High expression of hnRNPA1 promotes cell invasion by inducing EMT in gastric cancer

Abstract

Advanced gastric cancer (GC) has a poor prognosis and its treatment strategies are not very efficient. Heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1) has emerged as a plausible GC marker, however the role and molecular mechanism of hnRNPA1 in cell invasion and migration remains unknown. In the present study, the gene expression across normal and tumor tissue (GENT) database was used to evaluate the mRNA expression of hnRNPA1 in various types of cancer. Western blot analysis (WB) and immunohistochemistry (IHC) were performed to detect the protein expression of hnRNPA1 in GC tissues and adjacent non‑tumor tissues. The expression of multiple oncogenes was detected by western blot analysis and quantitative RT‑PCR in hnRNPA1 overexpressing GC cells. Soft agar colony formation, EdU incorporation, wound healing and invasion assays were applied to verify the role of hnRNPA1 in anchorage‑independent cell growth, migration and invasion in GC cells. Epithelial‑to‑mesenchymal transition (EMT) markers were detected by immunofluorescence, western blot analysis and IHC in vitro. A nude mice model of metastasis carcinoma was established to confirm the role of hnRNPA1 during EMT in vivo. Our results revealed that hnRNPA1 was significantly upregulated in GC tissue. HnRNPA1 overexpression significantly induced cell growth, migration and invasion ability in GC cells. In addition, hnRNPA1 promoted EMT of GC cells in vitro and in vivo. These findings indicated that hnRNPA1 is highly expressed in GC and promoted invasion by inducing EMT transition in GC cells. Thus, hnRNPA1 may be a potential therapeutic target for GC.

Figure 1.

HnRNPA1 expression in GC is higher than in normal cells. (A) GENT database was used to find the hnRNPA1 mRNA expression in various types of cancer. Boxes represent the median and 25th and 75th percentiles, dots represent outliers, red boxes represent tumor tissues, green boxes represent normal tissues and red and green dashed lines represent the average value of all tumor and normal tissues, respectively. HnRNPA1 mRNA expression in gastric tissue, represented by blue dotted lines. (B) Resected tumors and adjacent non-tumor tissue specimens were lysed and hnRNPA1 protein expression was detected by western blot analysis. C, gastric carcinoma; N, normal gastric tissues (n=7). (C) In two selected patient cases, higher expression of hnRNPA1 in tumor tissues was confirmed by IHC. Scale bars, 100 µm. (D) Western blotting was used to access the expression of hnRNPA1 in stable transfectants. (E) The expression of multiple oncogenes in vector and hnRNPA1 stable cells was detected by qPCR. **P<0.05.

Figure 2.

HnRNPA1 facilitates the malignant biological behavior of GC cells. (A) HnRNPA1 expression was detected by western blot analysis in BCG823 and AGS cells. (B and C) HnRNPA1-silenced GC cells and scr siRNA cells were plated individually in soft agar culture dishes with complete culture medium. Cell colonies were visualized after 12 days. *P<0.05. (D) DNA synthesis of the BCG823 cells was assessed using an EdU incorporation assay after transfection at 48 h. ***P<0.01, between hnRNPA1 and Scr siRNA. (E) Monolayer BCG823 cells transfected with the indicated siRNA were wounded and images at different time-points were captured. **P<0.05, ***P<0.01, ****P<0.001. (F) The invasive potential of the BCG823 and AGS cells transfected with Scr siRNA or hnRNPA1 siRNA was evaluated by invasion chamber and assessed by invasion index. **P<0.05. These experiments were repeated at least three times.

Figure 3.

HnRNPA1 regulates EMT in vitro. (A) Morphology of stable vector and hnRNPA1 transfected cells (BCG823 and AGS) as visualized under a phase-contrast microscope. (B) BCG823 cells carried with hnRNPA1 or vector stable cells stained with rhodamine-phallotoxin were visualized by fluorescent microscopy to access the expression of F-actin. (C) BCG823 transfected with hnRNPA1 or vector cells were stained by E-cadherin (red) and vimentin (green) primary antibody and then second antibodies individually, fluorescence microscope visualized the expression changes of E-cadherin and vimentin. (D) E-cadherin, Snail and vimentin expression in BCG823 stable cells were detected by western blotting. (E) Two GC tissues with lymph node metastatic sites were stained by hnRNPA1 as the representative IHC results. These experiments were repeated three times with identical findings. Scale bars represent 50 µm in A and B; 20 µm in C and 100 µm in E.

Figure 4.

HnRNPA1 promotes tumor metastasis in vivo. (A) Mice were orthotopically transplanted with the indicated cells (n=3 in each group). Representative images of metastatic loci in the lungs are displayed in yellow dotted lines. (B) H&E staining was used to verify the metastatic cancer tissues from sacrificed mice. (C) The expression of E-cadherin in tumors derived from BCG823 cells was determined by IHC. (D) qPCR was performed to evaluated the expression of E-cadherin in different group of tumor tissues. **P<0.01, Scr-shRNA vs. hnRNPA1-shRNA. Scale bars represent 200 µm in B and 100 µm in C.