2.1 Patients and samples
Sera from 7 LUAD patients and 7 normal control was used to detect the levels of miRNAs through miRNA microarray. In order to reduce the heterogeneity of samples, the sera of two participants were combined into a serum sample. Sera from 30 LUAD patients and 30 normal controls matched in age and gender were used to validate the expression of miR-625-3p by qRT-PCR. The patients with LUAD were obtained from the First Affiliated Hospital of Zhengzhou University from November, 2017 to September 2019. All patients were newly diagnosed as LUAD by histology and did not undergo any treatment. Sera of normal healthy were collected from health examination population without any pulmonary-related diseases or cancers from Zhengzhou Hospital of Traditional Chinese Medicine. Written informed consent had been obtained from all subjects before the study began. The serum samples were separated by centrifugation at 3000rpm for 5min and stored at -80℃ till further analysis.
2.2 Agilent miRNA Microarray
Human miRNA microarray 2.0 from Agilent Technologies including 2549 miRNAs were applied to screen candidate miRNAs. The data has been uploaded to the GEO database (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc =GSE157074)[12]. We analyzed the differentially expressed miRNAs between 7 LUAD serum samples and 7 normal serum samples matched by age and gender.
Publicly available miRNA-Seq data of LUAD tissue (n=521) with corresponding clinical information and adjacent noncancerous tissue (n=46) were directly downloaded from the TCGA data portal (http://cancergenome.nih.gov/). The R package “edgeR” was used to distinguish differentially expressed miRNAs between LUAD tissues and adjacent noncancerous tissues. The threshold of log2|FC|≥1 and P <0.05 was considered significantly. The R package “survive” was used to analyze the relationship between miR-625-3p and the prognosis of patients with LUAD.
2.3 Cell culture and transfection
Human NSCLC cell lines (A549, H358, CALU-3, H1299 and PC-9) were purchased from the Chinese National Infrastructure of Cell Line Resource. CALU-3 cells were maintained in DMEM (BI, Israel) medium with 10% FBS, and H358, A549, H1299 and PC-9 cells were cultured in RPMI-1640 medium (BI, Israel) supplemented with 10% FBS (BI, Israel). Cells were incubated in a humidified atmosphere containing 5% CO2[13]. MiR-625-3p mimic/mimic NC (10 nM, RiboBio, China) and miR-625-3p inhibitor/inhibitor NC (50 nM, RiboBio, China)were transfected into the cells via Lipofectamine 3000 (Invitrogen, United States) according to the manufacturer’s instructions[14]. After 24-48 h transfection, cells were harvested for further experiments. The lentivirus vector, hsa-miR-625-3p and KLF9 lentivirus or negative control lentivirus, were obtained from Genechem (Shanghai, China) with a titer of 107IU/ml. LUAD cells were transfected with individual types of lentivirus at a multiplicity of infection (MOI) of 10 in the presence of 2 g/ml puromycin.
2.4 RNA isolation and Quantitative real-time PCR
Total RNA was extracted from cells or serum using RNAiso Plus (TaKaRa, Japan) according to the manufacturer’s protocol. MicroRNA or mRNA was converted to cDNA applying PrimeScriptTM RT reagent Kit (TaKaRa, Japan), and PCR was performed using the miScript SYBR Green PCR Kit (TaKaRa, Japan)[15]. The miR-625-3p and U6 primers were employing the Bulge-Loop miRNA qRT-PCR Primer Set (RiboBio, China) according to the manufacturer's instructions[16]. Each sample was performed in triplicate. GAPDH or U6 was used as internal control. Relative quantification values were calculated via the 2-ΔΔCt method.
2.5 CCK8 cell proliferation assay
Cell proliferation was determined by Cell counting kit-8 assay (CCK8) (Meilunbio, China) at 0, 24, 48, 72 h according to the manufacturer's instructions[17]. Cells were seeded on 96-well plate (2×103 per well for H1299 cell line and 3×103 per well for CALU-3 cell line). 100 µL CCK-8 mixture (CCK8:DMEM/RPMI1640 medium=1:9) was added into each well for each measurement, and incubated for additional 2 h at 37℃. The growth curve was drawn according to the absorbance at 450 nm. Six multiple wells were detecting for cell viability in each treatment groups[18]. Three independent experiments were conducted.
2.6 Transwell migration and invasion assays
Invasion assays were performed by using a Transwell chamber with 8mm pores (Corning, United States) with Matrigel (BD, United Stated) according to the manufacturer’s instructions. When the confluence of cells reached 75%-80%, trypsin was used to digest cells. After that, we resuspended 105 cells with 200 µL serum - free medium and added to the upper chamber, meanwhile, 600 µL medium with 20% FBS was added to lower chamber. The Transwell chamber was then placed into the incubator. After 36 h, the culture medium was removed from the upper chamber and Matrigel was wiped away[18]. Invading cells at the bottom of chamber were fixed with 4% methanol and stained with 0.5% crystal violet (Solarbio, China) for 30 min. Six different fields of microscope in each group were acquired, and then count the number of invasive cells. For the Transwell migration assay, the process is similar to Transwell invasion analysis, except that the interior of the chamber is free of Matrigel. Three independent experiments were conducted.
2.7 Western Blot
Cells were harvested at 48h after transfected with miRNA NC, miR-625-3p mimics and miR-625-3p inhibitor, and extracted total protein applying RIPA assay buffer (Solarbio, China). The protein concentration was measured by BCA assay kit (Solarbio, China). The proteins were separated by 10% SDS-PAGE and then transferred to a polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with primary antibody at 4℃ overnight, and then reacted with the secondary antibody at room temperature for 2 h. The information for primary antibodies was as follows, anti-RASSF8 (1:500, Santa Cruz, Unite State), anti-KLF9 (1:500, Santa Cruz, Unite State), and anti-GAPDH (1:500, Santa Cruz, Unite State). The secondary antibodies, goat-mouse antibody (1:5000) was purchased from R&D Systems (Minneapolis, MN). The membrane was visualized using High Performance Luminol Substrate Solution (APExBIO, United States) according to manufacturer’s instructions. GAPDH was used as an internal control and for normalization.
2.8 Luciferase reporter assay
The wild-type (WT) or the mutant (MUT) KLF9 3’-UTR binding sequences of miR-625-3p (GeneChem, China) were synthesized and cloned into the pGL3 promoter vector. 293T cell line were co-transfected with miR-625-3p mimic and pGL3 vector using Lipofectamine 3000. After 48h of transfection, luciferase activity was measured using the Dual-Glo Luciferase Assay System (Promega, United States) according to the manufacturer’s protocol.
2.9 Animal experiments
Four-week-old female athymic nude mice (BALB/c Nude) were purchased from Vital River, Beijing, China. All mice were housed and maintained at our animal facility under pathogen-free conditions according to institutional guidelines. Control cells and miR-625-3p or KLF9 overexpression cells with SV40 vector (1.0×107 cells) were suspended in 100 μL PBS buffer and were subcutaneously inoculated into the flank of the mice. Five mice were in each group. Mice were monitored every 5 days for tumor growth, and tumor size was measured with a caliper. Tumor volume (V) was calculated according to the formula: V=ab2/2. After 15 days of the inoculation, mice were sacrificed by cervical dislocation and tumor weight was measured. All experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals, with the approval of the Henan Institute of Medical and Pharmaceutical Science, Zhengzhou, China (2020-49).
2.10 Hematoxylin and eosin (H& E) and Immunohistochemistry (IHC) staining
For IHC assay, Lung cancer tumor tissue and normal control tissue samples of nude mice were fixed in formalin and embedded in paraffin and 4 µm thick tissue sections were cut. Then paraffin sections were incubated with primary antibodies against KLF9 CD31, Ki67, E-ca and Vimentin at 37℃ for 60 mins, secondary antibodies at 37℃ for 15 mins and horseradish enzyme labelled streptavidin solution for 10 min, then strained by DAB and hematoxylin. Finally, we observed the slides through a microscope.
2.11 Statistical analysis
All experimental assays were performed in triplicate. All data were expressed as the mean ± standard deviation (SD) error of the mean. Student’s t-test was performed to analyze the difference between the two groups, and a one-way analysis of variance (ANOVA) test was performed between three or more groups. Transwell migration and invasion assay data were analyzed by Image J. P<0.05 was considered as a statistically significant difference.