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XLSX file, 12K, Phosphorylation of CAMKII in liver cancer patients.

XLSX file, 11K, Sphere formation (2 independent trials).

PDF file, 1752K, Figure S1. (Related to Figure 2) BBM suppresses the xenograft growth of Huh7 and SK-Hep-1 cells; Figure S2. (Related to Figure 3) BBM is localized in cytoplasm and affects cell survival; Figure S3. (Related to Figure 4) BBM suppressed the CD133+ population in Huh7 cells; Figure S4. (Related to Figure 5B). The chemical inhibitor of CAMKII, KN93, suppresses CD133+ population in Huh7 cells; Figure S6. (Related to Figure 6C). The knockdown of CAMKIIgamma suppresses liver cancer growth.

PDF file, 51K.

XLSX file, 9K, IC50s of berbamine on liver cancer cells.

To explore the genetic etiology for a child featuring mental retardation and speech delay.Clinical data of the child was collected. DNA was extracted from peripheral blood samples of the child and members of his pedigree. Whole exome sequencing was carried out for the child, and candidate variants were verified by Sanger sequencing. Prenatal diagnosis was provided for his mother upon her subsequent pregnancy.The child has mainly featured mental retardation, speech delay, ptosis, strabismus, photophobia, hyperactivity, and irritability. Whole exome sequencing revealed that he has harbored a pathogenic heterozygous variant of the KAT6A gene, namely c.5314dupA (p.Ser1772fs*20), which was not detected in either of his parents. The child was diagnosed with Arboleda-Tham syndrome. The child was also found to harbor a hemizygous c.56TG (p.Leu19Trp) variant of the AIFM1 gene, for which his mother was heterozygous and his phenotypically normal maternal grandfather was hemizygous. Pathogenicity was excluded. Prenatal diagnosis has excluded the c.5314dupA variant of the KAT6A gene in the fetus.The heterozygous c.5314dupA (p.Ser1772fs*20) variant of the KAT6A gene probably underlay the Arboleda-Tham syndrome in this child. Above finding has enabled genetic counseling and prenatal diagnosis for this pedigree.

Cohen syndrome (CS) is an autosomal recessive congenital disorder characterized by mutation in the vacuolar protein sorting 13 homolog B (VPS13B; formerly COH1) gene. In the current study, a Chinese family has two young sibling cases having a developmental delay, physical obesity, high myopia, and a special face, which suspected to be CS. The purpose of the study was to identify variants and further analyze their pathogenicity for CS. Next-generation sequencing (NGS) revealed a compound heterozygous mutation in VPS13B gene in the proband, which comprises a frameshift mutation in NM_017890.4: c.10076_10077delCA (p.T3359fs*29) and a putative splice site mutation in c.6940 + 1G > T. Both Minigene assay in vitro and splicing assay in vivo confirmed that the splicing mutation in c.6940 + 1G > T generates a frameshift transcript with whole exon 38 skipping. Eventually, quantitative real-time PCR demonstrated that either of the two mutations can lead to degradation of the VPS13B gene at the transcriptional level. Functional studies of variants identified in CS patients are essential for their subsequent genetic counseling and prenatal diagnoses and could also be the start point for new therapeutic approaches, currently based only on symptomatic treatment.

To analyze the genomic variation characteristics of fetal with abnormal serological screening, and to further explore the value of copy number variation (CNV) detection technology in prenatal diagnosis of fetal with abnormal serological screening.7617 singleton pregnant women who underwent amniocentesis for prenatal diagnosis solely due to abnormal Down's serological screening were selected. According to the results of serological screening, the patients were divided into high risk group, borderline risk group and single abnormal multiple of median (MOM) group. CMA and CNV-Seq were used to detect the copy number variation of amniotic fluid cell genomic DNA and combined with amniotic fluid cell karyotype analysis for prenatal diagnosis. Outpatient revisit combined with telephone inquiry was used for postnatal follow-up.Among 7617 amniotic fluid samples, aneuploidy was detected in 138cases (1.81%) by CMA and CNV-Seq, 9 cases of aneuploid chimerism were detected by amniotic fluid cell karyotype analysis, and 203 cases of fetus carrying pathogenic and likely pathogenic CNV (P/LP CNV) were detected, the variant of uncertain significance (VUS) was detected in 437 cases (5.7%), the overall abnormal detection rate was 10.33%. The detection rate of aneuploidy by CMA and CNV-Seq in three group were 123 cases (2.9%), 13 cases (1.3%) and 2 cases (0.4%), respectively,and showing no significant difference (χCompared with the traditional chromosome karyotype, CMA and CNV-Seq can improve the detection rate of pathogenic and likely pathogenic CNV. CMA and CNV-seq can be used for first tier diagnosis of pregnant women in the general population with abnormal Down's serological screening.

Prospective research have shown that whole exome sequencing (WES) may be considered when a diagnosis cannot be obtained using routine prenatal methods, e.g., chromosomal karyotyping and copy number variation sequencing, for fetuses with significant structural anomalies. WES can increase the diagnostic rate of genetic disorders in such fetuses by 8% - 10%. Prenatal WES has been gaining wide acceptance. However, due to the limitations of fetal phenotypic evaluation and complexity of ethical issues in prenatal diagnosis, to justify and standardize the application of prenatal WES and maximize its clinical utility has become an urgent need. In view of this, a consensus has been formed by referring to the latest guidelines, expert consensus and authoritative literature. This consensus has put forward suggestions on the suitable objects of prenatal WES, pre-test consultation, sampling and laboratory testing, results report, post-test consultation, pregnancy outcome follow-up, multidisciplinary consultation of difficult cases, preservation of prenatal WES samples and data information.

To analyze the clinical features and genetic variant in a patient with Usher syndrome.Whole exome sequencing was carried out for the patient. Suspected variants were validated by Sanger sequencing of her parents and fetus.The proband was found to harbor compound heterozygous variants c.17_18insA (p.Tyr6Ter*) and c.4095_4096insA (p.Arg1366Lys fs*38) of the PCDH15 gene (NM_033056), which were respectively inherited from her father and mother. The same variants were not detected in 100 healthy controls. Based on the guidelines of the American Society of Medical Genetics and Genomics, both variants were predicted to be pathogenic (PVS1+PM2+PP4). By prenatal diagnosis, her fetus was found to carry the c.4095_4096insA variant. After birth, the child has passed neonatal hearing screening test, and no abnormal auditory and visual function was found after the first year.The compound heterozygous variants c.17_18insA (p.Tyr6Ter*) and c.4095_4096insA (p.Arg1366Lys fs*38) of the PCDH15 gene probably underlay the Usher syndrome is this proband.

To explore the genetic basis for a Chinese pedigree affected with dyschromatosis symmetrica hereditaria (DSH).PCR and Sanger sequencing were carried out for the proband, and suspected variant was validated by Sanger sequencing in the pedigree.The proband was found to harbor a novel variant of c.1352delA (p.N451Mfs*13) of the ADAR (NM_001111) gene. The same variant was found in her affected mother and sister, but not in her unaffected father, uncle, and 100 healthy individual.The novel variant of the ADAR gene probably underlay the pathogenesis of DSH in this pedigree.

To explore the genetic basis for a pedigree affected with hereditary spastic paraplegia type 4 (HSP4).Peripheral venous blood samples were taken from members of the four-generation pedigree and 50 healthy controls for the extraction of genomic DNA. Genes associated with peripheral neuropathy and hereditary spastic paraplegia were captured and subjected to targeted capture and next-generation sequencing. The results were confirmed by Sanger sequencing.DNA sequencing suggested that the proband has carried a heterozygous c.1196CG variant in exon 9 of the SPAST gene, which can cause substitution of serine by threonine at position 399 (p.Ser399Trp) and lead to change in the protein function. The same variant was also detected in other patients from the pedigree but not among unaffected individuals or the 50 healthy controls. Based on the ACMG 2015 guidelines, the variant was predicted to be possibly pathogenic.The c.1196CG variant of the SPAST gene probably underlay the HSP4 in this pedigree.

Congenital hand malformations is rare and characterized by hand deformities. It is highly heterogeneous, both clinically and genetically, which complicates the identification of causative genes and mutations. Recently, targeted next-generation (NGS) sequencing has been successfully used for the detection of heterogeneous diseases, and the use of NGS also has contributed significantly in evaluating the etiology of heterogeneous disease. Here, we employed targeted NGS to screen 248 genes involved in genetic skeletal disorders, including congenital hand malformations. Three pathogenic mutations located in the GJA1, ROR2 and TBX5 genes were detected in three large Chinese families with congenital hand malformations. Two novel mutations were reported, and a known causative mutation was verified in this Chinese population. This is also the first report that the same panel of targeted NGS was employed to perform molecular diagnosis of different subtypes of congenital hand malformations. Our study supported the application of a targeted NGS panel as an effective tool to detect the genetic cause for heterogeneous diseases in clinical diagnosis.

Donohue syndrome (DS), a rare autosomal recessive disease which represents severe insulin resistance, pre- and postnatal growth retardation, hypertrichosis, and dysmorphic features, is caused by mutations in the insulin receptor (INSR) gene. Here, we have reported the clinical, molecular, and biochemical characterizations of a patient with DS. In this article, we have also reported a case with 2 novel INSR mutations and the DS phenotype. Using next-generation sequencing (NGS), we screened 27 known genes involved in inherited maturity-onset diabetes of the young (MODY) and identified compound heterozygous mutations in the INSR gene in the patient with DS, c.62T>G (p.L21R) and c.2563G>T (p.V855F). The positive correlation of these mutations with DS was further validated by Sanger DNA sequencing of his lineal consanguinity, indicating that these pathogenic mutations were inherited maternally and paternally, respectively. Therefore, our finding expanded the number of reported cases of this rare disease and the mutation spectrum of INSR mutation, suggesting that NGS is an accurate, rapid, and cost-effective method for the genetic diagnosis of this rare disease.

To detect variants of ARSA gene in a child featuring late infantile metachromatic leukodystrophy (MLD).PCR and Sanger sequencing was carried out for the patient and her parents.The patient had typical features of MLD including ARSA deficiency, regression of walking ability, and demyelination. Compound heterozygous variants of the ARSA gene, namely c.960GA and c.244CT, were detected in the patient, for which her mother and father were respectively heterozygous carriers. ARSA c.960GA was known to be pathogenic, while ARSA c.244CT was a novel variant. The same variants were not detected among 50 healthy controls.The compound heterozygous variants c.960GA and c.244CT of the ARSA gene probably underlie the MLD in this patient.

To explore the genetic basis for a fetus suspected for congenital nephrotic syndrome of Finland (CNF).Genomic DNA was extracted from peripheral and umbilical cord blood samples derived from both parents and the fetus. Potential variants were detected by using next-generation sequencing. Suspected variants were confirmed by Sanger sequencing.The fetus was found to carry compound heterozygous variants c.1440+1GA and c.925GT of the NPHS1 gene, which were respectively inherited from its mother and father.Identification of the compound heterozygous NPHS1 variants has enabled diagnosis of CNF in the fetus and genetic counseling for the affected family.

To carry out prenatal diagnosis for a fetus with ultrasonographic abnormality.Chromosomal karyotyping and array comparative genomic hybridization (array-CGH) analysis were applied for the diagnosis. Peripheral blood samples were also taken from the parents for chromosome karyotyping analysis.The fetal karyotype showed additional material of unknown-origin attached to Yq. Array CGH analysis confirmed that the material was derived from 3q22.1q29. The father was found to carry a balanced translocation 46, X, t(Y;3)(q12;q23) (which was diagnosed as 46,XY,Y≥18 elsewhere), whilst the mother was found to be normal.3q partial trisomy may present as malformation of multiple systems. Combination of chromosome karyotyping and array-CGH can provide reliable diagnosis for fetuses with abnormalities by ultrasonography.

To explore the genetic basis for a pedigree affected with Bartter's syndrome (BS).Panel-based next-generation sequencing (NGS) was carried out to detect mutation in BS-related genes SLC12A1, KCNJ1, BSND and CLCNKB. Sanger sequencing of MAGED2 gene and chromosomal microarray analysis (CMA) were also performed on the patient. Suspected mutation was validated in her family members.No pathogenic mutation was detected by NGS, while a 0.152 Mb microdeletion at Xp11.21 (54 834 585-54 986 301) was found in the male fetus, which removed the entire coding region of the MAGED2 gene. His mother was a heterozygous carrier of the deletion. His father and sister did not carry the same deletion.The loss of the MAGED2 gene may underlie the BS in this pedigree.

To analyze the clinical features and genetic mutations in a patient with mucolipidosis type II α/β by using next generation sequencing.Clinical data of the patient was collected. Genomic DNA of the patient and her parents was extracted by a standard method. The patient was subjected to targeted sequencing using an Ion Ampliseq panel, which included genes related to mucolipidosis and mucopolysaccharidosis. Suspected mutations were verified by Sanger sequencing.Compound heterozygous mutations, namely c.1284+1GT and c.1090CT (p.Arg364*), were detected in the patient, which were respectively inherited from her mother and father. No other disease-causing mutation was detected in the patient. GNPTAB c.1090CT was known to be pathogenic, while GNPTAB c.1284+1GT is a novel mutation. The same mutations were not detected among 50 healthy controls.The compound heterozygous mutations c.1284+1GT and c.1090CT (p.Arg364*) of GNPTAB gene probably account for the mucolipidosis type II α/β in the patient. NGS has a great value for the molecular diagnosis and typing of mucolipidosis.

To explore the clinical and genetic features of a patient suspected with Juvenile Parkinson's syndrome (JP).Clinical features of the patient were analyzed. Genomic DNA of the patient and his parents was extracted from peripheral blood samples and sequenced by exome capture sequencing. The nature and impact of detected mutations were predicted and validated.The patient displayed typical features including resting tremor, bradykinesia, rigidity, but with excellent response to low dose levodopa. DNA sequencing showed that she has carried compound heterozygous mutations of the Parkin gene, namely c.1381dupC and c.619-1GC, which were respectively inherited from his mother and father. Neither mutation was reported previously. Bioinformatic analysis predicted that both mutations are pathogenic.The patient has JP caused by mutations of the Parkin gene. Exome capture sequencing is an accurate and efficient method for genetic diagnosis of such disease.

Introduction Adams-Oliver syndrome (AOS) is a rare developmental disorder characterized by the combination of aplasia cutis congenita of the scalp vertex and terminal transverse limb defects. DOCK6 (Dedicator of cytokinesis 6) is one of the six identified AOS genes. Methods We performed targeted next-generation sequencing (NGS) of a child with an AOS phenotype. Sanger DNA sequencing further validated her lineal consanguinity. To explore the pathological features of the mutation, a minigene assay was used to investigate the effects of the mutation on splicing. Results Two compound heterozygous DOCK6 mutations (c.4106+2T>C and c.3063 C>G (p.Y1021*)) were identified in this family, and both mutations have not been reported previously. Sanger DNA sequencing indicated that the mutations were inherited maternally and paternally, respectively. The results of the minigene assay showed that the c.4106+2T>C mutation resulted in aberrant splicing and caused a four-nucleotide insertion in the transcript and a premature stop codon. Conclusions Our findings expanded the number of reported cases of this rare disease and the mutation spectrum of DOCK6 mutations, which can serve as the basis for prenatal diagnosis and genetic counseling.

To detect potential mutation of the WAS gene in a Chinese family affected with Wiskott-Aldrich syndrome.Peripheral blood samples were collected from the proband and his family members. All exons and flanking regions of the WAS gene were subjected to PCR amplification - Sanger sequencing as well as restriction endonuclease analysis. Plasma level of B-cell activating factor (BAFF) was also determined for all family members.A hemizygous mutation (c.257GA) of the WAS gene was identified in all patients from the family, for which the patient's mother was heterozygous. The same mutation was not found among healthy members of the family. Compared with unaffected members, all patients had a higher level of BAFF.The c.257GA mutation of the WAS gene probably underlies the Wiskott-Aldrich syndrome in this family.

OBJECTIVE To detect potential mutations of the EXT1 and EXT2 genes in a pedigree affected with hereditary multiple exostosis (HME). METHODS For a four-generation family with 7 affected individuals from 17 family members,genomic DNA was extracted from peripheral venous blood samples. All exons of the EXT1 and EXT2 genes were screened for potential mutation by PCR and Sanger sequencing. RESULTS A novel heterozygous frameshift mutation c.1202delT (p.I401Tfs*2)was found in exon 4 of the EXT1 gene in the proband and the other 6 affected individuals. The same mutation was not detected among the healthy members from the family. The mutation has given rise a truncated EXT1 protein with loss of 345 amino acids. CONCLUSION A novel frameshift mutation of the EXT1 gene has been identified in a pedigree affected with HME, which has enriched the mutational spectrum of the EXT1 gene and may facilitate genetic counseling and prenatal diagnosis for the family.

Background: Patients with Duchenne muscular dystrophy (DMD) usually have severe and fatal symptoms. At present, there is no effective treatment for DMD, thus it is very important to avoid the birth of children with DMD by effective prenatal diagnosis. We identified a de novo DMD gene mutation in a Chinese family, and make a prenatal diagnosis. Methods: First, multiplex ligation-dependent probe amplification (MLPA) was applied to analyze DMD gene exon deletion/duplication in all family members. The coding sequences of 79 exons in DMD gene were analyzed by Sanger sequencing in the patient; and then according to DMD gene exon mutation in the patient, DMD gene sequencing was performed in the family members. On the basis of results above, the pathogenic mutation in DMD gene was identified. Results: MLPA showed no DMD gene exon deletion/duplication in all family members. Sanger sequencing revealed c.2767_2767delT [p.Ser923LeufsX26] mutation in DMD gene of the patient. Heterozygous deletion mutation (T/-) at this locus was observed in the pregnant woman and her mother and younger sister. The analyses of amniotic fluid samples indicated negative Y chromosome sex-determining gene, no DMD gene exon deletion/duplication, no mutations at c.2767 locus, and the inherited maternal X chromosome different from that of the patient. Conclusion: The pathogenic mutation in DMD gene, c.2767_2767delT [p.Ser923LeufsX26], identified in this family is a de novo mutation. On the basis of specific conditions, it is necessary to select suitable methods to make prenatal diagnosis more effective, accurate, and economic.

To analyze a child with facial abnormalities with combined cytogenetic and molecular techniques and delineate its clinical phenotype.Neuropsychological profile of the child was analyzed. Color Doppler, CT and MRI were used for detecting the nodules in the body. Conventional peripheral blood karyotypes of the child and his parents were analyzed with G-banding. Array-comparative genomic hybridization (aCGH) was performed to detect minor structural chromosomal abnormalities.The child had mental retardation, maxillofacial dysmorphism on the right side, and irregular solid nodules on the back. The karyotypes of the child and his parents were all normal, while aCGH has identified a de novo constitutive 1.2 Mb deletion at 17q11.2 in the child. The aCGH results of his parents were normal.The de novo 17q11.2 microdeletion probably underlies the facial abnormalities and neurofibromatosis in the patient.

Liver cancer is the third leading cause of cancer deaths worldwide but no effective treatment toward liver cancer is available so far. Therefore, there is an unmet medical need to identify novel therapies to efficiently treat liver cancer and improve the prognosis of this disease. Here, we report that berbamine and one of its derivatives, bbd24, potently suppressed liver cancer cell proliferation and induced cancer cell death by targeting Ca2+/calmodulin-dependent protein kinase II (CAMKII). Furthermore, berbamine inhibited the in vivo tumorigenicity of liver cancer cells in NOD/SCID mice and downregulated the self-renewal abilities of liver cancer–initiating cells. Chemical inhibition or short hairpin RNA–mediated knockdown of CAMKII recapitulated the effects of berbamine, whereas overexpression of CAMKII promoted cancer cell proliferation and increased the resistance of liver cancer cells to berbamine treatments. Western blot analyses of human liver cancer specimens showed that CAMKII was hyperphosphorylated in liver tumors compared with the paired peritumor tissues, which supports a role of CAMKII in promoting human liver cancer progression and the potential clinical use of berbamine for liver cancer therapies. Our data suggest that berbamine and its derivatives are promising agents to suppress liver cancer growth by targeting CAMKII. Mol Cancer Ther; 12(10); 2067–77. ©2013 AACR.

Background & Aims Liver tumor, especially hepatocellular carcinoma (HCC), is closely associated with chronic inflammation. We previously showed that farnesoid X receptor knockout ( FXR − / − ) mice displayed chronic inflammation and developed spontaneous liver tumors when they aged. However, the mechanism by which inflammation leads to HCC in the absence of FXR is unclear. Because IFNγ is one of the most upregulated pro-inflammatory cytokines in FXR − / − livers, we generated IFNγ − / − FXR − / − double knockout mice to determine IFNγ's roles in hepatocarcinogenesis. Methods IFNγ − / − mice were crossed with an FXR − / − C57BL/6 background or injected i.p. with the hepatocarcinogen diethylnitrosamine (DEN). Hepatocarcinogenesis was analyzed with biochemical and histological methods. Results IFNγ deletion accelerated spontaneous hepatocarcinogenesis in FXR − / − mice and increased the susceptibility to DEN-induced hepatocarcinogenesis. IFNγ deletion enhanced activation of HCC promoters STAT3 and JNK/c-Jun, but abolished induction of p53 in IFNγ − / − livers after acute DEN-induced injury. Furthermore, hepatic p53 expression increased in aged wild type mice but not in aged IFNγ − / − and IFNγ − / − FXR − / − mice, while activation of STAT3 and JNK/c-Jun was enhanced in aged IFNγ − / − and IFNγ − / − FXR − / − mice. In addition, IFNγ inhibited liver cancer xenograft growth and impaired IL-6-induced STAT3 phosphorylation by inducing SOCS1/3 expression. Conclusions Increased IFNγ expression in FXR − / − livers represents a protective response of the liver against chronic injury and tumorigenesis. IFNγ suppresses hepatocarcinogenesis by inducing p53 expression and preventing STAT3 activation.

PNRC (Proline-rich Nuclear Receptor Coactivator) is a novel coactivator for multiple nuclear receptors. PNRC was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, functional analysis of the 5′ flanking region of the human PNRC gene revealed that the −123/+27 region is the minimal promoter of the human PNRC gene. Gel shift and ChIP analyses demonstrated the specific binding of RFX1 (Regulatory Factor X) protein to the human PNRC promoter region. In co-transfection experiments RFX1 was shown to repress promoter activity of PNRC gene in a dose-dependent manner. These results indicate that r RFX1 specifically bind to promoter region and negatively regulate the transcription of the human PNRC gene.

Nuclear receptor (NR) dependent transcriptional action requires recruitment of diverse factors characterized as coregulators. PNRC (proline-rich nuclear receptor coregulatory protein) is a member of coregulators that are capable of potentiating the transcriptional activity of NRs. Here we identified three human PNRC splicing variants designated PNRC1c, PNRC1d and PNRC1f. PNRC1c and PNRC1f are generated through alternative recognition of the 3'-splice site in exon 1, leading to in-frame deletion of 79 amino acids (aa) and an altered reading frame, respectively. PNRC1d is generated through the alternate promoter usage and forms a truncated protein containing C-terminus 142 aa of full-length PNRC. These isoforms differ in their abilities to bind NRs and potentiate NR mediated transcriptions. Moreover, PNRC1d can modulate the activity of full-length PNRC in enhancing ER mediated transcription. Our results suggest that PNRC exists as functionally distinct isoforms and alternative splicing serves as a regulatory mechanism of PNRC coactivator activity.

ERRalpha (estrogen receptor-related receptor alpha) is a member of the nuclear receptor superfamily. To further our understanding of the detailed molecular mechanism of transcriptional regulation by ERRalpha, we searched for ERRalpha-interacting proteins using a yeast two-hybrid system by screening a human mammary gland cDNA expression library with the ligand-binding domain (LBD) of ERRalpha as the "bait". Fast skeletal muscle troponin I (TNNI2), along with several known nuclear receptor co-activators, were isolated. We demonstrated that TNNI2 localizes to the cell nucleus and interacts with ERRalpha in co-immunoprecipitation experiments. GST pull-down assays also revealed that TNNI2 interacts directly with ERRalpha. Through luciferase reporter gene assays, TNNI2 was found to enhance the transactivity of ERRalpha. Combining mutagenesis and yeast two-hybrid assays, we mapped the ERRalpha-interacting domain on TNNI2 to a region encompassing amino acids 1-128. These findings reveal a new function for TNNI2 as a co-activator of ERRalpha.

PNRC (proline-rich nuclear receptor co-activator) was previously identified using bovine SF-1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC has been demonstrated to be a novel co-activator for multiple nuclear receptors. To understand the molecular mechanisms that regulate the expression of human PNRC gene, in this study, potential transcriptional start site was determined by 5' RACE analysis. Functional analysis of the 5' flanking region of the human PNRC gene by deletion mutagenesis, transient transfection and luciferase assays revealed that the -123/+27 region is the minimal promoter of the human PNRC gene. Within this promoter region, there is one putative binding site for the transcription factor NFY (nuclear factor Y). EMSA and ChIP analyses demonstrated the specific binding of NFY protein to the human PNRC promoter. Transient transfection and luciferase assays further revealed that over-expression of NFY represses promoter activity of PNRC gene in a dose-dependent manner. These results indicate that the transcription factor NFY specifically binds to promoter region of PNRC and negatively regulates the transcription of the human PNRC gene.

MicroRNAs (miRNAs) are a class of recently discovered noncoding RNA genes that post-transcriptionally regulate gene expression. It is becoming clear that miRNAs play an important role in the regulation of gene translation during development. However, in mammals, expression data are principally based on whole tissue analysis and are still very incomplete. We isolated CD34+CD38− hematopoietic stem cells (HSCs) from human umbilical cord blood, on the basis of cell-surface markers using fluorescence-activated cell sorting (FACS). Also, CD34+ subpopulation was FACS isolated as the control. Next, using microarray containing oligonucleotides corresponding to 517 miRNAs from human, mouse, and rat genomes, we obtained miRNA gene expression profiles of both subpopulations. We focused on the HSCs correlative miRNAs with comparison to the control. The miRNAs of particular interest were confirmed by real-time RT-PCR. HSCs-overexpressed hsa-miR-520h and underexpressed hsa-miR-129 were selected for target prediction and analysis. The result showed that EIF2C3 and CAMTA1, genes related to miRNAs processing or transcription regulation, were proved to be real targets for hsa-miR-129. And ABCG2, involved in stemness maintaining, a real target for hsa-miR-520h. Finally, we chose hsa-miR-520h, enriched in HSCs but low in CD34+ cells, for functional characterization, because of its possible role in differentiation of HSCs by regulating ABCG2. As a result, hsa-miR-520h transduction into CD34+ cells greatly increased number of different progenitor colonies in Colony-Forming-Cell assays, suggesting that hsa-miR-520h may promote differentiation of HSCs into progenitor cells by inhibiting ABCG2 expression. This study paves the way for identifying HSC-specific miRNAs and their roles in HSC development. J. Cell. Biochem. 104: 805–817, 2008. © 2008 Wiley-Liss, Inc.

We screened 60 known genes which are involved in inherited cataract in a pregnant woman with a four-generation family history of autosomal dominant congenital nuclear cataract through next-generation sequencing (NGS) and identified a heterozygous mutation, c.508dupC (p.L170fs), in the major intrinsic protein (MIP) gene. This mutation results in a frame-shift in MIP and has not been previously reported. The correlation of the mutation with disease was validated by Sanger sequencing of DNA from the other affected or unaffected members of the family. Therefore, our data expand the mutation spectrum of MIP mutation, and suggest that NGS is an accurate, rapid, and cost-effective method in the genetic diagnosis of congenital nuclear cataract.

CYP2E1, one of the cytochrome P450 mixed-function oxidases located predominantly in liver, plays a key role in metabolism of xenobiotics including ethanol and procarcinogens. Recently, down-expression of CYP2E1 was found in hepatocellular carcinoma (HCC) with the majority to be chronic hepatitis B virus (HBV) carriers. In this study, we tested a hypothesis that HBx may inhibit CYP2E1 gene expression via hepatocyte nuclear factor 4α (HNF4α). By enforced HBx gene expression in cultured HepG2 cells, we determined the effect of HBx on CYP2E1 mRNA and protein expression. With a bioinformatics analysis, we found a consensus HNF-4α binding sequence located on -318 to -294 bp upstream of human CYP2E1 promoter. Using reporter gene assay and site-directed mutagenesis, we have shown that mutation of this site dramatically decreased CYP2E1 promoter activity. By silencing endogenous HNF-4α, we have further validated knockdown of HNF-4α significantly decreased CYP2E1 expression. Ectopic overexpression of HBx in HepG2 cells inhibits HNF-4α expression, and HNF-4α levels were inversely correlated with viral proteins both in HBV-infected HepG2215 cells and as well as HBV positive HCC liver tissues. Moreover, the HBx-induced CYP2E1 reduction could be rescued by ectopic supplement of HNF4α protein expression. Furthermore, human hepatoma cells C34, which do not express CYP2E1, shows enhanced cell growth rate compared to E47, which constitutively expresses CYP2E1. In addition, the significantly altered liver proteins in CYP2E1 knockout mice were detected with proteomics analysis. Together, HBx inhibits human CYP2E1 gene expression via downregulating HNF4α which contributes to promotion of human hepatoma cell growth. The elucidation of a HBx-HNF4α-CYP2E1 pathway provides novel insight into the molecular mechanism underlining chronic HBV infection associated hepatocarcinogenesis.

Bcr-Abl tyrosine kinase inhibitors (TKIs) have been a remarkable success for the treatment of Ph+ chronic myeloid leukemia (CML). However, a significant proportion of patients treated with TKIs develop resistance because of leukemia stem cells (LSCs) and T315I mutant Bcr-Abl. Here we describe the unknown activity of the natural product berbamine that efficiently eradicates LSCs and T315I mutant Bcr-Abl clones. Unexpectedly, we identify CaMKII γ as a specific and critical target of berbamine for its antileukemia activity. Berbamine specifically binds to the ATP-binding pocket of CaMKII γ, inhibits its phosphorylation and triggers apoptosis of leukemia cells. More importantly, CaMKII γ is highly activated in LSCs but not in normal hematopoietic stem cells and coactivates LSC-related β-catenin and Stat3 signaling networks. The identification of CaMKII γ as a specific target of berbamine and as a critical molecular switch regulating multiple LSC-related signaling pathways can explain the unique antileukemia activity of berbamine. These findings also suggest that berbamine may be the first ATP-competitive inhibitor of CaMKII γ, and potentially, can serve as a new type of molecular targeted agent through inhibition of the CaMKII γ activity for treatment of leukemia.

Farnesoid X receptor (FXR) (nuclear receptor subfamily 1, group H, member 4) is a member of nuclear hormone receptor superfamily, which plays essential roles in metabolism of bile acids, lipid, and glucose. We previously showed spontaneously hepatocarcinogenesis in aged FXR−/− mice, but its relevance to human hepatocellular carcinoma (HCC) is unclear. Here, we report a systematical analysis of hepatocarcinogenesis in FXR−/− mice and FXR expression in human liver cancer. In this study, liver tissues obtained from FXR−/− and wild-type mice at different ages were compared by microarray gene profiling, histological staining, chemical analysis, and quantitative real-time PCR. Primary hepatic stellate cells and primary hepatocytes isolated from FXR−/− and wild-type mice were also analyzed and compared. The results showed that the altered genes in FXR−/− livers were mainly related to metabolism, inflammation, and fibrosis, which suggest that hepatocarcinogenesis in FXR−/− mice recapitulated the progression of human liver cancer. Indeed, FXR expression in human HCC was down-regulated compared with normal liver tissues. Furthermore, the proinflammatory cytokines, which were up-regulated in human HCC microenvironment, decreased FXR expression by inhibiting the transactivity of hepatic nuclear factor 1α on FXR gene promoter. Our study thereby demonstrates that the down-regulation of FXR has an important role in human hepatocarcinogenesis and FXR−/− mice provide a unique animal model for HCC study.

Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily and is the primary bile acid receptor. We previously showed that FXR was required for the promotion of liver regeneration/repair after physical resection or liver injury. However, the mechanism by which FXR promotes liver regeneration/repair is still unclear. Here we show that both hepatic-FXR and intestine-FXR contributed to promote liver regeneration/repair after either 70% partial hepatectomy or carbon tetrachloride-induced liver injury. Hepatic FXR, but not intestine FXR, is required for the induction of Foxm1b gene expression in liver during liver regeneration/repair. In contrast, intestine FXR is activated to induce FGF15 expression in intestine after liver damage. Ectopic expression of FGF15 was able to rescue the defective liver regeneration/repair in intestine-specific FXR null mice. Conclusion: These results demonstrate that, in addition to the cell-autonomous effect of hepatic FXR, the endocrine FGF15 pathway activated by FXR in intestine also participates in the promotion of liver regeneration/repair. (HEPATOLOGY 2012;56:2336–2343)

TGR5, an emerging G protein-coupled receptor, was identified as a membrane receptor for bile acids. The expression of TGR5 and its function are distinct from the previously identified nuclear bile acid receptor, farnesoid X receptor (FXR). These two bile acid receptors complement with each other for maintaining bile acid homeostasis and mediating bile acid signaling. Both receptors are also shown to play roles in regulating inflammation and glucose metabolism. An interesting finding for TGR5 is its role in energy metabolism. The discovery of TGR5 expression in brown adipocyte tissues (BATs) and the recent demonstration of BAT in adult human body suggest a potential approach to combat obesity by targeting TGR5 to increase thermogenesis. We summarize here the latest finding of TGR5 research, especially its role in energy metabolism and glucose homeostasis.

The farnesoid X receptor (FXR) is a bile acid (BA)-activated nuclear receptor that plays a major role in the regulation of BA and lipid metabolism. Although modulation of FXR expression has been reported, the mechanisms underlying the regulation of human FXR are yet unclear. Functional assays showed that the -150/+29 nucleotides region from the first nucleotide at the Exon I is the minimal promoter of the human FXR gene by the technique of serial deletion and point mutants of the 5'-flanking region. Chromatin immunoprecipitation analysis and electrophoretic mobility shift assay revealed that hepatic nuclear factor 1alpha (HNF1alpha) interacted with the region. Co-transfection of the promoter with HNF1alpha expression vectors enhanced promoter activity of FXR gene. Over-expression of HNF1alpha up-regulated FXR expression in HepG2 cells. These data indicate that (a) the identified HNF1alpha binding site serves as a positive regulatory sequence, (b) the binding site is functionally active both in vivo and in vitro, and (c) the transcription factor HNF1alpha that binds to this site plays an important role in the regulation of human FXR promoter activity.