Figure 3: Investigation of miR-193b-3p target genes in the HepG2 cells exposed to 0.01 Gy irradiation.

Rad51 protein levels were determined in response to irradiation in the NCTC, Hepa, and HepG2 cells. The cells were irradiated with 0.01 Gy (6.5 mGy/h), and the protein levels were determined 48 h post-irradiation (A) and at the indicated time points (B). (C) Following pretreatment with a miR-193b-3p mimic for 48 h, the Rad51protein levels were observed in response to irradiation in HepG2 cells. (D) Diagram of Rad51 mRNA showing the miR-193b-3p binding sites in the 3′untranslated region (UTR). The wild-type or mutant constructs were inserted into the psi-CHECK2 vector directly downstream of the luciferase gene. miR-193b-3p and its predicted seed binding site in the 3′UTR of Rad51 is shown in the top panel; the three miR-193b-3p MREs predicted to be located in the 3′UTR of Rad51 are shown in the middle panel; and the Rad51 3′UTR mutant without a seed binding region for miR-193b-3p is shown in the bottom panel. (E) HepG2 cells were co-transfected with either a miR-193b-3p mimic or a negative control (mock) and the Rad51 3′UTR containing wild-type or mutant MREs. Luciferase activity was measured 48 h after transfection using the dual-luciferase reporter assay system. The normalization of renilla luciferase expression was performed using the luciferase gene present on the psiCHECK2 vector. (F) Rad51 protein levels were observed in response to irradiation of the HepG2 cells pretreated with the HDACi NaB for 24 h. GAPDH was used as a loading control. Statistically significant differences between the non-irradiated and irradiated samples are indicated (**p < 0.01 and ***p < 0.001).