Downstream Target Analysis for miR-365 among Oral Squamous Cell Carcinomas Reveals Differential Associations with Chemoresistance
Abstract
:1. Introduction
2. Materials and Methods
2.1. Cell Lines and Tissue Culture
- SCC4 CRL-1624 Age: 55 years old Sex: Male
- Short tandem repeat (STR) confirmation: 92% Oral squamous cell carcinoma (OSCC)
- STR analysis:
- vWA: 15,17 TPOX: 8 TH01: 9.3 Penta_E: 14
- Penta_D: 12 FGA: 21,22 D8S1179: 14 D7S820: 9,11
- D5S818: 13 D3S1358: 18 D2S1338: 16,24 D21S11: 32.2
- D19S433: 12,14 D18S51: 15 D16S539: 12 D13S317: 11,13
- CSF1PO: 11 Amelogenin: X,Y
- SCC9 CRL-1629 Age: 25 years old Sex: Male
- STR confirmation: 100% (OSCC)
- STR analysis:
- vWA: 17 TPOX: 9,11 TH01: 8,9 Penta_E: 11
- Penta_D: 9 FGA: 20,25 D8S1179: 13 D7S820: 8
- D5S818: 12 D3S1358: 15 D2S1338: 19,21 D21S11: 28
- D19S433: 12,14 D18S51: 12,14 D16S539: 10,11 D13S317: 9
- CSF1PO: 11 Amelogenin: X,Y
- SCC15 CRL-1623 Age: 55 years old Sex: Male
- STR confirmation: 95% (OSCC)
- STR analysis:
- vWA: 15,17 TPOX: 8 TH01: 9,9.3 Penta_E: 7,13
- Penta_D: 9,13 FGA: 19,24 D8S1179: 10,13 D7S820: 10,11
- D5S818: 12 D3S1358: 1 D2S1338: 16,23 D21S11: 30,31.2
- D19S433: 15 D18S51: 16 D16S539: 12,15 D13S317: 9,14
- CSF1PO: 10,13 Amelogenin: X,Y
- SCC25 CRL-1628 Age: 70 years old Sex: Male
- STR confirmation: 100% (OSCC)
- STR analysis:
- vWA: 17,19 TPOX: 8,12 TH01: 8 Penta_E: 14,15
- Penta_D: 13 FGA: 20,24 D8S1179: 13 D7S820: 12
- D5S818: 12 D3S1358: 17 D2S1338: 17,19 D21S11: 30
- D19S433: 13,14 D18S51: 16 D16S539: 11,12 D13S317: 13
- CSF1PO: 10 Amelogenin: X
- CAL27 CRL-2095 Age: 56 years old Sex: Male
- STR confirmation: 93% (OSCC)
- STR analysis
- vWA: 14,17 TPOX: 8 TH01: 6,9.3 Penta_E: 7
- Penta_D: 9,10 FGA: 25 D8S1179: 13,15 D7S820: 10
- D5S818: 11,12 D3S1358: 16 D2S1338: 23,24 D21S11: 28,29
- D19S433: 14,15.2 D18S51: 13 D16S539: 11,12 D13S317: 10,11
- CSF1PO: 10,12 Amelogenin: X
2.2. Growth Assays
2.3. RNA Extraction and cDNA Synthesis
2.4. qPCR Screening
- Positive controls
- GAPDH forward, 5-′ATCTTCCAGGAGCGAGATCC-3′
- GAPDH reverse, 5′-ACCACTGACACGTTGGCAGT-3′
- Beta actin forward, 5′-GTGGGGTCCTGTGGTGTG-3′
- Beta actin reverse, 5′-GAAGGGGACAGGCAGTGA-3′
- Screening primers
- miR-16 forward, 5′-TAGCAGCACGTAAATATTGGCG-3′
- miR-16 reverse, 5′-TGCGTGTCGTGGAGTC-3′
- miR-365 forward, 5′-ATAGGATCCTGAGGTCCCTTTCGTG-3′
- miR-365 reverse, 5′-GCGAAGCTTAAAAACAGCGGAAGAGTTT-3′
- Downstream targets
- UBR3 (target score 95)
- UBR3 forward, 5′-TGGCTGTTCAAGGTTTCATAGG-3′
- UBR3 reverse, 5′-GGTGCCACTGCTTAGTTTTACC-3′
- NUDT12 (target score 93)
- NUDT12 forward, 5′-ATCCCTTGGTTACTCTAGGTGG-3′
- NUDT12 reverse, 5′-GGCTGGGCCAAATAATCCTTT-3′
- MOCS2 (target score 93)
- MOCS2 forward, 5′-AGTGCTGAAATAACAGGAGTTCG-3′
- MOCS2 reverse, 5′-TCCAAGCTCGACATATTCTTGAC-3′
- EPOR (target score 92)
- EPOR forward, 5′-TGGAGGACTTGGTGTGTTTCT-3′
- EPOR reverse, 5′-GCAACTCTAGGGGCACGAA-3′
- ZCCHC14 (target score 92)
- ZCCHC14 forward, 5′-CGGACGCATTTTATGTGGAGC-3′
- ZCCHC14 reverse, 5′-TCTGCGAGGACGGGATACC-3′
- IQCK (target score 91)
- IQCK forward, 5′-CTGCTCTACAGACTCGTCGTT-3′
- IQCK reverse, 5′-AGATTCTTGCTTGACGGCTCG-3′
- HOMEZ (target score 91)
- HOMEZ forward, 5′-CTGGACTGCGCTATCTCTGAA-3′
- HOMEZ reverse, 5′-CTGAAGGTTTTGAGCAGGTGT-3
- CPA3 (target score 91)
- CPA3 forward, 5′-GGGTTTGATTGCTACCACTCTT-3′
- CPA3 reverse, 5′-GCCAAGTCCTTTATGATGTCTGC-3′
- SLC24A3 (target score 90)
- SLC24A3 forward, 5′-TTGACCTCATGGACCTCGTAG-3′
- SLC24A3 reverse, 5′-GGCATCTTCGGAAGTCAGAGT-3′
- CCDC47 (target score 90)
- CCDC47 forward, 5′-TCTCCTCAACGGGTCATAATCA-3′
- CCDC47 reverse, 5′-GGTTCACTCTCAGTATCTCCCT-3′
2.5. Transfection and Overexpression of miR-365
2.6. Statistical Analysis
3. Results
4. Discussion
5. Conclusions
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Cell Line | Paclitaxel (Taxol) | Fluorouracil 5-FU | Cisplatin |
---|---|---|---|
SCC4 | −32.5% | −35.2% | −37.9% |
SCC9 | −3.3% * | −14.9% | −18.6% |
SCC15 | −62.7% | −68.3% | −65.4% |
SCC25 | −3.6% * | −10.9% | −11.9% |
CAL27 | −30.7% | −34.3% | −38.1% |
Average | −34.4% | −32.7% | −26.6% |
Range | −3.3% to −62.7% | −10.9% to −68.3% | −11.9% to −65.4% |
Cell Line | RNA Analysis [ng/uL] | cDNA Analysis [ng/uL] | A260:A280 Analysis |
---|---|---|---|
SCC4 | 224.3 ng/uL | 1557 ng/uL | 1.80 |
SCC9 | 164.4 ng/uL | 1461 ng/uL | 1.91 |
SCC15 | 294.4 ng/uL | 1252 ng/uL | 1.88 |
SCC25 | 234.2 ng/uL | 1151 ng/uL | 1.79 |
CAL27 | 155.4 ng/uL | 1257 ng/uL | 1.92 |
Average | 214.5 ng/uL | 1335.6 ng/uL | 1.86 |
Range | 155–294 ng/uL | 1151–1557 ng/uL | 1.80–1.92 |
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Yu, B.; Kruse, N.; Howard, K.M.; Kingsley, K. Downstream Target Analysis for miR-365 among Oral Squamous Cell Carcinomas Reveals Differential Associations with Chemoresistance. Life 2024, 14, 741. https://doi.org/10.3390/life14060741
Yu B, Kruse N, Howard KM, Kingsley K. Downstream Target Analysis for miR-365 among Oral Squamous Cell Carcinomas Reveals Differential Associations with Chemoresistance. Life. 2024; 14(6):741. https://doi.org/10.3390/life14060741
Chicago/Turabian StyleYu, Brendon, Nathaniel Kruse, Katherine M. Howard, and Karl Kingsley. 2024. "Downstream Target Analysis for miR-365 among Oral Squamous Cell Carcinomas Reveals Differential Associations with Chemoresistance" Life 14, no. 6: 741. https://doi.org/10.3390/life14060741