HAVCR1 Affects the MEK/ERK Pathway in Gastric Adenocarcinomas and Influences Tumor Progression and Patient Outcome

Abstract

The hepatitis A virus cellular receptor 1 (HAVCR1) gene as a sensitive and specific biomarker has been reported in various diseases. Especially, HAVCR1 overexpression promotes the development and progression of several human cancers. Hence, we aimed to detect the effects of HAVCR1 on gastric adenocarcinoma (GAC). We first determined the expression of HAVCR1 in GAC tissues compared with normal gastric tissues based on the Cancer Genome Atlas (TCGA) database using bioinformatics analysis methods. Then, we assessed the biological function of HAVCR1 in GAC cells using quantitative real-time reverse transcription-PCR (qRT-PCR), western blot, cell counting kit-8- (CCK-) 8, colony formation assay, wound healing assay, and transwell assay. Our results showed that HAVCR1 expression was upregulated in GAC tissues and positively associated with poor survival. Loss-of-function analyses indicated that knockdown of HAVCR1 inhibited the proliferation, colony formation, migration, and invasion of GAC cells. Furthermore, reduction of HAVCR1 in GAC cells can decrease the expression of phosphorylated MEK/ERK. These findings suggested that HAVCR1 may represent a potential biomarker for GAC prognosis, as well as a novel therapeutic target for GAC treatment.

Figure 1

HAVCR1 was upregulated in GAC tissues and cell lines, and high HAVCR1 expression was related with poor prognosis. (a) HAVCR1 expression was upregulated in GAC tissues (n = 375) compared with normal gastric tissues (n = 32). (b) Kaplan-Meier's survival analysis showed that the high regulation of HAVCR1 had a close relationship with the poor outcome of GAC patients, p = 0.012. (c) QRT-PCR reveals the expression levels of HAVCR1 in GAC cells (MKN-45 and AGS) and in a normal gastric cell line (GES-1). (d) Western blot analysis was performed to detect the expression of HAVCR1. (e) The gray value of protein bands was quantified. ^∗∗p < 0.01 versus GES-1 cells.

Figure 2

HAVCR1 expression was significantly decreased in MKN-45 and AGS cells using siRNA strategy. (a) QRT-PCR was used to assess the HAVCR1 mRNA expression in MKN-45 cells after si-HAVCR1#1 and si-HAVCR1#2 transfection, respectively, ^∗∗p < 0.01 versus the si-con group. (b and c) The HAVCR1 protein level was evaluated using MKN-45 cells that were transfected with si-HAVCR1#1 or si-HAVCR1#2. The gray value of the protein bands was quantified. ^∗∗p < 0.01 versus the si-con group. (d) QRT-PCR analysis showed the silencing efficiency of si-HAVCR1#1 and si-HAVCR1#2 in AGS cells, ^∗∗p < 0.01 versus the si-con group. (e and f) The expression of HAVCR1 in AGS cells was detected by western blot and quantified, ^∗∗p < 0.01 versus the si-con group.

Figure 3

Downregulation of HAVCR1 impaired the proliferation and colony formation of GAC cells. (a and b) A CCK-8 assay was used to measure the proliferation of MKN-45 and AGS cells. (c and d) A colony formation assay was conducted in MKN-45 and AGS cells to further determine the impact of HAVCR1 deficiency on clonogenic capability. And the number of clones was counted. ^∗∗p < 0.01 versus the si-con group.

Figure 4

Reduction of HAVCR1 attenuated the migration and invasion of GAC cells. A wound healing assay was implemented to assess the migration of (a) MKN-45 cells and (b) AGS cells. And the distance of the wound was also quantified. (c and d) The migration and invasion of GAC cells was further detected by transwell assays. ^∗∗p < 0.01 versus the si-con group.

Figure 5

Activation of MEK/ERK signaling pathway was associated with HAVCR1 expression. (a) The MEK/ERK related protein levels were examined by western blot analysis in MKN-45 cells after si-HAVCR1#1 transfection. (b) The gray values of protein bands were quantified. ^∗∗p < 0.01 versus the si-con group. (c) The effects of HAVCR1 deficiency in MEK, p-MEK, ERK, and p-ERK expression levels in AGS cells were measured by western blot. (d) The expression levels of the above proteins were quantified and normalized to GAPDH. One specific siRNA (si-HAVCR1#1) was used as an experimental group because of its higher knockdown efficiency, and nonspecific siRNA was used as a negative control group (si-con). ^∗∗p < 0.01 versus the si-con group.