healthmedicinet

Gastric cancer tissues

Gastric cancers and their morphologically normal tissue (located 3 cm away from the
tumor)were obtained between November 2011 and November 2014 from 50 gastric cancer
patients undergoing surgery at Cancer Hospital of Chinese Academy of Medical Sciences.
Tissue samples were cut into two parts, one was fixed with 10 % formalin for histopathological
diagnosis, and the other was immediately snap-frozen in liquid nitrogen, and stored
in liquid nitrogen until RNA extraction. This group consisted of 38 males and 12 females
with a median age of 58 years (range, 32–69 years). The use of the tissue samples
for all experiments was approved by all the patients and by Ethics Committee of the
institution.

Tissue RNA isolation and qRT-PCR

Total RNA was extracted from the cells and tissues using Trizol reagent (Invitrogen,
CA, USA), according to the manufacturer’s instructions. RT-qPCR assay was conducted
to detect the level of RNA transcripts. Briefly, cDNA was synthesised by M-MLV reverse
transcriptase (Invitrogen) from 5 ug of total RNA. Oligo (dT18) RT primer was used
for the reverse transcription of mRNA and lncRNA. Stem-poop RT primer was used for
the reverse transcription of miR-181a. Quantitative RT-qPCR was performed on the Bio-rad
CFX96 real-time PCR System (Bio-rad, Foster City, CA, USA) using KAPA PROBE FAST qPCR
Kits (Kapa Biosystems, MA, USA) and TaqMan probes (Invitrogen) with the following
cycling conditions: 95 °C for 10 min (initial denature); then 40 cycles of 95 °C for
15 sec, 60 °C for 60 sec. The miR-33b specific forward primer sequence was designed
on the basis of miRNA sequences obtained from the miRBase database. Human GAPDH and
U6 snRNA were used for mRNA/lncRNA and miRNA normalization, respectively.

Cell cultures and cell transfection

A total of 6 human gastric cancer cell lines MGC-803, HGC-27, MKN-45, SGC-7901, BGC-823
and AGS were examined in this study. The HGC-27, MKN-45, SGC-7901 cell lines were
provided by American Type Culture Collection (ATCC; Manassas, VA, USA), and were maintained
in RPMI 1640 medium (PAA) supplemented with 10 % FBS (PAA). The MGC-803, BGC-823 and
AGS cell lines were purchased from the Cell Resource Center of Institute of Basic
Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College
(Beijing, China), and was propagated in Dulbecco’s modified Eagle medium (Gibco; Invitrogen;
Life Technologies, Germany), supplemented with 10 % fetal bovine serum (FBS; PAA,
Pasching, Austria) and streptomycin (100 ?g/ml), penicillin (100 U/ml). The human
gastric cancer cell lines HGC-27 and MGC-803 were transfected with miRNA mimic, mimic
control, miRNA inhibitor, inhibitor control, (Scramble; GenePharma; Shanghai, China)
at a final concentration of 25 nmol/L using DharmaFECT 1 (Dharmacon; USA) in accordance
with the manufacturer’s instructions. The same cells were transfected with different
MEG3 constructs at a final concentration of 2 ?g/uL using Lipo2000 (Invitrogen; USA)
in accordance with the manufacturer’s instructions.

Cell proliferation assay

Hgc-27 and MGC-803 cells were incubated in 10 % CCK-8 (DOJINDO, Japan) diluted in
normal culture medium at 37 °C until visual color conversion occurred. Proliferation
rates were determined at 0, 24, 48, 72, 96 hours after transfection. The absorbance
of each well was measured with a microplate reader set at 450nM and 630nM. All experiments
were performed in quadruplicate.

Cell apoptosis assay

In order to detect the apoptosis of HGC-27 and MGC-803 cells, flow cytometric analysis
was applied with Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN Biotech, Nanjing,
China) according to the manufacturer’s instructions. The acquisition and analysis
were performed using MoFlow (Beckman Coulter, Atlanta, GA, USA).

Cell migration and invasion assays

HGC-27 and MGC-803 cells were grown to confluence on 12-well plastic dishes and treated
with miRNA mimics or Scramble. Then 24 hours after transfection, linear scratch wounds
(in triplicate) were created on the confluent cell monolayers using a 200 ?L pipette
tip. To remove cells from the cell cycle prior to wounding, cells were maintained
in serum-free medium. To visualize migrated cells and wound healing, images were taken
at 0, 24, 48 hours. A total of ten areas were selected randomly from each well and
the cells in three wells of each group were quantified.

For the invasion assays, after 24 hours transfection, 1?×?10
⁵
HGC-27or MGC-803 cells in serum-free media were seeded onto the transwell migration
chambers (8 ?m pore size; Millipore, Switzerland) which coated with the upper chamber
of an insert coated with Matrigel (Sigma-Aldrich, USA). Media containing 20 % FBS
were added to the lower chamber. After 24 hours, the noninvading cells were removed
with cotton wool, Invasive cells located on the lower surface of the chamber were
stained with May-Grunwald-Giemsa stain (Sigma-Aldrich, USA) and counted using a microscope
(Olympus, Tokyo, Japan). Experiments were independently repeated three times.

Luciferase reporter assay

HGC-27 cells were co-transfected with 0.4 ?g of pMIR constructs containing the wild
type MEG3 or diverse mutant MEG3, along with 0.02 ?g of the pRL-TK control vector
and miR-181a mimic or mimic control. Cells were harvested 48 h post-transfection and
assayed with Dual Luciferase Assay (Promega, WI, USA) according to the manufacturer’s
instructions. All transfection assays were carried out in triplicate.

Immunoblotting

Immunoblot analysis was carried out using standard methods. Proteins were separated
by 10 % SDS-PAGE, and transferred onto PVDF membranes (Millipore Corporation, Billerica
MA, USA). Membranes were blocked overnight with 5 % non-fat dried milk for 2 h and
incubated with anti-Bcl-2 antibody (Abcam, ab117115) at 1:2000 dilution; anti-GAPDH
antibody (Proteintech) at 1:50,000 dilution overnight at 4 °C. After washing with
TBST (10 mM Tris, pH 8.0, 150 mM NaCl, and 0.1 % Tween20), the membranes were incubated
for 2 h at room temperature with goat anti-rabbit antibody (Zsgb-bio, Beijing, China)
at 1:20000.

RNA pull-down by MS2-MBP

Maltose-binding protein (MBP)-affinity purification was used to identify miRNAs that
associated with lncRNA MEG3. The MS2-MBP protein was expressed and purified from E.
coli following a protocol from the Steitz lab. Three bacteriophage MS2 coat protein-binding
sites (5?-cgtacaccatcagggtacgagctagcccatggcgtacaccatcagggtacgactagtagatctcgtacaccatcagggtacg-3’)
were inserted downstream of MEG3 by site-directed mutagenesis using Stratagene’s QuikChange
Site-Directed Mutagenesis Kit. To obtain miRNAs associated with MEG3, HGC-27 cells
were transfected with MS2-containing MEG3 constructs, and 10 million cells were used
for each immunoprecipitation assay. The cells were harvested 48 h post-transfection
and subjected to RNA pull-down analysis as described elsewhere.