Differential Expression of MicroRNA MiR-145 and MiR-155 Downstream Targets in Oral Cancers Exhibiting Limited Chemotherapy Resistance
Conner Belnap,
Tyler Divis,
Karl Kingsley
et al.
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, RN… Show more
“…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].…”
Section: Discussionsupporting
confidence: 74%
“…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 ].…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
Expression of microRNAs, such as miR-365, is known to be dysregulated in many tumors, including oral cancers, although little is known about their role or functions. The objective of this project is to evaluate the downstream targets of miR-365 to determine any potential pathways or effects. Downstream targets for miR-365 (miRdatabase target scores > 90) were used for qPCR screening of oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, CAL27). Each oral cancer cell line expressed miR-365 downstream targets molybdenum cofactor synthesis-2 (MOCS2), erythropoietin receptor (EPOR), IQ motif containing-K (IQCK), carboxypeptidase A3 (CPA3), solute carrier family 24 member-3 (SLC24A3), and coiled-coil domain containing 47 (CCDC47)—although the expression levels varied somewhat. However, differential results were observed with ubiquitin protein ligase E3 component n-recognin-3 (UBR3), nudix hydrolase-12 (NUDT12), zinc finger CCHC-type containing-14 (ZCCHC14), and homeobox and leucine zipper encoding (HOMEZ). These data suggest that many of the miR-365 targets are expressed in the oral cancers screened, with the differential expression of UBR3, ZCCHC14, HOMEZ, and NUDT12, which may be correlated with chemoresistance among two specific oral cancer cell lines (SCC25, SCC9). These results suggest this differential expression may signal potential targets for patient treatment with tumors exhibiting miR-365 and chemotherapeutic resistance.
“…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].…”
Section: Discussionsupporting
confidence: 74%
“…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 ].…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
Section: Methodsmentioning
confidence: 99%
“…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.…”
Expression of microRNAs, such as miR-365, is known to be dysregulated in many tumors, including oral cancers, although little is known about their role or functions. The objective of this project is to evaluate the downstream targets of miR-365 to determine any potential pathways or effects. Downstream targets for miR-365 (miRdatabase target scores > 90) were used for qPCR screening of oral cancer cell lines (SCC4, SCC9, SCC15, SCC25, CAL27). Each oral cancer cell line expressed miR-365 downstream targets molybdenum cofactor synthesis-2 (MOCS2), erythropoietin receptor (EPOR), IQ motif containing-K (IQCK), carboxypeptidase A3 (CPA3), solute carrier family 24 member-3 (SLC24A3), and coiled-coil domain containing 47 (CCDC47)—although the expression levels varied somewhat. However, differential results were observed with ubiquitin protein ligase E3 component n-recognin-3 (UBR3), nudix hydrolase-12 (NUDT12), zinc finger CCHC-type containing-14 (ZCCHC14), and homeobox and leucine zipper encoding (HOMEZ). These data suggest that many of the miR-365 targets are expressed in the oral cancers screened, with the differential expression of UBR3, ZCCHC14, HOMEZ, and NUDT12, which may be correlated with chemoresistance among two specific oral cancer cell lines (SCC25, SCC9). These results suggest this differential expression may signal potential targets for patient treatment with tumors exhibiting miR-365 and chemotherapeutic resistance.
Epigenetic modulation of DNA and histones facilitated by and histone deacetylases (HDAC) is associated with the development and progression of many cancers, although less is known about DNA methyltransferase (DNMT) in oral cancers and the regulation of these targets. Using commercially available cell lines, oral squamous cell carcinomas (SCC4, SCC9, SCC15, SCC25, and CAL27), and normal gingival fibroblasts (HGF-1), growth assays and mRNA expression were evaluated using ANOVA. These results revealed homeostasis enzyme DNMT1 expression was significantly higher among slow-growing HGF-1 cells than among fast-growing oral cancers, p < 0.05. In contrast, DNMT3A and DNMT3B expression was significantly higher among oral cancers compared with HGF-1 cells, p < 0.05. However, differential expression of HDAC1 and HDAC2 was observed among SCC4, SCC25, and CAL27 cells. Further analysis of miR-152 (regulation and control of DNMT expression) and miR-21, miR-221, and miR-145 (regulation of HDAC expression) revealed all oral cancers produced miR-21, but none produced miR-221. However, differential expression of miR-145 (SCC15) and miR-152 (SCC25) suggested alternative epigenetic pathways and mechanisms of DNMT and HDAC regulation may be responsible for some of the observations revealed in this study.
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