The Study On Mechanism Of Micro RNA-320d Is Frequently Downregulated In DLBCL And Inhibits Cell Proliferation And Invasion Via Directly Targeting CDK6

Objective:To investigate the relationship between CDK6 expression and the clinical pathological features on diffuse large B cell lymphoma(DLBCL)and analyze the possible molecular mechanism of cell proliferation.Methods:(1)84 cases of DLBCL with follow-up data were collected from Shanxi Cancer Hospital,were studied by using immunohistochemical En Vision method for CDK6.(2)The target genes of mi R-320 d were predicted by using bioinformatics software.Dual-Luciferase Reporter assay was uesd to determine whether or not mi R-320 d combine with the specificity of CDK6 3'UTR.(3)Sh RNA knockdown of CDK6 was performed by using lentiviral transduction,whereas overexpression of mi R-320 d was also performed by lentiviral transduction.(4)Cell proliferation and apoptosis were quantitated by CCK-8 and Annexin V-APC/7-AAD, respectively.(5)Western blotting was used to detect the expression of CDK6 protein in DLBCL cells transfected with mi R-320 d lentiviral vector.Results:(1)The expression levels of CDK6 protein in DLBCL tissues were higher than in normal lymph nodes(P<0.05).(2)CDK6 was predicted as a candidate of target genes by bioinformatics software.Dual-Luciferase Reporter assay showed that mi R-320 d could target the 3'UTR of CDK6 gene.(3)We successfully constructed the mi R-320d-mimic and CDK6-sh RNA lentiviral vector. Mi R-320 d inhibited DLBCL cell proliferation(P<0.05). DLBCL cell apoptosis rate has no obvious change transfected with mi R-320d-mimic and CDK6-sh RNA, respectively.(4)The relative gray value of the CDK6 protein expression in mi R-320d-mimic group is 0.04±0.04, and in NC group is 0.87±0.05(P<0.05), suggesting that the expression of endogenous CDK6 protein was suppressed by mi R-320 d.Conclusion:The CDK6 protein was high in DLBCL tissues,indicated that CDK6 was involved in the occurrence and development process of DLBCL. Mi R-320 d is frequently downregulated in DLBCL and inhibits cell proliferation via directly targeting CDK6.