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. 2024 Feb 10;25(4):2167.
doi: 10.3390/ijms25042167.

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

Conner Belnap  1 , Tyler Divis  2 , Karl Kingsley  3 , Katherine M Howard  3
Affiliations

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

Conner Belnap et al. Int J Mol Sci. .

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.

Keywords: biomarkers; chemotherapy resistance; microRNA expression; oral cancer.

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Conflict of interest statement

The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Comparison of baseline (control) growth with experimental treatment among oral cancer cell lines. The administration of all three chemotherapy agents (Cisplatin, 5-FU, Taxol) inhibited growth in all cell lines with the least inhibition observed among SCC25 and SCC9 cells (−3.3% to −18.6%) at concentrations of 1.0, 5.0 and 10.0 ng/mL over three days. Moderate inhibition was observed among SCC4 and CAL27 cells (−32.5% to −44.3%), while the most inhibited (least chemoresistant) cell line was SCC15 (−62.7% to −68.3%). The results were statistically significant (experimental assays compared with baseline growth) except SCC25 (−3.6% p = 0.078) and SCC9 (−3.3%, p = 0.063) under Taxol administration, denoted by *. Error bars represent standard deviation (SD) derived from three independent experiments, n = 8 wells per experimental assay.
Figure 2
Figure 2
Heatmap analysis of qPCR screening for oral cancer microRNA expression. Oral cancer cell lines upregulated miR-16, miR-21, miR-125, miR-133, miR-365, miR-720, and miR-1246, while downregulation was observed with miR-140, miR-152, miR-218, miR-221, or miR-224. CT = cycle threshold value; N.D. = not detected (below the limit of detection at CT 40).
Figure 3
Figure 3
Heatmap analysis of qPCR revealed differential expression of microRNAs among oral cancers. Differentially upregulated microRNAs included miR-27, miR-124, miR-135, miR-143, miR-210, miR-222, miR-320, miR-375, miR-424, miR-494, and miR-654. Differential expression in SCC15 included miR-145 upregulation (only observed among SCC15 cells) and miR-155 downregulation (observed in all other cell lines except SCC15). CT = cycle threshold value; N.D. = not detected (below the limit of detection at CT 40).
Figure 4
Figure 4
Relative quantification (RQ) of microRNA data. Relative quantity (RQ) normalized to positive control miR-16 CT values was relatively consistent among all cell lines ranging between 0.75 and 2.11, including miR-21 (0.75 to 1.17), miR-125 (1.1 to 1.5), miR-133 (1.25 to 2.0), miR-365 (1.01 to 1.86), miR-720 (1.25 to 2.11), miR-1246 (1.5 to 1.94), miR-27 (1.06 to 1.42), miR-124 (1.25), miR-135 (1.57 to 1.78), miR-143 (1.38), miR-210 (1.11), miR-222 (1.25 to 1.43), miR-320 (1.29 to 1.57), miR-375 (1.06 to 1.42), miR-424 (1.1 to 2.0), miR-494 (1.1 to 1.42), miR-654 (1.29 to 1.43), miR-145 (1.18), and miR-155 (1.22 to 1.57).
Figure 5
Figure 5
Heatmap of qPCR screening and analysis of miR-145 downstream targets. All cell lines upregulated MBTD1 and FSCN1, while downregulation was observed with CLCN3, FLI-1, MRTFB, DAB, SRGAP1, or ABHD17C. Differential expression was observed with TRIM2, ADD3, and ABCE1 with SCC15-specific upregulation observed with KCNA4 and SRGAP2, with downregulation observed with FAM135A. CT = cycle threshold value; N.D. = not detected (below the limit of detection at CT 40).
Figure 6
Figure 6
Heatmap of qPCR screening and analysis of miR-155 downstream targets. All cell lines (except SCC4) upregulated OLFML3, TBR1, BACH1, ZNF652, IRF2-BP2, and ZIC3, while downregulation was observed with MARCH1, IKBIP, ACTL7A, CHAF1A, MPEG1, FOS, CDX1, JARID2, and KDM5B. No differential or SCC15-specific expression was observed among any of the miR-155 downstream targets analyzed. CT = cycle threshold value; N.D. = not detected (below the limit of detection at CT 40).
Figure 7
Figure 7
Relative quantification (RQ) of mRNA data. Relative quantity (RQ) normalized to positive control GAPDH CT values ranged between 1.11 and 2.01, including miR-145 downstream targets MBTD1 (1.11 to 1.89), FSCN1 (1.4 to 1.84), ADD3 (1.13), ACE1 (1.25), TRIM2 (1.15 to 1.55), FAM135A (1.12 to 1.4), KCNA4 (1.4), and SRGAP2 (1.65) and miR-155 downstream targets OLFML3 (1.56 to 2.01), TBR1 (1.52 to 1.95), BACH1 (1.28 to 1.8), ZNF652 (1.44 to 1.89), IRF2 (1.52 to 1.89), and ZIC3 (1.28 to 1.85).
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Grants and funding

This research received no external funding. The APC was funded by the Office of Research at the University of Nevada, Las Vegas—School of Dental Medicine and the Department of Advanced Education—Orthodontic Dental Residency Program.