Mirna inhibitor nc

The circLOC101928570 overexpression plasmids and empty vector were constructed and designed by GeneChem (Shanghai, China). The miR-150-3p mimics, miR-150-5p mimics, miR-150-3p inhibitor, miR-150-5p inhibitor, miRNA mimics NC, and miRNA inhibitor NC were chemically synthesized by Riobio (Guangzhou, China). The pCDH-CMV-MCSEF1- GFP + Puro (CD513B-1) vector was used as a negative control plasmid, and the pCDH-MYB plasmid was fragmented and inserted into the pCDH-CMV-MCSEF1-GFP + Puro (CD513B-1) vector with BamHI and NotI restriction sites. The vector construction results were verified by direct sequencing. The sequence used was 5 ‘-CCGGAATTCCGGGAAAGCGTCACTTGGGGAAAA-3’. PLKO.1-puro (Addgene plasmid # 8453) was used to design short hairpin RNA, and the cells were transfected with Lipofectamine 3000 (Invitrogen, United States). The transfection process lasted 48 h.

hBMSCs were seeded in the 6-well plate at a density of 6×10⁴, and the cells reached 70% confluence after about 48 h. According to the manufacturer’s instructions, miR-619-5p mimic and miRNA mimic NC (RiboBio, Guangzhou, China) were transfected with Lipofectamine 3000 transfection reagent (Invitrogen, US) at a concentration of 50 nM, and miR-619-5p inhibitor, miRNA inhibitor NC (RiboBio, Guangzhou, China) at a concentration of 100 nM. RNA was extracted 24 h after transfection, the protein was extracted 48 h for detection, and osteogenic induction solution was added for osteogenic induction differentiation. Transfection was performed again after 3 days. MiR-619-5p mimic and miR-619-5p inhibitor sequence are listed in Supplementary Table S5.

Liver cancer cells were inoculated in culture dishes 24h prior to transfection. For enhance or decreased expression of HOTAIR and miR-145-5p. Short hairpin RNA (shRNA) (5'-GAACGGGAGTACAGAGAGA-3') sequences targeting human HOTAIR and a non-target sequence (sh-NC) (5'-TTCTCCGAACGTGTCACGT-3') were constructed by GenePharma (Shanghai, China). A full-length human HOTAIR expression vector was purchased from Addgene (LZRS-HOTAIR, #26110). The negative control for LZRS-HOTAIR is LZRS vector. Si-NC (sence 5'-UUCUCCGAACGUGUCACGUTT-3', antisense 5'-ACGUGACACGUUCGGAGAATT-3'), Si-EZH2 (sense 5'-GCUAAGGCAGCUGUUUCAGTT-3', antisense 5'-CUGAAACAGCUGCCUUAGCTT-3'), miR-145 mimics (sence 5'-GUCCAGUUUUCCCAGGAAUCCCU-3', antisence 5'-CAGGUCAAAAGGGUCCUUAGGGA-3'), miRNA mimics NC (sence 5'-UUUGUACUACACAAAAGUACUG-3', antisense 5'-AAACAUGAUGUGUUUUCAUGAC-3'), miR-145 inhibitor (5'-AGGGAUUCCUGGGAAAACUGGAC-3'), and miRNA inhibitor NC (5'-CAGUACUUUUGUGUAGUACAAA-3') were synthesized from Ribobio (Guangzhou, China). NUAK1 inhibitor (HTH-01-015) was purchased from MCE (Shanghai, China). The vectors and microRNAs were transfected into SNU-387 and HepG2 cells using lipofectamine 3000 reagents following the instructions (invitrogen, USA).