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MiR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines.

ABSTRACT: Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two cell lines (SNU449 and SNU423); the proliferation of the other 5 treated cell lines was similar to control oligonucleotide. A pre-miR-199a-3p oligonucleotide formulated with chemical modifications to enhance stability while preserving processing, reduced cell proliferation in SNU449 and SNU423 to the same extent as the commercially available pre-miR-199a-3p oligonucleotide. Furthermore, only the duplex miR-199a-3p oligonucleotide, and not the guide strand alone, was effective at reducing cell viability. Since a CD44 variant was essential for c-Met signaling [V. Orian-Rousseau, L. Chen, J.P. Sleeman, P. Herrlich, H. Ponta, CD44 is required for two consecutive steps in HGF/c-Met signaling, Genes Dev. 16 (2002) 3074-3086] and c-Met is a known miR-199a-3p target, we hypothesized that miR-199a-3p may also target CD44. Immunoblotting confirmed that only the two HCC lines that were sensitive to the effects of pre-miR-199a-3p were CD44+. Direct targeting of CD44 by miR-199a-3p was confirmed using luciferase reporter assays and immunoblotting. Transfection of miR-199a-3p into SNU449 cells reduced in vitro invasion and sensitized the cells to doxorubicin; both effects were enhanced when hyaluronic acid (HA) was added to the cell cultures. An inverse correlation between the expression of miR-199a-3p and CD44 protein was noted in primary HCC specimens. The ability of miR-199a-3p to selectively kill CD44+ HCC may be a useful targeted therapy for CD44+ HCC.

SUBMITTER: Henry JC

PROVIDER: S-EPMC3039123 | biostudies-literature | 2010 Dec

REPOSITORIES: biostudies-literature

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miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines.

Henry Jon C JC Park Jong-Kook JK Jiang Jinmai J Kim Ji Hye JH Nagorney David M DM Roberts Lewis R LR Banerjee Soma S Schmittgen Thomas D TD

Biochemical and biophysical research communications 20101103 1

Previous work by us and others reported decreased expression of miR-199a-3p in hepatocellular carcinoma (HCC) tissues compared to adjacent benign tissue. We report here a significant reduction of miR-199a-3p expression in 7 HCC cell lines. To determine if miR-199a-3p has a tumor suppressive role, pre-miR-199a-3p oligonucleotides were transfected into the HCC cell lines. Pre-miR-199a-3p oligonucleotide reduced cell proliferation by approximately 60% compared to control oligonucleotide in only two ...[more]

PMID: 21055388

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Project description:Objective: The long noncoding RNA (lncRNA) nuclear paraspeckle assembly transcript 1 (NEAT1) has emerged as a novel player in hepatocellular carcinoma (HCC). Hypoxia is a common characteristic of the microenvironment of HCC. This study aimed to investigate whether lncRNA-NEAT1 is induced by hypoxia in HCC, and the mechanism that underlies LncRNA-NEAT1 function. Methods: The expression changes of lncRNA-NEAT1 in HCC cell lines under hypoxic conditions were examined by quantitative reverse transcription-polymerase chain reaction (qRT-PCR). The regulatory effect of HIF-1α on lncRNA-NEAT1 was confirmed with chromatin immunoprecipitation (ChIP) and luciferase reporter assays. The function of lncRNA-NEAT1 on HCC cell growth under hypoxic conditions was determined by CCK-8 assay and flow cytometry. lncRNA -NEAT1 was predicted to serve as a competing endogenous RNA (ceRNA) within microRNA (miRNA)/mRNA axes based on microarray data, public HCC-related datasets and integrative bioinformatics analysis, and the miR-199a-3p/UCK2 axis was selected and validated by qRT-PCR, western blotting, RNA immunoprecipitation, and luciferase reporter analyses. The role of miR-199a-3p/UCK2 in HCC and its functional association with lncRNA-NEAT1 were assessed both in vitro and in vivo. Results: LncRNA-NEAT1 expression was significantly induced by hypoxia in SNU-182 and HUH7 cells. HIF-1α was shown to regulate lncRNA-NEAT1 transcription. Under hypoxic conditions, lncRNA-NEAT1 maintained the growth of HCC cells and inhibited apoptosis and cell cycle arrest. LncRNA-NEAT1 was predicted to regulate a panel of HCC-associated miRNA-mRNA pairs consisting of 8 miRNAs and 13 mRNAs. LncRNA-NEAT1 was shown to function as a ceRNA of miR-199a-3p/UCK2 both in HCC cells under hypoxic conditions and in an animal model. Conclusion: LncRNA-NEAT1 is a hypoxia-responsive lncRNA in HCC cell lines Insilico evidence suggested that LncRNA-NEAT1 may sustainthe growth of HCC cells by regulating HCC-associated mRNAs that interact with tumor-suppressive miRNAs. The lncRNA-NEAT1/miR-199a-3p/UCK2 pathway may contribute to the progression of HCC cell lines in a hypoxic microenvironment and therefore may represent a novel therapeutic target for HCC.

Dataset Information

MiR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines.

Publications

miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines.

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