The Regulation Of MiRNA-21-5p In Human THP-1 Monocyte Proliferation

Human THP-1 monocytes derived from acute mononuclear leukemia patients,which have a certain similarity with human peripheral blood mononuclear cells, and can be proliferated in vitro. It can be used for acute monocytic leukemia (AMOL),mononuclear macrophages immune response ability and so on. Therefore, it is great significance for the occurrence of AMOL to explore the mechanism of THP-1 monocyte proliferation.MicroRNA (miRNA) is a highly conserved non-coding small molecule RNA with a length of about 22 nucleotides. MiRNA can bind to the 3'-UTR of the target mRNA and exert its function at the post-transcriptional level. MiR-21 is one of the earlier discovered and widespread miRNA. Studies have shown that miR-21 is highly expressed in almost all solid tumors and hematological malignancies, which may play a role of oncogene.In this paper, the proliferation mechanism of THP-1 human monocytes was studied by using qPCR, western blotting and double luciferase reporter gene methods.First of all, overexpression or inhibition miR-21 stable THP-1 cell lines were built by lentivirus vectors. Secondly, we explored the effects of miR-21 on THP-1 cell proliferation by MTT assay and detection of proliferating cell nuclear antigen (PCNA)by using western blotting. Thirdly, the effect of miR-21 on cell cycle was further examined by flow cytometry. Finally, to predict and verify the target gene of miR-21 by using TargetScan software and dual luciferase reporter gene methods. The results are as follows:1. The negative control (NC), miR-21 overexpression (LV-mir-21) and miR-21-5p inhibition (Anti-miR-21-5p) stable THP-1 cell lines were built by lentivirus infection (MOI value is 50) and screening by 2 ?g/mL puromycin. The miR-21-5p expression of LV-mir-21 raises about 6 times compared with the NC cells,indicating that miR-21 infection THP-1 human monocyte cells was successful built.2. MTT assay showed that overexpression of miR-21 could significantly promote the proliferation of THP-1 mononuclear cells and inhibition of miR-21-5p expression significantly inhibited cell proliferation. PCNA protein results were similar to the results of MTT assay. These results showed that miR-21-5p could significantly promote the proliferation of THP-1 mononuclear cells.3. Flow cytometry results showed that overexpression of miR-21 could promote THP-1 cells into the S phase and G2/M phase of the cell cycle and promote cell proliferation. The result of miR-21-5p inhibition is opposite to the overexpression of miR-21.4. The target gene of miR-21-5p was predicted by TargetScan software, and select KLF6 and BCL11B which related to cell proliferation as candidate genes. The 3'-UTR or 3'-UTR random mutant sequence of predicted target genes were constructed to psiCHECK-2 (a eukaryotic expression vector). The luciferase activity was detected after co-transfeced with the negative control/miR-21-5p mimics/miR-21-5p inhibitor into 293 cells. The results showed that the luciferase activity of the BCL11B plasmid was significantly lower than that of the control group (p<0.05),while the luciferase activity of the KLF6 plasmid was not significantly different(p>0.05). Indicates that BCL11B is a target gene of miR-21-5p and KLF6 is not its target gene. Western blotting results also showed that the overexpression of miR-21 resulted in the expression level of BCL11B protein is significant decreased (p<0.01),while the expression level of KLF6 protein did not change significantly between different groups (p>0.05). It is further illustrated that the negative relationship between BCL11B and miR-21-5p.In conclusion, overexpression of miR-21 promotes THP-1 cells into S phase and G2/M phase of cell cycle. MiR-21-5p promotes THP-1 monocyte proliferation by targeting BCL11B. It is helpful to explore the mechanism of acute monocytic leukemia and provide a certain theoretical basis.