PRELP has prognostic value and regulates cell proliferation and migration in hepatocellular carcinoma

Abstract

Purpose: Hepatocellular carcinoma (HCC) is an aggressive and prevalent tumor threatening human health. A previous study suggested low PRELP (proline/arginine-rich end leucine-rich repeat protein) expression was associated with poor patient survival in pancreatic ductal adenocarcinoma (PDAC). However, the role of PRELP in HCC has not yet been illuminated. Methods: PRELP expression analyses were carried out using transcriptomic datasets from the Integrative Molecular Database of Hepatocellular Carcinoma (HCCDB). The correlations between PRELP expression and clinicopathological features, and prognostic analyses were performed with a tissue microarray (TMA) and immunohistochemistry (IHC). The endogenous expression and in vitro roles of PRELP were investigated in cultured HCC cell lines. The potential mechanisms were characterized by a Gene Set Enrichment Analysis (GSEA) and gene-gene correlation analyses. Results: We found that PRELP mRNA expression was dramatically decreased in HCCs in comparison with that in adjacent normal tissues (NTs) or hepatic cirrhosis. IHC staining showed that PRELP was down-regulated in HCCs, which mainly located in cytoplasm, and was also found in nuclei. The correlation analyses revealed that PRELP expression was relevant to later p-stages (p= 0.028) and tumor size (p= 0.001). The overall survival (OS) and relapse free survival (RFS) time was shorter in HCC patients with lower PRELP expression levels than that with higher PRELP expression levels. Overexpression of PRELP inhibited, while knockdown of PRELP promoted proliferation and migration of HCC cells. For potential mechanisms, PRELP may inhibit progression of HCCs by interacting with integrin family members and the extracellular microenvironment. Conclusion: Our findings demonstrated that overexpression of PRELP correlates with better patient survival and inhibits both cell proliferation and migration in HCC. Therefore, PRELP can serve as a potential prognostic biomarker and therapeutic target which deserves further investigation.

Keywords: PRELP; hepatocellular carcinoma; integrin; migration; prognosis; proliferation.

Figure 1

PRELP was downregulated in HCCs. The comparisons of PRELP mRNA expression between HCCs and adjacent NTs or hepatic cirrhosis in 12 transcriptomic datasets (A-L). The comparison of PRELP mRNA expression among HCCs, adjacent NTs and hepatic cirrhosis (M), or together with healthy hepatic tissues (N). PRELP mRNA expression levels in different stages during HCC progression (O). Abbreviations: HCC: hepatocellular carcinoma; NTs: normal tissues; NS: not significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001.

Figure 2

Down-regulation of PRELP was associated with poor prognoses. Representative IHC staining images of PRELP in adjacent NTs and HCCs (200 x and 400x magnification) (A). The intensity of PRELP expression in IHC staining were evaluated by H-score (B) and categorized as low or high protein expression. Patients with low PRELP expression tend to have shorter OS time (C), as well as RFS time (D), which was confirmed by analysis of ICGC-LIRI-JP (E). Abbreviations: IHC: immunohistochemistry; OS: overall survival; RFS: relapse free survival; HR: hazard ratio; ****: p < 0.0001; ICGC-LIRI-JP: the Liver Cancer-RIKEN, JP Project from the International Cancer Genome Consortium.

Figure 3

Endogenous PRELP expression in a normal hepatic cell line and seven HCC cell lines. PRELP mRNA and protein were respectively examined by qPCR and WB in L02 and 7 HCC cell lines (A and B). PRELP expression was normalized by L02 cell line. The mRNA and protein expression of PRELP was respectively determined by qPCR and WB in SMMC-7721and HCCLM3 cells (C and D), which were both infected by corresponding lentiviruses. Abbreviations: NS: not significant; *: p<0.05; **: p<0.01; ***: p<0.001; ****: p<0.0001 vs. vector control or scramble control; qPCR: quantitative real-time polymerase chain reaction; WB: western-blotting; shRNA: short hairpin RNA.

Figure 4

Overexpression of PRELP inhibited HCC cell proliferation and migration. CCK8 assay was used to examine proliferation ability of HCC cells (SMMC-7721 and HCCLM3) infected with lentiviruses carrying scramble control or shPRELPs vector control or PRELP (A and B). Overexpression of PRELP decreased the mean number of colonies in the colony formation assay while down-regulation of endogenous PRELP increased colony formation ability of HCC cells (C and D). Wound healing assay was used to evaluate the migratory ability of these two cells. The representative images (left) and quantitative results(right) of wound healing assay both indicated that overexpressed PRELP inhibited cell migration (E and F). The same results of HCC cells were showed by transwell migration assay. Representative micrographs (left) and quantitative results (right) of transwell migration assay both indicated that overexpressed PRELP could limit the migratory ability of HCC cells. Abbreviations: CCK8: Cell counting Kit‑8; NS: not significant; **: p < 0.01; ***: p < 0.001; ****: p < 0.0001 vs. vector control or scramble control; shRNA: short hairpin RNA.

Figure 5

PRELP was associated with ECM Receptors and cell adhesion pathways. High PRELP expression was positively associated with cell-adhesion-activated gene signatures (ECM_RECEPTOR_INTERACTION and CELL_ADHESION_ MOLECULER_CAMS) in GSEA of transcriptomic data from HCCs (A and B) and adjacent NTs (C and D) in TCGA-LIHC. Abbreviations: TCGA-LIHC: The Liver Hepatocellular Carcinoma Project of the Cancer Genome Atlas; ES: enrichment score; FDR: false discovery rate; GSEA: gene set enrichment analysis.

Figure 6

Expression of PRELP positively correlated with most integrin family members. Gene-gene correlation analyses between PRELP and selected integrin family members (A-L). Abbreviations: ITG: integrin.