Previous studies have demonstrated that a number of HOX genes, a family of transcription
factors with key roles in early development, are up-regulated in head and neck squamous
cell carcinoma (HNSCC) and other cancers. The loci of several Homeobox (HOX) genes
also contain microRNAs (miRs), including miR-196a.
Global miR expression and expression of all 39 HOX genes in normal oral keratinocytes
(NOKs), oral pre-malignant (OPM) and HNSCC cells was assessed by expressionmicroarray
and qPCR and in tissues by immunohistochemistry (IHC) and qPCR of laser microdissected
(LCM) tissues. Expression of miR196a and HOXB9 was reduced using anti-miR-196a and
siRNA, respectively. Expression microarray profiles of anti-miR196a and pre-miR196a
transfected cells were compared to parental cells in order to identify novel targets of miR-
196a. Putative miR196a targets were validated by qPCR and were confirmed as binding to
the 3’UTR of miR196a by a dual luciferase reporter assay combined with mutational analysis
of the miR-196a binding site. RESULTS
miR-196a and HOXB9 are highly expressed in HNSCC compared to NOKs, a pattern also
seen in HNSCC tissues by HOXB9 IHC and qPCR of miR-196a in LCM tissue. Knock-down
of miR-196a expression decreased HNSCC cell migration, invasion and adhesion to fibronectin,
but had no effect on proliferation. Furthermore, knock-down of HOXB9 expression
decreased migration, invasion and proliferation but did not alter adhesion. We identified a
novel primary mRNA transcript containing HOXB9 and miR196a-1 as predicted from in-silico
analysis. Expression array analysis identified a number of miR196a targets, including MAMDC2 and HOXC8.We confirmed that MAMDC2 is a novel miR-196a target using a
dual luciferase reporter assay with the effect abolished on mutation of the binding site.
These results show that miR-196a and HOXB9 are overexpressed, perhaps co-ordinately,
as HNSCC develops and exert a pro-tumourigenic phenotype in HNSCC and OPM cells.
S1 Fig. Schematic diagram of the putative primary transcript and the length of the PCR
product for both the primers utilised in the nested PCR analysis.
S2 Fig. Expression of all HOX genes analysed by qPCR, showing data in the full panel of
cell cultures tested, including normal (black), OPM (red) and HNSCC (blue), arranged by
group (A-D) and in numerical order.
S3 Fig. Expression of putative miR196a targets suggested form investigation in other cancers,
as assessed by qPCR in anti-miR196a transfected D19 and B16 cells, and pre-miR
196a transfected OKF4 cells in panel D. A: Keratin V; B: Anexin A1; C S100A9; D: HOXC8.
Only HOXC8 shows significant changes in expression and this only in D19 (p<0.01). The data
does not support regulation of KRT5, ANXA1 or S100A9 by miR196a in HNSCC.
S1 Table. Clinical details of the HNSCC and OPM cell lines used in this study. All cell lines
are HPV negative.
S2 Table. Clinical and pathological details of the 25 HNSCC samples in the TMA used for
HOXB9 IHC. FOM = Floor of mouth, RM = retromolar, BM = buccal mucosa.
S3 Table. Clinical pathological details of HNSCC samples used for Laser capture microdissection
FOM = Floor of mouth, RM = retromolar, BM = buccal mucosa.
S4 Table. A full list of qPCR primers used in this study.
S5 Table. Gene ontology enrichment analysis demonstrating significantly enriched GO biological
processes on manipulation of miR-196a expression. The total number of significantly
differentially expressed genes entered into this analysis was 353. Analysis generated by analysis
of the gene list in DAVID (http://david.abcc.ncifcrf.gov).