Lipofectamine tm 2000 transfection kit

The sequences for the siRNA targeting circ_0003865 were sense: 5′-AGUUACACAGAUUCAGAUCCAUU-3′, antisense: 5′-AAUGGAUCUGAAUCUGUGUAACU-3′; its negative control, sense: 5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGAGAATT-3′; the miR-3653-3p mimics, sense: 5′-CUAAGAAGUUGACUGAAG-3′, antisense: 5′-UCAGUCAACUUCUUAGUU-3′; and mimics NC (the same as negative control); the miR-3653 inhibitors: 5′-CUUCAGUCAACUUCUAG-3′ and inhibitor NC: 5′-CAGUACUUUUGUGUAGUACAA-3′. All oligos were synthesized by the GenePharma Biotech company (Shanghai, China). As designated, the above sequences were transfected into human BMSCs using the Lipofectamine TM 2000 transfection kit (Invitrogen, USA) according to the manufacturer’s instructions. For overexpression of circ_0003865, the sequences of circ_0003865 were synthesized and ligated into the LV5 plasmid to construct the recombinant LV5-circ_0003865 vector. LV5 and LV5-circ_0003865 lentiviruses were packaged by the Vigene Biosciences company (Jinan, Shandong, China) and infected into BMSCs according to the manufacturer’s instructions.

All expression vectors were designed and synthesized by Shanghai Gene Pharma Co., Ltd., including miR-21 mimics, si-FGF1, sh-FGF1, pmirGLO-FGF1-3′UTR wild type (Wt), pmirGLO-FGF1-3′UTR mutant type (Mut), and blank vector, pmirGLO-NC. At 24 h before transfection, the cells were digested with trypsin, and then, the cells were transfected with expression vectors when the cell fusion reached about 80% according to specific operation steps in the kit instructions. Subsequently, the cells were cultured in an incubator with 5% CO₂ at 37°C for 48 h, and the culture medium was replaced every 6 h. Quantitative real time polymerase chain reaction (qRT-PCR) was adopted to determine the transfection results. Cells not transfected were used as a control group. The Lipofectamine TM2000 transfection kit (item number: 35050) was purchased from the Invitrogen Company in the United States.

Human ovarian cancer epithelial cell line OVCAR3 and human normal ovarian epithelial cell line IOSE80 were purchased from ATCC cell bank in the United States. Cell culture reagents (DEME medium, fetal bovine serum, streptomycin penicillin, trypsin) were purchased from Gibco, USA. Cell proliferation activity assay Kit CCK-8 was purchased from Tongren Institute of Chemistry, Japan. miRNA extraction kit, miRNA reverse transcription and fluorescence quantitative kit, and Lipofectamine TM 2000 transfection kit were all purchased from Invitrogen, USA. Mir-30b-3p, mimics and mimic control miRNAs were synthesized by Shanghai Gemar Pharmaceutical Technology Co., LTD., China. Mir-30b-3p and U6 primers were designed and synthesized by bioengineering (Shanghai) Co., LTD., China. ECL chemiluminescence reagent and BCA protein concentration detection kit were purchased from Shanghai Biyuntian Biotechnology Co., LTD., China. The dual luciferase report detection system is the product of Promega, USA.

The interactions between miR-3653-3p and circ_0003865 or the 3′ UTR region of the GAS1 gene were verified using the Dual-Luciferase Reporter Assay System (Promega) according to the manufacturer’s instructions. Briefly, the circ_0003865 sequences and its mutant version, as well as the GAS1 3′ UTR sequences and its mutant version, were amplified by RT-PCR and were ligated into PmirGLO plasmids. The plasmids were transfected into 293T cells using the Lipofectamine TM 2000 transfection kit (Invitrogen, USA) according to the manufacturer’s instructions, along with miR-3653-3p mimics or a negative control. Subsequently, the cells were lysed using the Passive Lysis Buffer and analyzed using the GloMax-20/20 luminometer (#E5311; Promega). At least three biological replicates were conducted for comparison of luciferase activities between groups.