NLRC5 Promotes Endometrial Cancer Progression By Regulating NF-κB Pathway-mediated Mismatch Repair Gene Deficiency

Endometrial cancer(EC)is the sixth most common cancer in women worldwide.The Cancer Genome Atlas of the United States(TCGA)classifies endometrial cancers into 4 distinct molecular subgroups,of which the microsatellite instability(MSI-H)type accounts for 17-33% of all endometrial cancers.Studies have demonstrated that patients with MSI-H EC have enhanced sensitivity to immunotherapy.Therefore,mobilizing MSI in endometrial cancer is a new idea for the treatment of endometrial cancer,and understanding the upstream e vents that control d-MMR is particularly important for formulating comprehensive strategies and guiding clinical cancer treatment.Studies have shown that NLRC5 is a transactivator of HLA class I gene transcription,and tumors can exert immune escape by targeting and inhibiting the expression of NLRC5.In this study,we explored the relationship between NLRC5 and MMR genes in endometrial cancer,and explored the role of NLRC5 in end ometrial cancer.Method:(1)NLRC5 overexpression lentivirus was added in appropriate titers to the EC cell lines HEC-1B and Ishikawa,then Western blot and q RT-PCR was using to detect whether NLRC5 was overexpressed in cell lines.MMR genes expression were detected by Western Blot and qrt-PCR.(2)Animal xenograft models of subcutaneous transplantation of HEC-1B cell lines were established,and tumor proliferation capacity in vivo was measured by tumor volume.The proliferation capacity was determined by CCK8 kit in vi t r o.The migration and invasion ability of cell lines were detected by the transwell experiment.(3)The activation of NF-κB pathway in cell lines were detected by Western blot and cell immunofluorescence experiments.(4)Endometrial cancer cell lines transfected with NLRC5 overexpressing lentivirus were treated with NF-κB activator lipopolysaccharide.The expression levels of p65,p-p65,MSH6,MSH2,MLH1,and PMS2 in the cells were detected by Western blot and q RT-PCR.The proliferation ability of HEC-1B and Ishikawa cell lines was detected by CCK-8 kit,the lateral migration ability was detected by wound-healing experiment,and the longitudinal migration ability and invasion ability were detected by Transwell assay(chamber without or with Matrigel).Results:(1)Results of Western blot and q RT-PCR experiments showed that the expression of NLRC5 was significantly up-regulated after the EC cell lines HEC-1B and Ishikawa were stably transfected with NLRC5-overexpressing lentivirus.Meanwhile,the expressions of MSH6,MSH2,MLH1,and PMS2 were inhi bited(all P<0.05).(2)After 4 weeks feeding of BALB/c nude mice after subcutaneous implantation of HEC-1B and HEC-1B-NLRC5-OE cell lines,tumors was removed and then photographed.NLRC5 was observed to significantly promote the proliferation of tumors in vivo.Result of CCK8 kit showed that NLRC5 significantly promoted the proliferation of HEC-1B and Ishikawa cell lines(both P<0.05).Results of transwell showed that the overexpression of NLRC5 significantly promoted the migration and invasion abilities of HEC-1B and Ishikawa(both P<0.05)(3)Result of cellular immunofluorescence assay showed that after NLRC5 was overexpressed,there was no difference in the expression of total p65 i,but the expression of phosphorylated p65 was significantly reduced.The results of western blot confirmed this result(P<0.05).(4)After adding lipopolysaccharide(LPS)to activate NF-κB pathway in the cell lines overexpressed NLRC5,the activation of NF-κB was first verified by the results of western blot.The results of q RT-PCR and western blot showed that LPS reversed the inhibition of NLRC5 on the m RNA and protein expression of MSH6,MSH2,MLH1,and PMS2(all P<0.05).Next,result of CCK-8 kit showed that LPS reduced the protumor effect of NLRC5 on cell lines.Then the results of transwell assay showed that LPS inhibited the promoting effect of NLRC5 on EC cell migration and invasion.Conclusions:(1)NLRC5 induces MMRd in EC and promotes tumor proliferative capacity in vitro and in vivo.(2)Investigation of the regulatory mechanism of the protumor effect of NLRC5 on EC revealed that NLRC5 suppresses the NF-κB pathway in vitro and in vivo.(3)NLRC5 promotes EC progression by regulating the NF-κB pathway-mediated MMRd.