Mimic nc

The mimic NC and mimic miR-328-3p were obtained from Dharmacon (Lafayette, CO, USA), inhibitor NC and inhibitor miR-328-3p were obtained from Life Technologies. Oe-CPT1A, oe-NC and si CPT1A were purchased from GenePharm (Shanghai). The transfection sequences in each group are shown in Table 1. Cells were plated in six-well at 60–70% density and transduced using Lipofectamine 3000 transfection Kit (Invitrogen) following the manufacturer’s instructions.Table 1

The sequences of primers and oligonucleotides used in this study.

Primers
Hsa-miR-328-3p F	5’-CTGGCCCTCTCTGCCC-3’
Hsa-miR-328-3p R	5’-GTGCAGGGTCCGAGGT-3’
CPT1A F	5’-CTGGACAATACCTCGGAGCC-3’
CPT1A R	5’-AACGTCACAAAGAACGCTGC-3’
GAPDH F	5’-GTCAAGGCTGAGAACGGGAA-3’
GAPDH R	5’-AAATGAGCCCCAGCCTTCTC-3’
U6 F	5′-CUGGCCCUCUCUGCCCUUCCGU-3′
U6 R	5′-CTCGCTTCGGCAGCACATATA-3′

siRNAs targeting sequence
Si CPT1A 1#	5′-GGATGGGTATGGTCAAGATCT-3′
Si CPT1A 2#	5′-TGCGCCGATCGTGGCCCACCTT-3′

Firstly, the sequence fragment of LDOC1 3′-untranslated region (3′UTR) was artificially synthesized and introduced into the psiCHECK-2 vector (Promega, Madison, WI, USA). The mutation sites in the complementary sequence of seed sequences were designed on the wild type (WT) of LDOC1. Subsequently, all luciferase reporter plasmids, such as LDOC1 3′UTR-WT and LDOC1 3′UTR-mutant (MUT), were obtained. All aforementioned plasmids were co-transfected with miR-4532 mimic (2 nM, Dharmacon, Lafayette, CO, USA) or mimic-NC (2 nM, Dharmacon, Lafayette, CO, USA) into 4 × 10⁵ HEK-293 T cells (CRL-1415, Shanghai Xin Yu Biotech Co., Ltd., Shanghai, China) and CD34⁺ HSCs, respectively. After 48 h, the cells were lysed and the luciferase activity was determined using Dual-Luciferase Reporter Assay kits (RG005, Beyotime Biotechnology, Shanghai, China) in the Glomax20/20 luminometer fluorescence detector (Promega, Madison, WI, USA). The experiment was repeated three times to obtain the mean value [24 (link)].

The cervical cancer cells were plated in a 6-well plate at a density of 3 × 10⁵ cells/well. The cells were transfected with a negative control of siRNA (si-NC), siRNA targeting has-circ-0000515 (si-hsa-circ-0000515), negative control of miR-326 mimic (mimic-NC), miR-326 mimic, miR-326 inhibitor, negative control of miR-326 inhibitor (inhibitor-NC), si-hsa-circ-0000515 + miR-326 inhibitor, negative control of over-expression plasmid (OE-NC), over-expression plasmid of ELK1 (OE-ELK1), or OE-ELK1 + si-hsa-circ-0000515 using lipofectamine 2000 (Invitrogen), when the cell confluence reached 90%. PLenti-GIII-CMV was purchased from Applied Biological Materials (ABM, Canada). The miR-326 mimic, miR-326 inhibitor, si-hsa-circ-0000515, ELK1 OE-ELK1 and their corresponding negative controls (si-NC, mimic-NC, inhibitor-NC and OE-NC) were all purchased from Dharmacon (Lafayette, CO, USA). The sequences of the three siRNAs against hsa_circ_000515 are as follows: “GAGGTGAGTTCCCAGAGAA (siRNA sequence 1), CCGGAGCTTGGAACAGACT (siRNA sequence 2), CCTTTGCCGGAGCTTGGAA (siRNA sequence 3) and the sequence of si-NC is AAGTCGGGTCAAGAGAAGC. The medium was renewed 6 h after transfection. The cells were harvested 36 - 48 h after transfection.