J-R Yang1, M-X Shi, Y Zeng. 1. First Affiliated Hospital of Kunming Medical University, Yunnan Hematology Research Center, Kunming, China. wuvh163@gmail.com.
Abstract
OBJECTIVE: The long noncoding RNA (lnc) HAND2-AS1 is down-regulated and microRNA-1275 (miR-1275) is up-regulated in many types of cancers. However, their expressions and the relationship between HAND2-AS1 and miR-1275 in chronic myeloid leukemia (CML) remain unknown and to be further investigated. PATIENTS AND METHODS: Bone marrow samples from 30 CML patients and 10 healthy donors were collected; HAND2-AS1 and miR-1275 were detected by RT-PCR. The correlation between HAND2-AS1 and miR-1275 was analyzed. After lentiviral (LV) HAND2-AS1 and miR-1275 inhibitors were respectively transfected into KCL22 and K562 cells, the expressions of HAND2-AS1 and miR-1275 were detected by RT-PCR. MTT assay was performed to evaluate cell viability and mRNA and proteins levels of Bcl-2, Caspase-3, MMP-2, MMP-9 were detected by RT-PCR and Western blot (WB). Luciferase reporter assay was performed to explore the binding site of HAND2-AS1 and miR-1275. RESULTS: Results showed that HAND2-AS1 was significantly downregulated than healthy control (p<0.05), and HAND2-AS1 expressions on stages of AP and BP were much lower than that of CP (p<0.05). The miR-1275 expression was significantly upregulated than healthy control (p<0.05), and the expressions in stages of accelerated phase (AP) and blast phase (BP) were much higher than that in the stage of chronic phase (CP) in CML (p<0.05). Furthermore, HAND2-AS1 was negatively correlated with miR-1275 in CML, but not in healthy control (p<0.05). After lentiviral HAND2-AS1 transfection, the HAND2-AS1 expression was significantly up-regulated while miR-1275 was significantly down-regulated (p<0.05). Then, the cell proliferation was inhibited after 72 h. Furthermore, the mRNA and protein levels of Bcl-2, MMP-2 and MMP-9 were significantly down-regulated (p<0.05), while the expression of Caspase-3 was significantly up-regulated (p<0.05). Luciferase reporter assay showed that HAND2-AS1 was a target gene of miR-1275. And after treating with miR-1275 inhibitor, HAND2-AS1 was significantly upregulated (p<0.01) and cell proliferation was inhibited (p<0.01). Furthermore, the expressions of Bcl-2, MMP-2, and MMP-9 were significantly decreased, while Caspase-3 was significantly increased (p<0.01). CONCLUSIONS: HAND2-AS1 was downregulated and miR-1275 was upregulated in CML, and HAND2-AS1 inhibited proliferation and promoted apoptosis of CML cells by sponging with microRNA-1275, which might be a novel therapeutic target for CML.
OBJECTIVE: The long noncoding RNA (lnc) HAND2-AS1 is down-regulated and microRNA-1275 (miR-1275) is up-regulated in many types of cancers. However, their expressions and the relationship between HAND2-AS1 and miR-1275 in chronic myeloid leukemia (CML) remain unknown and to be further investigated. PATIENTS AND METHODS: Bone marrow samples from 30 CMLpatients and 10 healthy donors were collected; HAND2-AS1 and miR-1275 were detected by RT-PCR. The correlation between HAND2-AS1 and miR-1275 was analyzed. After lentiviral (LV) HAND2-AS1 and miR-1275 inhibitors were respectively transfected into KCL22 and K562 cells, the expressions of HAND2-AS1 and miR-1275 were detected by RT-PCR. MTT assay was performed to evaluate cell viability and mRNA and proteins levels of Bcl-2, Caspase-3, MMP-2, MMP-9 were detected by RT-PCR and Western blot (WB). Luciferase reporter assay was performed to explore the binding site of HAND2-AS1 and miR-1275. RESULTS: Results showed that HAND2-AS1 was significantly downregulated than healthy control (p<0.05), and HAND2-AS1 expressions on stages of AP and BP were much lower than that of CP (p<0.05). The miR-1275 expression was significantly upregulated than healthy control (p<0.05), and the expressions in stages of accelerated phase (AP) and blast phase (BP) were much higher than that in the stage of chronic phase (CP) in CML (p<0.05). Furthermore, HAND2-AS1 was negatively correlated with miR-1275 in CML, but not in healthy control (p<0.05). After lentiviral HAND2-AS1 transfection, the HAND2-AS1 expression was significantly up-regulated while miR-1275 was significantly down-regulated (p<0.05). Then, the cell proliferation was inhibited after 72 h. Furthermore, the mRNA and protein levels of Bcl-2, MMP-2 and MMP-9 were significantly down-regulated (p<0.05), while the expression of Caspase-3 was significantly up-regulated (p<0.05). Luciferase reporter assay showed that HAND2-AS1 was a target gene of miR-1275. And after treating with miR-1275 inhibitor, HAND2-AS1 was significantly upregulated (p<0.01) and cell proliferation was inhibited (p<0.01). Furthermore, the expressions of Bcl-2, MMP-2, and MMP-9 were significantly decreased, while Caspase-3 was significantly increased (p<0.01). CONCLUSIONS:HAND2-AS1 was downregulated and miR-1275 was upregulated in CML, and HAND2-AS1 inhibited proliferation and promoted apoptosis of CML cells by sponging with microRNA-1275, which might be a novel therapeutic target for CML.
Authors: Priyanka Gokulnath; Tiziana de Cristofaro; Ichcha Manipur; Tina Di Palma; Amata Amy Soriano; Mario Rosario Guarracino; Mariastella Zannini Journal: Int J Mol Sci Date: 2020-06-05 Impact factor: 5.923