Sh circhipk3

Specific small interfering RNA (siRNA) against circHIPK3 (si-circHIPK3) and siRNA scrambled control (si-NC), and short hairpin RNA objecting circHIPK3 (sh-circHIPK3) and shRNA scrambled control (sh-NC) were purchased from GenePharma (Shanghai, China). The miR-876-5p mimic (miR-876-5p) and its negative control (miR-NC), and miR-876-5p inhibitor (anti-miR-876-5p) and its negative control (anti-miR-NC) were designed and acquired from Thermo Fisher Scientific (Waltham, MA, USA). HGC-27 and AGS cells were transfected with plasmids or oligonucleotides using Lipofecamine2000 (Invitrogen, Carlsbad, CA, USA). The sequences for si-circHIPK3, si-NC, sh-circHIPK3 and sh-NC were as follows: si-circHIPK3: 5′-CUACAGGUAUGGCCUCACA-3′, si-NC: 5′-UUCUCCGAACGUGUCACGUTT-3′, sh-circHIPK3: 5′-ccggCUACAGGUAUGGCCUCACAttcaagagaTGTGAGGCCATACCTGTAGTTTTTTGGTACC-3′, sh-NC: 5′- ccggUUCUCCGAACGUGUCACGUTTttcaagagaAATCGTGACACGTTCGGAGAATTTTTTGGTACC-3′. For overexpression of PIK3R1 (PIK3R1), the primers were used for amplification and then cloned in the mammalian expression pcDNA3.1 vector (Invitrogen), PIK3R1 F-5′-CCGGAATTCATGAGTGCTGAGGGGTACCAGTAC-3′; and R-5′-CCGCTCGAGATCGCCTCTGCTGTGCATATATA-3′.

The vector used to construct the lentiviral vectors was pLV-enhanced GFP (EGFP)-N (lentivirus gene overexpression vector) and pSIH1-H1-copGFP (lentivirus gene silencing vector of shRNA fluorescent expression). The lentiviral vectors of lenti-CircHIPK3, lenti-LncGAS5, lenti-GATA-3, sh-CircHIPK3 (lenti-siCircHIPK3), and sh-LncGAS5 (lenti-siLncGAS5) were constructed by GenePharma Technology (Shanghai, China). Lentiviral packaging was performed using 293T cells. The cells were cultured in RPMI 1640 complete medium containing 10% FBS and sub-cultured every other day. The virus was collected and CD4⁺ T cells were classified into the following groups according to different transfections: OVA + lenti-GFP (transfected with empty vector), OVA + lenti-CircHIPK3 (transfected with CircHIPK3 overexpression lentiviral vector), OVA + lenti-CircHIPK3 (transfected with LncGAS5 overexpression lentiviral vector) or OVA + lenti-GATA-3 (transfected with GATA-3 overexpression lentiviral vector). For the administration of lentiviral vectors, mice were intranasally administered with 2 × 10⁶ IFUs of the lentiviral vectors or empty lentiviral vectors 2 days before day 0^{14 (link)}.