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. 2014 Aug 1;9(8):e103698.
doi: 10.1371/journal.pone.0103698. eCollection 2014.

MicroRNA-21 (miR-21) regulates cellular proliferation, invasion, migration, and apoptosis by targeting PTEN, RECK and Bcl-2 in lung squamous carcinoma, Gejiu City, China

Long-Feng Xu  1 , Zhi-Ping Wu  2 , Yan Chen  2 , Qi-Shun Zhu  1 , Shoeleh Hamidi  3 , Roya Navab  4
Affiliations

MicroRNA-21 (miR-21) regulates cellular proliferation, invasion, migration, and apoptosis by targeting PTEN, RECK and Bcl-2 in lung squamous carcinoma, Gejiu City, China

Long-Feng Xu et al. PLoS One. .

Abstract

Background: In South China (Gejiu City, Yunnan Province), lung cancer incidence and associated mortality rate is the most prevalent and observed forms of cancer. Lung cancer in this area is called Gejiu squamous cell lung carcinoma (GSQCLC). Research has demonstrated that overexpression of miR-21 occurs in many cancers. However, the unique relationship between miR-21 and its target genes in GSQCLC has never been investigated. The molecular mechanism involved in GSQCLC must be compared to other non-small cell lung cancers in order to establish a relation and identify potential therapeutic targets.

Methodology/principal findings: In the current study, we initially found overexpression of miR-21 occurring in non-small cell lung cancer (NSCLC) cell lines when compared to the immortalized lung epithelial cell line BEAS-2B. We also demonstrated that high expression of miR-21 could increase tumor cell proliferation, invasion, viability, and migration in GSQCLC cell line (YTMLC-90) and NSCLC cell line (NCI-H157). Additionally, our results revealed that miR-21 could suppress YTMLC-90 and NCI-H157 cell apoptosis through arresting cell-cycle at G2/M phase. Furthermore, we demonstrated that PTEN, RECK and Bcl-2 are common target genes of miR-21 in NSCLC. Finally, our studies showed that down-regulation of miR-21 could lead to a significant increase in PTEN and RECK and decrease in Bcl-2 at the mRNA and protein level in YTMLC-90 and NCI-H157 cell lines. However, we have not observed any remarkable difference in the levels of miR-21 and its targets in YTMLC-90 cells when compared with NCI-H157 cells.

Conclusions/significance: miR-21 simultaneously regulates multiple programs that enhance cell proliferation, apoptosis and tumor invasiveness by targeting PTEN, RECK and Bcl-2 in GSQCLC. Our results demonstrated that miR-21 may play a vital role in tumorigenesis and progression of lung squamous cell carcinoma and suppression of miR-21 may be a novel approach for the treatment of lung squamous cell carcinoma.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. The expression level of miR-21 in the immortalized normal lung epithelial cell line BEAS-2B and non-small cell lung cancer cell lines.
The expression level of miR-21 was detected by quantitative real-time PCR. miRNA abundance was normalized to U6 as a reference gene, the expression values are normalized to BEAS-2B (n = 6, *p<0.05, **p<0.01 versus BEAS-2B). Cells images are under 100× microscopic fields.
Figure 2
Figure 2. miR-21 targets gene and cell transfection efficiency were determined.
A. The 3′-UTR of PTEN-mRNA, RECK-mRNA and Bcl-2-mRNA are targets for miR-21 and the seed matching sequences were marked in red. The miRNA targets were predicted by Target Scan Human Release 6.2 (http://www.targetscan.org). B. The expression of green fluorescent protein (GFP) transfected with pGCMV/EGFP-hsa-miR-21 or pGCMV/EGFP-hsa-miR-NC for 48 h was observed using a fluorescent inverted microscope (40×). C. The expression level of miR-21 was analyzed by quantitative real-time PCR at 48 h post-transfection shown in the bar graphs. miRNA abundance was normalized to U6 as a reference gene (n = 3, **p<0.01 versus corresponding control). NC means negative control (transfected with pGCMV/EGFP-hsa-miR-NC plasmid), RQ means relative quantitation.
Figure 3
Figure 3. miR-21 regulated PTEN, RECK and Bcl-2 expression in NSCLC cells.
YTMLC-90 and NCI-H157 cells were transfected with pGCMV/EGFP-hsa-miR-21 or pGCMV/EGFP-hsa-miR-NC for 48 h. A. The mRNA expression levels of PTEN, RECK and Bcl-2 were detected by quantitative real-time PCR in 48 h transfected cells. miRNA abundance was normalized to GADPH (n = 3, *p<0.05, **p<0.01 versus corresponding control). B. PTEN, RECK and Bcl-2 protein level were determined after 48 h by western blot assay. The β-actin level was also measured as a reference gene and BEAS-2B was served as a control, NC means negative control (transfected with pGCMV/EGFP-hsa-miR-NC plasmid), RQ means relative quantitation.
Figure 4
Figure 4. Down-regulation of miR-21 inhibited cell proliferation and viability.
A. Cell proliferation ability was detected by MTS assay. The proliferation rate of cells transfected with pGCMV/EGFP-hsa-miR-21 interference plasmid was inhibited compared with cells in the other groups (n = 6, **p<0.01, ***p<0.001 versus corresponding control). B. Morphologic changes of YTMLC-90 and NCI-H157 cells was observed in response to miR-21 inhibition. Cells images are under 100× microscopic fields. NC means negative control (transfected with pGCMV/EGFP-hsa-miR-NC plasmid), RQ means relative quantitation.
Figure 5
Figure 5. Inhibition of miR-21 expression weakened the cell migration ability (100×).
Cell migration was detected using the wound healing assays. Uniform scratches were made in YTMLC-90 and NCI-H157 cells and the serial photographs were obtained at regular time of post-transfection. NC means negative control (transfected with pGCMV/EGFP-hsa-miR-NC plasmid).
Figure 6
Figure 6. Knockdown of miR-21 suppressed cell invasion (200×).
The number of invasive cells was determined by trans-well invasion assays and enumerated under the inverted microscope. The average number of invasion cells was calculated from 5 random views. Invasion cells were stained with 0.1% crystal violet and appeared in purple. NC means negative control (transfected with pGCMV/EGFP-hsa-miR-NC plasmid) and data represent means of quintuplicates ± SD (n = 5, *p<0.05 versus corresponding control).
Figure 7
Figure 7. Down-regulation of miR-21 induced NSCLC cell apoptosis.
A. Flow cytometry (FCM) method was used to analyze the apoptosis rate of cells in each group at a qualitative level. The early apoptotic cells were marked by red boxes. B. The percent of the apoptotic cells in each group. Data represent means of triplicates ± SD (n = 3, **p<0.01 versus corresponding control). NC means negative control (transfected with pGCMV/EGFP-hsa-miR-NC plasmid). C. The cell morphology of apoptotic cells relative to the normal cell morphology on a quantitative level (10000×). Normal cells were from the un-transfected groups, and the apoptotic cells were from the pGCMV/EGFP-hsa-miR-21 interference plasmid transfected groups at 48 h post-transfection. These images were collected by the transmission electron microscope (TEM), formula imagenucleolus, formula imagemitochondria, formula imagekaryotheca, formula imagehigh density chromatin, formula imageapoptotic body, formula imagecell vacuolation.
Figure 8
Figure 8. miR-21 inhibition induced cell-cycle arrest at G2/M phase.
A. Cell number in each phase of cell cycle and apoptosis was detected by flow cytometry method. B. The percent of cell number at different phases of cell cycle (G0/G1, S and G2/M). NC means negative control (transfected with pGCMV/EGFP-hsa-miR-NC plasmid) and data represent means of triplicates ± SD (n  = 3, *p<0.05 versus corresponding control).
Figure 9
Figure 9. The regulatory mechanisms between miR-21 and PTEN/RECK/Bcl-2 in non-small lung cell carcinoma cells transfected with pGCMV-hsa-miR-21.
At the post-transcriptional level, PTEN/RECK/Bcl-2 expression level was regulated by miR-21. Knockdown of miR-21 activated PTEN and RECK, and repressed Bcl-2 at the protein level. PTEN, RECK and Bcl-2 may have single or combined effect on NSCLC cell proliferation, migration, invasion, and apoptosis at the protein level.
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This work was supported by the Science and Technology Project of the Yunnan Health Department (Grant No. 2011WS0065) and the Research and Talent Training Open Foundation of Life science college, Yunnan University (Grant No. 2013S213). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.