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. 2022 Apr 18;13(4):361. doi: 10.1038/s41419-022-04818-5

Fig. 3. miR-661 enhances tumorigenicity of ESCC by reducing PPM1L expression.

Fig. 3

A The top five miRNAs as microRNA response elements for circFAM120B. B RIP assays using an anti-AGO2 antibody, followed by qRT-PCR analysis, confirm the interactions between circFAM120B and miR-661. C A schematic representation of the 3’-UTR of circFAM120B with the predicted target site for miR-661, as well as the mutant sites of circFAM120B. Luciferase reporter analysis was performed to evaluate the binding between miR-661 and circFAM120B. Reporter constructs containing either circFAM120B-wt or circFAM120B-mut were co-transfected into HEK293T cells, along with miR-661 or miR-NC mimics. D The proliferation of ESCC cells transfected with miR-661 mimics or inhibitors was evaluated by CCK-8 assay (n = 5 biologically independent replicates). E Colony formation assays were performed in ESCC cells transfected with miR-661 mimics or inhibitors (n = 3 biologically independent replicates). F Analysis of proliferating ESCC cells transfected with miR-661 mimics or inhibitors by EdU assay (n = 3 biologically independent replicates). G Migration and invasion of ESCC cells transfected with miR-661 mimics or inhibitors were evaluated by Transwell assays (n = 3 biologically independent replicates). H Venn diagram showing the potential mRNAs targeted by miR-661. I A schematic representation of the 3’-UTR of PPM1L with the predicted target site for miR-661 and the mutant sites of PPM1L. Luciferase reporter analysis was performed to evaluate the binding between miR-661 and PPM1L. Reporter constructs containing either PPM1L-wt or PPM1L-mut were co-transfected into HEK293T cells, along with miR-661 mimics or miR-NC. J Expression of PPM1L was assessed by qRT-PCR in ESCC cells transfected with miR-661 mimics or inhibitors. *P < 0.05, **P < 0.01, ***P < 0.001.