Sirna design software

Human UC cell lines T24T and RT4 were cultured (see above) to 70% confluence and transfected using lipofectamine 2000 (Invitrogen) with siRNAs specific for PKM1 (corresponding to exon 9 sequence of the human PKM gene), PKM2 (corresponding to exon 10 of the human PKM gene) or PKM (corresponding to regions common to PKM1 and PKM2). The designations, siRNA sequences and their efficiency ranking scores (based on Web-based Invitrogen siRNA design software) were: (1) for PKM1, PKM1-a: 5’-GCGUGGAGGCUUCUUAUAAdTdT-3’ (4 stars); PKM1-b: 5’-CGUGGAGGCUUCUUAUAAGdTdT-3’ (3.5 stars); PKM1-c: 5’-GAGGCUUCUUAUAAGUGUU dTdT-3’ (2.5 stars)); (2) for PKM2, PKM2-a: 5’-CCAUAAUCGUCCUCACCAA dTdT-3’ (4 stars); PKM2-b: 5’-AGGCAGAGGCUGCCAUCUA dTdT-3’ (3.5 stars); PKM2-c: 5’-GCCAUAAUCGUCCUCACCA dTdT-3’ (2.5 stars); (3) for PKM, PKM-T-a: 5’-GCUGUGGCUCUAGACACUAdTdT-3’ (5 stars); and PKM-T-b: 5’-GGACCUGAGAUCCGAACUGdTdT-3’ (5 stars). siRNA specific for EGFP (5’-CUUACGCUGAGUACUUCGA dTdT-3’) was used as a negative control. The specificity of the siRNAs and the extent of target knockdown were determined by RT-PCR using primers specific for PKM2 or PKM1. Twenty-hours after transfection, the cells were collected and subject to WST-1 cell proliferation assay (Sigma-Aldrich, St. Louis, MO).

According to mRNA sequence of human BMP2 gene (NM_001200), siRNA (sense: 5′‐GCAACAGCCAACUCGAAAU dTdT‐3′; Anti‐sense: 5′‐AUUUCGAGUUGGCUGUUGC dTdT‐3′) was designed using a siRNA design software (Invitrogen Inc., Carlsbad, CA, USA). The target sequence corresponding to the siRNA sequence was 19 bases and the G/C ratio was close to 50%. A homology comparison verified that the siRNA had no common sequence with other genes. The sense sequence of siRNA was added by two TT prominent ends in the 3′ end. The chemical synthesis and modification were performed by Guangzhou RiboBio Co., Ltd. (Guangzhou, China).

Small INTERFERing RNA (siRNA) molecules were designed from the nucleotide sequence obtained from P. rapae using Invitrogen siRNA design software (http://rnaidesigner.thermofisher.com/rnaiexpress/) (Supplementary file 1B) and were synthesized by Invitrogen. siRNA for the negative control was used in transfection experiments as a control. Hemocytes were isolated as described above and were attached to the surface of 96-well tissue culture plates and incubated in Grace’s medium. The monolayers of hemocytes were treated with 2.4 ng siRNA and 0.7 μl siRNA transfection reagent INTERFERin (Polyplus-transfection SA, France) according to the instructions from the manufacturer, and incubated at 27°C.

IL-1β and TNF-α siRNAs were designed using siRNA design software of Invitrogen Company, and their homology was blasted in GenBank sequence database to confirm the specificity of the sequences, so as not to cause the silencing of other similar genes. The specific RNA interference fragments targeting IL-1β and TNF-α were synthesized by Invitrogen (sequences are shown in Table 1). The two siRNAs were dissolved and mixed with Entranster^TM-R4000 (Engreen Biosystem Co., Ltd. Beijing, China). The final concentration of both siRNAs was 120 nmol/L for injection in the further therapy experiments.