MicroRNA-320d Inhibit Proliferation Ability Of DLBCL Cells By Targeting CDK6

Objective:To investigate the impact of mi R-320 d overexpression on DLBCL cell proliferation and analyze the possible molecular mechanism.Methods:1.To transfect mi R-320d- mimic and mi R-320d-inhibitor lentiviral vector into human DLBCL cell line OCI-LY1.Then detect the proliferation ability by CCK-8 experiments.2.To predict the target gene of mi R-320 d using Target Scan, mi Randa and mi RDB,then construct the inhibitor lentiviral vector of target gene.3.To extract total RNA of OCI-LY1, OCI-LY1/ mi R-320 d cells to to detect the CDK6 m RNA level using real-time quantitative PCR technology. Then extract total protein of OCI-LY1, OCI-LY1/mi R-320 d and OCI-LY1/NC cells to detect the CDK6 protein level using western blotting.4.To con-transfect 3’UTR-NC+mi RNA-NC, 3’UTR-NC+mi RNA-320 d mimic;CDK63’UTR+mi RNA-NC,CDK63’UTR+mi RNA-320dmimic;CDK63’UTR-MU+mi RNANC,CDK63’UTR-MU+mi RNA-320 dmimic then detect the luciferase activities to determine whether or not mi R-320 d combine with the specificity of CDK6 3’UTR.5.To transfect CDK6-inhibitor lentiviral vector into human DLBCL cell line OCI-LY1. Then detect the proliferation ability by CCK-8 experiments and detect cell apoptosis by Annexin V/FITC experiments.Results:1.We successfully screened and forecasted hsa-mi R-320 d which may be specificity combined with CDK6 3’UTR, and then constructed the mi R-320d-mimic,mi R-320d-inhibitor lentiviral vector and CDK6- sh RNA lentiviral.2.OCI-LY1/mi R-320d-mimic group cells’ proliferation ability was markedly decreased compared to control group( P<0.05). Mi R-320 d inhibited DLBCL cell proliferation. OCI-LY1/CDK6-sh RNA group cells’ proliferation ability was decreased(P<0.05). DLBCL cell apoptosis rate was no obvious change transfecting with mi R-320d-mimic and CDK6-sh RNA, respectively.3.The CDK6 m RNA relative expression value in OCI-LY1/mi R-320d-mimic group is0.21±0.04, while the OCI-LY1/NC group is 1.0±0.22. The relative gray value of the CDK6 protein expression in OCI-LY1/ mi R-320d-mimic group is 0.43±0.07, and in OCI-LY1/NC group is 0.61±0.09. These results suggest that mi R-320 d can inhibit the expression of CDK6 m RNA and protein in OCI-LY1 and demonstrated that mi R-320 d regulate the expression of CDK6 at the post-transcriptional level.4.Relative luciferase activity value of experimental group is 0.37±0.01 and the value of control group is 1±0.06. These results demonstrated that CDK6 is a direct functional target of mi R-320 d.Conclusion:Upregulation of mi R-320 d expression can inhibit the ability of malignant proliferation of DLBCL cell. The mechanism is concerned with that mi R-320 d could inhibit the expression of CDK6 at post-transcriptional level. This study maybe provides a new ideas and methods for the DLBCL gene therapy.