Loss Of PBLD Expression Correlates With The Malignant Progression Of HCC And The Exploration Of The Underlying Molecular Mechanism

Hepatocellular carcinoma (HCC) ranks as the sixth most common cancer and the third leading cause of cancer-related mortality worldwide, accounting for approximately 75%-90% of malignant tumors in adult livers. The prognosis of HCC is poor because the disease is often at a fairly advanced stage at the time of diagnosis. Surgical treatments including liver resection and liver transplantation are effective therapies for patients with early HCC at present. However, the clinical outcome is still unsatisfactory due to its high recurrence rate after surgery. Although more and more molecular markers with high sensitivity and specificity for HCC have been proposed, none has justified its routine use in clinical practice till now. Hence, a better understanding of more molecular markers and pathways that contribute to the progression and recurrence of HCC is essential for the development of more effective, targeted therapies.PBLD, also termed as MAWBP, was first identified from a human liver cDNA library using a yeast two-hybrid technique by Iriyama et al.. As the only representative of phenazine biosynthesis-like protein family, PBLD is widely expressed in human tissues, such as brain, heart, lung, liver, pancreas, kidney, and placenta. Its expression is elevated in several diseases processes, including insulin resistance, folate deficiency, and hypotension. Recent accumulating genomic and proteomic data suggested that decreased expression of phenazine biosynthesis-like domain-containing protein (PBLD) was frequently involved in hepatocellular carcinoma (HCC). However, there is lack of systematical investigation focusing on its expression pattern, clinical relevance, and biological function.As powerful molecular techniques at present, gene expression profiling and proteomics analysis have been extensively used to improve the identification of new biomarkers leading to early diagnosis and more effective therapies, and the definition of the mechanisms associated with HCC progression. Using gene expression profiling and proteomics analysis several studies have found that PBLD was frequently decreased in HCC tissues relative to adjacent non-tumorigenic liver tissues which suggested a potential role of PBLD in hepatic tumorgenesis. However, there have been no further researches focused on its expression pattern, biological function and molecular mechanism. In the present study, we investigated the expression of PBLD in HCC tissues after curative resection by quantitative real-time PCR, immunohistochemistry, and western blotting. Also we analyzed in vitro the role of PBLD in HCC cells growth and metastasis. Furthermore, using microarray analysis, we described the gene expression profiling of PBLD overexpression HepG2 cell line and screened the potential targeted molecular and progression-related signaling pathways of PBLD.Methods1. The frequency of PBLD expression in HCC and its significance in patients with HCC(1) Tissue collection:Fresh HCC tissue samples, together with matched adjacent non-tumorous tissues, were collected from a total of 108 patients, who underwent surgical resection at our institute between 2008 and 2011.All enrolled subjects gave their written informed consent.(2) Exploration of PBLD expression in HCC tissues:We first examined the frequency of PBLD mRNA expression by real time PCR detection on larger sample sets. To further confirm the expression of PBLD, we carried out Western blotting and immunohistochemical analyses with 48 pairs of HCC and adjacent non-tumorous tissues.(3) The association between PBLD expression and prognosis of patients with HCC:To investigate the significance of PBLD expression in HCC, PBLD expression levels detected by qRT-PCR were correlated with specific clinicopathologic features of 108 patients with HCC. Kaplan-Meier and multivariate analysis were further used to analyze the significance of PBLD in the recurrence-free survival and overall survival of HCC patients. Moreover, this prognostic value of PBLD was further validated in another independent cohort.2. The biological function exploration of PBLD in HCC cell lines(1) The effect of PBLD on HCC cell proliferation: We chose HCC cell lines HepG2 and Huh7 for PBLD-targeted overexpression and chose HL-7702 and BEL-7402 for PBLD-targeted knockdown. We determined the effects of PBLD on cell growth in HCC cell lines in vitro by CCK-8 proliferation assay and colony-forming assay.(2) The effect of PBLD on HCC cell migration and invasion: To investigate the effects of PBLD on the migration (without Matrigel) and invasion of HCC cells, we carried out transwell migration and invasion assays.(3) The effect of PBLD on HCC cell cycles: Cell cycle distribution was analyzed by flow cytometry. HepG2 and Huh7 cells stably transfected with PBLD or empty vector were trypsinized, fixed in 70% ethanol, and incubated with propidium iodide (PI) along with RNase A.3. The effect of PBLD in tumor growth and angiogenesis in vivo(1) To explore whether the level of PBLD expression could affect the tumor growth, PBLD_pEGFP-N1 or empty vector stably-transfected HepG2 cells were respectively inoculated into the left flank of 4-to 6-week-old BALB/c male nude mice. Tumor volumes were examined every 5 days and calculated using the formula: length×width2/2. Mice were sacrificed by cervical dislocation after 30 days, and the tumors were frozen or fixed for immunofluorescence staining and western blot.(2) To explore the expression of CD31 immunohistochemistry staining was performed to analyze the effect of PBLD on vascularization of HepG2 cells in nude mice.4. Molecular mechanism examination(1) Microarray analysisBased on the observations, we performed Illumina’s whole-genome expression arrays to identify the downstream genes mediating the effects of PBLD in HepG2 cells. Data was collected using the Illumina Genome Studio software. Functional annotation was carried out using gene lists submitted to a variety of on-line software tools.(2) PBLD inhibited EMT of HCC cellsAnalysis of microarray data prompted us to further confirm the effect of PBLD overexpression on the functional networks. Using immunofluorescence staining, we detected the EMT markers (E-cadherin and N-cadherin) in two groups. Western blotting analysis was employed to investigate the two markers of HepG2-derived xenografts harboring PBLD_pEGFP-N1 or control empty vector as well as in the PBLD overexpression or knockdown HCC cells. Moreover, the association of PBLD and E-cadherin was further explored in 82 cases of HCC tissues by real time PCR assay.(3) PBLD inhibited the expression of VEGFUsing immunofluorescence staining, we detected the expression fo VEGF in HepG2-PBLD and HepG2-vector cells Then cck8 assay was used to detect whether VEGF could enhance the proliferation rate of HepG2-PBLD in vitro. Furthermore, western-blot was used to detect the influence of PBLD in VEGF and HIF1α expression both in normoxia and hypoxia environment.Results1. PBLD is downregulated in HCCPBLD mRNA expression was significantly decreased in HCC tissues compared with that in adjacent non-tumorous tissues, which was detected in 84.3% (91/108) of HCC patients, and PBLD mRNA expression in moderate to poor-differentiated HCC tissues was more frequently downregulated than that in well-differentiated tissues (P = 0.002). Western blotting and immunohistochemical analyses showed that its expression was also significantly decreased in HCC tissues at protein levels relative to non-tumorous tissues (P< 0.05). Surrounding non-tumorous liver tissues displayed extensively positive staining for PBLD, whereas HCC tissues showed variable proportions of PBLD-positive cells that gradually decreased with the grades of differentiation from well differentiated to poorly differentiated. Meanwhile, PBLD expression was decreased in 41 of 48 cases of HCC tissues as compared with matched adjacent non-tumorous tissues by western blot analysis.2. Decreased PBLD expression predicts poor prognosis in HCC patientsHigh PBLD expression was closely correlated with tumor differentiation (P= 0.017) and tumor stage (P= 0.038). Kaplan-Meier analysis revealed that decreased expression of PBLD expression was significantly associated with both shorter RFS (HR 0.447,95% CI 0.260-0.769, P= 0.004) and OS (HR 0.384,95% CI 0.165-0.894, P= 0.027). Moreover, multivariate analysis suggested that PBLD was an independent prognostic factor for both RFS (HR 0.502,95% CI 0.282-0.891, P= 0.019) and OS (HR 0.364,95% CI 0.138-0.957, P= 0.040). The prognostic role of tumor expression of PBLD was further validated in an independent cohort of 90 HCC patients. Kaplan-Meier analysis showed that PBLD was significantly associated with RFS (P= 0.012) and OS (P= 0.032). Multivariate analysis also indicated that PBLD was a powerful prognostic marker for recurrence (HR 0.490,95% CI 0.285-0.841, P= 0.010) and survival (HR 0.528,95% CI 0.303-0.922, P= 0.025)3. PBLD inhibited the growth, migration and invasion of HCC cells in vitroWe determined the effects of PBLD overexpression and knockdown on cell growth in HCC cells in vitro. The results of the CCK-8 proliferation assay showed that PBLD upregulation significantly inhibited the proliferation rate of HCC cells (P<0.01). Colony formation analysis consistently showed that PBLD significantly decreased the quantity and size of HepG2 and Huh7 cell colonies (816.67 vs 316.67, P<0.001; 736.33 vs 257.00,P<0.001). Cell cycle analysis revealed that PBLD overexpression caused a considerable inhibition of cell cycle progression, a characteristic decreased G1 phase, leading to a selective accumulation of cells in the S phase compared with control in Huh7 cells and G2/M phase arrest in HepG2 cells. We then tested the effects of PBLD silencing in relation to cell proliferation in the PBLD overexpressing cell lines HL-7702 and Bel-7402 in vitro. The results of the CCK-8 proliferation assay showed that PBLD downregulation significantly enhanced the proliferation rate of HCC cells (P<0.05). Transwell migration and invasion assays HepG2 and Huh7 cells migrated slower and had less ability to invade through the Matrigel-coated inserts when PBLD was upregulated. Taken together, the results mentioned above suggest that PBLD may be a potent tumor suppressor gene, and more investigation still need in vivo.4. Upregulated expression of PBLD retards tumor growth and angiogenesis in vivoThe xenograft tumors formed in HepG2_PBLD group were substantially smaller than those in the control group. We also found that the expression of CD31 (3.4 vs 5.4, P<0.05) which indicated vascularization was significantly inhibited in tumor xenografts formed from cells transfected with PBLD_pEGFP-N1 using immunohistochemistry staining. Immunofluorescence staining showed that the levels of PBLD expression in tumor tissues formed from PBLDPEGFP-N1 transfected cells were higher than those from empty vector (pEGFP-N1) transfected cells. These results in vivo experiments further indicate that overexpression of PBLD could inhibit HCC cell growth.5. Molecular mechanism analysis(1) PBLD inhibits multiple signaling pathways associated with tumor progression.Compared with the cells stably transfected with empty vector, specific deregulations of 289 known genes (96 upregulated genes; 193 downregulated genes) were identified in HepG2-PBLD cells. Concordant with the results presented in Figure 3A, gene set enrichment analysis (GSEA) showed that these deregulated genes were associated with cell cycle, cell adhesion, proliferation, and several tumorigenesis-related signaling pathways. Pathway analysis of differentially expressed genes further highlighted the functional networks, including MAPK, NF-kB, EMT, angiogenesis and others. From the heat map, a clear stepwise decrease in average gene expression can be seen from HepG2-PBLD to control cells.(2) PBLD inhibited EMT of HCC cells.Immunofluorescence staining and western-blot assays showed that the expression of E-cadherin was significantly enhanced in PBLD overexpression cell lines and xenografts while the expression of N-cadherin was decreased. Real time PCR detection in 82 cases of HCC tissues further conformed that the expression of PBLD was closely correlated with E-cadherin (r=0.61, P=0.000). Western-blot detection showed that PBLD could significantly inhibit the expression of snail(3) PBLD inhibited ERK/MAPK pathway.The expression of pERK was significantly inhibited by PBLD overexpression in HCC cells and xenograft tumors. The ERK pathway inhibitor U0126 could suppress the growth, migration and invasion of HCC cells.(4) PBLD inhibited the angiogenesis of HCC cells by HIF1 a/VEGFThe expression of VEGF was significantly inhibited when PBLD was overexpressed in HepG2 by immunofluorescence staining. Furthermore, we also proved that the proliferation rate of HepG2_PBLD was enhanced after treated with VEGF by CCK-8 assay in vitro. Western-blot showed that PBLD could significantly suppressed the expression of HIF1a and VEGF both in normoxia and hypoxia environment.Conclusion1. PBLD was frequently downregulated in HCC.2. Decreased PBLD expression predicted poor prognosis in HCC patients.3. PBLD inhibited the growth, migration and invasion of HCC cells in vitro. Upregulated expression of PBLD retards tumor growth and angiogenesis in vivo.4. PBLD inhibited multiple signaling pathways associated with tumor progression PBLD such as EMT, ERK/MAPK and so on.