Downregulation of CLDN7 due to promoter hypermethylation is associated with human clear cell renal cell carcinoma progression and poor prognosis.
Downregulation of CLDN7 due to promoter hypermethylation is associated with human clear cell renal cell carcinoma progression and poor prognosis.
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启动子高甲基化引起的 CLDN7 下调与人透明细胞肾细胞癌进展和不良预后相关
DOI：
10.1186/s13046-018-0924-y
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发表时间：
2018-11-14
期刊：
影响因子：
--
通讯作者：
Zhou L
中科院分区：
文献类型：
--
作者：
Li Y;Gong Y;Ning X;Peng D;Liu L;He S;Gong K;Zhang C;Li X;Zhou L
背景转移是肾细胞癌（renal cell carcinoma，RCC）的主要死亡原因.细胞与细胞粘附的丧失，包括紧密连接（TJ）是转移过程中的初始步骤。Claudin-7（CLDN 7）是TJ的主要成分。然而，其临床意义及其调控肾脏肿瘤的发生仍然知之甚少。方法共120例新鲜透明细胞肾细胞癌（ccRCC）标本和144例原发性RCC和邻近的非恶性肾组织石蜡标本来自北京大学第一医院泌尿外科。使用生物信息学数据挖掘、定量实时PCR（qRT-PCR）、蛋白质印迹和免疫染色来确定ccRCC组织和细胞系中CLDN 7的表达。分析北京大学第一医院ccRCC患者CLDN 7表达和启动子DNA甲基化状态的临床意义和癌症基因组图谱。此外，还进行了CLDN 7的甲基化特异性PCR、亚硫酸氢盐基因组测序和去甲基化分析。通过使用MTS测定和EdU掺入测定检查细胞增殖、通过体外伤口愈合测定和transwell迁移测定检查细胞迁移、通过transwell侵袭测定检查细胞侵袭以及通过流式细胞术检查细胞凋亡来研究CLDN 7的生物学功能。进行小鼠模型实验以确认CLDN 7对体内肿瘤生长和转移的影响。CLDN 7功能的分子机制进行了研究，使用基因集富集分析（GSEA）和高通量cDNA测序（RNA-Seq），并证实了qRT-PCR，Western blot和免疫染色在体外和体内。CLDN 7可以帮助预测ccRCC患者的侵袭性肿瘤状态和不良预后。有趣的是，CLDN 7启动子的高甲基化与晚期ccRCC状态和不良预后相关。此外，CLDN 7的过表达在体外和体内均诱导细胞凋亡，抑制ccRCC细胞的增殖、迁移和侵袭能力。此外，GSEA和RNA-Seq结果表明，CLDN 7在癌症相关信号通路和（上皮-间充质转化）EMT相关通路中具有负面影响。这些结果通过qRT-PCR，Western印迹和免疫staining.ConclusionsWe验证了CLDN 7作为ccRCC抑制剂的先前未描述的作用，并表明CLDN 7的损失增强EMT和肿瘤进展。CLDN 7可以作为肿瘤进展中的功能性肿瘤抑制因子以及ccRCC患者中的潜在生物标志物和靶标。
BackgroundMetastasis is the primary cause of death in renal cell carcinoma (RCC). Loss of cell-to-cell adhesion, including tight junctions (TJs) is the initial step in the process of metastasis. Claudin-7 (CLDN7) is a major component of TJs. However, the clinical significance and its regulation of kidney tumorigenesis remain poorly understood.MethodsA total of 120 fresh clear cell RCC (ccRCC) specimens and 144 primary RCC and adjacent nonmalignant renal paraffin specimens were obtained from Department of Urology, Peking University First Hospital. Expression of CLDN7 in ccRCC tissues and cell lines were determined using bioinformatic data mining, quantitative real-time PCR (qRT-PCR), Western blotting and immunostaining. The clinical significance of CLDN7 expression and promoter DNA methylation status was analyzed in ccRCC patients from Peking University First Hospital and The Cancer Genome Atlas. Additionally, the methylation specific-PCR, bisulfite genomic sequencing and demethylation analysis of CLDN7 were performed. Biological functions of CLDN7 were investigated by examining cell proliferation using MTS assays and EdU incorporation assays, cell migration by in vitro wound healing assays and transwell migration assays, cell invasion by transwell invasion assays, and cell apoptosis by flow cytometry. Mouse model experiments were performed to confirm the effects of CLDN7 on tumor growth and metastasis in vivo. The molecular mechanism of CLDN7 function was investigated using gene-set enrichment analysis (GSEA) and high-throughput cDNA sequencing (RNA-Seq) and confirmed by qRT-PCR, Western blot and immunostaining in vitro and in vivo.ResultsOur findings revealed that CLDN7 is frequently downregulated via hypermethylation of its promoter in ccRCC. CLDN7 can help predict aggressive tumor status and poor prognosis in ccRCC patients. Interestingly, hypermethylation of the CLDN7 promoter was related to advanced ccRCC status and poor prognosis. Moreover, overexpression of CLDN7 induced cell apoptosis, suppressed proliferation, migration and invasion abilities of ccRCC cells both in vitro and in vivo. Additionally, GSEA and RNA-Seq results showed that CLDN7 had negative effects in cancer-associated signaling pathways and (epithelial-mesenchymal transition) EMT-related pathways. These results were validated by qRT-PCR, Western blot and immunostaining.ConclusionsWe have demonstrated a previously undescribed role of CLDN7 as a ccRCC suppressor and suggest that loss of CLDN7 potentiates EMT and tumor progression. CLDN7 may serve as a functional tumor suppressor in tumor progression and a potential biomarker and target in patients with ccRCC.
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影响因子：
8
作者：
Nguyen N;Fernando SD;Biette KA;Hammer JA;Capocelli KE;Kitzenberg DA;Glover LE;Colgan SP;Furuta GT;Masterson JC
通讯作者：
Masterson JC
影响因子：
8
作者：
Bhat AA;Pope JL;Smith JJ;Ahmad R;Chen X;Washington MK;Beauchamp RD;Singh AB;Dhawan P
通讯作者：
Dhawan P
影响因子：
254.7
作者：
Siegel, Rebecca;Ma, Jiemin;Jemal, Ahmedin
通讯作者：
Jemal, Ahmedin
EMT，癌症干细胞和耐药性：癌症战争中新兴的邪恶轴。
影响因子：
8
作者：
Singh, A.;Settleman, J.
通讯作者：
Settleman, J.
影响因子：
4.5
作者：
Choi YD;Kim KS;Ryu S;Park Y;Cho NH;Rha SH;Jang JJ;Ro JY;Juhng SW;Choi C
通讯作者：
Choi C