Role And Mechanism Of MiR-378 In The Proliferation Of Myelodysplastic Syndrome Cell

Objective To detect miR-378 expression level in the bone marrow samples of myelodysplastic syndrome(MDS) patients, construct the lentiviral vector overexpressing miR-378 to investigate the effect of miR-378 on the growth of MDS cell line SKM-1 both in vitro and in vivo. Further, explore the potential target gene of miR-378 participating in the pathogenesis of MDS.Methods1. For miR-378 expression analysis, bone marrow samples were obtained from 20 MDS patients. Of these patients, 5 had RA, 3 had RARS, 2 had RCMD, 5 patients with RAEB, 1 with 5q- syndrome, 1 patient had secondary AML, and 3 with MDS-U. Nonmalignant bone marrow samples were collected from 13 randomly selected healthy people. Mononuclear cells were separated from bone marrow using Ficoll gradient centrifugation. q RT-PCR was conducted to observe the expression of miR-378 in MDS ptaitens and healthy controls.2. To obtain SKM-1 cells overexpressing miR-378, the premiRNA of miR-378 was cloned into the GV209 vector to construct a recombination lentiviral vector carrying miR-378(LV-miR-378). Then, SKM-1 cells were transfected with LV-miR-378 and lentivirus expressing empty vector(LV-control). The transfection efficiency was evaluated using flow cytometry, and the expression of miR-378 in each group was examined by real-time PCR. CCK-8 assay and Annexin V/7-AAD staining were conducted to detect the effect of miR-378 on SKM-1 cell proliferation and apoptosis. Cell cycle distribution was determined with the use of flow cytometer. Then, Western blot was performed to detect the expression of apoptosis-related proteins.3. To observe the effect of miR-378 on tumor growth in vivo, the NOD/SCID mice were subcutaneously injected with LV-miR-378-treated SKM-1 cells, LV-control-treated SKM-1 cells and normal SKM-1 cells, respectively. Tumor volume and weight were calculated and cell apoptosis in situ was evaluated using TUNEL assay.4. Bioinformatics algorithms TargetScan and PicTar were conducted to predict the potential target genes of miR-378. For dual-luciferase repoter assay, the wild-type and mutated 3’UTR of candidate target gene was cloned into the dual luciferase reporter vectors. We further conducted qRT-PCR and Western blot to determine whether overexpression of miR-378 could influence the expression of target gene.Results1. qRT-PCR result showed that the expression of miR-378 was obviously decreased in BM-MNCs of MDS patients in contrast with healthy controls(P<0.05). The recombinant lentiviral vector carrying miR-378 was successfully constructed, and miR-378 was stably expressed in SKM-1 cells after infection with LV-miR-378. The infection efficiency of lentiviral vector was more than 70%, and miR-378 was significantly upregulated in SKM-1 cells infected with LV-miR-378(P<0.05).2. CCK-8 assay showed that upregulated miR-378 expression could significantly inhibit proliferation activity of SKM-1 cells, OD value of miR-378-overexpressing group was significantly reduced compared to those of negative control group and non-infected group(P<0.05). The apoptotic rate was obviously higher in the miR-378 group [(12.90 ± 3.72) % ] than in the negative control group [(12.90±3.72)%] and the blank control group [(2.78±1.04)%]. In addition, upregulation of miR-378 increased the percentage of of SKM-1 cells in G1/G0 phase, decreased the proportion of cells in S phase(P<0.05). We also found that overexpression of miR-378 could activate both intrinsic and extrinsic apoptosis pathway, increasing the expression of cleaved-caspase-3, cleaved-caspase-8, cleaved-caspase-9 and Bax protein.3. Overexpression of miR-378 significantly reduced the volume and weight of xenograft tumors. TUNEL assay revealed the proportion of apoptotic cells was higher in the miR-378 overexpressing group [(37.08 + 7.65)%] than in the LV-control-treated group [(17.49 + 3.13)%, P=0.001] and the untreated SKM-1 group [(16.93 + 2.95)%, P=0.001)].4. Both bioinformatic analyses indicated that anti-apoptotic protein gene BCL2L2 is a putative target of mi R-378. Overexpression of mi R-378 resulted in a remarkable diminution of luciferase activity in SKM-1 cells transfected with BCL2L2 wild-type 3’UTR vector(P<0.05), and overexpression of mi R-378 in SKM-1 cells inhibited the expression of BCL2L2 protein, whereas the expression of BCL2L2 m RNA was not affected obviously(P>0.05). In addition, we found that mi R-378 could reduce the expression of CDC40, which is another target gene of mi R-378.ConclusionThis study identified the expression of mi R-378 was commonly decreased in MDS patients. Functional studies showed that enforced expression of mi R-378 in MDS cells could inhibit cell growth via inducing apoptosis and cell cycle arrest. Further, we found that mi R-378 palyed a tumor-suppressive role in MDS cells partly through inhibiting the expression of BCL2L2 and CDC40.