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ObjectiveTo investigate the inhibitory effects and mechanism of the compound Phyllanthus urinaria Ⅱ （CPU Ⅱ）on the growth of transplanted hepatocellular carcinoma Hep3B2.1-7 （Short for Hep3R） cells in nude mice. MethodAfter the establishment of a xenograft model of hepatocellular carcinoma Hep3B cells in mice， the model mice were randomly divided into a model group， a high-dose CPU Ⅱ group （57.5 g·kg-1）， a low-dose CPU Ⅱ group （28.75 g·kg-1）， and a 5-fluorouracil （5-FU） group （0.025 g·kg-1）， with eight mice in each group. The mice in the high- and low-dose CPU Ⅱ groups were treated with drugs by gavage， once per day， and those in the model group were treated with the same volume of normal saline. The mice in the 5-FU group were treated by 5-FU by intraperitoneal injection， once every other day. After 28 days of administration， mice were sacrificed， and transplanted tumors were collected. Immunohistochemistry （IHC） was used to detect the expression of proliferating cell nuclear antigen （PCNA） of tumor tissues. Terminal-deoxynucleotidyl transferase-mediated nick end labeling （TUNEL） was used to detect cell apoptosis of tumor tissues. The mRNA expression of miR-122 and insulin-like growth factor 1 receptor （IGF-1R） in tumor tissues was detected by Real-time quantitative PCR （Real-time PCR）. The protein expression of CCAAT/enhancer-binding protein α （C/EBPα）， hepatocyte nuclear factor-4α （HNF-4α）， and IGF-1R in tumor tissues was detected by Western blot. ResultThe tumor suppression rates of the high- and low-dose CPU Ⅱ groups and the 5-FU group were 74.90%， 63.62%， and 64.15%， respectively. Compared with the model group， the CPU Ⅱ groups and the 5-FU group showed reduced weight （P<0.01） and volume of tumors （P<0.01）， decreased PCNA positive cells， shallow staining， increased apoptosis cells of transplanted tumor tissues （P<0.05， P<0.01）， increased expression of mRNA expression of miR-122 （P<0.01）， down-regulated mRNA expression of IGF-1R （P<0.01）， and up-regulated protein expression of C/EBPα and HNF-4α in nude mouse transplanted tumor tissues （P<0.01）. The expression of IGF-1R protein in the high-dose CPU Ⅱ group was down-regulated （P<0.05）. Compared with the low-dose CPU Ⅱ group， the high-dose CPU Ⅱ group showed increased apoptotic cells （P<0.01）， up-regulated mRNA expression of miR-122 （P<0.01）， and increased expression of C/EBPα and HNF-4α proteins （P<0.01）. ConclusionCPU Ⅱ has an obvious inhibitory effect on the growth of transplanted hepatocellular carcinoma Hep3B cells in nude mice. The mechanism of action is related to enhancing the expression of transcription factors HNF-4α and C/EBPα， thereby promoting the expression of miR-122 and inhibiting the expression of its target gene IGF-1R.
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Objective To investigate the roles and mechanisms of hepatocyte nuclear factor 4α (HNF4α) in chenodeoxycholic acid (CDCA) induced gastric intestinal metaplasia (IM).Methods After the immortalized gastric mucosal epithelial cells GES-1 were stimulated with CDCA at different concentration,the changes of HNF4α,caudal-related homeobox 2 (CDX2) and trefoil factor family 3 (TFF3) expressions at mRNA and protein levels in GES-1 cells and gastric cancer cell lines (AGS,SGC7901 and BGC823) were detected by real time-polymerase chain reaction (RT-PCR) and Western blotting.After GES-1 were transfected with HNF4α short hairpin RNA (shRNA) or control shRNA,and followed by CDCA stimulation,the expressions of HNF4α,CDX2 and TFF3 at protein level were determined by Western blotting.HNF4α was overexpressed in GES-1 cells and SGC7901 cells,and HNF4α was silenced in BGC823 cell line and AGS by lentiviral vector system.The expressions of HNF4α,CDX2 and TFF3 at mRNA and protein levels were tested by RT-PCR and Western blotting.Luciferase reporter assay was used to analyze the regulation role of HNF4α on CDX2.T test was performed for statistical analysis.Results The expressions of HNF4α in GES-1,SGC7901,BGC823 and AGS cells at mRNA level were 1.00 ± 0.12,263.01±10.23,848.01±18.13 and 3 049.86±91.75,respectively.The mRNAlevels of HNF4α in AGS,BGC823 and SGC7901 cells were all higher than that of GES-1 cells,and the differences were statistically significant (t=33.23,46.72 and 25.62,all P＜0.01).The expressions of HNF4α in GES-1,SGC7901,BGC823 and AGS at protein level were consistent with mRNA level.The expressions of CDX2 and TFF3 at protein level of HNF4α shRNA transfected group were lower than those of non-HNF4α shRNA transfected group.In GES-1 cells,the expressions of HNF4α,CDX2 and TFF3 of HNF4α overexpressed group at mRNA level were 16 281.839 ± 1 843.017,6.275 ± 0.137 and 17.310± 1.533,respectively;which were all higher than those of overexpressed control group (1.000 ± 0.048,1.000 ± 0.012 and 1.000±0.108,respectively),and the differences were statistically significant (t =8.83,38.29 and 10.61,all P＜0.01).In AGS cells,the expressions of HNF4α,CDX2 and TFF3 of HNF4α silenced group at mRNA level were 0.021 ± 0.001,0.088 ± 0.007 and 0.074 ± 0.002,respectively,which were lower than those of silenced control group (1.000 ± 0.108,1.000 ± 0.131 and 1.000 ± 0.122),and the differences were statistically significant (t=9.09,6.93 and 7.57,all P＜0.01).In GES-1 overexpressed cells and AGS silenced cells,the expressions of HNF4α,CDX2,TFF3 at protein level were consistent with mRNA level.In double reporter plasmid containing the CDX2 promoter CDX2 1 (-2 000～-1 bp) and CDX2-2 (-1 510～1 bp),after transfected with HNF4α shRNA,the activities were 0.387 ± 0.013 and 0.533 ± 0.040,respectively,which were lower than those of HNF4α shRNA transfected control group (0.605 ± 0.012 and 0.882 ± 0.019),and the differences were statistically significant (t =21.49 and 13.53,both P＜0.01).Conclusion HNF4α may be involved in bile acid induced intestinal metaplasia by upregulating the expression of CDX2.
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Objective To establish a luciferase reporter gene system for detecting the activity of hepatocyte nuclear factor 4α (HNF4α), so as to screen the small molecule compounds regulating the activity of HNF4α. Methods HNF4α was purified by affinity chromatography. The direct interaction of DNA fragment or small molecule compounds to the HNF4α was determined by protein thermal shift assay. The constructing recombinant plasmid pGL3-NINJ1-3p or pGL3-NINJ1-9p, which contained three copies or nine copies of the HNF4α response element in the Ninjurin 1 (NINJ1) promoter, was transfected into hepatoma carcinoma cells. The transcriptional activity of HNF4α was detected by dual-luciferase reporter gene assay. The expressions of HNF4α and its down-stream genes were analyzed in hepatoma carcinoma cells treated with small molecular compound luteolin or alverine by real-time quantitative PCR. The changes of HNF4α transcriptional activity of cells treated with luteolin or alverine were estimated by luciferase reporter gene assay. Results Protein thermal shift confirmed that the HNF4α response element in NINJ1 promoter bound to HNF4α protein. In the hepatoma carcinoma cells with overexpression of HNF4α, both pGL3-NINJ1-3p and pGL3-NINJ1-9p could detect the alteration of the transcriptional activity of HNF4α, and pGL3-NINJ1-9p was more sensitive than pGL3-NINJ1-3p (P<0.01). Luteolin and alverine, both directly interacting with HNF4α, down-regulated and up-regulated the expression of HNF4α target genes, respectively. Moreover, pGL3-NINJ1-9p could validate the effect of luteolin or alverine on the transcriptional activity of HNF4α. Conclusion We successfully establish a detection system for HNF4α activity in hepatoma carcinoma cells by the reporter gene vector pGL3-NINJ1-9p. This system is a tool for screening small molecule compounds that regulate HNF4α activity.
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In our preliminary studies, we observed zolmitriptan (ZOL) treatment led to induction of CYP3A2 in male not female rats. To figure out the reason is of great significance for drug-drug interactions and personalized administration. Since growth hormone (GH) is known as the major mechanistic determinant of sexually-dimorphic gene expression like CYP3A2 in rat liver, the impacts of ZOL on both plasma GH levels in non monosodium glutamate (MSG)-treated rats and CYP3A2 expression in GH depleted MSG-treated rats were studied. ZOL was shown to partially suppress GH levels in both genders. Furthermore, CYP3A2 protein and mRNA level declined in male not female MSG-treated rats. In order to study the possible molecular events involved in the depression of GH and gender-selective induction on rat CYP3A2 by ZOL, the mRNA and protein level (whole protein and nuclear protein) of hepatocyte nuclear factor 4α (HNF4α) was investigated. Nuclear accumulation of HNF4α was observed in the normal male not female rat liver tissue following ZOL treatment. However, this kind of nuclear translocation did not occur in rat hepatocytes and MSG-treated rats. These findings demonstrated CYP3A2 inducibility by ZOL was gender-selective. GH and HNF4α may play an important role in CYP3A2 induction.
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Objective: To investigate the possible protective effects of sophocarpine on mucosal injury and epithelial barrier disruption on dextran sulfate sodium (DSS)-induced acute colitis. Methods: Male BALB/c mice were randomly divided into three groups. The mice in normal group were given normal water, and those in model and sophocarpine-treated groups were given 2.5% DSS for 6 d to induce acute colitis. Sophocarpine (30 mg/kg) was ip administered once daily during the study period. Severity of colitis was evaluated by disease activity index (DAI), histological injury and inflammatory cytokine production including tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and monocyte chemoattractant protein-1 (MCP-1). The colonic barrier disruption was assessed by testing the expression of junctional adhesion molecule-1 (JAM-1), E-cadherin (E-CAD), and desmocollin-2 (DSC-2) in colon mucosa. Expression of HNF4α in colon mucosa was detected by immunohistochemistry staining and real-time RT-PCR, respectively. Results: Compared with normal group, DAI, colonic shortening, and histopathological injury in model group were elevated (P < 0.05), but reduced in sophocarpine-treated group (P < 0.05). Compared with model group, the mRNA expression of inflammatory cytokines (TNF-α, IL-1β, MCP-1) were obviously lower in sophocarpine-treated group (P < 0.05), while the cellular junction proteins (E-CAD, JAM-1, and DSC-2) were higher (P < 0.05). The expression of HNF4α at mRNA and protein levels was decreased significantly in model group, but increased apparently in sophocarpine-treated group. Conclusion: Sophocarpine can enhance the expression of HNF4α, promote the expression of colonic intrecellular junctions, thus, maintain the integrity of the colonic barrier and inhibit the colitis process. We suggest that sophocarpine could enhance the production of cellular junction proteins to protect the intestinal barrier fuction, at least partly, in HNF4α-dependent pathway.
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Hepatocyte nuclear factor 4α is a transcription factor of the nuclear hormone receptor superfamily and plays a very important role in the differentiation and development of the digestive system as well as the polarity regulation,which is considered to be a tumor suppressor gene.It also plays a very important role in digestive tumorigenesis,invasion and metastasis of hepatic carcinoma,colon cancer and gastric cancer,which is closely associated with epithelial-to-mesenchymal transition,the regulation of cell proliferation and wnt signaling pathway.HNF4α expression and its regulation mechanism in the digestive system tumors are now the research focus.
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Objective To investigate the clinical significance of hepatocyte nuclear factor 4α(HNF4α)in rectal cancer and its relationship with prognosis.Methods Real-time PCR was designed to detect the expression of HNF4αon mRNA level and the immunohistochemistry was used to determine the expression of HNF4αon protein level in rectal cancer tissue.The relationship between HNF4αexpression and clinical characteristics was also analysed.The Kaplan-Meier method was used for univariate analysis and a Cox proportional hazards regression model was performed for multivariate analysis.Results HNF4αwas low expressed both on mRNA (t=6.092,P<0.001)and protein level (χ2 =15.230,P<0.001)in rectal cancer tissue.HNF4αexpression on protein level was related with the clinical stage (χ2 =48.311,P<0.001),depth of invasion (χ2 =23.911,P<0.001),histological differentiation (χ2 =20.787,P<0.001),lymph node metastasis (χ2 =39.064,P<0.001)and distant metastasis (χ2 =5.146,P=0.04),while age and gender were not relevant.The cumulative 3-year overall survival of patients with low HNF4αexpression (43.8%)was much worse than the patients with high HNF4αexpression (95 .5%),and the difference was statistically sig-nificant (P<0.001).Univariate analysis revealed that HNF4αexpression (χ2 =28.778,P<0.001),differ-entiation (χ2 =26.680,P<0.001 ),clinical stage (χ2 =32.702,P<0.001 ),depth of invasion (χ2 =6.226,P=0.013),lymph node invasion (χ2 =15.270,P<0.001)and distant metastasis (χ2 =21.817, P<0.001)were statistically significant worse predictors for rectal cancer,whereas age and gender were not rel-evant.The multivariate Cox proportional hazard analysis revealed that HNF4αlow expression (RR=6.084, P=0.028)was independent prognostic markers for 3-year overall survival in the patients with rectal cancer. Conclusion HNF4αwas closely related to the tumorigenesis and progression of rectal cancer,which is an independent prognostic marker for rectal cancer,and which may be an effective target for the therapy of rectal cancer.