Cell culture and reagents
The human CRC cell lines SW480, HCT116, HCT15, HCT8, HT29 were purchased from the American Type Culture Collection (ATCC), SW620, Lovo, RKO and immortalized colon epithelial cell lines NCM460 were obtained from Cell Repository of the Chinese Academy of Sciences (Shanghai). HCT116 and HT29 were cultured in McCoys’5A (Hyclone) supplemented with 10% fetal bovine serum (FBS, BI), SW480, SW620, RKO and NCM460 were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) with 10% FBS, and HCT15 and HCT8 were maintained with RPMI-1640(Hyclone). Cells were treated with Global methylation inhibitors 3-Deazaadenosine (DAA, B6121, APExBIO), the influence on the expression of targets was identified by qRT-PCR and western blot.
Lentiviruses expressing shMETTL14 and shCON (empty vector), pcDNA3.1-METTL14 (METTL14-OE) and empty Vector (METTL14-NC) were purchased from Hanbio Biotechnology Co.Ltd (Shanghai). Stable METTL14 knockdown experiment was carried out in SW480 and HCT116 cells, while METTL14 overexpression was conducted in SW620 cells. Transfected cells were selected using puromycin (3μg/ml) for 7 days or more. Small interfering RNAs (siRNA) targeted HuR, YTHDF2, ARRDC4 and corresponding negative controls (siNC) were synthesized by GenePharma Company (Shanghai) and infected cells utilizing Lipofectamine 2000 reagent (Invitrogen). Relevant applied sequence was presented in Table S1.
Cells or frozen tissue were collected and lysed in pre-cooled RIPA buffer (BB-3201, BestBio) containing protease inhibitor cocktail (HY-K0010, MedChemExpress) and PMSF (HY-B0496, MedChemExpress) on ice for 30 min. Equal Protein samples were added in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and then were transferred to 0.45μm polyvinylidene difluoride (PVDF) membranes (IPVH00010, Merck Millipore). Soaked in 5% non-fat milk at room temperature for 2 h, the membranes were then incubated with primary antibodies at 4°C overnight. After washed 3 times with TBST, these membranes were incubated with HRP-conjugated secondary antibodies for 1 h at room temperature. Immunoblots signals were detected using Pierce™ ECL Western Blotting Substrate (32106, Thermo Fisher Scientific). Primary antibodies were listed as: anti-METTL14 (HPA038002, Sigma-Aldrich), anti-HuR (#12582, CST), anti-IGF2BP1 (22803-1-AP, Proteintech), anti-YTHDC1 (14392-1-AP, Proteintech), anti-YTHDF1 (17479-1-AP, Proteintech), anti-YTHDF2 (24744-1-AP, Proteintech), anti-YTHDF3 (25537-1-AP, Proteintech), anti-ARRDC4 (HPA042109, Sigma-Aldrich), anti-LaminB1 (66905-1-lg, Proteintech), anti-PSAT1 (A6707, ABclonal), anti-HSP90AA1 (A13501, ABclonal), anti-ANXA1 (A1118, ABclonal), anti-ZEB1 (21544-1-AP, Proteintech), anti-Snail (13099-1-AP, Proteintech), anti-Slug (#9585, CST), anti-Twist (25465-1-AP, Proteintech), anti-TCF4 (22337-1-AP, Proteintech), anti-GAPDH (60004-1-lg, Proteintech) or β-actin (66009-1-lg, Proteintech) were used as the internal control.
Quantitative real-time PCR (qRT-PCR) and RNA stability assay
Total RNA of cells was extracted by TRIzol Regent (Invitrogen) according to the previous protocols followed by cDNA synthesis with PrimeScript™ RT Master Mix (RR036A, TAKARA). mRNA expression levels were measured by TB Green® Premix Ex Taq™ II Kit (RR820A, TAKAR) on Applied Biosystems StepOnePlus. Relative RNA amount of each group was calculated using the 2–ΔΔCt method with normalization by GAPDH. For RNA stability assay, cells were seeded in 6-well plates overnight, RNA decay rate was measured using actinomycin D (HY-17559, MedChemExpress) at 5μg/ml and cells were harvested after incubation at indicated time. The primers used in this study were presented in Table 2.
Transwell migration and invasion assays were conducted in 24-well plates. 4×104 cells in 200μl serum-starved medium were seeded into the upper chamber (8.0 μm pore size filter, Corning) with or without coated Matrigel (BD, Bioscience), while 600μl medium containing 10% FBS was placed into the lower chamber. After incubation in 37°C for 48 h, cells passed through the membrane were immobilized by methyl alcohol and stained with 0.2% crystal violet solution. Subsequently, the penetrated cells were photographed and calculated under Olympus microscope.
1×106 cells in complete medium were cultured in 6-well plates overnight. The confluent monolayers of cells were scratched with a fine pipette tip and photographed. After 48 h, cells migration into the wound was visualized and measured under Olympus microscope and compare it with the size of the initial wound.
Cell viability assay
Cell viability were explored by Cell Counting Kit-8 (CCK-8, Dojindo Laboratories) according to the manufacturer’s instruction. In brief, 3000 cells were seeded into 96-well plates and incubated at 37°C overnight. Six multiple wells were applied for assessment of each time point. Subsequently, 10μl CCK8 reagent was added into different groups of CRC cells and incubated in 37°C for another 2 h. Absorbance was captured at 450nm daily to evaluate the proliferation potential of cells. All experiments were conducted in triplicate.
CRC tissue specimens and clinical data
A total of 72 CRC and corresponding adjacent normal samples were collected after surgical resection in The First Affiliated Hospital of USTC between January 2010 and December 2015. Another cohort including 14 paired CRC and paracancerous were collected in 2019 and underwent protein extraction for western blot analysis of METTL14 and HuR expression. Informed consent had been obtained from each patient before our study and those who received local or systemic treatment were not taken into this study. Our study was approved by the Human Research Ethics Committee of The First Affiliated Hospital of USTC. The clinicopathological characteristics of these enrolled patients were listed in Table1.
Immunohistochemistry (IHC) analysis
The 4μm-thick serial-sectioned CRC and adjacent normal tissue were subjected to IHC staining according to previous protocol. Briefly, following deparaffinization, rehydration and antigen retrieval, sections were conjugated with primary antibodies at 4°C overnight. After incubation with secondary and development of Diaminobenzidine (DAB), the staining scores of target proteins were evaluated blindly by two independent pathologists by multiplication of the staining intensity grade (0, 1, 2 or 3 indicated negative, weak, moderate or strong stains, respectively) and proportion of positive stains (0, 1, 2, 3 or 4 implied positive areas of 0–5%, 6–25%, 26–50%, 51–75% or 76–100%, respectively)
MeRIP-sequencing projects and subsequent data analysis were supported by Genesky Biotechnologies Inc. (Shanghai, China). Total RNA was isolated from transfected SW480 cells followed by poly (A)+ RNA purification and fragmentation using NEBNext Poly (A) mRNA Magnetic Isolation Kit (New England Biolabs, UK). Concentration of RNA was detected on Nanodrop 2000 (Thermo Fisher Scientific, USA) and the integrality was guaranteed by Agilent 2100 Bioanalyzer (Agilent Technologies, USA). Dynabeads Protein A (Thermo Fisher Scientific, USA) was mixed with rabbit anti-m6A antibody (Synaptic system, Germany) at 4°C for 2 h in advance, then fragmented mRNA was incubated with the mixture for another 2 h to precipitate m6A-enriched RNAs. Qualified samples underwent Library Pooling and Sequencing using Illumina HiSeq 2500 machines. Following quality filter, the raw sequence data was mapped to human genome GRCh37/hg19 utilizing the HISAT2 software (v2.0.5) and the results were subjected to analyzed bioinformatically and statistically.
m6A dot blot assay
Total RNA was extracted as described above and the concentration was determined by NanoDrop one (Thermo Fisher scientific, USA). Samples continuously diluted (200ng/μl,100ng/μl and 50ng/μl) were denatured at 95 °C for 5min and loaded on the Amersham Hybond-N+ membrane (GE Healthcare, USA). Afterwards the membrane was crosslinked by UV light for 10 min followed by blocking with 5% BSA, special m6A antibody (ab, Abcam) was hatched with the membrane at 4°C overnight. 0.02% Methylene blue staining of the membrane was regarded as RNA loading control. Following incubation with HRP-conjugated anti-rabbit immunoglobulin G for 1 h at room temperature, the signal of dot blot was detected referencing western blot.
RNA immunoprecipitation (RIP)
RIP experiments were conducted in SW480 and HCT116 cells with the Imprint® RNA Immunoprecipitation Kit (RIP-12RXN, Sigma-Aldrich) following the manufacturer’s illustrations. Briefly, 4μg specific antibodies against rabbit immunoglobulin G (I5006, Sigma-Aldrich), METTL14, YTHDF2 or m6A were incubated with cell lysates overnight at 4 °C with rotation, then 20μl washed magnetic beads were added to each reaction and incubated at 4°C for 2 h. After washed 6 times, interested RNAs in the immunoprecipitation complex were purified for further analysis by qRT-PCR. Relative enrichment of RNA was normalized to the input.
RNA pull-down assay
RNA pull down assay was employed to identified the endogenic binding of RNAs and proteins in this study. 3μg Biotin-labeled probe was incubated with 2mg cell lysates containing cocktail and Ribonuclease Inhibitor at 4°C overnight on a rotator, then 20μl pre-cleared streptavidin magnetic beads (88816, ThermoFisher Scientific) were placed into the lysate for precipitation of RNA-protein complex. The beads were washed with lysis buffer for 4 times and boiled with protein loading buffer for 10min. Immunoblotting assay was used to determine the efficiency of co-precipitated proteins. The biotin probe sequence: CAUAGAUUGGAAUAGCUUCUC, negative control: GAGAAGCUAUUCCAAUCUAUG.
Chromatin immunoprecipitation (ChIP) assay
ChIP assay was carried out in SW480 and HCT15 cells with SimpleChIP® Enzymatic Chromatin IP Kit (#9003, CST) following the manufacturer’s instruction. 1×107 cells crosslinked by 37% formaldehyde (final concentration was 1%) were lysed in SDS lysis buffer and sonicated to break into DNA fragments with 200-900bp optimal length. Then the lysate was centrifuged and the supernatant was diluted with ChIP buffer for incubation with anti-HuR antibody or isotype control IgG (#2729, CST) at 4°C overnight. Next, 30μl ChIP-grade Protein G magnetic beads were added to the DNA-protein complexes rotating for another 2 hours. After washed and eluted, the immunoprecipitates were de-crosslinked at 65°C for 3h or overnight. Subsequently, DNA was purified and subjected to qRT-PCR analysis.
Subcutaneous xenograft model was performed using 5-weeks old male BALB/c athymic nude mice. Equal amount of HCT116 cells (2×106) stably expressed of relevant plasmids were injected into the right flank of mice, tumor bulks was monitored once a week after injection and volumes were counted as 0.5 × a2 × b (a and b respectively indicated short and long diameter of tumor). Four weeks later, mice were sacrificed and the weight of deprived tumors from each group were obtained. To explore the metastasis abilities of transfected HCT116 cells in vivo, 1×106 cells in 100μl PBS were injected into the tail of mice and the metastatic foci on liver of mice were analyzed after six weeks. The liver tissue was immobilized by formaldehyde, embedded in paraffin and subjected to HE staining.
All of the statistical analyses in this study were operated with the SPSS 19.0 (SPSS, Inc., USA) and GraphPad Prism 5.0 (GraphPad, Inc., USA). Experiments were repeated at least three times and data were showed as the means ± SD using a two-tailed Student’s t test or one-way ANOVA. The correlation between METTL14 protein levels of tissue and clinicopathologic characteristics was identified by Chi-square test. Independent prognostic factors of multiple multivariate analysis were explored with the Cox regression model. Overall survival curve was depicted with Kaplan–Meier analysis and the difference were detected via log-rank test. A P-value < 0.05 was considered as statistically significant.