miR-138-1* regulates aflatoxin B1-induced malignant transformation of BEAS-2B cells by targeting PDK1

Yun Wang, Zhan Zhang, Yudong Zhang, Huanqiang Wang, Minghui Ji, Hengsen Xu, Chao Wang, Zhenzhen Sun, Weimin Gao, Shou-Lin Wang

SUPPORTING INFORMATION

Materials and Methods

Primer design and synthesis

The primers for miRNAs and mRNA were designed by Generay Biotechnology (Shanghai, China), and the sequences are listed in Table S1 and Table S2.

RT-qPCR analysis

Total RNA was extracted using TRIzol.Before analysis, the purity of total RNA were judged by OD260/280 ratio (1.9~2.0) using a spectrophotometer (NanoDrop ND-1000), and the RNA integrity (separated by 28S and 18S) was determined through 1% formaldehyde denaturing gel electrophoresis.Then the total RNA were reverse transcribed using PrimeScriptTM miRNA qPCR Starter Kit Ver.2.0 (TaKaRa, Japan). RT-qPCR was performed with the ABI PRISM 7300HT instrument (Applied Biosystems, Carlsbad, USA) using the primers designed by Generay Biotechnology (Shanghai, China). The following PCR parameters of miRNAs were used: 95℃ for 30 s, followed by 40 cycles of 95℃ for 5 s,55℃ for 30 s, and 72℃ for 30 s. The values were normalized to RNU6B in miRNA expression analysis. All the data were analyzed with respect to a calibrator sample using the 2-ΔΔCt method..

Figure legends

Supplementary Fig. 1. Time-course change of miR-138-1* during the malignant transformation in B-2A13 cells treated by AFB1. The miRNA abundance was normalized against an endogenous RNU6B RNA control. Statistical analysis was performed using Student’s t-test. Error bars represent S.E.M. ns, P > 0.05; **, P < 0.01; ***, P < 0.001, compared with miR-control.

Table S1 Forward primers of miRNAs

miRNA / Sequence(5’-3’)
hsa-miR-1271 / F / ACACTCCAGCTGGGCTTGGCACCTAGCAAGC
hsa-miR-125b-1* / F / ACACTCCAGCTGGGACGGGTTAGGCTCTTGG
hsa-miR-138 / F / ACACTCCAGCTGGGAGCTGGTGTTGTGAATCA
hsa-miR-708 / F / ACACTCCAGCTGGGAAGGAGCTTACAATCTAG
has-miR-296-3p / F / ACACTCCAGCTGGGGAGGGTTGGGTGGAGGC
hsa-miR-183 / F / ACACTCCAGCTGGGTATGGCACTGGTAGAAT
hsa-miR-382 / F / ACACTCCAGCTGGGGAAGTTGTTCGTGGTGG
hsa-miR-432 / F / ACACTCCAGCTGGGTCTTGGAGTAGGTCATTG
hsa-miR-370 / F / ACACTCCAGCTGGGCAGGTCACGTCTCTGCA
hsa-miR-335 / F / ACACTCCAGCTGGGTCAAGAGCAATAACGAAA
hsa-miR-494 / F / ACACTCCAGCTGGGAGGTTGTCCGTGTTGTCT
hsa-miR-421 / F / ACACTCCAGCTGGGATCAACAGACATTAATTG
hsa-miR-138-1* / F / ACACTCCAGCTGGGGCTACTTCACAACACCA
hsa-miR-181b / F / ACACTCCAGCTGGGAACATTCATTGCTGTCGG
hsa-miR-425 / F / ACACTCCAGCTGGGAATGACACGATCACTCCC
hsa-miR-181a / F / ACACTCCAGCTGGGAACATTCAACGCTGTCGG
hsa-miR-105 / F / ACACTCCAGCTGGGTCAAATGCTCAGACTCCT
hsa-miR-200b / F / ACACTCCAGCTGGGTAATACTGCCTGGTAAT

F, forward.

Table S2 Primer pairs for PDPK1 and GADPH

Gene / Sequnece (5´-3´)
PDPK1 / F / TCGTCCTCCTCCTCACACTCCCT
R / GCCTGCTTCTCCAACAACAACCTCTT
GADPH / F / GCCGAGGGCCCACTAAAG
R / AGCATCAAAGGTGGAAGAATGG

F, forward; R, reverse.

Table S3Expression changes of differential miRNAs between P50 B-2A13 cells and P50 B-Vector cells

miRNA / Microarray / RT-qPCR
Fold changea / p / Fold changea / p
Down-regulated
hsa-miR-1271 / -2.2092 / 0.0295 / -9.3028 / 0.0008
hsa-miR-200b / -2.3864 / 0.0008 / -4.9619 / 0.0001
hsa-miR-296-3p / -2.0265 / 0.0428 / -3.4832 / 0.0011
hsa-miR-125b-1* / -2.3481 / 0.0049 / -3.2056 / 0.0007
hsa-miR-138-1* / -1.8735 / 0.0134 / -3.0980 / 0.0037
hsa-miR-708 / -3.6362 / 0.0060 / -2.6422 / 0.0007
hsa-miR-105 / -3.4870 / 0.0118 / -1.5242 / 0.0109
hsa-miR-138 / -2.6976 / 0.0244 / 1.3649 / 0.0031
Up-regulated
hsa-miR-425 / 1.8526 / 0.0343 / 3.9402 / 0.0012
hsa-miR-183 / 3.2434 / 0.0458 / 3.1387 / 0.0002
hsa-miR-335 / 3.4570 / 0.0281 / 2.8580 / 0.0134
hsa-miR-432 / 12.9967 / 0.0039 / 2.7313 / 0.0099
hsa-miR-421 / 2.3697 / 0.0430 / 2.4400 / 0.0042
hsa-miR-181a / 1.6615 / 0.0393 / 2.1544 / 0.0001
hsa-miR-382 / 10.9258 / 0.0022 / 1.9563 / 0.0032
hsa-miR-181b / 2.1446 / 0.0042 / 1.4459 / 0.0017
hsa-miR-494 / 2.7453 / 0.0040 / 1.1790 / 0.0094
hsa-miR-370 / 5.2863 / 0.0044 / -3.0324 / 0.0022

a Fold changes were presented as B-2A13/B-Vector.

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