Short hairpin rnas shrna

Four human LUAD cell lines, NCI-H1975, H1299, A549, and H23, and the human bronchial epithelioid cell line 16-HBE, were supplied by Procell (Wuhan, China). All cell lines were maintained in RPMI-1640 medium (Hyclone) supplemented with 10% FBS (S660JJ, BasalMedia, Shanghai, China) and 1% streptomycin/penicillin (P1400, Solarbio, Beijing, China) in a humidified atmosphere of 5% CO2 at 37 °C. Small interfering RNAs against CHCHD4 (si-CHCHD4-1, si-CHCHD4-2, and si-CHCHD4-3) and their negative control (si-NC) were purchased from RiboBio (Guangzhou, China). Short hairpin RNAs (shRNAs) for CHCHD4 (sh-CHCHD4), and shRNA negative control (sh-NC) were obtained from Genechem (Shanghai, China). The pcDNA3.1-USF1 vector, pcDNA3.1-MYC vector and their empty vector were constructed by Tsingke Biotechnology Co., Ltd. Transfection was performed using Lipofectamine 3000 (Invitrogen, USA) according to the manufacturer's instructions.

The ovarian epithelial cell line (IOSE80) and OC cell lines (A2780, HEY-T30, SKOV3, OVCAR-3, ES2, and OV-1063) were obtained from the American Type Culture Collection (USA). OC cells were cultured in DMEM containing 10% FBS at 37 °C, whereas IOSE80 cells were cultured in RPMI-1640 medium containing 10% FBS at 37 °C. Short hairpin RNAs (shRNAs) targeting circRNF144B, FBXL11, and their negative control (NC) were acquired from GeneChem (Shanghai, China) and subcloned into pSuper-retro-puro. MiR-342-3p mimic, inhibitor, and NC were also acquired from Genechem. Small interfering RNAs (siRNAs) targeting FBXL11 and NC were accessed from HANBIO (Shanghai, China). We constructed pCMV5-circRNF144B, pCMV5-HA-FBXL11, and pCMV5-flag-Beclin-1 to overexpress circRNF144B, HA-FBXL11, and Beclin-1. Polybrene reagent (Sangon Biotech Co., Ltd; Shanghai, China) was used to perform transfection. After transfection with shRNAs for 48 h, OC cells were selected for 10 days under puromycin treatment to obtain stably low-expressing cells; OC cells were selected for 14 days under geneticin reagent (G418) treatment to obtain high-expressing cells after transfection with plasmids for 48 h.

MiR-199a-3p and miR-199a-5p mimics and inhibitors were purchased from RiboBio Co. Ltd (Ribo, China) and short hairpin RNAs (shRNAs) knocking down DUSP5 and MAP3K11 were purchased from Shanghai Genechem Co. Ltd (Genechem, China) (Table 2). To overexpress DUSP5 and MAP3K11, a full-length coding sequence (CDS) was amplified, and the EcoRI-HF and XhoI sites (NEB, USA) were used to insert the CDS product into pCMV-myc vector (Invitrogen, NY, USA). Before transfection, 2*10⁵ cells were seeded into each well of 6-well plates. After 24 h incubation in growth medium without penicillin–streptomycin, the cells were transiently transfected with miRNA mimics and inhibitors using riboFECT™ CP (Ribo, China), or with plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA). The cells were incubated for an additional 24–48 h for the following experiments.Table 2

The target sequence of DUSP5-shRNAs and MAP3K11-shRNAs

Name	Target sequence	Start site	GC (%)
DUSP5-shRNA1#	ctGAGTGTTGCGTGGATGTAA	620	47.37
DUSP5-shRNA2#	ccTGTCCTTCTGTGTGCTTAT	2213	42.11
DUSP5-shRNA3#	gaTAGGCCATTTGCAGACACT	1224	47.37
MAP3K11-shRNA1#	atTGAGAGTGACGACATGGAG	1250	52.63
MAP3K11-shRNA2#	tcTTCCCGTCCAACTATGTGT	774	47.37
MAP3K11-shRNA3#	ctTAGGAGGAGTCACAGCATA	3071	47.37