Moreover, we found that ITGA6 manifestation was higher in NSCLC cells than in adjacent noncancerous lung tissues, and its manifestation was inversely correlated with miR-302a-5p manifestation in the NSCLC cells samples (Number 4E and ?and4F).4F). pLV3 vector (GeneChem, Shanghai, China) to generate pLV3-miRNA-302a-5p and pLV3-NC, respectively. Then, the recombinant lentiviral plasmids were cotransfected into HEK293T cells with the packaging and envelope vectors. At 48 h after transfection, the lentivirus-containing medium was collected and filtered. Animal experiments The animal experiments were conducted in accordance with protocols authorized by the Institutional Animal Care and Use Committee of Jinshan Hospital, Fudan University. Approximately 107 A549 cells overexpressing miR-302a-5p or miR-NC were injected into 6-8-week-old woman athymic nude mice. Tumor growth was monitored every 5 days, and at each time point, tumor volume was determined as (width2 size)/2. Thirty days later on, the mice were euthanized, the tumors were photographed, and the tumor weights were determined. Immunohistochemical assay Freshly isolated cells from mice injected with A549 cells overexpressing miR-302a-5p or miR-NC were fixed in formalin. The fixed cells were inlayed in paraffin, sectioned (4-m-thick), and incubated with main antibodies against ITGA6 and Ki-67 (Proteintech, Wuhan, China) over night. Then, the sections were incubated with the secondary antibody and stained having a 3,3-diaminobenzidine answer. Statistical analysis All analyses were performed using GraphPad Prism (Version 5.0; GraphPad Software, Inc, California, 10Panx USA). Data are indicated as the mean SD and were analyzed using College students test or ANOVA. The correlation between the manifestation levels of ITGA6 and 10Panx miR-302a-5p was assessed by Pearsons correlation analysis. Variations with values less than 0.05 were considered significant. Results MiR-302a-5p is definitely downregulated in NSCLC cell lines and cells To explore the part of miR-302a-5p in NSCLC, the manifestation of miR-302a-5p was analyzed in 30 combined NSCLC and adjacent non-cancerous lung tissue samples by qRT-PCR. miR-302a-5p manifestation was significantly reduced the NSCLC cells than in adjacent noncancerous lung cells (Number 1A). Moreover, the manifestation of miR-302a-5p was reduced the NSCLC cell lines (A549, SPC-A1, Itgb5 Personal computer-9, SK-MES-1, and H1299) than in HBE cells (Number 1B). These results suggest that overexpression of miR-302a-5p 10Panx inhibited the progression of NSCLC. Open in a separate windows Number 1 MiR-302a-5p is definitely downregulated in NSCLC cells samples and cell lines. A. Quantification of miR-302a-5p manifestation in 30 combined human NSCLC samples and adjacent noncancerous lung tissue samples. B. Quantification of miR-302a-5p manifestation in NSCLC cell lines (A549, SPC-A1, Personal computer-9, H1299 and SK-MES-1) and normal HBE cells. *P < 0.05 expression levels in HBE cells. MiR-302a-5p inhibits NSCLC cell proliferation and the cell cycle in vitro The miR-302a-5p mimic or a negative control (miR-NC) was transfected into two 10Panx NSCLC cell lines (A549 and H1299), and the transfection effectiveness was determined by qRT-PCR (Number 2A). The CCK-8 assay showed the proliferation of NSCLC cells transfected with the miR-302a-5p mimic was obviously suppressed when compared to the proliferation of cells transfected with the bad control miRNA (Number 2B). Similarly, the EdU assays suggested that overexpression of miR-302a-5p decreased the numbers of EdU-positive nuclei in A549 and H1299 cells compared with the figures in the miR-NC-transfected cells (Number 2C). We also assessed whether miR-302a-5p affected cell cycle progression in A549 and H1299 cells. As determined by circulation cytometry, overexpression of miR-302a-5p improved the number of cells in G1 phase and decreased the number of cells in S phase (Number 2D). These results indicate that miR-302a-5p inhibited NSCLC cell proliferation and cell cycle progression in vitro. Open in a separate window Number 2 Overexpression of miR-302a-5p suppresses NSCLC cell proliferation and the cell cycle. A. Quantification of miR-302a-5p manifestation after transfection of a miR-302a-5p mimic or control miRNA (miR-NC) into A549 and H1299 cells. B. Proliferation of these cells was measured from the CCK-8 assay. C. An EdU incorporation assay was performed on miR-NC- or miR-302a-5p-transfected A549 and H1299 cells. D. miR-302a-5p mimic or miR-NC-transfected A549 and H1299 cells in each cell cycle phase were quantified by a flow-cytometric assay. *P < 0.05 miR-NC-transfected cells. MiR-302a-5p inhibits NSCLC cell migration and invasion in vitro Next, we examined the effects of miR-302a-5p within the migration and invasion of NSCLC cells in vitro. The wound-healing assay showed the migratory ability of A549 and H1299 cells transfected with the miR-302a-5p mimic was lower than that of cells transfected with miR-NC (Number 3A). Consistent with these results, the migration assay confirmed that transfection of the miR-302a-5p.