Patients and tissue samples
The frozen paired samples used for mRNA quantification were stocked in RNAlater solution (Invitrogen, Thermo Fisher Scientific, USA), and were acquired from 41 CRC patients with primary CRC tissues and matched adjacent normal tissues, who had undergone surgery between November 2012 and December 2017 at the Sixth Affiliated Hospital of Sun Yat-sen University (SYSU).
We also constructed tissue microarrays of primary CRC from 217 patients from the Sixth Affiliated Hospital of SYSU. None of these patients received chemotherapy, radiotherapy and other related treatment prior to surgical resection of the tumor. Among these patients, 122 patients had disease relapse or metastasis within 3 years after surgery. Our study was approved by the Institutional Ethics Committee of Sixth Affiliated Hospital, Sun Yat-sen University.
Cell culture and transfection
All cell lines (HCT15, HCT8, SW480, SW620, LOVO, DLD1, SW48, HCT116, 293T) used in this study were obtained from ATCC. Cell lines were typically cultured at 37℃ in an incubator with 5% CO2. SiRNAs used were as follows: siORMDL1#1: 5’- GGAGTTGGCTTGCTTCATA-3’, siORMDL1#2: 5’- CTGGCAAGTTTCTATACGA-3’. Negative scramble fragment (RiboBio Co., China) were used as control.
Quantitative real time PCR (qRT-PCR)
The mRNA was extracted from CRC cell lines and tissues with TRIzol (Invitrogen, CA, USA). The sequence of the PCR primers were as follows: ORMDL1, forward: 5’- TTC AGT GTT CCT GTT GCT TG-3’and reverse: 5’-AGC CTT GCT TTA CCC TGG TC-3’; β-actin, forward: 5′-TTG TTA CAG GAA GTC CCT TGC C-3′,and reverse 5′-TTG TTA CAG GAA GTC CCT TGC C-3′. β-actin was used for normalization. The siRNAs were transfected into CRC cells via Lipofectamine RNAiMAX (Invitrogen) and overexpression plasmids of ORMDL1 (Full length ORMDL1 cDNA inserted onto pEZ-M98 plasmid, FulenGen, Guangzhou, China ) were transfected with Lipofection 3000 (Invitrogen).
Cells were lysed and boiled for 10 minutes. Proteins were separated by SDS-PAGE, transferred to PVDF membrane, and detected with relevant antibodies for RhoA (1:1000 dilution, Cat#2117, Cell signaling technology), RhoB (1:1000 dilution, Cat#63876, Cell signaling technology), RhoC (1:1000 dilution, Cat#3430, Cell signaling technology), Slug (1:1000 dilution, Cat#9585, Cell signaling technology), Snail (1:1000 dilution, Cat#3879, Cell signaling technology), and ZEB1 (1:1000 dilution, Cat#3396, Cell signaling technology). Blotting for α-tubulin (1:20000, Cat#66031-1-Ig, Proteintech) was used as a loading control.
Hematoxylin and eosin (H&E) and immunohistochemistry assay
The tissue microarray and related clinicopathologic information were collected from Sixth Affiliated Hospital of SYSU. IHC for ORMDL1 was carried on the CRC tissue microarray slides. The slides were first incubated in 60℃ for 4h following deparaffinized in xylene, then rehydrated in alcohol. After heating in citrate buffer for 21 minutes, we used the IHC kit (cat# SP9000; ZSGB-Bio, Beijing, China) to block the endogenous peroxidase activity. Slides were bloked with 5% goat serum for 1hour (h), and incubated in the anti-ORMDL1 antibody (1:100, PHA065643, Atlas Antibodies) overnight at 4℃. Next day, after 3 times washed by PBST, slides were incubated with secondary antibody for 1h, then stained by DAB kit (cat# ZLT-9017; ZSGB-Bio). After experiments, slides were observed by microscope. The IHC scores were independently assessed by two pathologists. And IHC score of 6.3 was selected as the cutoff value to divide the high and low expression of ORMDL1.
Cell proliferation assay
HCT116 and DLD1 were selected to perform this experiment. We separately seeded 5000 DLD1 cells and HCT116 cells after transfection with ORMDL1 overexpress plasmid or siORMDL1 into 96-well plates. The real-time cell analyzer (RTCA, xCELLigence system, ACEA Biosciences, Inc.) and IncuCyte Essens Bioscience incubator (Essens Bioscience, Birmingham, UK) were used to monitor cell growth. The live cells were recorded automatically every 30 minutes.
Colony formation assay
500 cells were seeded into 6-well plate, and incubated for 8 days at 37℃ incubator. Then colonies were fixed with 4% paraformaldehyde and stained with 0.1% crystal violet.
After transfection, cells were seeded into 24-well plates and incubated until 100% confluence. A scratch was made in the cell layer using 10ul pipette tip, then HCT116 were cultured in FBS-free McCoy's 5A and DLD1 in FBS-free RPMI-1640. Live cell photos were collected by the IncuCyte Essens Bioscience incubator every 30 minutes.
8-µm transwell filters (Coring, NY, USA) were used to perform the invasion assay. The upper chambers were covered with Matrigel (Corning, NY, USA), and placed in the 37℃ incubator for 1h. Then 8x104 DLD1 cells were seeded into the upper chamber with RPMI-1640 and we added 700ul RPMI-1640 with 10% FBS to the 24-well plates. After 24h, we wiped the upper chamber with cotton swabs, and stained cells in the other side of the membrane with crystal violet for 5min. The other cell line HCT116 was conducted with the same method. But it was cultured with McCoy's 5A medium and was planted for 10x104 cells.
In vivo subcutaneous tumor growth assay and H&E and IHC assay
The shORMDL1 plasmid (ORMDL1 specific shRNA sequence same as siORMDL1#1 inserted onto pLKD-CMVEGFP-2APuro-U6-shRNA plasmid, Obio Tech, Shanghai, China) or empty control plasmid were stably transfected into DLD1 cells used lentivirus transfection method, and subjected to stable selection by puromycin. The BALB/c nude mice were injected with 4x106 stable shORMDL1 DLD1 cells in each mouse subcutaneously, and each group had 7 mice. Tumor sizes were measured every 3 days then calculated as volume = 1/2 (length x width2). After 15 days, mice were scarified and tumors were measured then collected. All specimens were fixed by formalin and embedded by paraffin for slide preparation. Slides were stained by H&E and anti-Ki67 accordingly.
Immunofluorescence (IF) staining
DLD1 cells layered on confocal-compatible slides were transfected with control or siORMDL1 reagent, then fixed 24 hours afterwards with 4% paraformaldehyde. After being incubated with anti-ORMDL1 antibody (1:100, PHA065643, Atlas Antibodies) and following secondary antibody according to product manual, all slides were observed and photoed (Leica SP8, Mannheim, Baden-Wuerttemberg, Germany).
All statistical analysis was performed using GraphPad Prism 8.0 (Chicago, IL, USA) or SPSS 25.0 (California, USA). Association between ORMDL1 expression and clinical variables were analyzed by Chi-square tests. Overall survival was analyzed using Kaplan-Meier analysis and the p value was calculated by log-rank test. COX regression analyses model was used to evaluate univariate and multivariate survival analyses. Differences between groups were analyzed by a two-tailed Student’s t-test or a Mann-Whitney U-test.
HCT116 cells with transilent transfection of either ORMDL1 overexpression plasmid or knockdown (siORMDl1 and siControl) reagent, were collected 48 hours after transfection. Cell medium was collected as well for contamination detection for mycoplasma. Whole transcriptome deep sequencing (RNA seq) was conducted by GeneSeed Company (Guangzhou, China) with HiSeq X10 PE150 platform. Differential genes were generally defined as the difference between the two groups more than 1.5 times and p < 0.05.