miRNA-155-5p Targets ZNF703 and Suppresses Metastasis in Oral Cancer Cells

Fumihiko Uchida DDS, PhD, Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
Shogo Hasegawa DDS, PhD, Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
Osamu Baba , oral and maxillofacial surgery, Tsukuba university, Tsukuba, Japan
Takaaki Ito MD, DDS , Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
Masanobu Yamatoji D.D.S., Ph.D., Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
Naomi I. Kanno DDS, PhD, Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
Kenji Yamagata D.D.S., Ph.D., Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
Toru Yanagawa M.D., D.D.S., PhD., Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
Hiroki Bukawa MD, DDS, PhD, Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
MicroRNAs (miRNAs) are small, non-coding RNA molecules that regulate gene expression post-transcriptionally and are reported to be involved in multiple steps of tumor metastasis. It is well recognized that lymph node metastasis strongly correlates with poor survival in patients with oral cancer. In Nguyen et al. mRNA microarray analysis for identification of a predictive gene expression signature of cervical lymph node metastasis in oral cancer, 33 mRNAs were identified as the most underexpressed in human oral tumor versus controls. The purpose of the present study is to examine miRNA expression profiling on high metastatic potential human oral cancer cell lines and make integrated analysis of microRNA and mRNA with tumor metastasis in oral cancer.

     The human OSCC cell lines (SAT, HO1-u-1, SAS, HSC-3, HSC-3-M-3, OSC-19, and KON) were purchased from Japanese Collection of Research Bioresources. HSC-3, HSC-3-M-3, OSC-19, and KON are high metastatic potential cell lines. These cell lines were maintained in Dulbecco’s modified Eagle’s medium or RPMI1640 containing 10% heat-inactivated fetal calf serum (Nichirei Biosciences Inc.) and 1% penicillin-streptomycin in a humidified atmosphere with 5% CO2 at 37°C. For miRNA array experiments, total RNA from these cancer cell lines was isolated using an RNeasy Kit (Qiagen) and reverse-transcribed using the TaqMan microRNA Reverse Transcription Kit (Applied Biosystems). Subsequently, using a miRNA PCR array platform (Human Cancer Pathway Finder miScript miRNA PCR array, MIHS-102Z, Qiagen), we analyzed the miRNA profiles in these cell lines. After normalization to a set of housekeeping genes, differential expressions of the miRNAs were analysed. To identify the possible targets of these miRNA, we performed bioinformatics analysis by TargetScan algorithm and integrated analysis across the data of human cancer cell lines and mRNA microarray data (2) to identify miRNAs whose expression correlated with the inverse expression of mRNA targets predicted in silico. Next, we examined the endogenous expression of mRNA target of specific miRNA and the posttranslational protein in oral cancer cell lines by quantitative real-time PCR (qRT-PCR) and western blot analyses.

     In the miRNA PCR array, P values are calculated based on a Student t-test of the replicate 2^(Delta Ct) values for each gene in high metastatic potential cell line group comparing to the other cell line group. In mRNA expression analysis, one-way ANOVA was used for experimental designs with more than 2 experimental groupings (metastatic cell lines vs the other cell lines). These tests defined which probes were considered to be significantly differentially expressed based on a default P value of less than 0.05.

     miRNA-155-5p was found up-regulated (more than 7 fold, P=0.09) in high metastatic potential cell lines compared to the other cell lines. Results obtained from different searches by TargetScan algorithm predicted ZNF703 as a potential target for miRNA-155-5p. Through an integrative analysis of matching miRNA and mRNA expression data, candidate miRNA-mRNA interaction pairs were detected. By qRT-PCR and Western blot analyses, we confirmed down-regulation of ZNF703 in high metastatic potential cell lines. These data provided the evidence for an inverse correlation between the expression of miRNA-155-5p and ZNF703.

     By using an integrated miRNA and mRNA gene expression analysis, we demonstrated that the expression of miRNA can be associated with the inverse expression of a subset of predicted target mRNAs in oral cancer, leading to a more focused set of miRNA to functionally validate.

1. Regulation of microRNAs in cancer metastasis. Bouyssou JM, Manier S, Huynh D et al. Biochim Biophys Acta. 2014 22. pii: S0304-419X(14)00018-3. doi: 10.1016/j.bbcan.2014.02.002.

2. Identification of a predictive gene expression signature of cervical lymph node metastasis in oral squamous cell carcinoma. Nguyen ST, Hasegawa S, Tsuda H et al. Cancer Sci. 2007 ;98:740-6.

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