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. 2021 Mar 17;11:643613. doi: 10.3389/fonc.2021.643613

Figure 4.

Figure 4

The miR-650 regulates the expression of MGMT in the melanoma cells. (A) The schematic diagram showed the predicted binding site of MGMT-3′UTR and miR-650. A luciferase reporter gene assay was performed for the luciferase activity in A375 cells with wild-type and mutant MGMT-3′UTR. (B) The MGMT-3′UTR and GAPDH RNA levels were measured with a qRT-PCR assay after the transfection of biotinylated miR-650-wt or mutant in A375/DTIC cells. The histogram showed the relative ratio to the intraperitoneal input. (C) The expression of MGMT mRNAs was measured with the qRT-PCR assay in miR-650 transfected melanoma cells. (D) The protein levels of MGMT were analyzed in miR-650 transfected cells with the Western bolt assay. (E) Relative MGMT mRNA expression levels were compared with the A375 cells which were transfected with the miR-650 mimic or MGMT. (F) The percentage of cell apoptosis of the transfected cells indicated in (E) was analyzed with a flow cytometer after DTIC treatment for 48 h. (G) The transfected cells were treated with DTIC (25 μg/ml) for 3 days. Cell viability was detected with MTT assays. The data are presented as mean ± SD of at least three independent experiments, where *p < 0.01. DTIC, dacarbazine; miRNAs, micro RNAs; qRT-PCR, quantitative real-time PCR; MGMT, O6-methylguanine-DNA-methyltransferase.