Differential Expression of MicroRNA MiR-145 and MiR-155 Downstream Targets in Oral Cancers Exhibiting Limited Chemotherapy Resistance

Abstract

New evidence has suggested that non-coding microRNAs play a significant role in mediating and modulating chemotherapy resistance, particularly among oral cancers. One recent study found that the upregulation of miR-145 and the downregulation of miR-155 strongly correlated with a limited chemotherapy resistance to Cisplatin, 5-Fluorouracil, and Paclitaxel, although the mechanism(s) responsible for these observations remain unidentified. Using commercially available cell lines of oral squamous cell carcinoma, RNA was isolated, converted into cDNA, and subsequently screened for the expression of downstream targets of miR-145 and miR-155 using qPCR. These results demonstrated the upregulation of miR-21, miR-125, miR-133, miR-365, miR-720, and miR-1246, as well as the downregulation of miR-140, miR-152, miR-218, miR-221, and miR-224. This screening also confirmed the differential expression and regulation of mir-145 and miR-155 among the cell lines with limited chemotherapy resistance (SCC15). In addition, several downstream targets of these specific microRNAs were upregulated by all oral cancer cell lines, such as MBTD1 and FSCN1, or downregulated in all cell lines, such as CLCN3, FLI-1, MRTFB, DAB, SRGAP1, and ABHD17C. However, three miR-145 downstream targets were identified in the least chemotherapy-resistant cells, exhibiting the differential upregulation of KCNA4 and SRGAP2, as well as the downregulation of FAM135A, with this expression pattern not detected in any of the other oral cancer cell lines. These data strongly support that the differential regulation of these three downstream targets may be related to the chemosensitivity of this oral cancer cell line. The potential involvement of these targets must be further investigated to determine how and whether mechanisms of these cellular pathways may be involved in the observed lack of chemotherapy resistance. These data may be important to design targets or treatments to reduce chemotherapy resistance and improve patient treatment outcomes.

Downstream Target Analysis for miR-365 among Oral Squamous Cell Carcinomas Reveals Differential Associations with Chemoresistance

“…The primary goal of this project was to assess previously identified downstream targets of miR-365 to determine any potential associations with oral cancer phenotypes and to identify potential pathways of interest. This study successfully confirmed differences in phenotypes between commercially available oral cancer cell lines, with more chemoresistance observed among SCC25 and SCC9 cells and more chemosensitivity among SCC15 cells [23,24]. In addition, miR-365 expression was more highly correlated with SCC25 and SCC9 cells, as well as CAL27 cells, suggesting that pathways associated with this microRNA may be involved with these observed differences [21,22].…”

“…All cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM:F12) with the addition of 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin antibiotic obtained from Fisher Scientific (Fair Lawn, NJ, USA). Cells were maintained in 25 cm 2 tissue culture-treated flasks in a biosafety level two (BSL2) cabinet with supplemental CO 2 at 5%, as previously described [ 21 , 22 , 23 , 24 ].…”

“…Following centrifugation, the pellet was washed with ethanol and resuspended in nuclease-free water. RNA was converted into cDNA using the ABgene reverse iT one-step RT kit from Fisher Scientific (Fair Lawn, NJ, USA) using the manufacturer protocol, as previously described [ 21 , 22 , 23 , 24 ]. This reaction consisted of 1.0 ug of total RNA combined with universal random forward and reverse primers from Invitrogen (Waltham, MA, USA) and Reddy Mix RT-PCR Master Mix with RTase Blend.…”

“…Amplification of microRNAs was performed with the TaqMan Advanced microRNA (miRNA) Assay conversion kit obtained from AppliedBiosystems (Waltham, MA, USA) using the manufacturer-recommended protocol, as previously described [ 21 , 22 , 23 , 24 ]. In brief, due to the low volume of microRNAs and the lack of poly-adenylation, this system uses a poly-adenylation reaction for use with total RNA extracted (1.0 ug) from any cell or exosome source.…”

“…Recent work from this group has identified miR-365 as a potential biomarker for rapidly growing and aggressive oral cancers [ 21 , 22 ]. Moreover, these observations with miR-365 have more recently been linked with chemotherapy resistance within these same oral cancer cell lines, suggesting that miR-365 and the corresponding downstream targets may be important mediators of oral cancer phenotypes [ 23 , 24 ]. However, only one study has identified a limited number of downstream targets of miR-365 in oral cancers.…”