Regulator of Calcineurin 1 Gene Isoform 4, Down-regulated in Hepatocellular Carcinoma, Prevents Proliferation, Migration, and Invasive Activity of Cancer Cells and Metastasis of Orthotopic Tumors by Inhibiting Nuclear Translocation of NFAT1
Regulator of Calcineurin 1 Gene Isoform 4, Down-regulated in Hepatocellular Carcinoma, Prevents Proliferation, Migration, and Invasive Activity of Cancer Cells and Metastasis of Orthotopic Tumors by Inhibiting Nuclear Translocation of NFAT1
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钙调神经磷酸酶 1 基因亚型 4 的调节剂，在肝细胞癌中下调，通过抑制 NFAT1 的核转位来防止癌细胞的增殖、迁移和侵袭活性以及原位肿瘤的转移
DOI：
10.1053/j.gastro.2017.05.045
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发表时间：
2017-09-01
期刊：
影响因子：
29.4
通讯作者：
Qin, Wenxin
中科院分区：
文献类型：
--
作者：
Jin, Haojie;Wang, Cun;Qin, Wenxin
背景与目的：唐氏综合征患者患许多实体瘤的风险较低，这促使人们在人类21号染色体（HSA 21）上寻找肿瘤抑制基因。我们的目的是确定和探讨肿瘤抑制剂对HSA 21在肝细胞癌（HCC）的潜在机制。方法：我们使用Affytelium HG-U133 Plus 2.0阵列（Affytelium，Santa Clara，CA）比较了14对原发性HCC和邻近非癌肝组织中HSA 21基因的表达。从中国某医院收集的108例HCC组织和邻近正常肝组织进行实时聚合酶链反应和免疫组织化学分析;钙调神经磷酸酶1（RCAN 1）亚型4（RCAN 1. 4）的表达水平与临床特征相关。我们在MHCC 97 H和HCCLM 3细胞中过表达来自慢病毒载体的RCAN1.4，并在SMMC 7721和Huh 7细胞中使用小干扰RNA敲低表达。在增殖、迁移和侵袭测定中分析细胞。将过表达RCAN1.4或RCAN1.4敲低的HCC细胞注射到裸鼠的肝脏或尾静脉中;定量肿瘤生长和肺转移的数量。我们进行了亚硫酸氢盐焦磷酸测序和甲基化特异性聚合酶链反应分析来分析CpG岛甲基化。我们测定了肝癌细胞中钙调神经磷酸酶的活性。结果：原发性肝癌组织中RCAN1.4 mRNA和蛋白水平均显著低于癌旁肝组织。RCAN1.4 mRNA水平的降低与晚期肿瘤、低分化、较大肿瘤和血管浸润显著相关。Kaplan-Meier生存分析显示，RCAN1.4 mRNA水平较低的HCC患者的总生存时间和复发时间均短于RCAN1.4 mRNA水平较高的HCC患者。在HCC细胞系中，RCAN1.4的表达显著降低了增殖、迁移和侵袭活性。与对照HCC细胞相比，过表达RCAN1.4的HCC细胞形成较小的异种移植肿瘤，具有较少的转移和血管。在肝癌细胞中，RCAN1.4通过降低钙调磷酸酶活性和阻断活化T细胞核因子（NFAT 1）的核转位来抑制胰岛素样生长因子1和血管内皮生长因子A的表达。用钙调磷酸酶抑制剂环孢菌素A孵育的HCC细胞核NFAT 1水平降低。HCC细胞中RCAN1.4 5'调控区的CpG岛高度甲基化，这降低了其表达。结论：RCAN1.4在肝癌组织中表达较非肿瘤肝组织下调。RCAN1.4在体外可防止细胞增殖、迁移和侵袭; HCC细胞中过表达的RCAN1.4可通过抑制钙调磷酸酶活性和NFAT 1的核转位来防止异种移植肿瘤的生长、血管生成和转移。
BACKGROUND & AIMS: Individuals with Down syndrome have a low risk for many solid tumors, prompting the search for tumor suppressor genes on human chromosome 21 (HSA21). We aimed to identify and explore potential mechanisms of tumor suppressors on HSA21 in hepatocellular carcinoma (HCC). METHODS: We compared expression of HSA21 genes in 14 pairs of primary HCC and adjacent noncancer liver tissues using the Affymetrix HG-U133 Plus 2.0 array (Affymetrix, Santa Clara, CA). HCC tissues and adjacent normal liver tissues were collected from 108 patients at a hospital in China for real-time polymerase chain reaction and immunohistochemical analyses; expression levels of regulator of calcineurin 1 (RCAN1) isoform 4 (RCAN1.4) were associated with clinical features. We over-expressed RCAN1.4 from lentiviral vectors in MHCC97H and HCCLM3 cells and knocked expression down using small interfering RNAs in SMMC7721 and Huh7 cells. Cells were analyzed in proliferation, migration, and invasion assays. HCC cells that overexpressed RCAN1.4 or with RCAN1.4 knockdown were injected into livers or tail veins of nude mice; tumor growth and numbers of lung metastases were quantified. We performed bisulfite pyrosequencing and methylation-specific polymerase chain reaction analyses to analyze CpG island methylation. We measured phosphatase activity of calcineurin in HCC cells. RESULTS: RCAN1.4 mRNA and protein levels were significantly decreased in primary HCC compared with adjacent noncancer liver tissues. Reduced levels of RCAN1.4 mRNA were significantly associated with advanced tumor stages, poor differentiation, larger tumor size, and vascular invasion. Kaplan-Meier survival analysis showed that patients with HCCs with lower levels of RCAN1.4 mRNA had shorter time of overall survival and time to recurrence than patients whose tumors had high levels of RCAN1.4 mRNA. In HCC cell lines, expression of RCAN1.4 significantly reduced proliferation, migration, and invasive activity. HCC cells that overexpressed RCAN1.4 formed smaller xenograft tumors, with fewer metastases and blood vessels, than control HCC cells. In HCC cells, RCAN1.4 inhibited expression of insulin-like growth factor 1 and vascular endothelial growth factor A by reducing calcineurin activity and blocking nuclear translocation of nuclear factor of activated T cells (NFAT1). HCC cells incubated with the calcineurin inhibitor cyclosporin A had decreased nuclear level of NFAT1. HCC cells had hypermethylation of a CpG island in the 5' regulatory region of RCAN1.4, which reduced its expression. CONCLUSIONS: RCAN1.4 is down-regulated in HCC tissues, compared with non-tumor liver tissues. RCAN1.4 prevents cell proliferation, migration, and invasion in vitro; overexpressed RCAN1.4 in HCC cells prevents growth, angiogenesis, and metastases of xenograft tumors by inhibiting calcineurin activity and nuclear translocation of NFAT1.