Elevated UHRF1 expression contributes to poor prognosis by promoting cell proliferation and metastasis in hepatocellular carcinoma

Abstract

Ubiquitin-like with plant homeodomain and ring finger domains, 1 (UHRF1) is overexpressed in a variety of tumor tissues and is negatively correlated with prognosis of patients with cancers, yet so far, a comprehensive study of UHRF1 in hepatocellular carcinoma (HCC) has not been conducted. The present study was designed to explore the expression of UHRF1, associated clinical implications, and its possible functions in HCC. Reverse transcription-polymerase chain reaction and immunohistochemical staining were used to detect UHRF1 expression in HCC specimens including cancerous and noncancerous tissues. Associations of UHRF1 expression with demographic and clinicopathologic features in HCC were analyzed, and the effects of RNA interference of UHRF1 on cell proliferation, cell cycle, apoptosis, and migration were investigated in vitro and in vivo. UHRF1 mRNA and protein expression were both upregulated and negatively correlated with prognosis in HCC patients. Furthermore, inhibition of proliferation, migration, invasion, and epithelial-mesenchymal transition progression were observed in vitro and in vivo after UHRF1 knockdown, moreover, G2/M arrest was detected in HCC cells. In conclusion, elevated UHRF1 expression contributes to poor prognosis by promoting cell proliferation and metastasis in HCC.

Keywords: HCC; UHRF1; invasion; prognosis; proliferation.

Figure 1. UHRF1 expression is elevated and correlates with poor prognosis in HCC

A and B. Comparison of the expression levels of UHRF1 mRNA (n = 80) between HCC cancerous tissues (CT) and the matched noncancerous tissues (NT); C. Comparison of the expression levels of UHRF1 protein between CT and NT (n = 102); D. Kaplan-Meier analysis post-operative relapse-free survival time between high and low UHRF1 mRNA expression; E. Kaplan-Meier analysis post-operative relapse-free survival time between positive and negative UHRF1 protein expression; F. UHRF1 protein is localized in cell nuclei: 1, negative staining in noncancerous tissues; 2, negative staining in cancerous tissues; 3 and 4, positive staining in cancerous tissues. G. mRNA expression of UHRF1 in the HCC cell lines and the immortalized hepatic cell line LO2. Columns, mean of three or five independent data; bars, SEM. *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant.

Figure 2. Depletion of UHRF1 expression in HCC cells

A. The transfection efficiency of UHRF1 siRNAs was assayed by qRT-PCR in HCCLM3 and HepG2 cells after transfected with for 48 h; B. The transfection efficiency of UHRF1 siRNAs was assayed by immunoblotting; C. UHRF1 depletion efficiency was assessed under an inverted fluorescence microscope after HCCLM3 cells were transfected with shRNAs for 48 h; D. The transfection efficiency of UHRF1 shRNAs was confirmed by FACS analysis; E. The transfection efficiency of UHRF1 shRNAs was confirmed by qRT-PCR. Columns, mean of three independent data; bars, SEM. ***P < 0.001, NS = not significant.

Figure 3. UHRF1 depletion inhibits tumor growth in vitro and in vivo

A. HCCLM3 and HepG2 cells were transfected with siRNAs for 48 h followed by CCK-8 assay to detect cell proliferation at 0 h, 24 h, 48 h, 72 h, and 96 h after transfection; B. HCCLM3 cells that were transfected with shRNAs were implanted into nude mice through subcutaneous injection, tumor volume differed between the sh-UHRF1 group and the sh-NC group; C. tumor weight differed between the sh-UHRF1 group and the sh-NC group; D. Tumor growth curves revealed a difference between the sh-UHRF1 group and the sh-NC group; E and F. H&E and Ki-67 IHC staining of tumor nodules and quantitative analysis; G. qRT-PCR analysis of UHRF1 mRNA expression in tumor nodules. Columns, mean of three or five independent experiments; bars, SEM. *P < 0.05, **P < 0.01, ***P < 0.001, NS = not significant.

Figure 4. UHRF1 knockdown leads to cell cycle arrest, but does not induce apoptosis in HCC cells

A. Changes of cell cycle were determined by FACS analysis in HCCLM3 and HepG2 cells after transfected with siRNAs for 48 h; B. Quantification of cells in the different cell cycle phases; C. Western blot analysis of cyclin expression; D. Apoptosis changes were determined by FACS analysis.

Figure 5. UHRF1 silencing inhibits migration and invasion in HCC cells, and restrains EMT progression in vitro and in vivo

A. Transwell migration and invasion assays were carried out in transfected HCCLM3 cells after transfected with siRNAs for 48 h; B. Transwell migration and invasion assays were carried out in transfected HepG2 cells after transfected with siRNAs for 48 h; C. Western blot analysis of EMT markers in HCC cells; D. IHC detection of EMT markers in the tumor nodules. Columns, mean of three independent data; bars, SEM. ***P < 0.001, NS = not significant.