Hsa-miR-125a-3p and hsa-miR-125a-5p are downregulated in non-small cell lung cancer and have inverse effects on invasion and migration of lung cancer cells

Abstract

Background: Two mature microRNAs (miRNAs), hsa-miR-125a-3p and hsa-miR-125a-5p (collectively referred to as hsa-miR-125a-3p/5p), are derived from 3' and 5' ends of pre-miR-125a, respectively. Although impaired regulation of hsa-miR-125a-5p has been observed in some tumors, the role of this miRNA in invasion and metastasis remains unclear, and few studies have examined the function of hsa-miR-125a-3p. In order to characterize the functions of hsa-miR-125a-3p/5p in invasion and metastasis of non-small cell lung cancer (NSCLC), we investigated the relationships between hsa-miR-125a-3p/5p expression and lymph node metastasis in NSCLC tissues. We also explored the impact of expression of these miRNAs on invasive and migratory capabilities of lung cancer cells.

Methods: Expression of hsa-miR-125a-3p/5p in NSCLC tissues was explored using real-time PCR. The relationships between hsa-miR-125a-3p/5p expression and pathological stage or lymph node metastasis were assessed using the Spearman correlation test. For in vitro studies, lung cancer cells were transfected with sense and antisense 2'-O-methyl oligonucleotides for gain-of-function and loss-of-function experiments. Transwell experiments were performed to evaluate cellular migration and invasion.

Results: Expression of hsa-miR-125a-3p/5p was lower in NSCLC tissues than in adjacent normal lung tissues (LAC). Furthermore, the results from the Spearman correlation test showed a negative relationship between hsa-miR-125a-3p expression and pathological stage or lymph node metastasis and an inverse relationship between hsa-miR-125a-5p expression and pathological stage or lymph node metastasis. In vitro gain-of-function experiments indicated that hsa-miR-125a-3p and hsa-miR-125a-5p function in an opposing manner, suppressing or enhancing cell migration and invasion in A549 and SPC-A-1 cell lines, respectively. These opposing functions were further validated by suppression of hsa-miR-125a-3p and hsa-miR-125a-5p expression in loss-of-function experiments.

Conclusion: Hsa-miR-125a-3p and hsa-miR-125a-5p play distinct roles in regulation of invasive and metastatic capabilities of lung cancer cells, consistent with the opposing correlations between the expression of these miRNAs and lymph node metastasis in NSCLC. These results provide new insights into the roles of miR-125a family members in the development of NSCLC.

Figure 1

Relative expression of hsa-miR-125a-3p in lung cancer tissues. The relative expression of hsa-miR-125a-3p decreased in 52 NSCLCs in comparison to corresponding LACs. The data are representative of three independent experiments, and the relative expression values were calculated using the equation RQ = 2^-ΔΔCt.

Figure 2

Relative expression of hsa-miR-125a-5p in lung cancer tissues. The relative expression of hsa-miR-125a-5p decreased in 52 NSCLCs in comparison to corresponding LACs. The results are representative of three independent experiments, and the relative expression values were calculated using the equation RQ = 2^-ΔΔCt.

Figure 3

Mean levels of hsa-miR-125a-3p and hsa-miR-125a-5p in lung cancer tissues. (A) The mean level of hsa-miR-125a-3p relative expression in 52 NSCLCs. (B) The mean level of hsa-miR-125a-5p relative expression in 52 NSCLCs. The values represent the means of three replicates, and the relative expression values were calculated using the equation RQ = 2^-ΔΔCt.

Figure 4

Prediction of hsa-miR-125a-3p and hsa-miR-125a-5p target genes. Predictions were conducted using microrna.Org, TargetScanHuman 5.1, and MiRBase webservers. Microrna.org and MiRBase predictions are based on the miRanda algorithm, while TargetScanHuman5.1 predictions are based on the TargetScan algorithm.

Figure 5

Relative expression of hsa-miR-125a-3p and hsa-miR-125a-5p in different lung cancer cell lines. (A) The relative expression levels of hsa-miR-125a-3p in four lung cancer cell lines, particularly the NCI-H460 cell line, were all lower than that of the HBE cell line. (B) The relative expression levels of hsa-miR-125a-5p in four lung cancer cell lines, particularly the A459 cell line, were all lower than that of the HBE cell line. The results are representative of three independent experiments, and the relative expression values were calculated using the equation RQ = 2^-ΔΔCt.

Figure 6

Relative levels of hsa-miR-125a-3p and hsa-miR-125a-5p were increased by transfection with sense 2'-O-methyl oligonucleotides. (A) The relative levels of hsa-miR-125a-3p were significantly increased in the sense-3p-transfected group. (B) The relative levels of hsa-miR-125a-5p were significantly increased in the sense-5p-transfected group. The results are representative of three independent experiments, and the relative expression values were calculated using the equation RQ = 2^-ΔΔCt.

Figure 7

Effects of gain-of-function of hsa-miR-125a-3p/5p on migration and invasion of lung cancer cells. Migration assay results showed that the number of A549 cells that migrated through the microporous membrane was significantly decreased in the sense-3p group (p < 0.001). In contrast, the number of migratory SPC-A-1 cells was significantly increased in the sense-5p group (p < 0.001, bar = 20 μm). Invasion assay results showed that the number of A549 cells that invaded through the Matrigel was significantly decreased in the sense-3p group (p < 0.001). However, the number of invasive SPC-A-1 cells significantly increased in the sense-5p group (p < 0.001, bar = 20 μm). The results are representative of three independent experiments.

Figure 8

Effects of loss-of-function of hsa-miR-125a-3p/5p on migration and invasion of lung cancer cells. Migration assay results showed that the number of A549 cells that migrated through the microporous membrane was significantly increased in the antisense-3p group (p < 0.001). However, the number of migratory SPC-A-1 cells was significantly decreased in the antisense-5p group (p < 0.001, bar = 20 μm). Invasion assay results showed that the number of A549 cells that invaded through the Matrigel was significantly increased in the antisense-3p group (p < 0.001). In contrast, the number of invasive SPC-A-1 cells was significantly decreased in the antisense-5p group (p < 0.001, bar = 20 μm). The results are representative of three independent experiments.