Sh mettl3

Short hairpin RNAs (shRNAs) against circUHRF2, METTL3, and DDX27 were provided by GenePharma (Shanghai, China). The sequences for shRNAs were shown as follows: sh-circUHRF2-1: 5′-GTATGATGATTGGAAAATGGA-3′; sh-circUHRF2-2: 5′-GATGATTGGAAAATGGATATA-3′; sh-METTL3-1: 5′-GCCTTAACATTGCCCACTGAT-3′; shMETTL3-2: 5′-GCAAGTATGTTCACTATGAAA-3′; sh-DDX27-1: 5′-GCAGAGGAAAGGTCTCAGTTT-3′; sh-DDX27-2: 5′-GCAGGAATTTGACTTGGCCTT-3′; sh- negative control (NC): 5′-TTCTCCGAACGTGTCACGT-3′. The full-length sequences of the circUHRF2 gene (circBase ID: hsa_circ_0002359) were inserted into the pCD5-ciR vector (GENESEED, Guangzhou, China) to establish the circUHRF2 over-expression plasmid. CRC cells were transfected with these segments using Lipofectamine 3000 (Thermo Fisher), according to the user’s guide. For transient transfection assay with shRNAs and plasmids, cells were harvested at 48 h for further experiments. For animal experiments, SW620 cells were stably infected with lentiviruses carrying sh-METTL3 or sh-circUHRF2 (GenePharma) in the presence of 8 μg/mL polybrene (GenePharma). Stably transfected or infected cell clones were chosen by appropriate antibiotics (puromycin, 2–5 μg/mL, Sigma, Saint Louis, MO, USA) for at least one week after virus infection or plasmid transfection.

Small interfering RNA targeting METTL3 (si-METTL3), and negative control (NC) were purchased from RiboBo (Guangzhou, China). The METTL3 interfering sequence was 5′-GCTGCACTTCAGACGAATT-3′. Human METTL3, MYC over-expression plasmids (OE-METTL3, OE-MYC), and negative control vector (Ctrl) were purchased from GenePharma (Shanghai, China). The plasmid vectors expressing shRNA that targets human METTL3 (sh-METTL3) and the scrambled shRNA (sh-Scr) were purchased from GenePharma (Shanghai, China). The shRNA sequences were as follows: sh-METTL3: 5′-GCACTTGGATCTACGGAATCC-3′, sh-Scr: 5′-GCTTCGCGCCGTAGTCTTA-3′. Plasmids for expression of wild-type METTL3 (METTL3-WT) and methyltransferase catalytic mutant METTL3 (METTL3-Mut) were kindly provided by Dr. Jun-Ju He (Central South University, China). Transfections were performed using Lipofectamine 2000 (Invitrogen, USA) for small interfering RNA and Lipofectamine 3000 (Invitrogen, USA) for plasmid according to the manufacturer's protocols.

The human pancreatic cancer cell lines, BxPC-3 (with epithelial properties) and PANC-1 (with more mesenchymal properties), were cultured in DMEM medium supplemented with and 10% FBS and 1% penicillin/streptomycin solution. The cells were cultured in an incubator with 5% CO₂ at 37°C. Human MALAT1 cDNAs were subcloned into pcDNA3 vector. Human METTL3 cDNAs were subcloned into pLenti-C-mGFP vector. Specific small hairpin RNAs (shRNAs) targeting METTL3 (shMETTL3) or MALAT1 (shMALAT1) and the control shRNA (shNC) were obtained (GenePharma, Shanghai, China).

The human gastric cancer cell lines, HGC-27 and BGC-823, were cultured in RPMI-1640 medium supplemented with 1% penicillin-streptomycin and 10% fetal bovine serum. The cells were maintained in a humidified incubator with 5% CO₂ at 37°C. Human METTL3 cDNAs were subcloned into pLenti-C-mGFP vector. The pLenti-C-mGFP vector was used as an empty vector control. Specific small hairpin RNAs (shRNAs) targeting METTL3 (shMETTL3) and the control shRNA (shNC) were obtained (GenePharma, Shanghai, China). Lipofectamine 2000 was used for transfection as described before (20 (link)).

The siRNAs for METTL3 and YTHDF1, and lentivirus for METTL3 knockdown were synthesized by GenePharma (Shanghai, China). The sequences were as follows: siMETTL3#1 (sense: 5′-GCUACCUGGACGUCAGUAUTT-3′, antisense: 5′-AUACUGACGUCCAGGUAGCTT-3′); siMETTL3#2 (sense: 5′-GGUUGGUGUCAAAGGAAAUTT-3′, antisense: 5′-AUUUCCUUUGACACCAACCTT-3′); siYTHDF1 (sense: 5′-GGAGAAUAACGACAACAAATT-3′, antisense: 5′-UUUGUUGUCGUUAUUCUCCTT-3′); and shMETTL3 (5′-GCAAGAATTCTGTGACTATGG-3′).
The pEX-3-METTL3 expression plasmid was synthesized by GenePharma (Shanghai, China). The pcDNA3.1-SLC7A11 expression plasmid was synthesized by Genomeditech (Shanghai, China).

In this study, lipopolysaccharide (LPS, #L2880) and MIF antagonist (ISO-1, #475837) were bought from Sigma-Aldrich (Missouri, USA). Cycloheximide (CHX, #HY-12320) and 3-Deazaadenosine (DAA, #HY-W013332) were purchased from MedChemExpress (MCE, New Jersey, USA). VX765 (#inh-vx765i) was purchased from Invivogen (California, USA). The sh-IGF2BP1, sh-METTL3, sh-MIF, sh-E2F1, and sh-NC were obtained from Genepharma company (Shanghai, China). Lentiviruses expressing sh-IGF2BP1 and nonsense control (NC) were purchased from Tsingke (Nanjing, China). The E2F1 overexpression vector was constructed using p-ENTER, while the overexpression plasmids for MIF or METTL3 were constructed in pcDNA3.1.