MicroRNA-194 inhibits cell invasion and migration in hepatocellular carcinoma through PRC1-mediated inhibition of Wnt/ß-catenin signaling pathway.

BACKGROUND: Hepatocellular carcinoma (HCC) is a commonly occurring malignancy accompanied by significant mortality rates. More recently, extensive investigations into microRNA (miRNA) expression profiles have been conducted to identify their ability to inhibit tumors. Thus, this study explored the role of miR-194 in epithelial-mesenchymal transition (EMT), cell invasion and migration through Wnt/ß-catenin signaling pathway by binding to protein regulator of cytokinesis 1 (PRC1) in HCC. METHODS: Initially, HCC related microarray data were retrieved and analyzed, and regulatory miRNAs of PRC1 were predicted accordingly. Next, the roles of miR-194, PRC1, and Wnt/ß-catenin signaling pathway in HCC were determined, with relationship between PRC1 and miR-194 being verified subsequently. The role of miR-194 in cell EMT, migration, proliferation and invasion was evaluated through gain- and loss- function studies. Finally, tumor xenograft in nude mice was induced to assess tumor growth of HCC. RESULTS: miR-194 affected HCC development in Wnt/ß-catenin signaling pathway with putative binding sites to PRC1. MiR-194 could target PRC1. MiR-194 was downregulated while PRC1 was upregulated in HCC tissues. Additionally, miR-194 elevation and PRC1 silencing could suppress EMT, growth, proliferation, invasion, and migration in HCC cells by inactivating Wnt/ß-catenin signaling pathway. CONCLUSION: Taken together, this study demonstrated that miR-194 inhibited EMT, cell invasion and migration through inactivation of PRC1-dependent Wnt/ß-catenin signaling pathway.

Pseudogene integrator complex subunit 6 pseudogene 1 (INTS6P1) as a novel plasma-based biomarker for hepatocellular carcinoma screening.

In this study, we aimed to determine whether the pseudogene integrator complex subunit 6 pseudogene 1 (INTS6P1) in plasma could be used as a novel approach to screen for and detect hepatocellular carcinoma (HCC). We explored the clinical role of INTS6P1: First, the expression level of INTS6P1 was measured in a cohort of 33 HCC tissue samples and adjacent normal liver tissue, next, the INTS6P1 expression was detected in the culture medium and tumor cells in a cellular experiment, and last, the diagnostic performance of INTS6P1 was examined in an independent cohort of 100 people. The expression level of INTS6P1 was remarkably downregulated in the HCC tissues compared with that in the normal liver tissues (p = 0.0066). In plasma, the INTS6P1 levels were significantly decreased in HCC patients compared with non-HCC patients (p < 0.01). Additionally, we inferred that INTS6P1 might be a prospective biomarker for screening HCC patients in which the serum-AFP levels were lower than 20 ng/ml by the area under the curve-receiver operating characteristic (AUC-ROC) analysis (p < 0.05). Pseudogene INTS6P1 could be used as a novel HCC plasma-based biomarker and might improve the accuracy of HCC screening.

Retinoic acid receptor-related receptor alpha (RORalpha) is a prognostic marker for hepatocellular carcinoma.

Retinoic acid receptor-related receptor alpha (RORalpha) has been proven to play a tumor suppressive role in certain types of solid tumors. However, the clinical characteristic of RORalpha has not been reported by far. This study investigated the expression of RORalpha in hepatocellular carcinoma (HCC) and evaluated its relationship with clinical parameters and prognosis in HCC patients. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and Western blot analyses were performed to detect RORalpha expression levels in 20 paired HCC and corresponding adjacent non-cancerous tissues. Immunohistochemistry was performed on 100 archived paraffin-embedded HCC samples. Statistical analyses evaluated the correlations between RORalpha expression and clinicopathological features. qRT-PCR showed that RORalpha mRNA expression was significantly down-regulated in tumors compared to the adjacent non-cancerous tissues, and Western blots found that RORalpha protein expression was also reduced in tumor tissues. Immunohistochemical assays revealed that decreased RORalpha expression was present in 65 % of HCC patients. Correlation analyses showed that RORalpha expression was significantly correlated with serum alpha fetoprotein (AFP, p = 0.005), pathology grade (p < 0.001), tumor recurrence (p = 0.008), and vascular invasion (p < 0.001). Kaplan-Meier analysis revealed that patients with low RORalpha expression levels had a shorter overall and disease-free survival than patients with high expression (p < 0.001 and p = 0.002, respectively). Multivariate regression analysis indicated that RORalpha was an independent predictor for overall survival and disease-free survival. In conclusion, the results of our study showed that down-regulated RORalpha expression was associated with poorer prognosis in HCC patients. RORalpha may be a new potential prognostic marker for HCC patients.

[Identification and preliminary analysis of a novel full-length cDNA encoding retinoid X receptor 2 from Schistosoma japonicum].

OBJECTIVE: To clone and preliminarily analyze the full-length cDNA encoding retinoid X receptor 2 (RXR2) from Schistosoma japonicum. METHODS: The rapid amplification cDNA ends (RACE)was applied to get a full-length cDNA encoding retinoid X receptor 2 from S. japonicum (SjRXR2). The transcription of SjRXR2 was detected by real-time PCR. By bioinformatical technology, the gene structure was analyzed and the antibody epitope was predicted. The polyclonal antibodies were raised in mice immunized with the synthesis peptide. Western blot was applied to detect its expression in the worm. RESULTS: The full-length cDNA of SjRXR2 was 5 960 bp and contained an open reading frame encoding a 1 435 amino acid which had a predicted molecular weight 159 kDa. Bioinformatical analysis indicated that SjRXR2 had a highly conserved DNA binding domain (DBD) and a moderate conserved ligand binding domain (LBD). The relative mRNA (s) of SjRXR2 with higher expressions at Day 21 and 42 were evaluated in five different S. japonicum developmental stages. The Western blot analysis showed that polyclonal antibodies were able to specifically recognize the protein with molecular around 150 kDa from the extract of S. japonicum. CONCLUSION: A full-length cDNA encoding retinoid X receptor 2 (RXR2) from S. japonicum is obtained which provides preliminary information for further investigation of SjRXR2 functions in S. japonicum.

Gardenia jasminoides attenuates hepatocellular injury and fibrosis in bile duct-ligated rats and human hepatic stellate cells.

AIM: To investigate the anti-hepatofibrotic effects of Gardenia jasminoides in liver fibrosis. METHODS: Male Sprague-Dawley rats underwent common bile duct ligation (BDL) for 14 d and were treated with Gardenia jasminoides by gavage. The effects of Gardenia jasminoides on liver fibrosis and the detailed molecular mechanisms were also assessed in human hepatic stellate cells (LX-2) in vitro. RESULTS: Treatment with Gardenia jasminoides decreased serum alanine aminotransferase (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 146.6 ± 15 U/L vs 77 ± 6.5 U/L, P = 0.0007) and aspartate aminotransferase (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 188 ± 35.2 U/L vs 128 ± 19 U/L, P = 0.005) as well as hydroxyproline (BDL vs BDL + 100 mg/kg Gardenia jasminoides, 438 ± 40.2 µg/g vs 228 ± 10.3 µg/g liver tissue, P = 0.004) after BDL. Furthermore, Gardenia jasminoides significantly reduced liver mRNA and/or protein expression of transforming growth factor ß1 (TGF-ß1), collagen type I (Col I) and α-smooth muscle actin (α-SMA). Gardenia jasminoides significantly suppressed the upregulation of TGF-ß1, Col I and α-SMA in LX-2 exposed to recombinant TGF-ß1. Moreover, Gardenia jasminoides inhibited TGF-ß1-induced Smad2 phosphorylation in LX-2 cells. CONCLUSION: Gardenia jasminoides exerts antifibrotic effects in the liver fibrosis and may represent a novel antifibrotic agent.

[Evaluation on the immuno-protective efficacy of the recombinant antigen SjPGAM-SjEnol against Schistosoma japonicum in mice].

OBJECTIVE: To construct and express the recombinant plasmid pET32a-SjPGAM-SjEnol and evaluate its immuno-protective efficacy against the infection of Schistosoma japonicum in mice. METHODS: The peptides of SjPGAM and SjEnol containing the multivalent epitopes with higher binding capacity of human MHC II and mouse H2-dII but low homology with the host were analyzed and screened through bioinformatics. The corresponding nucleotide sequence of selected epitopes was spliced and the recombinant plasmid pET32a-SjPGAM-SjEnol was constructed and expressed in Escherichia coli BL21 cells. The antigenicity of the recombinant protein was detected by Western blotting and the protective effect induced with the recombinant was evaluated in mice. 55 BALB/c mice were randomly divided into 5 groups each with 11. Mice from groups A, B and C were injected with a mixture of recombinant protein (27 microg) pET32a-SjPGAM-SjEnol (A), pETL28a-SjPGAM (B) and pET28a-SjEnol (C) respectively together with 206 adjuvant, mice from groups D and E received adjuvant or PBS only, all injected for three times at two-week intervals. Mice were then challenged with 40 +/- 2 cercariae of S. japonicum at two weeks after the last vaccination, and sacrificed for perfusion by 6 weeks post infection. Adult worms were collected, the number of eggs in a gram of liver tissue was counted, and the rates of worm reduction and egg reduction were calculated. Serum samples were collected before vaccination, every one week after each inoculation and before sacrifice, and specific IgG was detected by ELISA. RESULTS: The sequences encoding the 96-147 aa of SjPGAM and 233-312 aa of SjEonl were chosen for constructing the recombinant plasmid, a cDNA fragment with the length of 447 bp was amplified by PCR. The recombinant plasmid was expressed in E. coli with a molecular weight of Mr 33,000. Western blotting revealed that the fusion protein was recognized by the rabbit serum specific to SjSWAP, and showed an adequate antigenicity. Vaccination experiment showed that when compared with those of the blank control, the worm reduction rate in group A was 39.7%, significantly higher than that of groups B (18.5%) and C (14.7%) (P < 0.05). The liver egg reduction rate in group A was 64.9%, also higher than that of groups B (47.5%, P < 0.05) and C (30.5%, P < 0.01). ELISA showed that the serum specific IgG in group A (2.372 +/- 0.268) was much higher than that of groups D (0.490 +/- 0.138) (P < 0.01 and E (0.220 +/- 0.088) (P < 0.01). CONCLUSION: The recombinant plasmid pET32a-SjPGAM-SjEnol has been constructed, and recombinant protein pET32a-SjPGAM-SjEnol induces higher immune-protection against S. japonicum than that of SjPGAM and SjEonl.

[Immune protection of tegument protein rSj29 against Schistosoma japonicum in mice].

OBJECTIVE: To clone, express and characterize a tegument protein gene of Schistosoma japonicum (Sj29) , and investigate the immune protection of the recombinant protein against S. japonicum in mice. METHODS: The gene coding for Sj29 protein was amplified by PCR, and the sequence was analyzed by bioinformatics tools. Partial fragment of Sj29 gene was subcloned into the prokaryotic expression vector pET28c(+). The recombinant plasmid was transformed into E. coli BL21 (DE3) and induced the recombinant with IPTG. The recombinant protein (rSj29) was purified by His-binding-resin affinity chromatography and characterized by Western blotting. Three groups each with 10 BALB/c mice were immunized subcutaneously three times (two weeks interval) respectively with 100 microl recombinant rSj29 (0.1 mg/ml) , adjuvant or PBS. At the 15th day after the final inoculation, each mouse was challenged by 40 +/- 2 cercariae of S. japonicum. At the 53rd day after infection, the mice were sacrificed to obtain the number of adult worms, number of eggs in liver and feces. Serum samples were collected at pre-immunization and certain time after immunization, and were analyzed for IgG by ELISA. The localization of rSj29 in worms of different developmental stages was demonstrated by immunofluorescent technique. mRNA expression level of Sj29 gene in worms of different developmental stages and three groups after infection was detected by quantitative real-time PCR. RESULTS: A 576 bp Sj29 gene fragment was obtained. The recombinant protein rSj29 with Mr 22,900 was expressed in the form of inclusion body. The recombinant rSj29 can be recognized by sera of mice immunized with rSj29 and sera of infected mice. The number of adult worms (15.4 +/- 5.9), number of hepatic eggs (40,143.3 +/- 2,995.9) and number of fecal eggs (3,803.9 +/- 110.9) in recombinant protein group were significantly higher than those of PBS control group (20 +/- 3.4, 49,318.1 +/- 6,648.3, 5,238.1 +/- 303.5, respectively) (P < 0.05) . There was a high level of specific IgG against rSj29 (maximum dilution 1:32000) in recombinant protein group. Immunohistochemical analysis showed the Sj29 protein expressed on the surface of different stages of S. japonicum. mRNA level of Sj29 was the highest at the 32nd day post-infection. CONCLUSION: The recombinant protein rSj29 induces certain degree of protective immunity in mice.