BCORL1 is an independent prognostic marker and contributes to cell migration and invasion in human hepatocellular carcinoma

Abstract

Background: The deregulation of E-cadherin has been considered as a leading cause of hepatocellular carcinoma (HCC) metastasis. BCL6 corepressor-like 1 (BCORL1) is a transcriptional corepressor and contributes to the repression of E-cadherin. However, the clinical significance of BCORL1 and its role in the metastasis of HCC remain unknown.

Methods: Differentially expressed BCORL1 between HCC and matched tumor-adjacent tissues, HCC cell lines and normal hepatic cell line were detected by Western blot. The expression of BCORL1 was altered by siRNAs or lentivirus-mediated vectors. Transwell assays were performed to determine HCC cell invasion and migration.

Results: Increased expression of BCORL1 protein was detected in HCC specimens and cell lines. Clinical association analysis showed that BCORL1 protein was expressed at significant higher levels in HCC patients with multiple tumor nodes, venous infiltration and advanced TNM tumor stage. Survival analysis indicated that high expression of BCORL1 protein conferred shorter overall survival (OS) and recurrence-free survival (RFS) of HCC patients. Multivariate Cox regression analysis disclosed that BCORL1 expression was an independent prognostic marker for predicting survival of HCC patients. Our in vitro studies demonstrated that BCORL1 prominently promoted HCC cell migration and invasion. Otherwise, an inverse correlation between BCORL1 and E-cadherin expression was observed in HCC tissues. BCORL1 inversely regulated E-cadherin abundance and subsequently facilitated epithelial-mesenchymal transition (EMT) in HCC cells. Notably, the effect of BCORL1 knockdown on HCC cells was abrogated by E-cadherin silencing.

Conclusions: BCORL1 may be a novel prognostic factor and promotes cell migration and invasion through E-cadherin repression-induced EMT in HCC.

Fig. 1

BCORL1 is abnormally up-regulated in human HCC. a Representative Western blot analysis showed the expression of BCORL1 in the HCC (T) and matched nontumor tissues (NT). Quantitative analysis indicated that BCORL1 protein was significantly up-regulated in HCC tissues as compared with that in nontumor tissues. n = 86; ^* P < 0.05 by t test. b The expression levels of BCORL1 protein in HCC cell lines (HepG2, Hep3B, MHCC97H and HCCLM3) and a normal hepatic cell line (LO2). n = 3; ^* P < 0.05 by ANOVA. c The expression levels of E-cadherin mRNA in HCC cell lines and a normal hepatic cell line. n = 3; ^* P < 0.05 by ANOVA

Fig. 2

Prognostic value of BCORL1 for HCC patients. Kaplan-Meier overall survival (OS) and recurrence-free survival (RFS) curves of HCC patients in accordance with their expression status of BCORL1 protein. High expression of BCORL1 protein conferred a worse 5-year OS and RFS for HCC patients. The expression of BCORL1 was divided into low level group (n = 43) and high level group (n = 43) based on the cutoff value which was defined as the median protein level of the 86 HCC samples detected by Western blot

Fig. 3

BCORL1 increases migrated and invaded HCC cells. a HCCLM3 cells with BCORL1 siRNA and scrambled siRNA transfection, respectively, were subjected to immunoblotting for BCORL1. n = 6; ^* P < 0.05 by t test. b Transwell migration assays showed that BCORL1 knockdown inhibited cell migration in HCCLM3 cells. As assessed by Transwell invasion assays, HCCLM3 cell invasion was inhibited by BCORL1 knockdown. ^* P < 0.05 by t test; n = 3. c Hep3B cells that were infected with BCORL1 or empty vector (EV) lentiviruses, were detected by Western blot. n = 6; ^* P < 0.05 by t test. d BCORL1 overexpression increased the number of migrated and invaded cells as measured by Transwell assays in Hep3B cells. ^* P < 0.05 by t test; n = 3

Fig. 4

BCORL1 is inversely correlated with E-cadherin in HCC. a The positive expression rate of E-cadherin in BCORL1 positive HCC specimens was significantly lower than that in BCORL1 negative cases. ^* P < 0.05 by Pearson chi-squared test. b Representative immunohistochemical staining showed weak staining of E-cadherin (III) in BCORL1 positive-expressing tumor (I) and strong staining of E-cadherin (IV) in BCORL1 negative-expressing tumor (II). Scale bar: 50 μm. c qRT-PCR analysis of E-cadherin mRNA expression in HCCLM3 cells with BCORL1 siRNA or scrambled siRNA transfection. ^* P < 0.05 by t test, n = 3. d BCORL1 knockdown increased the level of E-cadherin protein and reduced the expression of vimentin and N-cadherin in HCCLM3 cells. ^* P < 0.05 by t test, n = 6. e qRT-PCR analysis of E-cadherin mRNA expression in Hep3B cells with BCORL1or empty vector (EV) lentiviruses infection. ^* P < 0.05 by t test, n = 3. f BCORL1 overexpression decreased the level of E-cadherin protein and up-regulated the expression of vimentin and N-cadherin in Hep3B cells. ^* P < 0.05 by t test, n = 6

Fig. 5

Immunofluorescent staining of E-cadherin in HCC cells. HCC cells that were treated with corresponding vectors were subjected to immunofluorescence for E-cadherin. BCORL1 knockdown increased E-cadherin immunofluorescence densities in HCCLM3 cells and BCORL1 overexpression reduced the expression of E-cadherin in Hep3B cells. DAPI stain (blue) was used to identify nuclei. Scale bar: 20 μm

Fig. 6

E-cadherin knockdown abolishes the anti-metastatic effect of BCORL1 knockdown. a Scrambled siRNA or E-cadherin siRNA was transfected into BCORL1 down-regulating HCCLM3 cells and confirmed by Western blot. n = 6; ^* P < 0.05 by t test. b E-cadherin knockdown increased cell migration and invasion in BCORL1 down-regulating HCCLM3 cells. n = 3, ^* P < 0.05 by t test