RRC ID 67200
Author Jin H, Wang C, Jin G, Ruan H, Gu D, Wei L, Wang H, Wang N, Arunachalam E, Zhang Y, Deng X, Yang C, Xiong Y, Feng H, Yao M, Fang J, Gu J, Cong W, Qin W.
Title 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.
Journal Gastroenterology
Abstract 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 overexpressed 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.
Volume 153(3)
Pages 799-811.e33
Published 2017-9-1
DOI 10.1053/j.gastro.2017.05.045
PII S0016-5085(17)35687-1
PMID 28583823
MeSH Adult Aged Animals Calcineurin / metabolism* Carcinoma, Hepatocellular / chemistry Carcinoma, Hepatocellular / genetics* Carcinoma, Hepatocellular / pathology* Carcinoma, Hepatocellular / secondary Cell Line, Tumor Cell Movement Cell Proliferation Chromosomes, Human, Pair 21 CpG Islands / genetics DNA Methylation DNA-Binding Proteins Disease-Free Survival Down-Regulation Female Gene Expression Humans Insulin-Like Growth Factor I / genetics Insulin-Like Growth Factor I / metabolism Intracellular Signaling Peptides and Proteins / analysis Intracellular Signaling Peptides and Proteins / genetics* Liver / chemistry Liver Neoplasms / chemistry Liver Neoplasms / genetics* Liver Neoplasms / pathology* Male Mice Middle Aged Muscle Proteins / analysis Muscle Proteins / genetics* NFATC Transcription Factors / genetics NFATC Transcription Factors / metabolism* Neoplasm Grading Neoplasm Invasiveness Neoplasm Staging Neoplasm Transplantation Protein Isoforms / genetics Protein Transport / drug effects RNA, Messenger / analysis* Regulatory Sequences, Nucleic Acid Signal Transduction Survival Rate Tumor Burden Vascular Endothelial Growth Factor A / metabolism
IF 17.373
Human and Animal Cells HuH-7