Pmscvpuro

Plasmid pDONR221-SLC3A2_STOP was a gift from RESOLUTE Consortium & Giulio Superti-Furga (Addgene plasmid # 161379, RRID:Addgene_161379) (Girardi et al., 2020 (link)). Plasmid pENTR223-1 GCN2 (NM_001013703) was purchased from Transomic Technologies and pLX303 was a gift from David Root (Addgene plasmid # 25897, RRID:Addgene_25897). GCN2 coding sequence was subcloned into the lentiviral vector pLVX-IRES-Hygromycin (Clontech, Cat. #632185) using an In-Fusion HD Cloning plus kit (TaKaRa, Cat. #638920) according to the manufacturer’s protocol yielding plasmids pLVX-GCN2-hygro. The SLC3A2 (4F2) coding region was subcloned into the lentiviral vector pLX303 by Gateway cloning using Gateway LR Clonase II Enzyme Mix (Invitrogen, Cat. #11791-020) according to the manufacturer’s protocol yielding plasmid pLX303-4F2-blast. Plasmid pMSCV-GADD34-puro contains the human PPP1R15A (GADD34, NM_014330.5) gene subcloned into the NcoI/EcoRI sites of the retroviral vector pMSCV-puro (Clontech Laboratories, Mountain View, CA).

A DNA fragment containing the hsa-miR-214 precursor with 300 bp flanking sequence of each side was amplified into retroviral transfer plasmid pMSCV-puro (Clontech, Palo Alto, CA). Retroviral production and infection were performed as standard procedure. The open reading frames (ORFs) of CDK3, CDK6 and E2F2 generated by PCR amplification were cloned into the mammalian expression vector pcDNA3.1 (Invitrogen). 3′-UTRs of CDK3, CDK6 and E2F2 were amplified and then cloned into the downstream of the luciferase gene in a modified pGL3 control vector (Promega, Madison, WI). miR-214 mimics, anti-miR-214 oligonucleotides and their relative control oligonucleotides were purchased from Invitrogen. For depletion of genes, FlexiTube GeneSolution were used to knockdown for CDK3(GS1018, GIAGEN), CDK6(GS1021, GIAGEN), E2F2(GS1870, GIAGEN). QGY-7703/vector cells and QGY-7703/miR-214 HCC cells were infected with PMSCV-neo-luc2(QGY-7703/vector-luc cells and QGY-7703/miR-214-luc) to be tracked in vivo through bioluminescent imaging assay.

A cDNA including the hsa-miR-188 precursor with 500 bp of flanking genomic sequence on each side was cloned into the retroviral transfer plasmid pMSCV-puro (Clontech, Mountain View, CA, USA). The open reading frame (ORF) of PTEN was inserted into the mammalian expression vector pcDNA 3.1 (Invitrogen). The 3′-UTR of PTEN was placed downstream of the luciferase gene in a pmirGLO dual-luciferase miRNA target expression vector (Promega, Madison, WI, USA). The primers used are listed in Additional file 4: Table S3. MiR-188-5p mimic, miR-188-5p inhibitor, and their control oligonucleotides were obtained from GenePharma (Shanghai, China). PTEN-TSB, a customized target site blocker (TSB) to block miR-188-5p binding to PTEN, and non-targeting control TSB LNA oligonucleotides were purchased from Exiqon (Vedbaek, Denmark). Transfection of plasmids or oligonucleotides was performed using X-tremeGENE reagent (Roche Diagnostics, GmbH, Mannheim, Germany) according to the manufacturer’s instructions.

Flag-RNH1 was further sub-cloned into retroviral vector pMSCVpuro (Clontech). Retroviral vector pMSCVpuro-Flag–RNH1 was co-transfected with the helper plasmids VSV-G and Hit60 into HEK293T cells using PEI transfection reagent. Culture supernatants containing recombinant viral particles were harvested and used to infect THP1 cells. To establish stable cell lines, THP1 cells were selected with puromycin (5 μg ml⁻¹) 3 d after infection.

The miR-500a-3p expression plasmid was generated by cloning the genomic pre–miR-500a-3p gene, with a 300-bp sequence on each flanking side, into retroviral transfer plasmid pMSCV-puro (Clontech Laboratories Inc.) to generate plasmid pMSCV- miR-500a-3p. pMSCV- miR-500a-3p was cotransfected with the pIK packaging plasmid in 293FT cells using the standard calcium phosphate transfection method, as previously described [25 (link)]. Thirty-six hours after the co-transfection, supernatants (MOI: 20) were collected and incubated with cells to be infected for 24 h in the presence of polybrene (2.5 μg/ml). After infection, puromycin (1.5 μg/ml) was used to select stably transduced cells over a 10-day period. The 3’UTR of SOCS2, SOCS4 and PTPN11 were amplified and cloned downstream to the luciferase gene in a modified pGL3 control vector (Promega, USA), and the list of primers used in clone reactions was presented in Additional file 1: Table S1. Anti-miR-500a-3p, small interfering RNA (siRNA) for SOCS2, SOCS4 and PTPN11 knockdown and respective control RNA (50 nM) were synthesized and purified by RiboBio. The sequence of anti-miR-500a-3p is cagaauccuugcccaggugcau. Transfection of miRNAs, siRNAs, and plasmids was performed using Lipofectamine 3000 (Life Technologies) according to the manufacturer’s instructions.