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Construction of the Over-Expressing GPR183 Lentiviral Vector and the Effect on Apoptosis of Cutll1 Cells
Zhang Hang, Shu Yi, Lü Wenqiong, Zhang Jia, Zhang Hongyang, Tang Shi, Zou Lin*
Center for Clinical Molecular Medicine, Children’s Hospital, Chongqing Medical University, Key Laboratory of Pediatrics in Chongqing, Ministry of Education Key Laboratory of Child Development and Disorders, Key Laboratory of Pediatrics in Chongqing, Chongqing International Scienceand Technology Cooperation Center for Child Development and Disorders, Chongqing 400014, China
Abstract: To construct the human GPR183 over-expressing lentiviral vector and investigate the effect of GPR183 on apoptosis of human T-ALL cell line Cutll1 cells， the human GPR183 cDNA sequence was amplified by PCR and linked to the lentivirus vector pEZ-Lv201 and the GPR183-overexpressing lentivirus plasmid was constructed then lentivirus was packaged by using 293T cells. The Cutll1 cells were infected with lentivirus and the GPR183 expression level was detected by q-PCR and Western blot. The CCK-8， PI and Annexin V staining were used to study the growth， cell cycle and apoptosis of the Cutll1 cell with (Cutll1-GPR183) or without (negative control) GPR183 overexpression. The plasmid confirmation PCR and sequencing data indicate the human GPR183- overexpressing lentivirus plasmid was constructed. The GFP+ Cutll1 cells were detected after lentivirus infection. Consistently， the q-PCR and Western blot results showed the expression of GPR183 was elevated in Cutll1-GPR183 cells (P<0.01). The CCK-8 and Annexin V staining data showed the proliferation of Cutll1-GPR183 was inhibited (P<0.001) and the apoptosis of Cutll1-GPR183 was significant increase (P<0.01)， while the cell cycle of two groups had no difference. Overall， our results indicated that GPR183 could inhibit the growth and promote the apoptosis of T-ALL Cutll1 cells and laid the foundation for the follow-up study of mechanisms.
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