MicroRNA 133B targets pro-survival molecules MCL-1 and BCL2L2 in lung cancer

Abstract

Lung cancer is the most frequent cause of cancer-related death in this country for men and women. MicroRNAs (miRNAs) are a family of small non-coding RNAs (approximately 21-25nt long) capable of targeting genes for either degradation of mRNA or inhibition of translation. We identified aberrant expression of 41 miRNAs in lung tumor versus uninvolved tissue. MiR-133B had the lowest expression of miRNA in lung tumor tissue (28-fold reduction) compared to adjacent uninvolved tissue. We identified two members of the BCL-2 family of pro-survival molecules (MCL-1 and BCL2L2 (BCLw)) as predicted targets of miR-133B. Selective over-expression of miR-133B in adenocarcinoma (H2009) cell lines resulted in reduced expression of both MCL-1 and BCL2L2. We then confirmed that miR-133B directly targets the 3'UTRs of both MCL-1 and BCL2L2. Lastly, over-expression of miR-133B induced apoptosis following gemcitabine exposure in these tumor cells. To our knowledge, this represents the first observation of decreased expression of miR-133B in lung cancer and that it functionally targets members of the BCL-2 family.

Figure 1. MiRNA expression in human lung cancer

(A) Heat map representing significantly altered miRNAs between 8 cases of paired adenocarcinomas (T) and uninvolved adjacent lung (N) and 4 non-paired adenocarcinoma cases. (B) Downregulated and upregulated miRNAs with fold changes and p values. Out of 198 detected miRNA, 41 miRNA were differentially expressed between tumor and adjacent lung using a false discovery rate of 1/198 or .005. Among these miRNA, 27 were upregulated and 14 downregulated. Of note, miR-133B was the lowest expressed miRNA in adenocarcinomas compared to adjacent uninvolved lung tissue (28.62 fold downregulated p=2.33E-05)

Figure 2. Relationship between miR-133B and MCL-1/BCL2L2

(A) Location of predicted 3’UTR target sites for miR-133B in both MCL1 and BCL2L2 based on TargetScan 5.1 and PicTar prediction programs. (B) Representation of in situ expression of miR-133B in human lung tissue. Adenocarcinoma demonstrated no expression while in normal lung, miR-133B localizes to bronchial epithelium (arrow head), as a negative control no expression was evident of mock miRNA in normal lung. (C) qRT-PCR miRNA analysis in 10 additional cases of adenocarcinoma and adjacent uninvolved lung demonstrates reduced miR-133B expression in tumor tissue ((N)=Normal (T)=Tumor). Data was presented as fold differences based on calculations of 2 ^(−ΔΔCt) . With the exception of case 6, all demonstrated statistically significant differences with p<.05 (*) (D) Representation of MCL1 and BCL2L2 protein expression in 8 paired cases of adenocarcinoma (T) adjacent uninvolved lung (N). Protein was normalized with β-actin (Sigma St. Louis, Missouri) and measured by densitometry by two independent researchers.

Figure 3. MiR-133B targets BCL2L2 and MCL1

(a) Lung cancer cell line screening. NSCLC cell lines demonstrated an inverse relationship between MCL1/BCL2L2 and miR-133B expression. (b) Effects of miR-133B on MCL-1 and BCL2L2 mRNA and protein. Transient transfection of pre-miR-133B (5–20 nM) resulted in increasing expression of mature miR-133B as measured by q-RT-PCR. (*p<.05). Transfection of pre-miR-133B in H2009 NSCLC cell lines resulted in reduced MCL1 and BCL2L2 protein expression at 48 hours. (p<.05) However, neither MCL-1 nor BCL2L2 mRNA were altered. Protein was assessed and scored by densitometry by two independent researchers and westerns conducted in triplicate. MRNA was normalized to GAPDH. (c) MiR-133B targets 3’UTRs of both MCL-1 and BCL2L2. Cells were transfected with 50 ng of psiCHECK-MCL-1 (WT=wild type or Mut=mutant) or -BCL2L2 (WT=wild type. Mut=mutant) and either 20 nM of scrambled pre-miR or pre-miR-133B. 48 hours following transfection, cells were assayed for both firefly and renilla luciferase using the dual luciferase glow assay . All transfection experiments were conducted in triplicate. * p value< .05.

Figure 4. Effects of miR-133B on Apoptosis

(A) Silencing of MCL-1 and BCL2L2. MiR-133B induction and MCL-1 and BCL2L2 siRNA both resulted in an increase in cleaved parp in NSCLC cells lines. (B) MiR-133B over-expression and MCL-1/BCL2L2 siRNA increased gemcitabine induced apoptosis. Cells were transfected with either pre-miR-133B/mock pre miR or MCL-1/BCL2L2 siRNA for 48 hours followed by 24 hour exposure to gemcitabine (25 nM). Both annexin and annexin/PI staining cells were included in our analysis. Flow cytometry was conducted in duplicate on two separate days. (C) Effects of miR-133B and gemcitabine on MCL-1 and BCL2L2 Expression. Transfection of H2009 cell line with pre-miR-133B followed by treatment with gemcitabine (25nM) decreased both MCL-1 and BCL2L2 protein .Westerns for MCL-1 and BCL2L2 and densitometry were conducted as previously described. *p<.05