Utility of Saliva in the Evaluation of Microrna Functions as a Tumor Suppressor in Oral Squamous Cell Carcinoma
microRNAs (miRNAs) are short non-coding RNA that consisted of 20-22 nucleotides. In the recent study, abnormal functions of miRNA cause human disease like some types of cancer by suppressing translation of the mRNA of the target gene have been demonstrated. In oral cancer, some oral cancer-specific miRNA has been investigated. [1] But there are few researches that investigate cancer-specific miRNA using saliva sample. Saliva is thought to contain useful information that influenced by surrounding environment. miRNAs are also known to exists in saliva and miRNA in human saliva have recently become an emerging field in research for diagnosis application and its potential role in biological implications of oral diseases. Purpose of this study is to investigate oral cancer-specific miRNA in saliva itself and its biological implications by compering with that of clinical indexes, another clinical samples and cell lines.
Saliva and serum were taken before any surgical treatment or chemo-radiotherapy from patients with oral cancer and from non-cancerous patients. When patient take surgical treatment, cancerous tissue and normal tissues that nearby the cancer site was collected.
SAS, KON, HO1u-1, HSC3, HSC3 M3, OSC19 and HaCaT cells were grown in DMEM High glucose, 10% FBS, 1% penicillin/streptomycin.
After extraction of total RNA from above samples, total RNAs were reverse-transcripted and cDNAs were underwent real-time PCR. The data from saliva and serum were analyzed with the method that provided by manufacturer (http://pcrdataanalysis.sabiosciences.com/mirna/arrayanalysis.php).
The data from cell lines and tissues were analyzed using man-whitney U test to search any significant difference of expression of miR-203 between cancer cell lines and HaCaT or cancerous tissue and normal tissue.
Formalin-Fixed Paraffin-Embedded (FFPE) specimens of biopsy were also collected for immunostaining. Immunostaining of biopsy sample using p-63 antibody was made and confirmed p-63 expression in the human cancerous tissues and compared with HE staining.
In the saliva samples, we found that miR-203 tend to shows low expression in the oral cancer patients but there is no such tendency in the serum samples. On the other hand, data analysis of cell line shows that miR-203 was significantly low expressed in KON, Ho-1-u-1, and HSC3M3. miR-203 in the cancer tissue was also have a same tendency of low expression compare with the normal tissue.
The immunostaing of biopsy tissues using p63 shows that p63 exist in the oral cancer tissue. p63 is well demonstrated in the usage of immunostaing of OSCC, differentiating moderately or well differentitation. [2] miR-203 was demonstrated that miR-203 promotes differentiation by repressing stemness as a result of directly repress the expression of p-63. Additionally, predominant p63 isoform,ΔNp63α was demonstrated that is an oncogene which drive stem cell proliferation and tumor genesis in skin cancer.
In this study, we indicate future prospects of using mi-R203 and saliva samples in the more efficient detection and management of oral cancer. However, study of more large number of clinical samples and make sure relationship between miR-203 and p-63 in the oral cancer is needed.
[1] Antonia K, Review of MicroRNA Deregulation in Oral Cancer. Part I Oral Maxillofac Res. 2011 Apr-Jun; 2(2): e1.
[2] Faridoni L, P73 expression in basal layers of head and neck squamous epithelium: a role in differentiation and carcinogenesis in concert with p53 and p63? Oncogene. 2001 Aug 30;20(38):5302-12.