Effect of miR-27a-3p on Proliferation and Migration in Non-small Cell Lung Cancer Cells

Objective To investigate the expression of miR-27a-3p in tumor tissue of patients with non-small cell lung cancer (NSCLC) and explore the effect of miRNA-27a-3p expression on invasion and migration of lung cancer cells. Methods The tumor tissues of 21 NSCLC patients and corresponding adjacent normal tissues were collected for this study and the differences of miR-27a-3p expression levels in tumor tissues and adjacent tissues were detected by real-time fluorescence quantitative PCR. The expression of miR-27a-3p in A549 cells was overexpressed and silenced by transfecting miR-27a-3p mimics and miR-27a-3p inhibitor. And the ability of proliferation and migration of A549 cells after transfecting were detected by MTT and cell scratch assaies. Results The expression levels of miR-27a-3p in tumor tissues of NSCLC patients were significantly higher than adjacent non-cancerous tissues. The proliferation and migration ability of A549 cells were enhanced significantly after overexpression of miR-27a-3p. The silencing of miR-27a-3p expression in A549 cells produces the opposite effect. Conclusion Compared with normal tissues, NSCLC tissues have high expression of miR-27a-3p. Overexpression of miR-27a-3p enhanced the proliferation and migration ability of lung cancer cells. Therefore, miR-27a-3p may be an important regulatory factor in the development of NSCLC.

. The relationship between miRNA and tumor has become one of the hot spots in the research of many scientists at present. Different miRNAs may exert stimulative or inhibitory function, which depended on the means of miRNAs, target or cellular environment (Feng et al., 2015). Calin

RNA extraction and qRT-PCR
According to the instructions of RNA extraction kit, the total RNA of the cancer tissue, corresponding para-cancerous tissue and the A549 cells were extracted with the method of TRIzol. Total RNA was used as the template for reversing transcribe RNA into cDNA adopting miRNA reverse transcription kit. In accordance with qRT-PCR Kit instructions for cDNA amplification, the U6 was used as the internal control for measuring the relative expression of miR-27a-3p.
The reaction conditions included an initial step at 95 ° C for 5 min followed by 40 cycles of 95 ° C for 30 s,

Cell proliferation assay
After digestion and centrifugation, A549 cells were Image Pro Plus6 was used to analyze the images and calculated the migration distance.

Statistical analysis
All data were presented as mean±SD and analyzed by Graphpad 6.0 statistical software. The one-way variance analysis were used to determine the differences between groups of tests, and the two groups were compared using t test. P <0.05 was considered statistically significant. showed that the expression of miR-27a-3p was down-regulated (Fig. 1A). The comparative analysis showed that the average expression level of miR-27a-3p in NSCLC tissues was significantly higher than that in adjacent non-cancerous tissues (P <0.01) ( Fig. 1B). We selected PCR products numbered 8, 13, and 21 for gel electrophoresis. The result of PCR gel electrophoresis showed that the expression levels of miR-27a-3p in NSCLC tissue samples were significantly higher than that in the paracancerous tissues (Fig. 1C).

MiR-27a-3p expression in the A549 cells
The expression levels of miR-27a-3p of A549 cells after transfection were detected by quantitative PCR (Fig. 2). We found that miR-27a-3p expression were significantly increased in cells transfected with miR-27a-3p mimics compared with control group (

Cells
In order to investigate the effect of miR-27a-3p on cell proliferation, we detected the proliferation ability of cells after transfection at different time points by MTT assay. In comparison with the control group, the proliferation ability of miR-27a-3p mimics group was significantly increased at 48 h, 72 h and 96 h (Fig. 3A), which was statistically significant (P <0.01). The proliferation ability of miR-27a-3p inhibitor group was significantly decreased (Fig. 3B), which was statistically significant as well (P <0.05). The results suggested that the expression level of miR-27a-3p was positively correlated with the proliferation of A549 cells.

Effect of miR-27a-3p on Migration of A549 Cells
We then investigated the role of miR-27a-3p in the regulation of invasion of A549 cells. Cell wound healing assay was used to examine the migration. And the results displayed that the percentage of healing area in the miR-27a-3p mimics group was significantly increased (P <0.01) (Fig. 4A). Cells transfected with miR-27a-3p showed a significant decreased percentage of the healing area for the scratched cells, which was statistically significant. (Fig. 4B). The migration rate of A549 cells was significantly increased after overexpression of miR-27a-3p. The silencing of miR-27a-3p significantly inhibited the migration of A549 cells. These data suggested an promotional effect of miR-27a-3p in A549 cell migration.  The molecular mechanism of miR-27a-3p functioning in NSCLC remains to be further studied.
In this study, we found that miR-27a-3p, the major subtype of miR-27a, was significantly elevated in Therefore, the miR-27a-3p, the major subtype of miR-27a, was an oncogene that played an important role in the development of NSCLC and may provide a potential therapeutic target for the treatment of lung cancer. This study was limited to the detection of clinical samples and cell functions. The molecular mechanism of miR-27a-3p in NSCLC needs further investigation.