Study On The Function Of LMNA R527C Recessive Genetic Mutation

Objective: To eliminate the interference of endogenous LMNA,we use lentiviral infection technology to construct the stable cell line with expressing LMNA R527 C mutation in 293 T LMNA knockout cells.Study the effects of LMNA mutations on cell proliferation,migration,cycle,apoptosis and related signaling pathways.Investigate the related pathway mechanism of LMNA R527 C mutation and the occurrence of laminopathy.Methods: Total RNA were extracted from the peripheral blood cells of patients,c DNA was obtained by RT-PCR,and ORF of LMNA mutation was got by PCR.Then,constructed recombinant plasmids form LMNA mutations and lentiviral expression plasmids by using Molecular cloning technology.Next,transfected three plasmid lentiviral packaging system in 293 T cells to generate and harvest lentiviral.Finally,lentiviral infected 293 T LMNA knockout cells,through puromycin and monoclonal screening,DNA sequencing and western blot identification to acquire stable cell line with expressing LMNA mutation.The cells were divided into five groups: normal 293 T cells as control group(293T),293 T LMNA knockout cells as knockout group(293T LMNAKO),293 T LMNA knockout cells expressing LMNA again as rescue group(293T LMNA),293 T LMNA knockout cells with expressing LMNA G608 G mutation as G608 G mutation group(293T LMNA G608G),and 293 T LMNA knockout cells with expressing LMNA R527 C mutation as R527 C mutation group(293T LMNA R527C).Knockout group studied the function of LMNA forward,rescue group verified the function of LMNA backward,and the G608 G mutant group was used as the dominant mutant control.Nucleus and cytoskeleton morphology were detected by using immunofluorescence experiments.CCK8 and plate monoclonal formation experiment were used to detect cell proliferation ability,cell Wound healing assay and Transwell assay to detect cell migration and invasion ability,flow cytometry to detect cell cycle and apoptosis,and Western blot to detect protein expression levels of related signaling pathways.Results: 1.Successfully obtained three stable cell lines with re-expressing LMNA,expressing LMNA G608 G mutation and expressing LMNA R527 C mutation in 293 T LMNA knockout cells.2.Compared with the control group,nuclear morphology and structure of knockout group,G608 G mutation group and R527 C mutation group were abnormal,mutant Lamin A was discontinuous and aggregation,and its location was also changed(p>0.05).3.Compared with the control group,the cytoskeleton morphology and structure of knockout group,G608 G mutation group and R527 C mutation group were abnormal,and the attachment of the cytoskeleton to the nucleus is disturbed(p>0.05).4.Cell proliferation ability: Knockout group,G608 G mutation group,R527 C mutation group accelerated the speeds of cell's proliferation ability compared with control group and rescue group(p<0.01).5.Cell migration ability: Compared with control group and rescue group,Knockout group,G608 G mutation group,R527 C mutation group prefer to migrate(p<0.01).6.Cell cycle and apoptosis:LMNA mutation had no obvious effect on cell cycle and apoptosis(p>0.05).7.Protein expression levels of related signaling pathways: Compared with the control group and rescue group,the Rb expression level of the mutant group cells decreased(p<0.01),p16 expression level increased(p<0.05),but CDK6,CDK4,CDK1 and Cyclin B1 expression level were no significant difference(p>0.05).EMT pathway related protein E-cadherin and Snail expression levels decreased(p<0.01),Vimentin expression levels increased(p<0.05),Twist expression levels were not significantly different(p>0.05),and NF-?b pathway related protein I?B? expression levels decreased(p <0.01),the expression levels of p50 and p65 increased(p<0.05).Conclusions: 1.LMNA R527 C mutation caused abnormal nuclear,and affected the connection between nucleus and cytoskeleton.2.LMNA R527 C mutation inhibited the expression level of Rb,up-regulated the p16 expression level,and enhanced cell proliferation and clone formation ability.Moreover,LMNA R527 C mutation effected NF-?B signaling pathway,p65 expression level increased,thereby inhibited E-cadherin expression level leading to accelerated cell migration.