Impact of Oncogenic Targets by Tumor-Suppressive miR-139-5p and miR-139-3p Regulation in Head and Neck Squamous Cell Carcinoma

Abstract

We newly generated an RNA-sequencing-based microRNA (miRNA) expression signature of head and neck squamous cell carcinoma (HNSCC). Analysis of the signature revealed that both strands of some miRNAs, including miR-139-5p (the guide strand) and miR-139-3p (the passenger strand) of miR-139, were downregulated in HNSCC tissues. Analysis of The Cancer Genome Atlas confirmed the low expression levels of miR-139 in HNSCC. Ectopic expression of these miRNAs attenuated the characteristics of cancer cell aggressiveness (e.g., cell proliferation, migration, and invasion). Our in silico analyses revealed a total of 28 putative targets regulated by pre-miR-139 (miR-139-5p and miR-139-3p) in HNSCC cells. Of these, the GNA12 (guanine nucleotide-binding protein subunit alpha-12) and OLR1 (oxidized low-density lipoprotein receptor 1) expression levels were identified as independent factors that predicted patient survival according to multivariate Cox regression analyses (p = 0.0018 and p = 0.0104, respectively). Direct regulation of GNA12 and OLR1 by miR-139-3p in HNSCC cells was confirmed through luciferase reporter assays. Moreover, overexpression of GNA12 and OLR1 was detected in clinical specimens of HNSCC through immunostaining. The involvement of miR-139-3p (the passenger strand) in the oncogenesis of HNSCC is a new concept in cancer biology. Our miRNA-based strategy will increase knowledge on the molecular pathogenesis of HNSCC.

Identification of miR-199-5p and miR-199-3p Target Genes: Paxillin Facilities Cancer Cell Aggressiveness in Head and Neck Squamous Cell Carcinoma

“…The miRNA precursors, negative control miRNA, and siRNAs were obtained from Invitrogen/Thermo Fisher Scientific (Waltham, MA, USA). The procedures used for transient transfection of miRNAs, siRNAs, and plasmid vectors have been described in our previous studies [ 16 , 17 , 24 ]. The reagents used are listed in Supplementary Table S2 .…”

“…RNA extraction from HNSCC cell lines and qRT-PCR were performed as described in our previous studies [ 16 , 17 , 24 ]. Briefly, total RNA was harvested using Trizol reagent and the PureLink™ RNA Mini Kit (Invitrogen/Thermo Fisher Scientific, Waltham, MA, USA).…”

“…The immunohistochemistry procedures have been described in our previous studies [ 16 , 17 , 24 ]. Briefly, paraffin sections of tumors were obtained from HNSCC patients who underwent surgical treatment at Chiba University hospital.…”

“…The Western blotting procedures have been described in our previous studies [ 16 , 17 , 24 ]. Briefly, cell lysates were loaded onto 4–15% polyacrylamide gels at 18 μg/well.…”

“…The guide strands of the miR-199 family share the same seed sequence (CCAGUGU), and the passenger strands share the seed sequence (CAGUAGU). Analysis of our miRNA expression signatures of HNSCC and OSCC, determined by RNA sequencing, showed that the miR-199 family is downregulated in cancer tissues [ 15 , 16 , 17 ]. Our previous study revealed that the miR-199 family members are downregulated in HNSCC tissues, and ectopic expression of these miRNAs markedly attenuated cancer cell migration and invasive abilities [ 16 ].…”