Overexpression of RALY promotes migration and predicts poor prognosis in hepatocellular carcinoma

Abstract

Introduction: RALY plays a critical role in promoting invasiveness and is associated with poor prognosis in different types of cancers. However, the prognostic value of RALY and its precise role in hepatocellular carcinoma (HCC) remain unknown.

Materials and methods: We detected the expression of RALY in 127 clinical HCC tissue samples and seven HCC cell lines by immunohistochemical staining and Western blotting. The prognostic value of RALY expression was assessed using the Kaplan-Meier method. The expression and prognostic value of RALY were also studied by bioinformatics analysis of data from the Gene Expression Omnibus and The Cancer Genome Atlas. The biological influence of RALY on HCC cell lines was studied using proliferation, transwell migration, and invasion assays in vitro.

Results: The expression of RALY in HCC tissues was significantly higher than that in adjacent normal liver tissues. Abnormally high expression of RALY was associated with tumor size (P=0.031), TNM stage (P=0.026), presurgical serum AFP levels (P=0.025), and vascular invasion (P=0.001). Kaplan-Meier analysis demonstrated that higher expression of RALY correlated with poorer overall survival and disease-free survival in HCC patients. High RALY expression was an independent adverse prognostic factor for overall survival (HR =2.559, 95% CI: 1.710-3.827, P<0.001) and disease-free survival (HR =2.053, 95% CI: 1.384-3.047, P<0.001) in HCC. Moreover, knockdown of RALY expression using a specific shRNA suppressed the proliferation, migration, and invasion capabilities of HCC cells in vitro. Knockdown of RALY expression in HCC cell lines resulted in upregulation of E-cadherin and downregulation of N-cadherin, vimentin, and snail.

Conclusion: Taken together, our results indicate that RALY represents a biomarker for the prognosis of patients with HCC and highlight the importance of RALY as an oncogene in HCC.

Keywords: RALY; hepatocellular carcinoma; invasion; migration.

Figure 1

Upregulation of RALY in HCC tissues compared with the levels in adjacent normal liver tissues. Notes: (A) The mRNA expression of RALY was significantly higher in HCC tissues than in normal liver tissues, according to an analysis of data from the GEO datasets (GSE25097, tumor, n=268; normal, n=243, P<0.001; GSE57958, tumor, n=39; normal, n=39, P<0.001). (B) Western blotting (WB) analysis of RALY expression in seven HCC cell lines (Huh7, SMMC-7721, Bel-7402, HepB3, HepG2, MHCC-97H, and MHCC-97L) and one immortal hepatocellular cell line (LO2). β-Actin served as the internal control; the right-side band is the relative fold change of RALY/β-actin detected by ImageJ software. (C) Protein expression of RALY in HCC tissues and adjacent noncancerous liver tissues determined by immunohistochemistry (IHC), captured at 200× magnification and 400× magnification under a microscope. Box plots are presented to compare RALY expression between HCC tissues and adjacent noncancerous liver tissues in the right-side band. The boxes represent the 25th to 75th percentiles of the observations, and the medians are indicated by the lines in the middle of the box. The scale bars indicate 50 µm for the 200× magnification and 25 µm for the 400× magnification. *P<0.05; **P<0.01; ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; GEO, Gene Expression Omnibus.

Figure 2

Protein expression of RALY in HCC tissues of different tumor sizes, TNM stages, AFP levels, and conditions of vascular invasion determined by immunohistochemistry (IHC). Notes: (A) The difference of the expression of RALY in HCC tissues with TS >5 cm and in HCC tissues with TS ≤5 cm was not significant (P=0.092). (B) The expression of RALY in HCC tissues with later TNM stages (III + IV) was significantly higher than that in HCC tissues with earlier TNM stages (I+II) (P=0.002). (C) The expression of RALY in HCC tissues with higher AFP levels (>200 ng/mL) was significantly higher than that in HCC tissues with lower AFP levels (≤200 ng/mL) (P=0.007). (D) The expression of RALY in HCC tissues without VI was significantly lower than that in HCC tissues with VI (P=0.006). The boxes represent the 25th to 75th percentiles of the observations, and the medians are indicated by the lines in the middle of the box. The scale bars indicate 25 µm. **P<0.01; NS represents no significant difference. Abbreviations: HCC, hepatocellular carcinoma; TS, tumor size; VI, vascular invasion.

Figure 3

Prognostic value of RALY expression in HCC patients. Notes: (A) Analysis of overall survival in 318 HCC patients from the TCGA database by RALY expression. (B) Analysis of disease-free survival in 318 HCC patients from the TCGA database by RALY expression. (C) Analysis of overall survival in 127 HCC patients from our center by RALY expression. (D) Analysis of disease-free survival in 127 HCC patients from our center by RALY expression. The P-value was calculated by the log-rank test. Abbreviations: HCC, hepatocellular carcinoma; TCGA, The Cancer Genome Atlas.

Figure 4

RALY knockdown suppressed the proliferation, migration, and invasion capabilities of HCC cells in vitro. Notes: (A) Western blotting showed that the RALY protein level was significantly decreased in the RALY shRNA-transfected Bel-7402 and MHCC-97H cells compared to the mock- or NC shRNA-transfected Bel-7402 and MHCC-97H cells. (B) The relative fold change in RALY/β-actin detected by ImageJ software. (C) The proliferation capabilities of HCC cells were detected by the CCK-8 assay, and RALY knockdown prominently reduced the proliferation potential of Bel-7402 and MHCC-97H cells. (D) The migration potential of HCC cells was detected by a transwell migration assay, and the migration abilities of the Bel-7402 and MHCC-97H cells were dramatically reduced after transfection with RALY shRNAs. (E) The invasion potential of HCC cells was detected by a transwell invasion assay, and the invasion abilities of the Bel-7402 and MHCC-97H cells were dramatically reduced after transfection with RALY shRNAs. Data represent the mean ± SEM of three independent experiments. ***P<0.001. Abbreviations: HCC, hepatocellular carcinoma; NC, negative control; CCK-8, Cell Counting Kit-8; SEM, standard error of the mean; WB, Western blotting.

Figure 5

RALY knockdown caused changes in EMT marker expression levels in HCC cells. Notes: (A) Western blot analysis of EMT-related protein expressions in Bel-7402 and MHCC-97H cells. (B) Relative expression levels of EMT markers in Bel-7402 and MHCC-97H cells. Data represent the mean ± SEM of three independent experiments. *P<0.05, **P<0.01, ***P<0.001. Abbreviations: EMT, epithelial-to-mesenchymal transition; HCC, hepatocellular carcinoma; SEM, standard error of the mean; NC, negative control; WB, Western blotting.