Mir 1246 inhibitor

The synthetic miR-1246 mimic (forward, 5′- AAUGGAUUUUUGGAGCAGG - 3′; reverse, 5′- UGCUCCAAAAAUCCAUU TT- 3′), miR-1246 inhibitor (5′- CCUGCUCCAAAAAUCCAUU- 3′), mimic control (forward, 5′-UUCUCCGAACGUGUCACGUTT-3′; reverse, 5′-ACGUGACACGUUCGGAGAATT-3′) and inhibitor control (5′-UUGUACUACACAAAAGUACUG-3′) were purchased from GenePharma (GenePharma Inc., Shanghai, China). The siRNAs for CADM1 was synthesized (Invitrogen Inc.) and the sequence was list in Additional file 1: Table S1. The nucleotide sequences were delivered into human HCC cell lines by Amaxa Nucleofector® following the manufacturer’s instructions. Briefly, cell pellets were collected by 90 × g centrifugation at room temperature for 10 min and resuspended in 100 μl of Nucleofector Solution to a final concentration of 1 × 10⁶ cells/100 μl. Each 100 μl sample was transferred into an Amaxa-certified cuvette and underwent nucleofection using the appropriate Nucleofector program. The program for transfecting HepG2 and SMMC7721 was T028. After nucleofection, each sample was immediately transferred from the cuvette to a culture plate in 2 ml of medium [14 (link)].

For the miR-1246 functional analysis, miR-1246 mimics and NC mimics (GenePharma, Shanghai, China) were transfected into chemo-sensitive K562 and HL-60 cells using Lipofectamine 2000 (Invitrogen; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocols. In the same way, chemo-resistant K562/ADM and HL-60/RS cells were transfected with miR-1246 inhibitor or NC inhibitor (GenePharma, Shanghai, China). 48 h after transfection, they were collected and subjected for further analysis.

EJ5-GFP plasmids were linearised by HindIII enzyme digestion and pCherry vectors were used to detect the
double-strand break (DSB) repair efficiency of nonhomologous end joining
(NHEJ).^{33 –35 (link)} miR-1246 mimic (sense:
5′-AAUGGAUUUUUGGAGCAGG-3′, antisense: 5′-UGCUCCAAAAAUCCAUUUU-3′); miR-NC (sense:
5′-UUCUCCGAACGUGUCACGUTT-3′, antisense: 5′-ACGUGACACGUUCGGAGAATT-3′); miR-1246
inhibitor (5′-CCUGCUCCAAAAAUCCAUU-3′); and inhibitor-NC
(5′-CAGUACUUUUGUGUAGUAGUACAA-3′) were purchased from GenePharma (Shanghai,
China).
Antibodies used in this study were as follows: anti-53BP1 (Abcam,
Cambridge, UK), anti-LIG4 (12695-1-AP; Proteintech Group Inc., Rosemont, IL, USA),
anti-CD63 (H-193, sc-15363; Santa Cruz Biotechnology, Santa Cruz, CA, USA),
anti-TSG101 (ab133586; Abcam, Cambridge, MA, USA), anti-GAPDH (TA309157; Beijing
Zhongshan Jinqiao Biotechnology Co., Ltd., Beijing, China), and anti-β-actin
(TA-09; Beijing Zhongshan Jinqiao Biotechnology Co., Ltd.).