基因启动子区存在转录因子RUNX2结合序列,且-688CpG位点DNA甲基化修饰影响RUNX2的结合。相关性分析显示,TP63基因DNA甲基化与肿瘤大小、病理分级、临床分期相关( P 均<0.05),与患者年龄无关( P >0.05)。结论: TP63基因启动子DNA甲基化抑制其表达,参与宫颈癌的发生发展。展开更多
BACKGROUND Pancreatic cancer(PC)is one of the deadliest cancers worldwide.PC metastasis involves a complex set of events,including epithelial-mesenchymal transition(EMT),that increase tumor cell invasiveness.Recent ev...BACKGROUND Pancreatic cancer(PC)is one of the deadliest cancers worldwide.PC metastasis involves a complex set of events,including epithelial-mesenchymal transition(EMT),that increase tumor cell invasiveness.Recent evidence has shown that hypoxia is a major EMT regulator in pancreatic cancer cells and facilitates metastasis;however,the mechanisms remain elusive.AIM To investigate the role of miR-301a in hypoxia-induced EMT in PC cells.METHODS Real-time PCR and Western blot analysis were used to detect the expression of miR-301a and EMT markers in PDAC cells cultured in hypoxic and normoxic conditions.Western blot analysis was used to detect the expression of EMT markers in PDAC cells with miR-301a overexpression.Wound healing assay and Transwell assay were used to detect the migration capabilities of PDAC cells with miR-301a overexpression and knockout.Luciferase assay was used to detect the miR-301a promoter and the 3’untranslated region activity of TP63.Orthotopic PC mouse models were used to study the role of miR-301a in metastasis of PDAC cells in vivo.In situ hybridization assay was used to detect the expression of miR-301a in PDAC patient samples(adjacent paratumor and paired tumor tissues).RESULTS Hypoxic environment could directly promote the EMT of PC cells.The expression level of miR-301a was increased in a HIF2αdependent manner in hypoxia-cultured CFPAC-1 and BxPC-3 cells.Overexpression of miR-301a enhanced the hypoxia-induced EMT of PC cells,while knocking out miR-301a result in the suppression of hypoxia-induced EMT.TP63 was a direct target of miR-301a and involved in the metastatic process of PC cells.Furthermore,miR-301a upregulation facilitated PDAC distant metastasis and lymph node metastasis in vivo.Additionally,miR-301a overexpression was indicative of aggressive clinicopathological behaviors and poor prognosis.CONCLUSION The newly identified HIF-2α-miR301a-TP63 signaling pathway may play a crucial role in hypoxia-induced EMT in PDAC cells.展开更多
文摘目的:探讨DNA甲基化影响TP63的表达及其与子宫颈癌的相关性。方法:收集宫颈癌患者40例(CC)和因子宫良性病变行全子宫切除术的45例患者的宫颈组织。采用全基因组DNA甲基化分析(WGBS)和RNA sequence联合筛查宫颈癌组织和正常宫颈组织(各3例)的DNA甲基化度和mRNA表达。采用亚硫酸氢盐测序法(BSP)和实时定量PCR(qPCR)法检测TP63基因启动子区DNA甲基化和RUNX2、TP63、survivin的mRNA表达。CHIP法检测RUNX2在宫颈组织中与TP63基因启动子的结合情况,并分析TP63基因DNA甲基化状态与SCC病理参数的关系。结果: WGBS和RNA-seq联合筛查发现,TP63启动子DNA甲基化水平升高与其mRNA表达减少相关。BSP结果证实,与正常宫颈组织比较,宫颈癌组织中TP63基因启动子区-688 CpG位点DNA甲基化率显著升高,差异有统计学意义[77.5%(31/40)vs 31.1%(14/45), P <0.05]。qPCR结果显示,与对照组比较,宫颈癌组织中RUNX2和survivin的mRNA表达上调,TP63 mRNA表达下调( P 均<0.05)。染色质免疫共沉淀技术(ChIP)证实,宫颈癌组织中TP63基因启动子区存在转录因子RUNX2结合序列,且-688CpG位点DNA甲基化修饰影响RUNX2的结合。相关性分析显示,TP63基因DNA甲基化与肿瘤大小、病理分级、临床分期相关( P 均<0.05),与患者年龄无关( P >0.05)。结论: TP63基因启动子DNA甲基化抑制其表达,参与宫颈癌的发生发展。
基金Supported by National Natural Science Foundation of China,No.81372640.
文摘BACKGROUND Pancreatic cancer(PC)is one of the deadliest cancers worldwide.PC metastasis involves a complex set of events,including epithelial-mesenchymal transition(EMT),that increase tumor cell invasiveness.Recent evidence has shown that hypoxia is a major EMT regulator in pancreatic cancer cells and facilitates metastasis;however,the mechanisms remain elusive.AIM To investigate the role of miR-301a in hypoxia-induced EMT in PC cells.METHODS Real-time PCR and Western blot analysis were used to detect the expression of miR-301a and EMT markers in PDAC cells cultured in hypoxic and normoxic conditions.Western blot analysis was used to detect the expression of EMT markers in PDAC cells with miR-301a overexpression.Wound healing assay and Transwell assay were used to detect the migration capabilities of PDAC cells with miR-301a overexpression and knockout.Luciferase assay was used to detect the miR-301a promoter and the 3’untranslated region activity of TP63.Orthotopic PC mouse models were used to study the role of miR-301a in metastasis of PDAC cells in vivo.In situ hybridization assay was used to detect the expression of miR-301a in PDAC patient samples(adjacent paratumor and paired tumor tissues).RESULTS Hypoxic environment could directly promote the EMT of PC cells.The expression level of miR-301a was increased in a HIF2αdependent manner in hypoxia-cultured CFPAC-1 and BxPC-3 cells.Overexpression of miR-301a enhanced the hypoxia-induced EMT of PC cells,while knocking out miR-301a result in the suppression of hypoxia-induced EMT.TP63 was a direct target of miR-301a and involved in the metastatic process of PC cells.Furthermore,miR-301a upregulation facilitated PDAC distant metastasis and lymph node metastasis in vivo.Additionally,miR-301a overexpression was indicative of aggressive clinicopathological behaviors and poor prognosis.CONCLUSION The newly identified HIF-2α-miR301a-TP63 signaling pathway may play a crucial role in hypoxia-induced EMT in PDAC cells.
