The microRNA-200 family targets multiple non-small cell lung cancer prognostic markers in H1299 cells and BEAS-2B cells

Abstract

Lung cancer remains the leading cause of cancer-related mortality for both men and women. Tumor recurrence and metastasis is the major cause of lung cancer treatment failure and death. The microRNA‑200 (miR-200) family is a powerful regulator of the epithelial-mesenchymal transition (EMT) process, which is essential in tumor metastasis. Nevertheless, miR-200 family target genes that promote metastasis in non-small cell lung cancer (NSCLC) remain largely unknown. Here, we sought to investigate whether the microRNA-200 family regulates our previously identified NSCLC prognostic marker genes associated with metastasis, as potential molecular targets. Novel miRNA targets were predicted using bioinformatics tools based on correlation analyses of miRNA and mRNA expression in 57 squamous cell lung cancer tumor samples. The predicted target genes were validated with quantitative RT-PCR assays and western blot analysis following re-expression of miR-200a, -200b and -200c in the metastatic NSCLC H1299 cell line. The results show that restoring miR-200a or miR-200c in H1299 cells induces downregulation of DLC1, ATRX and HFE. Reinforced miR-200b expression results in downregulation of DLC1, HNRNPA3 and HFE. Additionally, miR-200 family downregulates HNRNPR3, HFE and ATRX in BEAS-2B immortalized lung epithelial cells in quantitative RT-PCR and western blot assays. The miR-200 family and these potential targets are functionally involved in canonical pathways of immune response, molecular mechanisms of cancer, metastasis signaling, cell-cell communication, proliferation and DNA repair in Ingenuity pathway analysis (IPA). These results indicate that re-expression of miR-200 downregulates our previously identified NSCLC prognostic biomarkers in metastatic NSCLC cells. These results provide new insights into miR-200 regulation in lung cancer metastasis and consequent clinical outcome, and may provide a potential basis for innovative therapeutic approaches for the treatment of this deadly disease.

Figure 1

Computational prediction of miRNA and target lung cancer prognostic genes and experimental results focusing on miR-200 family. (A) Overview of bioinformatic prediction of miRNA target genes and functional assays. (B) Gene expression fold change of miR-200 in primary squamous cell lung cancer tumors vs. normal lung tissues in the patient cohort from Raponi et al(24). ^*Statistically significant at p≤0.05. Data presented as mean ± SEM.

Figure 2

Relative mRNA levels of the predicted targets of miR-200 in H1299. (A) Relative mRNA expression of predicted miR-200 target genes in H1299 cells infected with miR-scrambled and miR-200a, -200b and -200c. These targets were predicted with microRNA.org based on 3′-UTR and downregulation after microRNA transduction. (B) Relative mRNA expression level of E-cadherin in H1299 infected with miR-200a and -200c. Cells infected with miR-scr were used as a negative control. The mRNA expression levels were determined using qRT-PCR as described in Materials and methods. ^*Statistically significant at p≤0.05, n=3 (biological replicates). Data presented as mean ± SEM. Relative mRNA level was calculated after normalization to endogenous gene, UBC and relative to negative control, miR-scr.

Figure 3

Protein levels of the predicted molecular targets of miR-200 in H1299 cells after infection with miR-scr (scrambled) or with miR-200a, -200b or -200c. miR-scr was used as a negative control. (A) 3′-UTR sequences of the miR-200a, -200b and -200c putative binding sites of target genes is given in the 5′- to 3′-orientation. (B) Western blot analysis of protein levels in H1299 cells over-expressing miR-200a, -200b, -200c or miR-scr. One representative blot is shown. The experiments were repeated in three biological replicates. Tubulin was used as a loading control. (C) Semi-quantitative analysis of protein levels relative to negative control miR-scr. Protein level was determined as described in Materials and methods.

Figure 4

Relative mRNA and protein levels of the predicted molecular targets of miR-200 in BEAS-2B. (A) Relative mRNA expression of predicted miR-200 target genes in BEAS-2B cells infected with miR-scrambled and miR-200a, -200b and -200c. (B) Western blot analysis of protein levels in BEAS-2B cells overexpressing miR-200a, -200b, -200c or miR-scr. One representative blot is shown. The experiments were repeated in three biological replicates. Tubulin was used as a loading control. (C) Semi-quantitative analysis of protein levels relative to negative control miR-scr. Protein level was determined as described in Materials and methods. DLC1 protein was not detected in BEAS-2B in western blots.

Figure 5

Molecular network analysis of the miR-200 family and potential molecular targets with Ingenuity pathway analysis (IPA).

Figure 6

Proposed mechanisms of the miR-200 regulation in tumor initiation and metastasis.