Objective To explore the mechanism of long-chain non-coding RNA (LncRNA) MIR22HG to regulate cytotoxic T-lymphocyte protein 4 (CTLA4) through microRNA-9-3p (miR-9-3p) to inhibit prostate cancer.
Methods VCAP, PC3, LNCap and DU145 cells were cultured from Samples of 26 patients with prostate cancer and 26 patients with prostatic hyperplasia. MIR22HG and miR-9-3p mRNA levels in prostate cancer tissue, prostate hyperplasia tissue and 4 types of prostate cancer cells were determined by quantitative real-time polymerase chain reaction (qRT-PCR). The cells with higher expression of MIR22HG and miR-9-3p mRNA were screened. The selected cells were divided into OE-CTLA4 (CTLA4 overexpression group), OE-MIR22HG (MIR22HG overexpression group), miR-9-3p mimic (miR-9-3p overexpression group), OE-MIR22HG+miR-9-3p mimic (MIR22HG and miR-9-3p co-overexpression group), OE-NC+NC mimic (transfection control group) and NC mimic (miR-9-3p overexpression control group). TargetScan database and luciferase reporter gene experiments were used to verify whether MIR22HG and miR-9-3p and miR-9-3p and CTLA4 had targeted inhibition relationships. The mRNA expression of MIR22HG and miR-9-3p and the protein expression of CTLA4 were detected by qRT-PCR and Western blot. A total of 96 nude mice were taken, and they were divided into OE-NC+NC mimic group (transfection control), OE-MIR22HG group (MIR22HG overexpression) and OE-MIR22HG+miR-9-3p mimic group (MIR22HG and miR-9-3p co-overexpression) according to the intervention methods, with 32 mice in each group. The tumors were transplanted in situ and the changes in body mass and volume of the tumors were measured each week for 4 weeks after inoculation.
Results Among adenocarcinoma cells VCAP, PC3, LNCap and DU145 cells, PC3 cells showed a relatively low MIR22HG mRNA and a relatively high miR-9-3p mRNA, so PC3 cells were selected for the study. The MIR22HG in PC3 cells was successful overexpressed, luciferase reporter gene assay verified the targeted inhibition relationship between MIR22HG and miR-9-3p, and the expression of miR-9-3p in the overexpression of MIR22HG was significantly down-regulated (P < 0.05). TargetScan database predicted the targeting relationship between immune gene CTLA4 and miR-9-3p, luciferase reporter gene assay verified the targeted inhibition relationship between miR-9-3p and CTLA4, and the expression of CTLA4 overexpressing miR-9-3p was significantly up-regulated (P < 0.05). The weight of subcutaneous tumor in nude mice overexpressed MIR22HG was significantly decreased (P < 0.05). The weight and volume of subcutaneous tumors after OE-MIR22HG+miR-9-3p mimic group showed no significant differen compared with those of the OE-NC+NC mimic group (P>0.05).
Conclusion There is a negative regulatory relationship between MIR22HG and miR-9-3p, and there is a negative regulatory relationship between miR-9-3p and CTLA4. MIR22HG/miR-9-3p may achieve the purpose of immunotherapy for prostate cancer by inhibiting CTLA4.
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