Effect of Compound Phyllanthus urinaria Ⅱ on Growth and Expression of miR-122 and IGF-1R of Hepatocellular Carcinoma Hep3B Cells in Nude Mice / 中国实验方剂学杂志

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.

Research progress on sex hormone-binding globulin involved in the occurrence and development of diabetes mellitus / 中国基层医药

Sex hormone-binding globulin (SHBG) is significantly associated with abnormal glucose metabolism. Low SHBG level is a risk factor for insulin resistance and the occurence of diabetes mellitus. SHBG is negatively correlated with the risk of type 2 diabetes mellitus and plays an important role in regulating insulin resistance while predicting its development. The genotype of SHBG has been found to be closely related to the occurrence of diabetes mellitus. Fatty liver and DNA methylation are also important factors mediating the relationship between SHBG and type 2 diabetes mellitus. The change in SHBG level may be related to insulin resistance by influencing hepatocyte nuclear factor 4a and regulating glucose transporter.

Sortilin-induced lipid accumulation and atherogenesis are suppressed by HNF1b SUMOylation promoted by flavone of Polygonatum odoratum / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

This study aims to investigate the impact of hepatocyte nuclear factor 1β (HNF1b) on macrophage sortilin-mediated lipid metabolism and aortic atherosclerosis and explore the role of the flavone of Polygonatum odoratum (PAOA-flavone)-promoted small ubiquitin-related modifier (SUMO) modification in the atheroprotective efficacy of HNF1b. HNF1b was predicted to be a transcriptional regulator of sortilin expression via bioinformatics, dual-luciferase reporter gene assay, and chromatin immunoprecipitation. HNF1b overexpression decreased sortilin expression and cellular lipid contents in THP-1 macrophages, leading to a depression in atherosclerotic plaque formation in low-density lipoprotein (LDL) receptor-deficient (LDLR-/-) mice. Multiple SUMO1-modified sites were identified on the HNF1b protein and co-immunoprecipitation confirmed its SUMO1 modification. The SUMOylation of HNF1b protein enhanced the HNF1b-inhibited effect on sortilin expression and reduced lipid contents in macrophages. PAOA-flavone treatment promoted SUMO-activating enzyme subunit 1 (SAE1) expression and SAE1-catalyzed SUMOylation of the HNF1b protein, which prevented sortilin-mediated lipid accumulation in macrophages and the formation of atherosclerotic plaques in apolipoprotein E-deficient (ApoE-/-) mice. Interference with SAE1 abrogated the improvement in lipid metabolism in macrophage cells and atheroprotective efficacy in vivo upon PAOA-flavone administration. In summary, HNF1b transcriptionally suppressed sortilin expression and macrophage lipid accumulation to inhibit aortic lipid deposition and the development of atherosclerosis. This anti-atherosclerotic effect was enhanced by PAOA-flavone-facilitated, SAE1-catalyzed SUMOylation of the HNF1b protein.

Forkhead box A1 expression in thyroid carcinoma based on bioinformatics andclinical significance

Thyroid cancer, known to be common in women than men, accounts for 12% all types of cancers and ranks 9th with5,86,202 cases worldwide. Role of forkhead box A1 (FOXA1), also known as hepatocyte nuclear factor 3? and involved inthe oncogenesis and progression of several tumors such as gliomas, breast, stomach, lung, ovarian, and esophageal cancers,has not been elucidated well in thyroid carcinoma until now. Here, we analyzed the expression of FOXA1 in thyroidcarcinoma tissues and its effects on the biological characteristics of papillary thyroid carcinoma TPC-1 cells. The expressionlevels of FOXA1 in normal thyroid and thyroid carcinoma tissues were analyzed using UALCAN database(http://ualcan.path.uab.edu/index.html), and the correlations between FOXA1 expression levels and survival time of patientswith thyroid carcinoma were analyzed. Si-FOXA1 and si-NC were transfected into cells that were then divided into si-FOXA1 (transfected si-FOXA1), si-NC (transfected si-NC), and control (TPC-1) groups. Cell proliferation was determinedwith MTT assay, and invasion ability was measured by Transwell assay. Early apoptotic rate was detected by flowcytometry. The mRNA expression levels of p27Kip1, Cyclin D1 and Cyclin E were measured by qRT-PCR. The expressionlevel of FOXA1 was significantly higher in thyroid carcinoma tissues than that in normal thyroid tissues. UALCAN-basedanalysis indicated that patients with low expression level of FOXA1 had longer survival time than those with highexpression level of FOXA1 (P <0.05). The cell proliferation rate was significantly lower in si-FOXA1 group than those insi-NC group and control group at 24 h, 48 h and 72 h (P <0.05). The early apoptotic rate was significantly higher andnumber of invading cells was lower in si-FOXA1 group than those in si-NC and control groups (P <0.05). Si-FOXA1 grouphad higher mRNA expression level of p27Kip1 and lower expression levels of Cyclin D1 and Cyclin E than those of si-NCand control groups (P <0.05). Targeted inhibition of FOXA1 suppresses the proliferation and invasion and promotes theapoptosis of thyroid carcinoma cells, probably by regulating activation of the p27Kip1 pathway.

PXR activation impairs hepatic glucose metabolism partly via inhibiting the HNF4α-GLUT2 pathway

Drug-induced hyperglycemia/diabetes is a global issue. Some drugs induce hyperglycemia by activating the pregnane X receptor (PXR), but the mechanism is unclear. Here, we report that PXR activation induces hyperglycemia by impairing hepatic glucose metabolism due to inhibition of the hepatocyte nuclear factor 4-alpha (HNF4α)‒glucose transporter 2 (GLUT2) pathway. The PXR agonists atorvastatin and rifampicin significantly downregulated GLUT2 and HNF4α expression, and impaired glucose uptake and utilization in HepG2 cells. Overexpression of PXR downregulated GLUT2 and HNF4α expression, while silencing PXR upregulated HNF4α and GLUT2 expression. Silencing HNF4α decreased GLUT2 expression, while overexpressing HNF4α increased GLUT2 expression and glucose uptake. Silencing PXR or overexpressing HNF4α reversed the atorvastatin-induced decrease in GLUT2 expression and glucose uptake. In human primary hepatocytes, atorvastatin downregulated GLUT2 and HNF4α mRNA expression, which could be attenuated by silencing PXR. Silencing HNF4α downregulated GLUT2 mRNA expression. These findings were reproduced with mouse primary hepatocytes. Hnf4α plasmid increased Slc2a2 promoter activity. Hnf4α silencing or pregnenolone-16α-carbonitrile (PCN) suppressed the Slc2a2 promoter activity by decreasing HNF4α recruitment to the Slc2a2 promoter. Liver-specific Hnf4α deletion and PCN impaired glucose tolerance and hepatic glucose uptake, and decreased the expression of hepatic HNF4α and GLUT2. In conclusion, PXR activation impaired hepatic glucose metabolism partly by inhibiting the HNF4α‒GLUT2 pathway. These results highlight the molecular mechanisms by which PXR activators induce hyperglycemia/diabetes.

Effect of Shuangyu Tiaozhi Decoction on HNF1α/PCSK9/LDLR Signaling Pathway in Liver of Hypercholesterolemic Rats / 中国实验方剂学杂志

Objective:To observe the expression of hepatocyte nuclear factor 1<italic>α</italic> （HNF1<italic>α</italic>）， proprotein convertase subtilisin/kexin type 9 （PCSK9） and low-density lipoprotein cholesterol （LDLR） in hypercholesterolemia rat liver， and investigate the mechanism of Shuangyu Tiaozhi Decoction regulating cholesterol metabolism and attenuating hypercholesterolemia. Method:After providing a high-fat diet for 4 weeks， 40 SD rats were selected， 8 of which were randomly selected as normal group and fed a normal diet， and the remaining 32 rats were fed a high-fat diet. The rats successfully established as hypercholesterolemic model， were randomized into 4 groups： model group， low dose of Shuangyu Tiaozhi decoction group （7.8 g·kg^-1）， high dose of Shuangyu Tiaozhi decoction group （15.6 g·kg^-1）， and simvastatin group （4 mg·kg^-1）， with 8 rats in each group. The drugs were continuously given for 8 weeks. Serum total cholesterol （TC）， triglyceride （TG）， low-density lipoprotein cholesterol （LDL-C）， high-density lipoprotein cholesterol （HDL-C） were measured. The pathomorphological changes in liver were observed by hematoxylin and eosin （HE） staining. The immunohistochemistry was used to detect PCSK9 and LDLR expression in liver. The mRNA and protein expression levels of HNF1<italic>α</italic>， PCSK9 and LDLR were determined by Real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot. Result:Compared with normal group， the TC， TG， LDL-C levels in model group were significantly increased （<italic>P</italic><0.01）， the morphology showed obvious liver steatosis. The mRNA and protein expression of HNF1<italic>α</italic> and PCSK9 were increased （<italic>P</italic><0.05）， the mRNA and protein expression of LDLR was decreased （<italic>P</italic><0.05）. Compared with model group， the serum TC， TG， LDL-C levels were significantly lowered in the Shuangyu Tiaozhi decoction high-dose group （<italic>P</italic><0.01）， the serum TC， LDL-C levels were significantly lowered in the Shuangyu Tiaozhi decoction low-dose group and simvastatin group （<italic>P</italic><0.05，<italic>P</italic><0.01）， while no significant effect was observed on the serum HDL-C levels in each treatment group. The liver steatosis decreased in each treatment group. The mRNA and protein expression of HNF1<italic>α</italic> was obviously decreased in each treatment group （<italic>P</italic><0.05，<italic>P</italic><0.01）， the mRNA and protein expression of PCSK9 was obviously decreased in Shuangyu Tiaozhi decoction low and high-dose groups （<italic>P</italic><0.05，<italic>P</italic><0.01）， the mRNA expression of PCSK9 was significantly increased in the simvastatin group （<italic>P</italic><0.01）， while the protein expression showed a downward trend. The LDLR mRNA levels were significantly increased in each treatment group （<italic>P</italic><0.01）， the LDLR protein expression was significantly increased in Shuangyu Tiaozhi high-dose group （<italic>P</italic><0.01）， and showed an upward trend in Shuangyu Tiaozhi low-dose group and simvastatin group. Results of immunohistochemistry showed PCSK9 expression was weakly positive， the expression of LDLR was strongly positive in each treatment group. The therapeutic effect of Shuangyu Tiaozhi decoction high-dose group was more remarkable than simvastatin group， while there was no obvious difference between the Shuangyu Tiaozhi decoction low-dose group and simvastatin group. Conclusion:Shuangyu Tiaozhi decoction may reduce the blood lipid levels through HNF1<italic>α</italic>/PCSK9/LDLR signaling pathway， play an active role on regulating cholesterol metabolism and alleviating high-fat diet-induced hypercholesterolemia.

Expression of alpha-methylacyl-CoA racemase in vaginal gastric-type adenocarcinoma and uterine clear cell carcinoma

Background: Alpha-methylacyl-coenzyme A racemase (AMACR, P504S) is a commonly used marker in immunohistochemical diagnosis of prostate cancer. Recent studies identified P504S markers of the clear cell histotype in the ovary and/or endometrium. Gastric-type adenocarcinoma (GAS) is difficult to diagnose histologically, particularly when there is crossover with clear cell carcinoma (CCC). However, the significance of P504S for differentially diagnosing GAS and CCC is unclear. Aim: To evaluate P504S as a potential diagnostic marker of GAS and CCC. Settings and Design: We analyzed P504S expression in 48 cervical carcinomas (32 GAS and 16 CCC), as well as the expression of other markers including hepatocyte nuclear factor-1 beta (HNF-1?) and NapsinA. Material and Methods: The expression differences of HNF-1?, NapsinA, and P504S in GAS and CCC were detected by immunohistochemistry. Immunohistochemical histoscores based on the intensity and extent of staining were calculated. Results: The positive rates of HNF-1? in GAS and CCC were 90.32% and 75%, respectively. (?2 = 2.251, P = 0.663). The positive rates of NapsinA in GAS and CCC were 19.36% and 81.25%, respectively. (?2 = 47.332, P < 0.01). The positive rates of P504S in GAS and CCC were 16.13% and 81.25%, respectively. (?2 = 41.420, P < 0.01). HNF-1? was frequently expressed in GAS and CCC, while NapsinA and P504S were frequently expressed in CCC, and reduced or lost in GAS. Conclusion: NapsinA and P504S can be used to differentiate between GAS and CCC.

Effects of HNF1A inhibition on drug resistance of human pancreatic cancer PANC1 cells to gemcitabine plus abraxane / 中华胰腺病杂志

Objective To explore the effects of hepatocyte nuclear factor 1 homeobox A(HNF1A) on drug resistance of PANC1 cells to gemcitabine plus abaraxane and explore the potential mechanism. Methods 78 pancreatic cancer patients with locally advanced or distant metastasis who received gemcitabine plus abaraxane chemotherapy after surgery in Biliary and Pancreatic Surgery Department of Sun Yat-sen Memorial Hospital from March 2012 to May 2017 were enrolled. qPCR was used to detect HNF1A mRNA levels in pancreatic cancer tissue. The patients were divided into high-expression group ( n=39 ) and low-expression group (n=39) according to the median expression level of HNF1A, and the correlation of HNF1A expression with cancer clinicopathologic parameters and survival was analyzed. qPCR was used to detect HNF1A mRNA of 3 drug-sensitive cell lines (BxPC-3, CFPAC-1 and L3. 6pl) and 4 drug-resistant pancreatic cancer cell lines (PANC1, MIA PaCa-2, Hs766T and Mpanc96). Lentivirus with plasmids carrying HNF1AcDNA infection was used to establish HNF1A overexpressing PANC1 cells ( HNF1A group), and lentivirus with empty plasmids were used to infect PANC1 cells to construct the control group. The mRNA and protein expression of HNF1A and ATP binding cassette transporter family ABCC1 in HNF1A group and control group were measured by qPCR and Western Blot, respectively. The half inhibition concentration ( IC50 ) of gemcitabine plus abaraxane was detected by MTT, and cell apoptosis was examined by flow cytometry. Results Pancreatic cancer patients with high HNF1A expression had a better overall survival than those with low HNF1A expression (17. 9 months vs 12.4 months), and the difference was statistically significant (P<0.001). HNF1A low expression in pancreatic cancer tissue was significantly associated with advanced TNM stage, perineural invasion ( PNI) and short overall survival. The expression level of HNF1A was significantly down-regulated in drug-resistant PANC1 cells compared to drug-sensitive BxPC-3 cells by an average fold change of 6. 73, and the difference was statistically significant ( P<0. 001 ). In HNF1A group, the mRNA and protein levels of ABCC1 were significantly decreased compared with those in control group (0. 012 ± 0. 004vs 0. 047 ± 0. 008,0. 281 ± 0. 040 vs 0. 832 ± 0. 046,P=0. 003,P <0. 001). IC50 of HNF1A group to gemcitabine plus abraxane was decreased compared with that of control group [(26. 31 ± 2. 91)μmol/L vs (72. 63 ± 4. 07) μmol/L], and the cell apoptosis rate of HNF1A group was increased compared with that of control group [(40. 18 ± 1. 64)％ vs (21. 31 ± 1. 98)％], and the differences were statistically significant (P<0. 01). Conclusions HNF1A may induce resistance of pancreatic cancer cell to gemcitabine plus abraxane by downregulating ABCC1.

Expression and Significance of Foxa2 in Colorectal Polyps and Colorectal Cancer / 胃肠病学

Background: Forkhead box A2 (Foxa2) plays an important role in the proliferation and distant metastasis of colorectal cancer, but the expression of Foxa2 in colorectal polyps has not been reported yet. Aims: To investigate the expression and significance of Foxa2 in colorectal polyps and colorectal cancer. Methods: A total of 45 patients with hyperplastic polyps, 135 patients with adenomatous polyps and 45 patients with colorectal cancer and corresponding adjacent tissue from January 2018 to May 2019 at Renmin Hospital of Wuhan University were collected. Immunohistochemical SP staining was used to detect the expression of Foxa2. Expressions of Foxa2 mRNA and protein were determined by real-time fluorescence quantitative PCR and Western blotting, respectively. The relationships between the expression of Foxa2 and colorectal polyps, colorectal cancer were analyzed. Results: The positive expression rate of Foxa2 in normal colorectal mucosal tissue, hyperplastic polyps, adenomatous polyps and colorectal cancer was gradually increased (13.3%, 31.1%, 62.2%, 86.7%, respectively), and the difference was statistically significant (P<0.05). The expressions of Foxa2 mRNA and protein were also increased, and the differences were statistically significant (P<0.05). The expression of Foxa2 in adenomatous polyps was related to size of polyps and presence of pedicle (P<0.05); the expression of Foxa2 in colorectal cancer was related to differentiation, lymph node metastasis and TNM staging (P<0.05). Conclusions: The expression of Foxa2 is high in adenomatous polyps and colorectal cancer, and increases with the increase of risk of canceration. Therefore, detecting the expression of Foxa2 in colorectal polyps is helpful for the early detection of colorectal cancer.

Role and mechanisms of hepatocyte nuclear factor 4α in bile acid induced gastric intestinal metaplasia / 中华消化杂志

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.

Induction study of CYP3A2 in male rats by zolmitriptan / 药学学报

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.

Hepatocyte Nuclear Factor 4α Inhibits Expression of Vascular Endothelial Growth Factor in Human Hepatocellular Carcinoma Cell Lines and Tube Formation of Human Umbilical Vein Endothelial Cell / 胃肠病学

Background: Hepatocyte nuclear factor 4α (HNF4α) plays an important role in the development of liver,and studies demonstrate that it is correlated with the pathogenesis of hepatocellular carcinoma (HCC).However,the regulatory effect of HNF4α on expression of vascular endothelial growth factor (VEGF) in human HCC cell lines and tube formation of human umbilical vein endothelial cell (HUVEC) is not yet clear.Aims: To investigate the effect of HNF4α on expression of VEGF in human HCC cell lines and tube formation of HUVEC.Methods: Lentiviral vector overexpressed HNF4α was constructed,and then transfected into HepG2 and SMMC-7721 cells (experimental group),cells transfected with lentiviral blank vector and cells without transfection were served as negative control group and blank control group,respectively.The mRNA and protein expressions of HNF4α,VEGF were detected by qRT-PCR and Western blotting,respectively.The conditioned media of HepG2 and SMMC-7721 cells were co-cultured with HUVEC,and number of HUVEC tube formation was measured.Results: HepG2 and SMMC-7721 cells with stable overexpression of HNF4α were successfully established.Compared with negative control group and blank control group,mRNA and protein expressions of VEGF in experimental group were significantly decreased (P＜0.05),and number of HUVEC tube formation was significantly decreased (P＜0.05).Conclusions: HNF4α can significantly inhibit the expression of VEGF in HepG2 and SMMC-7721 cells and tube formation of HUVEC.

Expression of ER,HNF-1β and COX-2 in endometriosis-associated ovarian cancer and its clinical significance / 重庆医学

Objective To investigate the expression difference of estrogen receptor(ER),hepatocyte nuclear factor 1β(HNF-1β) and epoxy-2(COX-2) in different types of endometriosis-associated ovarian cancer (EAOC).Methods Forty-four patients with EAOC treated in our hospital from July 2011 to April 2016 were selected,including 17 cases of endometrioid carcinoma,21 cases of clear cell carcinoma and 6 cases of ovarian serous carcinoma.The expression of ER,HNF-1β and COX-2 in different types of EAOC were detected by immunohistochemistry.The correlation among expression levels of ER,HNF-1β and COX-2 was investigated by adopting Spearman rank correlation analysis.Results The positive rate of ER in endometrioid carcinoma was 100.0％,which was significantly higher than 9.5％ in the patients with clear cell carcinoma and 0％ in the patients with ovarian serous carcinoma(x2=4.305,P＜0.01),and the positive rate of HNF-1β in the patients with clear cell carcinoma and ovarian serous carcinoma were 85.7％ and 100.0％ respectively,which was significantly higher than 11.8％ in the patients with endometrioid carcinoma(x2 =3.585,P＜0.01),the positive rates of COX-2 in the patients with endometrioid carcinoma,clear cell carcinoma and ovarian serous carcinoma were 76.5％,81.0％ and 83.3％,respectively,and the difference was not statistically significant (x2 =0.744,P =0.104).The correlation analysis showed that ER was negatively correlated with HNF-1β expression level(r=-0.428,P＜0.01)and positively correlated with COX-2 expression level (r=0.204,P=0.013).Conclusion ER is mainly expressed in endometrioid carcinoma.HNF-1β is mainly expressed in clear cell carcinoma and ovarian serous carcinoma.The expression level of ER had a certain correlation to the expression of HNF-1β and COX-2,which may be closely related to EAOC progression.

Expression of ER,HNF-1β and COX-2 in endometriosis-associated ovarian cancer and its clinical significance / 重庆医学

Objective To investigate the expression difference of estrogen receptor(ER),hepatocyte nuclear factor 1β(HNF-1β) and epoxy-2(COX-2) in different types of endometriosis-associated ovarian cancer (EAOC).Methods Forty-four patients with EAOC treated in our hospital from July 2011 to April 2016 were selected,including 17 cases of endometrioid carcinoma,21 cases of clear cell carcinoma and 6 cases of ovarian serous carcinoma.The expression of ER,HNF-1β and COX-2 in different types of EAOC were detected by immunohistochemistry.The correlation among expression levels of ER,HNF-1β and COX-2 was investigated by adopting Spearman rank correlation analysis.Results The positive rate of ER in endometrioid carcinoma was 100.0％,which was significantly higher than 9.5％ in the patients with clear cell carcinoma and 0％ in the patients with ovarian serous carcinoma(x2=4.305,P＜0.01),and the positive rate of HNF-1β in the patients with clear cell carcinoma and ovarian serous carcinoma were 85.7％ and 100.0％ respectively,which was significantly higher than 11.8％ in the patients with endometrioid carcinoma(x2 =3.585,P＜0.01),the positive rates of COX-2 in the patients with endometrioid carcinoma,clear cell carcinoma and ovarian serous carcinoma were 76.5％,81.0％ and 83.3％,respectively,and the difference was not statistically significant (x2 =0.744,P =0.104).The correlation analysis showed that ER was negatively correlated with HNF-1β expression level(r=-0.428,P＜0.01)and positively correlated with COX-2 expression level (r=0.204,P=0.013).Conclusion ER is mainly expressed in endometrioid carcinoma.HNF-1β is mainly expressed in clear cell carcinoma and ovarian serous carcinoma.The expression level of ER had a certain correlation to the expression of HNF-1β and COX-2,which may be closely related to EAOC progression.

Establishment of a reporter gene system for activity detection of hepatocyte nuclear factor 4α / 第二军医大学学报

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.

Overexpression of hepatocyte nuclear factor 1α mediated by adeno-associated virus attenuates carbon tetrachloride-induced hepatic fibrosis in mice / 第二军医大学学报

To explore the influence of the hepatocyte-specific up-regulation of hepatocyte nuclear factor 1α (HNF1α) on mouse hepatic fibrosis induced by carbon tetrachloride (CCl4). Methods Eighteen C57/B6 male mice were randomly divided into normal group, AAV8-TBG-Ctrl group and AAV8-TBG-HNF1α group, with 6 mice in each group. Mice in the AAV8-TBG-Ctrl and AAV8-TBG-HNF1α groups were intraperitoneally injected with CCl4 to establish the hepatic fibrosis mouse model, and then the mice in the AAV8-TBG-HNF1α group were injected with AAV8-TBG-HNF1α carrying HNF1α gene under the control of the thyroid-binding globulin (TBG) promoter to specifically up-regulate expression of HNF1α in hepatocytes, while the mice in the AAV8-TBG-Ctrl group were injected with control vector AAV8-TBG. The expression of HNF1α was determined by immunohistochemistry and qPCR. The pathological changes and collagen deposition of liver tissues were detected by hematoxylin-eosin (H-E) staining and sirius red staining, respectively. Immunohistochemistry method was used to detect α-smooth muscle actin (α-SMA), and the expression of fibrosis related genes (typecollagen α1 chain[COL1A1], α-SMA), epithelial related genes (E-cadherin, Plakoglobin) and mesenchymal related genes (Vimentin, Slug and Twist1) in liver tissues were analyzed by qPCR. The cell proliferation and apoptosis in fibrotic livers were detected by immunohistochemistry and TdT-mediated dUTP Nick-End Labeling (TUNEL) method, respectively. Results Compared with the normal mice, CCl4 promoted collagen deposition and the expression of α-SMA in livers, and the expression of HNF1α was significantly decreased (P0.05). Conclusion Hepatocyte-specific up-regulation of HNF1α significantly improves CCl4-induced liver fibrosis in mice.

The role of hepatocyte nuclear factor 4 alpha in development and progression of liver diseases / 临床肝胆病杂志

Hepatocyte nuclear factor 4 alpha (HNF4α), a member of the nuclear receptor superfamily, has a high expression level in mature hepatocytes. HNF4α can regulate hepatocyte-specific gene expression at a transcriptional level, promote hepatocyte development and differentiation, participate in establishment and maintenance of hepatocyte polarity, and enhance the synthetic, metabolic, and detoxifying functions of the liver. Through inhibiting the activation of hepatic stellate cells, reversing epithelial-mesenchymal transition, and inhibiting the proliferation, invasion, and metastasis of hepatoma cells, HNF4α may be involved in the development and progression of various liver diseases including liver fibrosis, liver cirrhosis, and hepatocellular carcinoma. This paper elaborates on the biological functions of HNF4α, and summarizes and analyzes the research advances in the mechanisms of action of HNF4α in the pathological process of liver diseases, in order to provide references for further investigation of the potential targeted therapies for liver diseases.

Effect of hepatocyte nuclear factor 1α on expression profile of hepatocellular carcinoma cells HuH7 / 中华消化杂志

Objective To investigate the regulation effect of hepatocyte nuclear factor (HNF) 1α on the gene expression profile and the signal pathways in HuH7 cells.Methods The expression of HNF1α was increased or decreased in HuH7 cells by Lenti-virus carrying HNF1α or shHNF1α.The expression profile of the cells after treated was examined by microarray technology.The difference expressed gene regulated by HNF1α were screened and the pathway was analyzed with DAVID software and related analysis system.The regulation effect of HNF1α on transforming growth factor (TGF)β signal pathway was detected by reporter gene test and the regulation role of HNF1α on related genes of TGFβ signal pathway was determined by real-time polymerase chain reaction (PCR) and Western blotting assay.Results The expression of HNF1α in HuH7 cells was significantly up-regulated by Lenti-virus carrying HNF1α gene (Lenti-HNF1α) and which was down regulated by Lenti-virus with shHNF1α gene (LentishHNF1 α).Expression profile analysis revealed that 339 genes were positively up regulated two times by HNF1α and 325 genes were negatively down regulated two times.Signal pathway analysis revealed that HNF1α regulated drug metabolism,biosynthesis of unsaturated fatty acids and glycolysis/gluconeogenesis metabolism signal pathways.Moreover,it also involved in the regulation of TGFβ、nuclear factor (NF)-κB and p53 tumor-related signal pathways.Furthermore,Luciferase reportor gene experiment indicated that up-regulated HNF1α could inhibit the activation of TGFβ signal pathway.And the results of real-time PCR and Western blotting verified that up-regulated HNF1α could inhibit TGFβ signal pathway related gene c-myc and TGFβ1 and then inhibited the activation of TGFβ signal pathway.Conclusion HNF1α broadly affects the gene expression profile and the tumor genesis and development related signal pathways in HuH7 cells,furthermore,HNF1α can inhibit the activation of TGFβ signal pathway.

Protection of Sophocarpine on Colonic Barrier in DSS-induced Acute Colitis in Mice by Increasing Expression of HNF4α / 中草药·英文版

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.

Inhibitory effect of PEP-1-mediated recombinant hepatocyte nuclear factor 4 alpha transduction on hepatocellular carcinoma cells / 第二军医大学学报

Objective To investigate cell penetrating peptide (PEP-1)-mediated transduction of recombinant hepatocyte nuclear factor 4 alpha (HNF4α) protein into hepatocellular carcinoma (HCC) cells, and to observe the effect of the fusion protein P-HNF4α on HCC cells. Methods The expression vector pET28a-P-HNF4α was constructed. The prokaryotic expression condition of fusion protein P-HNF4α was optimized. Recombinant P-HNF4α carrying cell penetrating peptide PEP-1 was obtained by abundant expression, purified by affinity chromatography, and was concentrated and dialyzed. P-HNF4α was transduced into HCC cells. The transduction efficiency was analyzed by Western blotting analysis. Sub-cellular localization of P-HNF4α was detected by Western blotting analysis with nuclear and cytoplasmic extracts and confirmed by immunofluorescence assay. Real-time RT-PCR was used to examine the gene expression of HCC cells. The proliferation of HCC cells was detected with CCK-8 kit. The migration and invasion of HCC cells were detected by wound-healing assay and trans-well invasion assay, respectively. Results P-HNF4α was efficiently transduced into Huh7 cells and located in the nucleus as mediated by PEP-1. P-HNF4α significantly up-regulated the expression of characteristic hepatocyte markers and down-regulated the "stemness" genes in Huh7 cells (P<0.05 or P<0.01). Moreover, the proliferation (P<0.05), migration (P<0.001) and invasion (P<0.05) of HCC cells were significantly suppressed by fusion protein P-HNF4α. Conclusion P-HNF4α can induce the differentiation of HCC cells to mature hepatocytes and reduce the malignancy phenotype of HCC cells, suggesting that PEP-1-mediated HNF4α protein transduction may be a potential strategy for HCC differentiation therapy.