2.1 Cell culture and cell transfection
MKN74, MKN45 and HEK-293 cell lines were purchased from ATCC (American Type Culture Collection, Manassas, USA). And all cells were grown in Roswell Park Memorial InstituteRPMI-1640 medium with 10% fetal calf serum at 37℃ and in a 5% CO2 incubator.
Lentivirus particles were designed and purchased from Genechem (Shanghai, China), including LV-FGD5-AS1, LV-MiR-196a-5p-precursor, LV-anti-MiR-196a-5p, and controls. The vectors were as follows: Ubi-MCS-SV40-EGFP-IRES-puromycin used for FGD5-AS1 overexpressed, hU6-MCSUbiquitin-EGFP-IRES-puromycin was used for MiR-196a-5p up-regulation, hU6-MCSCMV- EGFP was used for MiR-196a-5p down-regulation. Lentivirus transfection was performed according to the manufacturer’s instructions. Lentivirus was transfected with Enhance Liquid and Polyberene as ratio provided by Genechem.
Mimics of miR-196a-5p was designed and purchased from GenePharma (Shanghai, China). Mimics was transfected with lipo2000 (Invitrogen, ThermoFisher, USA).
The BMP pathway inhibitor, LDN-193189 from Axon Medchem (Netherlands), was dissolved with DMSO in 100 nM and added into cells culturing in 6-well plates for 10 hours before cell lysed for protein collection.
2.2 Clinical and tissue samples
The clinical tissue cDNA chip was purchased in Shanghai Outdo Biotech Co., Ltd. (CGt No: cDNA-HStmA060CS01; Lot No:96*R100-M-201703xx-xx,). The 96-well plate was centrifuged before being performed with usage of SYBR Green real time PCR MasterMix (Takara (Japan)) and FGD5-AS1 primer. All the patients were gastric carcinoma diagnosed with pathological analysis. The tumor node metastasis (TNM) stage were assessed according AJCC Cancer Staging Manual.[23]
2.3 Cell migration assay
Cell metastasis was examined using transwell assay, co-culture chambers were purchased from BD Biosciences (San Jose, CA, USA). Serum-free medium are placed in top chambers, and medium containing 10% FBS was added to the bottom chambers. Cells were evenly suspended in top chambers. Then the 6-well plate with chambers were cultured for 24 h at 37 °C with 5% CO2. After incubation, the non-metastasis cells were gently removed from the top wells with a cotton-tipped swab and the chambers were fixed with methanol for 30 min. The chambers were then stained with crystal violet for another 30 min. Cell counting was facilitated by photographing the membrane through amicroscope (Zeiss) under a × 10 objective lens.
2.4 Cell proliferation assay
Cell counting Kit-8 (Genview, GK3607-100T) was used to detect cell proliferation. 5000 suspended cells were planted in 96-well plate with complete medium containing 10%FBS. After the suspended cells adherence in 8 hours, 10% CCK solution was added in the well. Incubating for another 40 min, then microplate reader was used to test the OD value at 450 nm, which will indicate the proliferation of cells.
2.5 Quantitative reverse transcription-PCR
Trizol purchased from Invitrogen (Calsbad, USA) was used to extract total cell RNA as standard protocol. RNA concentration was tested by Nanodrop (Invitrogen, USA). PrimeScriptTM RT Master Mix kits were used to synthesis cDNA; and Taqman MirNA assay kit (designed for microRNA) and SYBR® Select Master Mix kit (designed for total RNA) were used for RT-qPCR analysis on Bio-Rad CFX96 qPCR instrument (Bio‐Rad,Hercules, CA). All these kits were purchased from Takara (Japan). β-actin and U6 were used as reference genes.
2.6 Western blot assay
RIPA lysis (ThermoFisher, USA) was used for extracting total cell protein. Protein concentration was tested by BCA assay (ThermoFisher, USA). Equal amounts of protein were separated by SDS-PAGE gel as regular protocol. And the protein was transferred from gel to a PVDF membrane (Millipore). Then the PVDF membrane was blocked in 5% BSA-TBST (Sigma, USA) for 2 hours at room temperature, followed by primary antibody at 4-degree overnight. The membrane was washed 10 min 3 times with TBST, followed by secondary antibody at room temperature for 1 hour. After another washing cycle, the membrane was visualized by ultra-sensitive ECL kit. Results was calculated by Target protein/ β-actin based on band intensity tested by ImageJ. All these blots were cropped for better presentation in publication view with Image Lab (Bio-Rad Version 5.2) and labeled with Adobe Illustration. The full length of blots was shown in Supplementary Figures.
The related antibodies used were as follows:
E-cadherin (CST, Boston, USA,14472), N-cadherin (CST, Boston, USA, 13116), Vimentin (CST, Boston, USA, 5741), Snail (CST, Boston, USA), MMP9(CST, Boston, USA), smad2/3((abcam, USA, ab202445), p-smad2/3 (CST, Boston, USA),BMP4 (abcam, USA, ab124715),P-SMAD1/5/8(CST, Boston, USA, 13820), Anti-SMAD1/5/8 antibody (abcam, USA ,ab80255), and TGF-BETA1 (abcam, USA, ab179695), SMAD6(SANTA CRUZ, USA, sc-25321).
2.7 Dual luciferase reporter assay
Luciferase reporter assay. The binding sites of 3’UTR in SMAD6 were analyzed by Targetscan and were amplified by polymerase chain reaction (PCR) and inserted into the vector, which was designed and purchased by Genechem (Shanghai, China). Then transfected the 3’UTR plasmids and miR-196-5p mimics in to HEK 293 cells. Besides that, Renilla luciferase expression plasmid was co-transfected (Genechem Shanghai, China) as transfection control in all groups. After 24 hours, cells were lysed in 250 µl of Passive Lysis Buffer (Promega) and 20 µl were used to measure luciferase activity with the Luciferase Assay System (Promega). The different groups as follow: PGL3-NC + microup,PGL3-NC + microNC༌PGL3-SMAD6MUT + micro up༌PGL3-SMAD6MUT + micro NC༌PGL3-SMAD6WT + micro up༌PGL3-SMAD6WT + microNC. Each group has 6 parallel holes and the assay was repeated for 3 times.
2.8 Fluorescence in Situ Hybridization (FISH)
RNA-FISH was used to test location of lncRNA FGD5-AS1. The florescence probes of FGD5-AS1 were purchased from Genechem (Shanghai, China). 18S rRNA was the probe for cytoplasmic control. For analysis the MKN-74 cells were cultured on slides. The slides were fixed in absolute ethyl alcohol for 15 min (Sigma), and then cells were treated by cold 0.1% Triton-100x for 15 min before hybridized with probes overnight at 37℃. After washing with SSC/0.3%Tween20 buffer, the coverslip was dyed with DAPI and fluorescence test was conducted with laser scanning confocal microscope (Leica Application Suite, Germany )
2.9 Tumor xenografts in mice
Twelve 4-weeks old female J;NU Homozygous for Foxn1nu mice were obtained from the Medical Laboratory Animal Center of Xi’an Jiao Tong University and averagely separated into four cages randomly. The initial body weights of mice were 20 ± 0.4 g (mean ± STD). All nude mice were housed under specific pathogen-free conditions and the order of the cages changed every week by staff member to avoid position influence.
To test the function of lncFGD5-AS1 in vivo, we randomly separated these mice into lncFGD5-AS1 overexpressed group and control group (3 mice/cage; 2 cages/group). The stable transfected Lv-FGD5-AS1 MKN45 and LV-control MKN45 cells were counted and suspended in 50% matrigel (USA, Corning) followed by injecting subcutaneously with 106cells 0.1 ml in each mouse. These mice were observed under standard SPF housing condition and the length, width and height of the tumor were tested every three days following randomly order for 5 weeks.
Before final test, two mice from control group were sacrificed for cachexia (severe body weight loss with body weight of 12.3 g & 15.8 g) at the early of 4th week; two mice from FGD5-AS1 overexpressed group were excluded from the experiment for the tumor didn’t grow successfully. At final test time, each group had four 10-weeks old mice. Mice were anesthetized with isoflurane while harvesting tumor tissue and sacrificed with cervical dislocation. The xenograft tumor tissues were harvested and weighted immediately followed by extracting protein and RNA from these tissues according to the common protocol. The protein and RNA were employed for further western blot and RT-QPCR test.
The experiment was in accordance with the regulation of the Ethics Committee of Xi’an Jiao Tong University and the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health. The animal test was approved by the Ethics Committee of Xi’an Jiao Tong University and the ethics document was numbered as NO.G-271.
2.10 Statistics methods
The data in this article are shown as the means ± SEM. Data was collected from at least three independent experiments. Paired Student-test was used to test the difference between paired groups. Unpaired Student-test was used to test the difference between un-paired groups. Chi-square test was used to test the difference for binary variables. Wilcoxon paired test was used to test differences between paired patients’ samples because the difference value doesn’t obey normal distribution. Graphpad (GraphPad Software, La Jolla, CA, USA) was used to do the statistics analysis. Image J was used to collect intensity from western blot figure. P < 0.05 was marked as *; P < 0.01 was marked as **; P < 0.005 was marked as***, P < 0.001 was marked as ****.
2.11 Bioinformatics analysis
The target gene and binding site of miR-196a-5p was predicted by TargetScan (http://www.targetscan.org/vert_72/). The competition lncRNA and binding site of miR-196a-5p was predicted by Starbase (http://starbase.sysu.edu.cn/starbase2/index.php).
The Kaplan–Meier analysis was performed using the online Kaplan–Meier Plotter (http://www.kmplot.com) to estimate relapse-free survival curves of the 875 gastric cancer patients, and the median threshold was used as the cut-off point for the high and low groups of FGD5-AS1 expression.