AGER promotes proliferation and migration in cervical cancer

Abstract

The receptor for advanced glycation end products (AGER) is an oncogenic transmembranous receptor up-regulated in various human cancers. We have previously reported that AGER was overexpressed in squamous cervical cancer. However, mechanisms of AGER involved in the progression of cervical cancer are unknown. In the present study, we investigated the effects of AGER on biological behavior, including proliferation, apoptosis, and migration using multiple biological approaches. AGER protein primarily localized in the cytoplasm and cytomembrane of cervical squamous cancer cells. Blockage of AGER with multiple siRNAs suppressed proliferation, stimulated apoptosis, inhibited migration of cervical squamous cancer cells. Conversely, overexpression of AGER increased cell proliferation, migration, and inhibited cell apoptosis. These results indicate that AGER promotes proliferation, migration, and inhibits apoptosis of squamous cervical cancer and might function as a tumor promoter in cervical cancer. Our study provides novel evidence for a potential role of AGER in bridging human papillomavirus (HPV)-induced inflammation and cervical cancer.

Keywords: AGER; cervical cancer; migration; proliferation.

Figure 1. Immunocytochemical staining of AGER in cervical squamous cancer cells

(a) Negative control in MS751 cells (SP staining, ×400). (b) Positive staining of AGER protein in MS751 cells (SP staining, ×400). (c) Negative control in C33A cells (SP staining, ×400). (d) Positive staining of AGER protein in C33A cells (SP staining, ×400). (e) Negative control in Caski cells (SP staining, ×400). (f) Positive staining of AGER protein in Caski cells (SP staining, ×400). (g) Negative control in SiHa cells (SP staining, ×400). (h) Positive staining of AGER protein in SiHa cells (SP staining, ×400).

Figure 2. The expression of AGER mRNA and protein in human cervical squamous cancer cells

(a) The mRNA levels of AGER in four cervical squamous cancer cells were detected by qRT-PCR. GAPDH transcript was used for normalization. (b) The protein levels of AGER in cervical squamous cancer cells was detected by Western blot. GAPDH protein level was used to validate equal sample loading. Data presented were mean ± S.D. from triplicate experiments (*P<0.05).

Figure 3. The effect of AGER on the proliferation of cervical cancer cells evaluated by CCK-8 assay

(a) AGER cDNA and match vector were transfected into SiHa and Caski cells via lentivirus infection. Protein levels of AGER in AGER cDNA transfected, control vector transfected and NC cells by Western blot. GAPDH protein level was used to validate equal sample loading. (b) Cell proliferation was analyzed by CCK-8 assay. (c) Confirmation of AGER silencing in SiHa cells by Western blot. GAPDH protein level was used to validate equal sample loading. (d) Cell proliferation was analyzed by CCK-8 assay.

Figure 4. The effect of AGER on the apoptosis of cervical cancer cells

(a) AGER cDNA and match vector were transfected into SiHa and Caski cells via lentivirus infection. Apoptosis percentage was analyzed by Annexin V-APC/7-AAD staining. (b) SiHa cells were transfected with AGER siRNA. Apoptosis percentage was analyzed by Annexin V-FITC/PI staining. Each bar represents mean ± S.D. of triplicate experiments.

Figure 5. The effect of AGER on cervical cancer cells migration

(a,b) AGER cDNA and match vector were transfected into SiHa cells via lentivirus infection. Cell migration was evaluated by transwell migration assay (×200 magnification). (c,d) SiHa cells were transfected with AGER siRNA. Cell migration was evaluated by transwell migration assay (×200 magnification). Data presented were mean ± S.D. from triplicate experiments (*P<0.05).