Clinical tissue specimen

58 patients (42 males and 16 females; aged 58.69 ± 12.71) with lung cancer were enrolled in this study. The inclusion criteria: (1) The cancer is confirmed by postoperative pathological examination; (2) all patients have not undergone preoperative radiotherapy or chemotherapy. The exclusion criteria: (1) Patients with other types of tumors; (2) patients have undergone preoperative radiotherapy or chemotherapy. After lung tumor tissues and adjacent normal tissues were obtained and underwent histological diagnosis, tissues were frozen in liquid nitrogen for subsequent assays. This study had received the approval from the Ethics Committee of Ningde Municipal Hospital of Ningde Normal University. Written informed consent for tissue usage was signed by patients.

RT-qPCR

Extracting total RNA from clinical specimens and from various lung cancer cell lines was performed as previously described [11]. MiRNA expression was determined using the miRNA-specific TaqMan MiRNA Assay Kit (Applied Biosystems; Thermo Fisher Scientific, Inc., Waltham, MA, USA) following the protocol of the manufacturer. Detailed procedure was in accordance with a previous study [20]. All samples were evaluated in triplicate. The primer sequences used were shown as following:

miR-144-3p-F: 5′-TCCGATCATGTAGTAGATATTGACAT-3′,

miR-144-3p-R: 5′-GTGCAGGGTCCGAGGT-3′,

HGF-F: 5′-GTGTGCCACAACTCACAACTA-3′,

HGF-R: 5′-GGTCCTGGGTATTGGAGCA-3′,

U6-F: 5′-TCCGATCGTGAAGCGTTC-3′,

U6-R: 5′-GTGCAGGGTCCGAGGT-3′,

GAPDH-F: 5′-TGGACATCCGCAAAGACC-3′,

GAPDH-R: 5′-GGGACTTCCTGTAACGC-3′.

Cell transfection

Purchasing lung cancer cell lines from the American Type Culture Collection (ATCC, Manassas, VA, USA), the cells were maintained in Dulbecco’s Modified Eagle Medium (DMEM) that contained fetal bovine serum (10% FBS) and cultured at 37 °C under the conditions of 5% CO2 and saturated humidity. An inverted microscope was adopted to observe the growth of lung cancer cells. As A549 cells andNCI-H1299 cells reached 70–80% confluence, they were harvested with trypsin and sub-passaged. Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) was adopted to proceed transfection following the protocol of manufacturer. A549 and NCI-H1299 cell lines were divided into four groups: control group, miR-144-3p group, miR-144-3p + vector group and miR-144-3p + HGF group. A549 and NCI-H1299 cells were transfected with scrambled control mimics, miR-144-3p mimic, respectively or co-transfected with miR-144-3p plus vector or miR-144-3p plus HGF. Used sequences were shown as below:

miR-NC: 5′-UCACAGUGAACCGGUCUCUUU-3′,

miR-144-3p mimics: 5′-GGAUAUCAUCAUAUACUGUAAG-3′.

Luciferase reporter assays

Online databases, which included EIMMO and miRanda-mirSVR (microRNA.org), were used to predict the potential target genes of miR-144-3p. Reporter vectors that contained wild-type (WT) or mutant (Mut) HGF3′-untranslated region (UTR) were constructed. A549 cells were co-transfected with miR-144-3p mimics or negative control miRNA mimics (pMIR-Control), together with reporter vectors. According to the recommendations of the manufacturer, Luciferase activity was determined through the Dual-Luciferase Reporter Assay System (Promega, Madison, WI, USA).

Western blot

All of protein coming from transfected cells (about 2 × 106 cell each group) was extracted and subjected to sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE), then transferred on polyvinylidene difluoride membranes. This assay was carried out as previously described [11] with primary antibodies against HGF (1:500, Sigma-Aldrich; Merck KGaA). The protein samples were then cultured together with a secondary antibody (Abcam, Cambridge, MA, USA). GAPDH was chosen as the internal control.

Cell proliferation assay

The Cell Counting Kit-8 (CCK-8) assay was performed with an aim of detecting lung cancer cell proliferation. Briefly, transfected A549 cells (100 μl/well) and NCI-H1299cells (100 μl/well) were inoculated into 96-well plates with culturing condition maintained at 37 °C and 5% CO2. The proliferation of A549 and NCI-H1299 cells was measured at 3 days adopting CCK-8, following the protocol of the manufacturer. The optical density (OD) value at 490 nm was measured using an automatic microplate reader. The assay was performed in triplicate.

Transwell invasion

Detecting invasions of A549 and NCI-H1299 cells was achieved by Transwell invasion assay. Briefly, transfected A549 and NCI-H1299 cells were harvested and propagated (about 2 × 106 cells each group), then inoculated to the top chamber (105 cells/chamber) and maintained under standard conditions for 24 h. Subsequently, transmigrated-cells were fixed and dyed, and counted using an inverted microscope. The assay was performed in triplicate; five random visual fields were selected for each chamber.

Wound-healing assay

Detecting migration of A549 cells and NCI-H1299 cells was achieved by Wound-healing assay. Briefly, A549 and NCI-H1299cells were seeded into 6-well plates after transfection (2 × 105cells/well) and cultured under standard conditions. A wound was produced by a 200 μl sterile pipette tip to scrape monolayer-cell. Fresh medium was added and the plate was cultured for 24 h. Using an inverted microscope to gain the images, the area covered by cells due to migration into the artificial wound was observed.

Cell cycle measurement

Flow cytometry was adopted to observe cell cycle distribution. Transfected cells (2 × 106 cell each group) were suspended in PBS and stained with 10 μl Annexin V-FITC as well as 5 μl PI reagents about 15 min away from light at room temperature. Adopting FACSCalibur flow cytometer (BD Biosciences, Franklin Lakes, NJ, USA), we measured cell proportion in S-phase, followed by analyzing the obtained data with Cell Quest software (BD Biosciences).

Mouse xenograft model in vivo

Establishing a mouse xenograft model to explore the functional role of miR-144-3p in vivo, A549 cells were allowed to be plated in six-well plate, then transfected with miR-144-3p (miR-144-3p group) or control mimics (miR-NC group). Nude mice accepted the subcutaneous injection of these treated cells on the left flank (BALB/c, from Experimental Animal Center, Chinese Academy of Sciences). The above procedures were conducted based on the national (D.L.n.26, March 4, 2014) and international laws and policies (Directive 2010/63/EU). This study had received the approval from the Animal Experimental Ethics Committee, Ningde Municipal Hospital of Ningde Normal University. Mice were randomized into two groups with each group having 8 mice, which included control group (treated with control mimics) and miR-144-3p group (treated with miR-144-3p). When measuring tumor volume, the formula was: V = (L × W2) × 0.5, with L and W respectively representing the length and width. Mouse tumors were dissected out using a sterile scalpel, separated from skin carefully, weighed at day 21 post-inoculation, and then preserved in 4% paraformaldehyde for further analysis. The procedures were in accordance with the Guide for the Care and Use of Laboratory Animals (The 8th edition, NIH).

Immunohistochemistry

Immunohistochemical (IHC) staining of tumor tissues was conducted as previously described [21]. An antibody against HGF protein (1:100, Boster, Hubei, China) was incubated with the tissue samples, and the stained samples were examined under a light microscope.

Statistical analysis

Experimental data are presented as the mean ± standard deviation. When making multiple group comparisons, one-way analysis of variance was performed, while Student’s t-test was adopted to make a two-group comparison. It was considered to have statistical significance if P < 0.05.

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