Sh nc

XIST overexpression plasmid pcDNA-XIST and corresponding negative control pcDNA-NC, XIST interference plasmid sh-XIS #1, sh-XIS #2, sh-XIS #3, and corresponding negative control sh-NC, Antago mature rat miR-29b-3p, and corresponding negative control Antagomir-NC, NNMT interference plasmid sh-NNMT# 1, sh-NNMT# 2, sh-NNMT# 3, and corresponding negative control sh-NC were designed and synthesized by GenePharma (China). BMSCs were cultured for 24 h before transfection. pcDNA (1 μg/mL), shRNA (50 nM), or antago miRNA (100 nM) were transfected into BMSCs using Lipofectamine 2000 kit (Invitrogen, USA). Follow-up experiments were performed 48 h after transfection.

Small hairpin RNA (shRNA) lentiviral used for stable silencing of OLA1 (shOLA1) and the control non-targeting plasmid (shNC) were purchased from GenePharma (Nanjing, China) by inserting the following short-hairpin sequences into the pGLV3/H1/GFP/Puro vector:
5′-CCGGGAGGAAATGATTGGGCCCATTCTCGAGAATGGGCCCAAT CATTTCCTCTTTTTTG-3′ for sh-OLA1 and 5′-CCGGCAACAAGATGAAGAG CACCAACTCGAGTTGGTGCTCTTCATCTTGTTGTTTTTG-3′ for Small hairpin control (shNC). shRNA transfections and protocol were followed the recommendations by GenePharma (China). The shNC and shOLA1 vectors were transfected into MDA-MB-231 cells. The knockdown efficiency of the target gene was verified by qRT-PCR and western blot analysis.

To verify our hypothesis, we synthesized the following plasmids: short‐hairpin DNM3OS (sh‐DNM3OS) and its respective non‐targeting sequence (negative control; sh‐NC) (GenePharma, Shanghai, China). Cells were grown to 70%–80% confluence in 24‐well plates and transfected using Lipofectamine™ 3000 (Life Technologies Corporation, Carlsbad, CA) according to the manufacturer's instructions. Geneticin (G418; Sigma‐Aldrich, St. Louis, MO, USA) was used to select stably transfected cells. Three groups were designed to verify the effect of DNM3OS in httex1p‐Q74 cells: control, sh‐NC (transfected with empty plasmid) and sh‐DNM3OS.
miR‐196b‐5p mimics (mimic‐miR‐196b‐5p), miR‐196b‐5p inhibitors (inhibitor‐miR‐196b‐5p) and their respective non‐targeting sequences (mimic‐NC or inhibitor‐NC) were synthesized by GenePharma (Shanghai, China) and used to investigate the function of miR‐196b‐5p in httex1p‐Q74 cells. The httex1p‐Q74 cells were divided into five groups: control, mimic‐196b‐5p‐NC, mimic‐196b‐5p, inhibitor‐196b‐5p‐NC and inhibitor‐196b‐5p. To verify our hypothesis that DNM3OS functions by suppressing miR‐196b‐5p, httex1p‐Q74 cells were divided into five groups: control, sh‐DNM3OS‐NC + mimic‐miR‐196b‐5p‐NC, sh‐DNM3OS + mimic‐miR‐196b‐5p, sh‐DNM3OS‐NC + inhibitor‐miR‐196b‐5p‐NC and sh‐DNM3OS + inhibitor‐miR‐196b‐5p.

All cell transfections were performed by Lipofectamine 2000 reagent (Invitrogen, USA), including Sh-NC, Sh-METTL3#1, Sh-METTL3#2; Vector-OE, METTL3-OE; lncRNA METTL4-2-OE; Sh-NC, Sh-YTHDF1#1; and Sh-YTHDF1#2; (GenePharma, Shanghai, China). Cells were harvested at 48 h post-transfection for the following experiments.

The target sequence of SRC-3 for constructing lentiviral shRNA was: shSRC-3: 5′-AGACTCCTTAGGACCGCTT-3′, shNC: 5′-TTCTCCGAACGTGTCACGT-3′. HTR8/SVneo cells were transfected with shRNA and shNC (GenePharma, Shanghai, China), according to the manufacturer’s protocol. At 24 h after transfection, 5 µg/ml puromycin was added for three days to screen SRC-3-KD cells.