Effects Of Mechanically Sensitive Ion Channels Piezo1 And Kif18A On The Proliferation Of Articular Chondrocytes

Objective To investigate whether Piezo 1 can change the cytoskeleton morphology by regulating the expression of Kif18 A,and then affect cell division and proliferation.Methods The FAM38A-sh RNA lentiviral vector which interfered with the expression of Piezo1 was prepared and transfected into chondrocytes of osteoarthritis patients in vitro.The sequence which effectively interfered with the expression of Piezo1 was selected as the lentiviral interference sequence group(group C),and a blank control group(group A)was established.Lentiviral empty vector group(group B),lentiviral interference sequence(LV3-Piezo1-homo-3201)group(group C),Kif18 A drug blocking group(group D),lentiviral interference sequence(LV3-Piezo1-h omo-3201)+ Kif18 A drug blocking group(group E)..According to the pre-experiment results,multi-channel cell stretch stress loading system FX-4000T(Flexcell,USA)was used to periodically load each group for 0h,2 h,12 h,24 h,48 h.The morphological changes of cells were observed under light microscope.The cell proliferation was measured by cck-8.The transcription levels and protein expressi on levels of the Piezo1、β-tubulin and Kif18 A were detected by RT-PCR and West ern-Blot.Results The relative expression of the target gene of Piezo1 in the lentiviral vector LV3-Piezo1-homo-3201 was significantly decreased,and the interference effect was significant(P<0.05).The effect of viral vector itself on cell proliferation: the value-added rate of Kif18 A inhibitor group and Kif18 A inhibitor + FAM38A-shRNA interference group was significantly lower than that of blank control group and FAM38A-sh RNA interference group(P<0.05).The value-added rate of the blank control group increased first and then decreased,and the value-added rate of the FAM38A-sh RNA interference group remained basically unchanged.Under the 12 h stress,the value-added rate of Kif18 A inhibitor group and Kif18 A inhibitor + FA M38A-sh RNA interference group was significantly different(P<0.05),and there was no difference under the other afterburning time(P>0.05).The relative expression levels of Piezo1 in FAM38A-sh RNA interference group and Kif18 A inhibitor+FA M38A-shRNA interference group were significantly lower than the blank control group,lentiviral empty vector group and Kif18 A inhibitor group(P<0.01).The relative expression levels of the subgroups of genes at different stress times were small.The expression of Kif18 A inhibitor group was lower than that of blank control g roup and lentiviral empty vector group at 24 h and 48h afterburning period(P<0.05).The expression of Kif18 A in the Kif18 A inhibitor group and the Kif18 A inhibi tor + FAM38A-sh RNA interference group was significantly lower than that in the blank control group(P<0.01),and the Kif18 A inhibitor + FAM38A-sh RNA interference group had lower gene expression than the Kif18 A inhibitor.Group(P < 0.05).The expression of the target gene in the FAM38A-sh RNA interference group was significantly lower than that in the blank control group(P<0.05).The expression characteristics of β-Tubulin in each experimental group: There was no difference in the expression of genes in each group under stress of 24 h and 48h(P>0.05),and the expression levels of blank control group and lentiviral vector group were higher than 0h,12 h.The amount of expression under 24 h stress.The expression of the target gene in the Kif18 A inhibitor group and the Kif18 A inhibitor + FAM38A-sh RNA interference group was significantly lower than that in the blank control group(P<0.01),and the Kif18 A inhibitor + FAM38A-sh RNA interference group had lower gene expression than the Kif18 A inhibitor group.(P<0.05).The expres sion of the target gene in the FAM38A-sh RNA interference group was significantly lower than that in the blank control group(P<0.05).Conclusion LV3-Piezo 1-homo-3201 virus interference sequence can specifically interfere with Piezo 1 synthesis.Piezo 1,a mechanically sensitive ion channel,can sense biological stress and activate Kif18 A to destroy the cytoskeleton structure,thereby inhibiting cell division and proliferation,affecting the structure and function of articular cartilage.