Figure 4.

Enz reduces BRCA1 through miR-146a-5p in HCC cells. A) HA22T cells were infected with lentivirus pLKO control or shAR. SK-HEP-1 cells were infected with lentiviruses pWPI control or oeAR. Anti-Argonaute 2 RNA immunoprecipitation (RIP) was used to detect BRCA1 and BCL2 mRNA expression levels in Argonaute 2 complex by qRT-PCR. B) In pLKO and AR knocked down HA22T cells, the differential expression levels of 11 candidate miRNAs were detected using qRT-PCR (left). The miR-146a-5p expression level was detected in pWPI and oeAR SK-Hep-1 cells (right). C) PLKO control or shAR HA22T cells were transfected with miR-146a-5p inhibitor or control. The protein levels of BRCA1, BCL2, AR, and GAPDH were detected using western blot (left upper) and quantified by ImageJ software (left lower). HA22T cells were infected with lentiviruses pLV control or oemiR-146a-5p and expression levels detected using qRT-PCR (middle). BRCA1 and GAPDH protein levels were detected by western blot (right). D) The wild type (WT) or mutant BRCA1 3’UTR was subcloned into the psiCHECK™−2 Vector. SK-HEP-1 cells were co-transfected with WT or mutant vector, miR-146a-5p inhibitor or control, oemiR-146a-5p or pWPI for 2 days. Luciferase activities were presented in ratios of Renilla to Firefly luciferase reporter activities. E) HA22T (left) and SK-HEP-1 (middle) cells transfected with PLV control or oemiR-146a-5p were treated with different concentrations of Olaparib (OLA) for 72 h. HA22T (right) cells were transfected with miR-146a-5p inhibitor or control and treated with 20 μM Enz (ENZ) and different concentrations of OLA for 72 h. The MTT assay was performed to determine cell viability. Data are presented as mean ± SD or SEM. *P < 0.05, **P < 0.01, NS=Not Significant.