GEFT-Rac1/Cdc42 Affects Rhabdomyosarcoma Cell Pyroptosis And Its Biological Behavior By Regulating The GSDMD Pathway

Objective: To investigate the effect of GEFT-Rac1/Cdc42 in regulating the GSDMD signaling pathway on rhabdomyosarcoma(RMS)cell pyroptosis and its biological behavior.Methods:(1)Western blot and qRT-PCR were used to detect the expression of pyroptosis-related molecules such as Caspase-1 and GSDMD in RMS cells.(2)After treating RMS cells with nigericin(pyroptosis agonist),the changes of pyroptosis in RMS cells were detected by light microscopy and electron microscopy observation,ELISA,and flow cytometry.Western blot and q RT-PCR were used to detect the expression of pyroptosis-related molecules NLRP3,Caspase-1,and GSDMD.Immunofluorescence was employed to locate Caspase-1 and GSDMD molecules in RMS cells.(3)After GSDMD was inhibited by NSA(GSDMD inhibitor),the changes of pyroptosis in RMS cells were detected by light microscopy observation,ELISA,flow cytometry,and other methods.The effect of inhibiting GSDMD expression on the biological behavior of RMS cells was detected by CCK8,plate clone,Ed U,Transwell,AO,and MTT assays.(4)On the basis of the successful construction of overexpressing GEFT RMS cell stable transfection line and the successful interference of si RNA on GEFT expression in RMS cells in the early stage of the research group,light microscopy observation,ELISA,Western blot,and q RT-PCR were used to detect the changes of pyroptosisrelated molecules and the degree of pyroptosis in RMS cells in different groups.Finally,the changes of proliferation,invasion and migration,and viability of RMS cells in different treatment groups were detected by cell functional experiments such as CCK8,Transwell,and AO.Results:(1)The expression levels of pyroptosis-related molecules(NLRP3,Caspase-1,GSDMD)were lower in RMS cells.(2)Compared with the control group,after treatment with nigericin,pyroptotic vacuoles were observed in RMS cells under the light microscope.Pyroptotic bodies were seen under the electron microscope.ELISA showed that the release of LDH(pyroptosis indicator)increased.Flow cytometry showed the pyroptosis rate of RMS cells.Western blot results showed that NLRP3 protein expression,Caspase-1activation(cleaved Caspase-1),and GSDMD cleavage(GSDMD-N)were enhanced.Immunofluorescence co-localization experiments showed that nigericin cleaved GSDMD in the nucleus by inducing the nuclear translocation of Caspase-1,and then GSDMD-N was released into the cytoplasm.(3)Western blot and q RTPCR results showed that NSA could inhibit the expression of GSDMD and attenuate pyroptosis.Cell function assays showed that inhibiting GSDMD could promote the proliferation,invasion,migration,and antiapoptotic effects of RMS cells.(4)Western blot and q RT-PCR were used to detect the expressions of GEFT,Rac1,and Cdc42.The results showed that the m RNA and protein expression levels of Rac1 and Cdc42 were significantly increased after GEFT overexpression.The results of light microscopy and cytological assays also showed that after the GSDMD signaling pathway was inhibited by GEFT-Rac1/Cdc42,the pyroptosis of RMS cells was attenuated,and the proliferation,invasion,migration,and anti-apoptotic effects were enhanced.Conclusion:(1)Nigericin induces pyroptosis in RMS cells by activating the NLRP3/Caspase-1/GSDMD signaling pathway.(2)GEFT-Rac1/Cdc42 attenuates nigericin-induced pyroptosis and promotes the proliferation,invasion,migration,and anti-apoptotic effects of RMS cells possibly by inhibiting the GSDMD signaling pathway.