Sirna oligonucleotides

The small interfering RNA (siRNA) oligonucleotides targeting MIB2 were designed and synthesized by RiboBio (Guangzhou, China). Cells were seeded in the 6-well plates a day before the transfection in 60–70% confluence. Cells were transfected with siRNAs by Lipofectamine 2000 reagents (Invitrogen, USA) in medium with no FBS. The cells used for proliferation, migration, invasion assays were validation by qPCR and western blot after 48 h transfection. Sequences of the siRNAs are shown below:
Si-MIB2#1: GCTACATGCACAACAAGCA.
Si-MIB2#2: GGAGGTGCCAAACATCGAT.

The siRNA oligonucleotides targeting IFITM3 and universal negative control siRNA were designed and synthesized from Ribobio (Guangzhou, China), their sequences are: siIFITM3-1: 5′-CCCACGUACUCCAACUUCC [dT][dT]-3′, siIFITM3-2: 5′-UGUCCAAACCUUCUUCUCU [dT][dT]-3′. Cells were transfected with siRNAs (50 nM each well) using RNAiMax (Invitrogen, Carlsbad, CA, USA) following the manufacturer’s instructions. Transfected cells were treated with IFN-α2b (10,000 U/mL) or medium alone for 24 h and then infected with 1 PFU/cell of VTT-EGFP. At 24 h postinfection, the percentage of infected cells was determined by flow cytometry. Alternatively, IFITM3 expression or silencing was confirmed by Western blot.

Small interfering RNA (siRNA) oligonucleotides targeting PLXNC1 were designed and synthesized by RiboBio (Guangzhou, China). Cells were transfected with siRNAs using the Lipofectamine RNAiMAX reagent (Invitrogen) at a final concentration of 50 nM. Cells were used for RNA extraction, proliferation, migration, and immunoblotting assays after transfection for 48 h. The sequences for the PLXNC1 siRNAs used are listed in Table S1.

The H9c2 cells (ATCC, Manassas, VA, USA) were cultured in high-glucose Dulbecco’s modified Eagle’s medium (HyClone, Cat# SH30022.01, Logan, UT, USA) containing 10% fetal bovine serum and 1% penicillin/streptomycin at 37 °C in an incubator with 5% CO₂. For the ISO-induced cell model, the H9c2 cells were incubated with 30 μM ISO in a serum-free medium for 36 h.
Small interfering RNA (siRNA) oligonucleotides specific to circCacna1c and negative control siRNA (si-NC) were designed and synthesized by RiboBio (Guangzhou, China). The siRNA sequence for circCacna1c was 5′-UCCCAUAGUUGGAACCAGG-3′. The mmu-miR-29b-2-5p mimic (miR-mimic) and their corresponding negative controls (NC-mimic) were purchased from RiboBio. The sequence for miR-mimic was 5′-CUGGUUUCACAUGGUGGCUUAGAUU-3′. The siRNAs or miRNA mimics were transfected into H9c2 cells using the riboFECTTM CP transfection reagent (RiboBio, Cat# C10511-1, Guangzhou, China), according to the manufacturer’s instructions.

Small interfering RNA (siRNA) oligonucleotides which were specific to ADAM8 and Smad2/3 obtained from RiboBio Co., Ltd. SW480 cells with higher endogenous ADAM8 expression (as shown in Figure 4A) were cultured in six‐well plates until 60% confluence and transfected with 50 nmol/L of the indicated siRNA using riboFECT™ CP Reagent (RiboBio) according to the manufacturer's instructions. The efficiency of gene knockdown was analysed by Western blotting and qRT‐PCR after 48 hours.

The information of cell lines was shown in Supplementary Materials and Methods. Specific knockdown of circTADA2A-E6 was achieved using three siRNA oligonucleotides designed and synthesized by Ribobio, Guangzhou, China to target the back-splice junction (Supplementary Table 6). To efficiently circularize a circRNA transcript in cells, the mature sequence for circTADA2A-E6 (chr17, 35800605–35800763) was synthesized and cloned into pLCDH-CMV-MCS-EF1-copGFP-puro (Geneseed; Guangzhou, China, Supplementary Figure S7a). A luciferase reporter incorporating the circTADA2A-E6 sequence (phRluc-circTADA2A-E6; Supplementary Figure S7b) was constructed by subcloning the circTADA2A-E6 fragment into the 3′-UTR downstream region of Renilla luciferase in the pciCHECK^TM-2 vector (Geneseed; Guangzhou, China). Primer sequences for phRluc-circTADA2A-E6 subcloning are shown in Supplementary Table 6.