All immunohistochemical assays involving human tumor specimens were conducted according to the institutional guidelines of Jiangsu Province.
The human gastric cancer cell lines BGC-823, MGC-803, and SGC-7901 and the normal gastric epithelial cell line GES-1 were purchased from the Cell Biology Institute of the Chinese Academy of Science (Shanghai, China). All cells were grown in Dulbecco’s modified Eagle’s medium (Hyclone, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (Gibco, Carlsbad, CA, USA) and antibiotics (100 U/mL penicillin and 100 μg/mL streptomycin) (Invitrogen, Carlsbad, CA, USA). Cells were maintained in a humidified incubator at 37 °C with 5% CO2. Cells were grown on coverslips for immunofluorescence staining and in six-well plates (Costar, Corning, NY, USA) for RNA isolation and protein extraction.
Plasmids and siRNAs
The empty vector control pcDNA-3.1-HA-C and full-length human MICAL2 cDNA were purchased from YouBio (Changsha, China). The pEGFP-N1 vector containing the full-length Cdc42-Q61L (CA) insert was kept in this laboratory. The siRNAs (control siRNA or siRNA targeting MICAL2 [siMICAL2]) used in this study were synthesized and purified by GenePharma (Shanghai, China). The sequences of the siRNAs targeting MICAL2 were as follows: siMICAL2 #1, 5′-GAGAACGUGAACCAAGACATT-3′; siMICAL2 #2, 5′-GCAUAGAUCUUGAGAACAUTT-3′; siMICAL2 #3, 5′-GCAGCGACACGUGUUACUUTT-3′. Transfection (plasmids or siRNA) was performed using Lipofectamine 2000 following the manufacturer’s instructions. After 24 h of transfection, the cells were cultured in starvation medium overnight and then treated with cycloheximide (CHX) (Sigma-Aldrich, Saint Louis, MI, USA), LiCl (Sigma-Aldrich), MG-132 (Selleck Chemicals, Houston, TX, USA), or chloroquine diphosphate (Chlq) (MedChemExpress, Monmouth, IL, USA) at the indicated time points.
Wound healing assay
For the wound healing assay, cells were seeded in six-well plates until confluence. A wound was then made in the cell monolayer by scratching with a 10-µL pipette tip. After rinsing with PBS, the cells were treated with the indicated stimulator and allowed to migrate for 24 h. Images of wound areas were captured using an inverted phase-contrast microscope (Carl Zeiss Meditec, Jena, Germany) at 0 h and 24 h.
The transwell assay was performed using a 24-well cell culture insert with 8-μm pores. A total of 1×104 cells were seeded in the upper chamber in serum-free medium while medium containing 10% FBS was added to the lower chamber. After incubation at 37 °C for 48 h, the cells on the upper side of the membrane (non-migrated cells) were removed while those that had migrated to the underside of the membrane were fixed in 4% paraformaldehyde and stained with 0.1% crystal violet. The average number of cells was determined and scored under a fluorescence microscope (Carl Zeiss Meditec).
Cell lysates were prepared using RIPA lysis buffer (Beyotime, Shanghai, China). Cytoplasmic and nuclear protein fractions were obtained using a nuclear protein extraction kit from Beyotime following the manufacturer’s instructions. Protein concentrations were assessed using a BCA protein assay kit (Thermo Fisher Scientific). Equal amounts of cellular protein were separated by SDS–PAGE, transferred to a pure nitrocellulose membrane, blocked with 5% skimmed milk, and then incubated with primary antibodies targeting MICAL2 (Affinity, Cincinnati, OH, USA), GAPDH (Bioworld, Nanjing, China), E-cadherin (Proteintech, Wuhan, China), N-cadherin (BD Biosciences, New Jersey, USA), β-catenin, vimentin, Cdc42, and HA-tag (all from Cell Signaling Technology, Danvers, MA, USA) overnight at 4 °C. After washing and incubation with secondary antibody (HRP-conjugated normal rabbit IgG; Jackson, Lancaster, PA, USA), the bands were visualized using enhanced chemiluminescence reagent (FuDeBio, HangZhou, China) and analyzed using Quantity One software (Bio-Rad, Hercules, CA, USA).
Real-time quantitative PCR
Total cellular RNA was isolated using Trizol reagent (Invitrogen) and reverse-transcribed with HiScript Q RT SuperMix for qPCR (Vazyme, Nanjing, China). The sequences of the primers used were GAPDH: 5′-TCGGATCAACGGATTTGGT-3′ (sense) and 5′-TTCCCGTTCTCAGCCTTGAC-3′ (antisense); MICAL2: 5′-GGGGATTTCCCGCAGAATAAAC-3′ (sense) and 5′-GGCTGGGATGAAAATGGAACC-3′ (antisense); vimentin: 5′-AGTCCACTGAGTACCGGAGAC-3′ (sense) and 5′-CATTTCACGCATCTGGCGTTC-3′ (antisense); E-cadherin: 5′-ATTTTTCCCTCGACACCCGAT-3′ (sense) and 5′-TCCCAGGCGTAGACCAAGA-3′ (antisense); N-cadherin: 5′-AGCCAACCTTAACTGAGGAGT-3′ (sense) and 5′-GGCAAGTTGATTGGAGGGATG-3′ (antisense). Real-time PCR was carried out using AceQ qPCR SYBR Green Master Mix (High ROX Premixed) (Vazyme) in an ABI StepOne Real-Time PCR System (Applied Biosystems, Foster City, CA, USA). Relative mRNA expression levels were calculated using the 2-ΔΔCt method and StepOne Software v2.1 (Applied Biosystems).
Cells were placed on a glass coverslip, fixed in 4% paraformaldehyde for 20 min, treated with 0.2% Triton X-100 for 5 min, blocked with 1% BSA for 1.5 h, and then incubated with primary antibodies against β-catenin, E-cadherin, EEA1, Rab7 (all Cell Signaling Technology), and LAPM1 (Proteintech) overnight. After washing three times with PBS, the cells were incubated with Alexa-conjugated species-matched secondary antibody or TRITC-conjugated secondary antibody for 1 h. The nuclei were counterstained with DAPI (Beyotime). Images were captured using an Olympus BX51 microscope fitted with an Olympus DP70 digital camera (Olympus, Tokyo, Japan).
Rho GTPase pulldown assays were performed as previously described. Active Cdc42 was pulled down using PAK-CRIB beads. Briefly, cells were lysed, the lysates were centrifuged, the supernatants were transferred to new tubes containing beads pre-coupled with PAK-CRIB, and incubated under rotation at 4 °C for 30 min. The beads were subsequently washed and the proteins bound on the beads were separated by SDS–PAGE. The quantity of active Cdc42 was determined by western blot using the corresponding antibodies.
Co-immunoprecipitation (Co-IP) assay
Co-IP was conducted as previously described. In brief, a protein mixture was prepared and incubated with antibody-bound affinity beads (Beyotime). Once unbound proteins had been cleared using multiple washing steps, interacting proteins were detected via SDS–PAGE and western blot.
Clinicopathological analysis of MICAL2
The Oncomine and UALCAN databases were used to analyze the association between the mRNA or protein expression of MICAL2 and clinical parameters as well as the prognostic value of mRNA expression of MICAL2 in gastric cancer tissue. Patients with gastric cancer were divided into high and low MICAL2 expression groups based on the median values of MICAL2 mRNA expression.
Data were analyzed using SPSS version 19.0 (SPSS, Chicago, IL, USA) and are reported as means ± standard error of the mean. The Student’s t-test was used for comparisons between two groups. One-way ANOVA was utilized for comparisons among three or more groups. A P-value <0.05 was considered significant. All experiments were repeated independently at least three times.