NLRC5 promotes endometrial carcinoma progression by regulating NF-κB pathway-mediated mismatch repair gene deficiency

Abstract

The innate immune molecule NLR family CARD domain-containing 5 (NLRC5) plays a significant role in endometrial carcinoma (EC) immunosurveillance. However, NLRC5 also plays a protumor role in EC cells. Mismatch repair gene deficiency (dMMR) can enable tumors to grow faster and also can exhibit high sensitivity to immune checkpoint inhibitors. In this study, we attempted to determine whether NLRC5-mediated protumor role in EC is via the regulation of dMMR. Our findings revealed that NLRC5 promoted the proliferation, migration, and invasion abilities of EC cells and induced the dMMR status of EC in vivo and in vitro. Furthermore, the mechanism underlying NLRC5 regulated dMMR was also verified. We first found NLRC5 could suppress nuclear factor-kappaB (NF-κB) pathway in EC cells. Then we validated that the positive effect of NLRC5 in dMMR was restricted when NF-κB was activated by lipopolysaccharides in NLRC5-overexpression EC cell lines. In conclusion, our present study confirmed the novel NLRC5/NF-κB/MMR regulatory mechanism of the protumor effect of NLRC5 on EC cells, thereby suggesting that the NLRC5-mediated protumor in EC was depend on the function of MMR.

Keywords: Endometrial carcinoma; Mismatch repair gene deficiency; NLR family CARD domain-containing 5; Nuclear factor-kappaB.

Figure 1

NLRC5 promotes the migration and invasion of EC cells and enhances the proliferation of EC in vivo and in vitro. (A,B) The proteins expression levels of NLRC5 in HEC-1B and Ishikawa cell lines were examined by western blot (mean ± SD, n = 3). (C) The mRNA expression levels of NLRC5 in HEC-1B and Ishikawa cell lines were examined by qRT-PCR (mean ± SD, n = 3). (D) The proliferation of HEC-1B cells and Ishikawa cells was measured via CCK-8 at 24,48 and 72 h (mean ± SD, n = 3). (E,F) Transwell assay detect the ability of cell migration (Scale bar = 100 μm, mean ± SD, n = 3). (G,H) Transwell assay detect the ability of cell invasion (Scale bar = 100 μm, mean ± SD, n = 3). (I) Representative tumor images. J) The average tumor volume and weight of nude mice. (K) Representative hematoxylin and eosin (HE) stained images of tumors (Scale bar: up 100 μm and down 1000 μm). The corresponding brightfield images are included in the supplementary material. Data represent mean ± SD, n = 3, *P < 0.05 versus control, **P < 0.01 versus control, ****P < 0.0001 versus control group.

Figure 2

NLRC5 inhibits the expression of MMR genes in EC cells. (A) The correlation between NLRC5 and MSH6, MSH2 in EC was analyzed on GEPIA website. (B) The mRNA expression levels of NLRC5 in HEC-1B and Ishikawa cell lines were examined by qRT-PCR (mean ± SD, n = 3). (C) The protein expression levels of NLRC5 in HEC-1B and Ishikawa cell lines were examined by western blot (mean ± SD, n = 3). (D) The protein expression levels were quantified using the ImageJ software and normalized using β-actin protein levels (mean ± SD, n = 3). The corresponding brightfield images are included in the supplementary material. Data represent mean ± SD, *P < 0.05 versus control, **P < 0.01 versus control.

Figure 3

NLRC4 inhibits the expression of MSH2, PMS2, MLH1 genes in vivo. Representative images of results of immunohistochemical (IHC) detection of MSH2, PMS2, MLH1 gene expression of xenograft tumors. (Scale bar = 100 μm mean ± SD, n = 3). The protein expression levels were quantified using the Image J software. Data represent mean ± SD, *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control.

Figure 4

NLRC5 suppresses the NF-κB pathway in vivo and in vitro. (A) NLRC5-related pathways investigated by gene ontology (GO) terms and KEGG pathways enrichment scores. (B,C) The protein expression levels of p65 and p-p65 in HEC-1B and Ishikawa cell lines were examined by western blot. The protein expression levels were quantified using the ImageJ software and normalized using p65 protein levels (mean ± SD, n = 3). (D,E) Representative images of results of IHC detection of p65 and p-p65 protein expression of xenograft tumors. (Scale bar = 100 μm mean ± SD, n = 3). The corresponding brightfield images are included in the supplementary material. Data represent mean ± SD, *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control.

Figure 5

Activation of NF-κB pathway can inhibit the pro-tumor effect of NLRC5 on the proliferation, migration and invasion of EC cells. (A,B) The proliferation of HEC-1B cells and Ishikawa cells was measured via CCK-8 at 24,48 and 72 h. (C,D) Transwell assay detect the ability of cell migration (Scale bar = 100 μm, mean ± SD, n = 3). (E,F) Transwell assay detect the ability of cell invasion (Scale bar = 100 μm, mean ± SD, n = 3). (G) Wound healing detected the ability of cell migration(Scale bar = 100 μm, mean ± SD, n = 3). Data represent mean ± SD, ***P < 0.001 versus control, **P < 0.01 versus control, ****P < 0.001 versus control, ^##P < 0.01 versus NLRC5, ^###P < 0.001 versus NLRC5, ^####P < 0.0001 versus NLRC5.

Figure 6

NLRC5 induces dMMR status by suppress NF-κB pathway in EC. (A,B) The protein expression levels of p65 and p-p65 in HEC-1B and Ishikawa cell lines were examined by western blot. The protein expression levels were quantified using the ImageJ software and normalized using p65 protein levels (mean ± SD, n = 3). (C) The mRNA expression levels of MMR genes in HEC-1B and Ishikawa cell lines were examined by qRT-PCR (mean ± SD, n = 3). (D) The protein expression levels of MMR genes in HEC-1B and Ishikawa cell lines were examined by western blot. The protein expression levels were quantified using the ImageJ software and normalized using β-actin protein levels (mean ± SD, n = 3). The corresponding brightfield images are included in the supplementary material. Data represent mean ± SD, *P < 0.05 versus control, **P < 0.01 versus control, ***P < 0.001 versus control, ****P < 0.0001 versus control. ^#P < 0.05 versus NLRC5, ^##P < 0.01 versus NLRC5, ^###P < 0.001 versus NLRC5, ^####P < 0.0001 versus NLRC5.