miR-199a-3p targets CD44 and reduces proliferation of CD44 positive hepatocellular carcinoma cell lines.
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.
Department of Surgery, Ohio State University Medical Center, Columbus, OH, USA.
Jon C
JC
Henry
Jong-Kook
JK
Park
Jinmai
J
Jiang
Ji Hye
JH
Kim
David M
DM
Nagorney
Lewis R
LR
Roberts
Soma
S
Banerjee
Thomas D
TD
Schmittgen
D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D004317:Doxorubicin / Q000494:pharmacology; D019008:Drug Resistance, Neoplasm; D016147:Genes, Tumor Suppressor; D006801:Humans; D018960:Hyaluronan Receptors / Q000235:genetics*; D008113:Liver Neoplasms / Q000235:genetics / Q000378:metabolism / Q000473:pathology*; D035683:MicroRNAs / Q000235:genetics*; D009361:Neoplasm Invasiveness
D000903:Antibiotics, Antineoplastic; D018960:Hyaluronan Receptors; D035683:MicroRNAs; C531408:mirn199 microRNA, human; D004317:Doxorubicin