The cell lines used in this study (human alveolar type II epithelial carcinoma cell line A549 and human cervical cancer cell line HeLa) were maintained in our laboratory. HeLa cells and A549 cells were grown in DMEM (Hygclone) containing 10% FBS (Hygclone and PAN) and 1% penicillin-streptomycin. All cells were cultured at 37℃ under 5% CO2 in a humidified incubator. Cells were subjected to 60Co γ-ray irradiation at a dose rate of 85.69 cGy min–1 at room temperature.
The full sequence of LPAR5 was cloned into pcDNA3.1 to generate over expression plasmid.
For LPAR5 knockdown, two synthesized duplex RNAi oligos targeting human mRNA sequences from Sigma were used (si-LPAR5-1:5′- GCAGCUGCAUCUUCCUGAUGCUCAU-3′; si-LPAR5-2:CGCCUGCACUUGGUGGUCUACAGCU). A scrambled duplex RNA oligo (5′-UUCUCCGAACGUGUCACGU-3′) was used as RNA control. Transfected using Lipofectamine 2000 reagent (Invitrogen) with vector control, plasmid construct, siRNA negative control (siNC), or siRNAs according to the manufacturer’s instructions.
RNA extraction and quantitative RT-PCR analysis
Total RNAs were isolated using the TRIzol. 1ug RNA was reverse-transcribed into cDNA with ReverTra Ace qPCR RT Master Mix with gDNA Remover (TOYOBO; Code No.FSQ-301) according to the manufacturer’s instructions. Quantitative real-time PCR analysis was performed with 1μL cDNA using HUNDERBIRD SYBR qPCR Mix (TOYOBO; Code No.QPS-201). Actin was used as endogenous control.LPAR5 primers used for qRT-PCR,F:5′- GAGGTCTCTGCTGCTGAT-3′; R:5′- AGAACTGTTGGTTGAGGAG-3′
Western blot analysis and antibodies
Cells were ruptured with RIPA buffer containing 5 mM EDTA, PMSF, and phosphatase inhibitor cocktail. Cell extracts were centrifuged for 15 min at 12,000 × g and supernatants were then collected. About 40μg total proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred onto Nitrocellulose (NC) membranes, blocked with 5% non-fat milk at room temperature for 2 h, and incubated with primary antibodies. After washed three times, membrane was incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies (1: 4000 dilution) for 1 h at room temperature and the blot was visualized by using SuperSignalTM West Pico Plus Chemiluminescent Substrate (ThermoFisher Scientific; TL275133). The primary detection antibodies were as follows: anti-E-cadherin (CST, 24E10, 1 : 1000); anti-N-cadherin (CST, D4R1H, 1 : 1000); anti-vimentin (Abcam, ab8978, 1 : 1000); anti-GAPDH (Santa Cruz, USA; sc-25778, 1 : 1000); anti-ERK (Abcam, ab17942, 1 : 1000); anti-Snail (CST, L70G2, 1 : 1000) ;anti-MMP1 (NeoMarbers, 1536P8C7C, 1 : 1000) andanti-MMP9 (Proteintech, 10375-2-AP, 1 : 1000) . The protein expression was detected with an enhanced chemiluminescent reagent (Thermo, MA, USA).
Cells (1.5× 103) were seeded into 96-well plates and incubated the plates at 37 °C in a humidified 5% CO2 atmosphere. Cellular proliferation was measured with Cell Counting Kit-8 (DOJINDO；SJ608). Briefly, 10 μL/well CCK8 solution was added at the indicated times and then the cells were incubated at 37 °C for 3 h. The absorbance at 450 nm value was recorded by the microplate reader.
Cell Migration Assays
Cell migration was examined by wound-healing assays. After treatment of cells, scratches were made using sterile 10 μL-pipette tips, and bright-field microphotographs were taken at different times. The percentages of cell migration were quantitated, by the ImageJ software, measuring the width of the cell-free zone immediately after making the scratch.
Cell apoptosis analysis
To assess cell apoptosis, after treated with trypsin, the cells were collected and washed with PBS twice. 4μL of PI(Propidium Iodide) 4μL of FITC(Fluorescein Isothiocyanate) (DOJINDO；AD10) were added after the cells were pelleted and resuspended in 400μL of 1× binding buffer. Apoptosis was detected by flow cytometry after 15 minutes of incubation in the dark at room temperature.
Quantification and statistical analysis
Data are presented as the mean ± SEMs or SDs. Statistical analyses were performed in GraphPad Prism 6 (GraphPad Software, Inc.) using unpaired two-tailed Student’s t-test to compare differences between two groups with significance of P < 0.05. One-way analysis of variance with multiple comparisons tests was used to compare three or more groups with significance of P < 0.05.