Down-Regulation of MicroRNA-133b Suppresses Apoptosis of Lens Epithelial Cell by Up-Regulating BCL2L2 in Age-Related Cataracts

Abstract

BACKGROUND MicroRNA-133b (miR-133b) has been reported to be involved in many diseases, including ovarian cancer and osteosarcoma. Accumulating evidence suggests that miR-133b plays important roles in human disease. In this study, we aimed to investigate the molecular mechanism, including the potential regulator and signaling pathways, of BCL2L2. MATERIAL AND METHODS We first searched the online miRNA database (www.mirdb.org) using the "seed sequence" located within the 3'-UTR of the target gene, and then performed luciferase assay to test the regulatory relationship between miR-133b and BCL2L2. Western blot and real-time PCR were used to determine the expression of BCL2L2 in human samples or cells treated with miRNA mimics or inhibitors. Flow cytometry was conducted to evaluate the apoptosis status of the cells. RESULTS We validated BCL2L2 to be the direct gene using a luciferase reporter assay. We also conducted real-time PCR and Western blot analyses to study the mRNA and protein expression level of BCL2L2 among different groups (control: n=29, cataract: n=33) or cells treated with scramble control, miR-133b mimics, BCL2L2 siRNA, and miR-133b inhibitors, and identified the negative regulatory relationship between miR-133b and BCL2L2. We also conducted experiments to investigate the influence of miR-133b and BCL2L2 on the viability and apoptosis of cells. The results showed that miR-133b positively interfered with the viability of cells, while BCL2L2 negatively interfered with the viability of cells, and that miR-133b inhibited apoptosis while BCL2L2 accelerated apoptosis. CONCLUSIONS BCL2L2 was the virtual target of miR-133b, and we found a negative regulatory relationship between miR-133b and BCL2L2. MiR-133b and BCL2L2 interfered with the viability and apoptosis of cells.

Figure 1

We used online miRNA target prediction tools to search the regulatory gene of miR-133b; BCL2L2 was the candidate target gene of miR-133b in cells with the ‘seed sequence’ in the 3′UTR.

Figure 2

Luciferase activity reporter assay was conducted to verify BCL2L2 as the direct target gene of miR-133b (** denotes P<0.01).

Figure 3

To further explore the effects on the interaction between miR-133b and BCL2L2, we used real-time PCR to detective the expression of the mRNA of miR-133b and BCL2L2, and BCL2L2 protein was measured by densitometry analysis. The results showed a negative regulatory relationship between miR-133b and BCL2L2 between cataract and control (cataract =33, control, n=29).

Figure 4

The expression of miR-133b decreased in the recurrence group (A) compared with normal control group while the expression of BCL2L2 mRNA (B)and protein (C) increased in cataract group compared with control group (** denotes P<0.01).

Figure 5

When transfected with the prostate cancer stem cells with scramble control, miR-133b mimics, BCL2L2 siRNA, and miR-133b inhibitors, the expression level of BCL2L2 protein (upper panel) and mRNA (lower panel) treated with miR-133b mimics and BCL2L2 siRNA decreased, while cells treated miR-133b inhibitors increased (** denotes P<0.01).

Figure 6

Cells transfected with miR-133b inhibitors showed evidently downregulated viability (A), while cells transfected with miR-133b mimics and BCL2L2 siRNA showed comparably higher viability. Cells transfected with miR-133b mimics and BCL2L2 siRNA inhibited apoptosis while cells transfected with miR-124 inhibitors accelerated apoptosis (B) (** denotes P<0.01).