MicroRNA-328 is associated with (non-small) cell lung cancer (NSCLC) brain metastasis and mediates NSCLC migration

Abstract

Brain metastasis (BM) can affect ∼ 25% of nonsmall cell lung cancer (NSCLC) patients during their lifetime. Efforts to characterize patients that will develop BM have been disappointing. microRNAs (miRNAs) regulate the expression of target mRNAs. miRNAs play a role in regulating a variety of targets and, consequently, multiple pathways, which make them a powerful tool for early detection of disease, risk assessment, and prognosis. We investigated miRNAs that may serve as biomarkers to differentiate between NSCLC patients with and without BM. miRNA microarray profiling was performed on samples from clinically matched NSCLC from seven patients with BM (BM+) and six without BM (BM-). Using t-test and further qRT-PCR validation, eight miRNAs were confirmed to be significantly differentially expressed. Of these, expression of miR-328 and miR-330-3p were able to correctly classify BM+ vs. BM- patients. This classifier was used on a validation cohort (n = 15), and it correctly classified 12/15 patients. Gene expression analysis comparing A549 parental and A549 cells stably transfected to over-express miR-328 (A549-328) identified several significantly differentially expressed genes. PRKCA was one of the genes over-expressed in A549-328 cells. Additionally, A549-328 cells had significantly increased cell migration compared to A549 cells, which was significantly reduced upon PRKCA knockdown. In summary, miR-328 has a role in conferring migratory potential to NSCLC cells working in part through PRKCA and with further corroboration in additional independent cohorts, these miRNAs may be incorporated into clinical treatment decision making to stratify NSCLC patients at higher risk for developing BM.

Figure 1. Data analysis for SHC Discovery and SHC Validation cohorts

(A and B) Discovery and validation for BM− and BM+ using miR-328 and miR-330-3p in the SHC Discovery and SHC Validation cohorts, respectively. Sensitivity and specificity are 0.8571 and 0.8333 for the SHC Discovery cohort and 0.7500 and 0.8182 for the SHC Validation cohort. (C and D) ROC curve for SHC Discovery and SHC Validation cohorts, respectively.

Figure 2. Relative expression of miR-328 in the validation cohort

Box plot showing the relative expression of miR-328 in the validation set represents the interquartile range (25–75^th percentile) and the line within this box is the median value. Bottom and top bars of the whisker indicate the 10^th and 90^th percentiles, respectively. Outlier values are indicated (closed circles). Significance between the indicated classes of specimens was tested using a two-sample t-test assuming unequal variances. The expression was normalized to the expression of 5S-rRNA in all samples plotted as fold change to A549 miR-328 expression (used as a reference).

Figure 3. Gene and protein expression analysis in A549-empty and A549-328 cells

(A) Hierarchical Clustering of 363 Genes Filtered at p value < 0.05 and fold change ≥ 2; 1 and 2 represent the duplicate runs. (B) A representative VEGF/IL1 signaling pathway leading to cellular migration/adhesion/proliferation that might be affected as a result of miR-328 over-expression. Protein products of several of the differentially-expressed genes are involved in this signaling cascade (these proteins are marked in pink circles). (C) Western blot showing over-expression of PRKCA in A549-328 cells as compared to A549-empty cells. GAPDH was used as a loading control.

Figure 4. Role of miR-328 in NSCLC

(A) Migration of NSCLC cells. Graph showing migration of GFP-empty vector (Control) and miR-328 over-expressing cells for cell line (i) A549 and (ii) H1703 in modified microwell Boyden chamber assay. Both cell lines demonstrated significant increase in cell migration in miR-328 over-expression as compared to Control [p = 0.014 (A549) and 0.0006 (H1703)]. Plotted values are mean ± standard deviation of triplicate determinations. HPF: High Power Fields. (B) siRNA mediated knockdown of PRKCA. A549-328 cells were treated with specific PRKCA siRNAs (siPRKCA_1 or siPRKCA_2) or non-silencing control (NS) along with an Untreated (UT) control. Seventy-two hours later protein lysates were prepared and Western blot was performed. The results show successful knockdown of PRKCA protein expression. GAPDH was used as a loading control. (C) Effect of PRKCA knockdown on the migration of NSCLC cells. Graph showing migration of A549-328 cells vs. PRKCA silenced A549-328 cells in modified microwell Boyden chamber assay. PRKCA silencing in A549-328 cells demonstrated significant reduction in cell migration as compared to Untreated as well as Non-silencing control [*p < 0.005; ^#p<0.0001]. Plotted values are mean ± standard deviation of triplicate determinations. HPF: High Power Fields