Prognostic and predictive values of long non-coding RNA LINC00472 in breast cancer

Abstract

LINC00472 is a novel long intergenic non-coding RNA. We evaluated LINC00472 expression in breast tumor samples using RT-qPCR, performed a meta-analysis of over 20 microarray datasets from the Gene Expression Omnibus (GEO) database, and investigated the effect of LINC00472 expression on cell proliferation and migration in breast cancer cells transfected with a LINC00472-expressing vector. Our qPCR results showed that high LINC00472 expression was associated with less aggressive breast tumors and more favorable disease outcomes. Patients with high expression of LINC00472 had significantly reduced risk of relapse and death compared to those with low expression. Patients with high LINC00472 expression also had better responses to adjuvant chemo- or hormonal therapy than did patients with low expression. Results of meta-analysis on multiple studies from the GEO database were in agreement with the findings of our study. High LINC00472 was also associated with favorable molecular subtypes, Luminal A or normal-like tumors. Cell culture experiments showed that up-regulation of LINC00472 expression could suppress breast cancer cell proliferation and migration. Collectively, our clinical and in vitro studies suggest that LINC00472 is a tumor suppressor in breast cancer. Evaluating this long non-coding RNA in breast tumors may have prognostic and predictive value in the clinical management of breast cancer.

Keywords: LINC00472; breast; lincRNA.

Figure 1. Associations of LINC00472 expression with patient survival in turin study

A. Kaplan-Meier estimates for disease-free survival by LINC00472 expression. B. Kaplan-Meier estimates for overall survival by LINC00472 expression.

Figure 2. Meta-analysis of associations between LINC00472 expression and clinicopathological features of breast cancer

Summarized odds ratios were estimated using the random-effect model, and the odds ratio in each study was weighted with the variance of probe values (inverse-variance weighted method). A. Lower LINC00472 expression associated with high tumor grade (OR = 2.81; 95% CI: 2.24–3.53), positive lymph node (OR = 1.51; 95% CI: 1.22–1.87), or molecular subtypes of luminal B, Her2 positive and basal-like (OR = 3.80; 95% CI: 2.38–6.07). B. Higher LINC00472 expression associated with ER positive tumors (OR = 0.44; 95% CI: 0.35–0.57), PR positive tumors (OR = 0.44; 95% CI: 0.35–0.55), or Her2 negative tumors (OR = 2.62; 95% CI: 1.76–3.90).

Figure 3. Meta-analysis of associations between LINC00472 expression and patient survival

A. Overall survival: HR = 0.91 (95% CI: 0.57–1.45) between mid and low LINC00472 expression (Upper Figure), and HR = 0.41 (95% CI: 0.22–0.78) between high and low LINC00472 expression (Lower Figure). B. Disease-free Survival: HR = 0.69 (95% CI: 0.55–0.88) between mid and low LINC00472 expression (Upper Figure), and HR = 0.43 (95% CI: 0.31–0.59) between high and low LINC00472 expression (Lower Figure).

Figure 4. Effect of LINC00472 expression on breast cancer cell proliferation

A. GFP fluorescence images in MCF7 and SKBR3 cells transfected with pCDH or pCDH_LINC00472 vectors. B. RT-qPCR results of LINC00472 expression in MCF7 and SKBR3 cells transfected with pCDH or pCDH_LINC00472 or mock transfected. C. Cell growth inhibition by LINC00472 in MCF7 cells. Twenty hours after seeding in 96-well plates, cells were transiently transfected with pCDH or pCDH_LINC00472 vectors, and kept in culture for up to 96 hours. Absorbance at 450nm of each well, which was directly proportional to the number of living cells in the well, was measured by SpectraMax M3 Multimode Plate Reader. The y axis showed the relative absorbance of corresponding wells from different days compared to that from day 0. Error bars represent SEM, n = 15. P values were determined by the Mann-Whitney U test. D. Inhibition of cell growth by LINC00472 in SKBR3 cells. The experiment and analysis are same as to those in C.

Figure 5. Effect of LINC00472 expression on breast cancer cell migration

A. Microscopic views of cell migration before (0 hour) and after (24 hour) removal of stopper in SKBR3 and MCF7 cells transfected with pCDH or pCDH_LINC00472. Cells transfected with pCDH or pCDH_LINC00472 vectors formed monolayers in Oris 96-well plate and started to migrate to the exposed area after removing the stoppers in the well. Twenty-four hours later, the cells were fixed and stained with 0.1% crystal violet stain. The photomicrograph of the entire ‘wound’ area was taken under the IX71 inverted microscope with 4X objective lens. The representative wells were presented and the yellow circles indicated the areas previously occupied by stoppers. B. Measurements of absorbance after removal of stopper in SKBR3 and MCF7 cells transfected with pCDH or pCDH_LINC00472. With the detection mask, the absorbance at 590 nm wavelength of each well, which was directly proportional to the number of cells that migrated into the ‘wound’ area, was measured. The bar charts showed the average absorbance (y axis) from the wells with different cells after subtracting the background, the absorbance of the reference wells. Error bars represent SEM, n = 8. P values were determined by the Mann-Whitney U test.