miR-205-5p Targets Interferon Regulatory Factor 1 and Suppresses Metastasis in Oral Cancer Cells

Shogo Hasegawa DDS, PhD, Oral and Maxillofacial Surgery, University of Tsukuba, Tsukuba, Japan
Fumihiko Uchida 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
In 2012, 360 790 Japanese people died from cancer. Of these, 7163 people had oral cavity cancer and the major cause of death by cancer was metastasis. In our microarray analysis, we revealed that 52 mRNAs were significantly up-regulated and 33 mRNAs were significantly down-regulated in metastatic oral squamous cell carcinoma (OSCC). Recent evidences have indicated the role of microRNAs (miRNAs) in modulating the metastatic process in solid tumors. miRNAs are small, non-coding RNAs involved in post-transcriptional regulation of protein-coding genes in various biological processes. The purpose of the present study is to examine miRNAs 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 our mRNA microarray data 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 metastatic 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 non-metastatic 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-205-5p was found down-regulated (more than -4 fold, P<0.001) in metastatic cell lines compared to non-metastatic cell lines. Results obtained from different searches by TargetScan algorithm predicted interferon regulatory factor 1 (IRF1) as a potential target for miRNA-205-5p. 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 up-regulation of IRF1 in metastatic cell lines. These data provided the evidence for an inverse correlation between the expression of miRNA-205-5p and IRF1.

    In Summary, we have demonstrated that miR-205-5p can be associated with IRF-1 expression in metastatic OSCC cell-lines. 

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