NR0B1 augments sorafenib resistance in hepatocellular carcinoma through promoting autophagy and inhibiting apoptosis.

NR0B1 is frequently activated in hepatocellular carcinoma (HCC). However, the role of NR0B1 is controversial in HCC. In this study, we observed that NR0B1 was an independent poor prognostic factor, negatively correlated with the overall survival of HCC and the relapse-free survival of patients treated with sorafenib. Meanwhile, NR0B1 promoted the proliferation, migration, and invasion of HCC cells, inhibited sorafenib-induced apoptosis, and elevated the IC50 of sorafenib in HCC cells. NR0B1 was further displayed to increase sorafenib-induced autophagic vesicles and activate Beclin1/LC3-II-dependent autophagy pathway. Finally, NR0B1 was revealed to transcriptionally suppress GSK3ß that restrains AMPK/mTOR-driven autophagy and increases BAX-mediated apoptosis. Collectively, our study uncovered that the ectopic expression of NR0B1 augmented sorafenib-resistance in HCC cells by activating autophagy and inhibiting apoptosis. Our findings supported that NR0B1 was a detrimental factor for HCC prognosis.

NR0B1 suppresses ferroptosis through upregulation of NRF2/c-JUN-CBS signaling pathway in lung cancer cells.

Ferroptosis has demonstrated significant potential in treating radiochemotherapy-resistant cancers, but its efficacy can be affected by recently discovered ferroptosis suppressors. In this study, we discovered that NR0B1 protects against erastin- or RSL3-induced ferroptosis in lung cancer cells. Transcriptomic analysis revealed that NR0B1 significantly interfered with the expression of 12 ferroptosis-related genes, and the expression level of NR0B1 positively correlated with that of c-JUN, NRF2, and CBS. We further revealed that NR0B1 suppression of ferroptosis depended on the activities of c-JUN, NRF2, and CBS. NR0B1 directly promoted the expression of NRF2 and c-JUN and indirectly upregulated CBS expression through enhancing NRF2 and/or c-JUN transcription. Moreover, we showed that NR0B1 depletion restrained xenograft tumor growth and facilitated RSL3-induced ferroptosis in the tumors. In conclusion, our findings uncover that NR0B1 suppresses ferroptosis by activating the c-JUN/NRF2-CBS signaling pathway in lung cancer cells, providing new evidence for the involvement of NR0B1 in drug resistance during cancer therapy.

Lipoprotein glomerulopathy induced by ApoE Kyoto mutation in ApoE-deficient mice.

BACKGROUND: Lipoprotein glomerulopathy (LPG) is a rare autosomal dominant kidney disease that is most commonly caused by mutations in ApoE Kyoto (p.R43C) and ApoE Sendai (p.R163P). Differences in phenotype among the various ApoE mutations have been suggested, but the pathogenic role of ApoE Kyoto has not been validated in an animal model. This study intended to establish an ApoE Kyoto murine model and to further compare the pathologic differences between ApoE Kyoto and ApoE Sendai. METHOD: Male ApoE-deficient mice, 3 months of age, were divided into five groups, including the AD-ApoE Sendai, AD-ApoE Kyoto, AD-ApoE3, AD-eGFP, and ApoE (-/-) groups. The first four groups received recombinant adenovirus that contained the entire coding regions of the human ApoE Sendai and ApoE Kyoto, apoE3, and eGFP genes, respectively. Fasting blood and urine samples were collected at multiple time points. Lipid profiles and urine albumin-creatinine ratio were measured. Renal and aortic histopathologic alterations were analyzed. RESULTS: After virus injection, plasma human ApoE was detected and rapidly reached the maximum level at 4-6 days in the AD-ApoE Kyoto and AD-ApoE Sendai groups (17.4 ± 3.1 µg/mL vs.: 22.2 ± 4.5 µg/mL, respectively) and at 2 days in the AD-ApoE3 group (38.4 µg/mL). The serum total cholesterol decreased by 63%, 65%, and 73% in the AD-ApoE Kyoto, AD-ApoE Sendai and AD-ApoE3 groups, respectively. There were no significant changes in serum triglyceride and urinary albumin-creatinine ratio among the five groups. Typical lipoprotein thrombi with positive ApoE staining were detected in the AD-ApoE Kyoto and AD-ApoE Sendai groups. The Oil-red O-positive glomerular area tended to be higher in the AD-ApoE Kyoto group (9.2%) than in the AD-ApoE Sendai (3.9%), AD-ApoE3 (4.8%), AD-eGFP (2.9%), and ApoE (-/-) (3.6%) groups. The atherosclerotic plaque area in the aorta was lower in the group injected with various ApoE mutations than in the group without injection of ApoE mutation. CONCLUSIONS: In this animal study, we first established an ApoE Kyoto mutation murine model and confirmed its pathogenic role in LPG. Our results suggested that LPG may be more severe with the ApoE Kyoto than with the ApoE Sendai.

A rare mutant of OFD1 gene responsible for Joubert syndrome with significant phenotype variation.

Joubert syndrome (JBTS), a rare genetic disorder resulted from primary cilium defects or basal-body dysfunction, is characterized by agenesis of cerebellar vermis and abnormal brain stem. Both genotypes and phenotypes of JBTS are highly heterogeneous. The identification of pathogenic gene variation is essential for making a definite diagnosis on JBTS. Here, we found that hypoplasia of cerebellar vermis occurred in three male members in a Chinese family. Then, we performed whole exome sequencing to identify a novel missense mutation c.599T > C (p. L200P) in the OFD1 gene which is the candidate gene of X-linked JBTS (JBST10). The following analysis showed that the variant was absent in the 1000 Genomes, ExAC and the 200 female controls; the position 200 Leucine residue was highly conserved across species; the missense variant was predicted to be deleterious using PolyPhen-2, PROVEAN, SIFT and Mutation Taster. The OFD1 expression was heavily lower in the proband and an induced male fetus compared with a healthy male with a wild-type OFD1 gene. The in vitro expression analysis of transiently transfecting c.599T or c.599C plasmids into HEK-293T cells confirmed that the missense mutation caused OFD1 reduction at the protein level. And further the mutated OFD1 decreased the level of Gli1 protein, a read-out of Sonic hedgehog (SHH) signaling essential for development of central neural system. A known pathogenic variant c.515T > C (p. L172P) showed the similar results. All of these observations suggested that the missense mutation causes the loss function of OFD1, resulting in SHH signaling impairs and brain development abnormality. In addition, the three patients have Dandy-Walker malformation, macrogyria and tetralogy of Fallot, respectively, the latter two of which are firstly found in JBTS10 patients. In conclusion, our findings expand the context of genotype and phenotype in the JBTS10 patients.

CLOCK and BMAL1 stabilize and activate RHOA to promote F-actin formation in cancer cells.

Circadian genes control most of the physiological functions in cancer cells, including cell proliferation, migration, and invasion. The CLOCK and BMAL1 complex plays a central role in circadian rhythms. Previous studies have shown that circadian genes may act as oncogenes or tumor-suppressor genes. In addition, F-actin, regulated by RHOA, has been shown to participate in tumor progression. However, the roles of the CLOCK and BMAL1 genes in the regulation of tumor progression via the RHOA-ROCK-CFL pathway remain largely unclear. Here we first indicate that the rearrangement of F-actin is regulated by CLOCK and BMAL1. We found that CLOCK and BMAL1 can upregulate RHOA expression by inhibiting CUL3-mediated ubiquitination and activate RHOA by reducing the interaction between RHOA and RhoGDI. Consequently, CLOCK and BMAL1 control the expression of the components of the RHOA-ROCK-CFL pathway, which alters the dynamics of F-actin/G-actin turnover and promotes cancer cell proliferation, migration, and invasion. In conclusion, our research proposes a novel insight into the role of CLOCK and BMAL1 in tumor cells.

Clock1a affects mesoderm development and primitive hematopoiesis by regulating Nodal-Smad3 signaling in the zebrafish embryo.

Circadian clock and Smad2/3/4-mediated Nodal signaling regulate multiple physiological and pathological processes. However, it remains unknown whether Clock directly cross-talks with Nodal signaling and how this would regulate embryonic development. Here we show that Clock1a coordinated mesoderm development and primitive hematopoiesis in zebrafish embryos by directly up-regulating Nodal-Smad3 signaling. We found that Clock1a is expressed both maternally and zygotically throughout early zebrafish development. We also noted that Clock1a alterations produce embryonic defects with shortened body length, lack of the ventral tail fin, or partial defect of the eyes. Clock1a regulates the expression of the mesodermal markers ntl, gsc, and eve1 and of the hematopoietic markers scl, lmo2, and fli1a Biochemical analyses revealed that Clock1a stimulates Nodal signaling by increasing expression of Smad2/3/4. Mechanistically, Clock1a activates the smad3a promoter via its E-box1 element (CAGATG). Taken together, these findings provide mechanistic insight into the role of Clock1a in the regulation of mesoderm development and primitive hematopoiesis via modulation of Nodal-Smad3 signaling and indicate that Smad3a is directly controlled by the circadian clock in zebrafish.

Common AZFc structure may possess the optimal spermatogenesis efficiency relative to the rearranged structures mediated by non-allele homologous recombination.

The azoopsermia factor c (AZFc) region of human Y-chromosome is an essential genomic segment for spermatogenesis with frequent non-allele homologous recombination (NAHR). Recent case-control studies on the association of the NAHR-based AZFc structural mutations with spermatogenic failure produced inconsistent results. To more precisely evaluate their spermatogenesis effects, we investigated the correlation between the subdivided AZFc mutations and sperm production in 3,439 Han Chinese males. Our results showed that both partial AZFc deletion-only and primary duplication mutation presented a significant risk for decreased sperm production. Restoration of the reduced dosage of the AZFc content to the normal level had a milder effect, whereas an overdose of the AZFc content arising from multiple duplications of a partial AZFc-deleted structure produced a more serious consequence compared to the partial deletion-only mutation. Additionally, the AZFc-mutated structures with excessive NAHR-substrate showed a notably negative effect on spermatogenesis. These results suggest that the recurrent NAHR-based AZFc mutations may be associated with decreased spermatogenesis efficiency in present population. More significantly, our finding implies that the overdose of AZFc NAHR-substrate may produce an additional risk for the massive AZFbc deletions during the multi-stage division process of germ cells and thus impair the global spermatogenesis efficiency in the carriers.

[Identification and functional analysis of a testis-specific E3 ubiquitin ligase gene Rnf148 in mouse].

OBJECTIVE: To investigate the temporal and spatial features of mouse Rnf148 gene expression and the function of RING finger domain of Rnf148 protein. METHODS: The whole RNA was extracted from different tissues of adult mice, embryo in four developmental stages, and testes of postnatal mice respectively. RT-PCR and Northern blotting analysis were used to investigate the expression of Rnf148 gene in the above tissues. The in vitro expression vector for GST-Rnf148 fused protein was constructed, which encompassing the entire RING domain of Rnf148 protein. GST-Rnf148 fused protein was expressed in Escherichia coli. BL21(DE3) cells and purified with glutathione-sepharose 4B. In vitro ubiquitination assay was performed to analyze whether GST-Rnf148 fused protein possess the function of E3 ubiquitin ligase. RESULTS: The Mice Rnf148 mRNA expression was only observed in testis, and Northern blotting confirmed that there was only one 1.2 kb mRNA band present in mice testis. Rnf148 mRNA started to appear in the testis of day 21 mice, and then increased dramatically and reached to the highest level in day 25, and continued to express thereafter. GST-Rnf148 fused protein was induced and purified, in vitro ubiquitination reaction showed that the recombinant protein has E3 ubiquitin ligase activity. CONCLUSION: Rnf148 gene is specifically expressed in mice testis.

[Identification and expression analysis of Macaca mulatta piwil4 gene].

To investigate the structure and expression pattern of rhesus monkey PIWIL4 protein, homologous comparison and reverse transcription PCR (RT-PCR) were carried out to identify rhesus monkey piwil4. The expression of piwil4 mRNA was tested in rhesus monkey heart, brain, colon, epididymis and testis, and the result showed that piwil4 mRNA was expressed in these rhesus monkey tissues. Bioinformatic analysis suggested that the rhesus PIWIL4 protein shared 97% identity in amino acids and the same domains such as PAZ and Piwi with the human PIWIL4 (HIWI2) protein. The immunohistochemical result indicated that PIWIL4 proteins had the same localization in adult testes of the two species, but the distribution of these proteins was altered dynamically at different developmental stages in rhesus monkey testes. PIWIL4 protein was expressed in the nucleus of convoluted seminiferous tubules in infant monkey testes, whereas it was expressed in the cytoplasm of adult monkey testes. The results suggest that piwil4 gene play a similar role in rhesus and human, and different localizations of PIWIL4 protein in infant monkey and adult monkey testes suggest that it functions differently at different developmental stages.

Human RING finger protein ZNF645 is a novel testis-specific E3 ubiquitin ligase.

A large number of testis-specific genes are involved in the complex process of mammalian spermatogenesis. Identification of these genes and their roles is important for understanding the mechanisms underlying spermatogenesis. Here we report on a novel human RING finger protein, ZNF645, which contains a C3HC4 RING finger domain, a C2H2 zinc-finger domain, and a proline-rich region, indicating that it has a structure similar to that of the c-Cbl-like protein Hakai. ZNF645 was exclusively expressed in normal human testicular tissue. Immunohistochemical analysis confirmed that ZNF645 protein was present in spermatocytes, round and elongated spermatids, and Leydig cells. Immunofluorescence staining of mature sperms further showed that the ZNF645 protein was localized over the postacrosomal perinuclear theca region and the entire length of sperm tail. An in vitro ubiquitination assay indicated that the RING finger domain of the ZNF645 protein had E3 ubiquitin ligase activity. Therefore, we suggest that ZNF645 might act as an E3 ubiquitin-protein ligase and play a role in human sperm production and quality control.

c.822+126T>G/C: a novel triallelic polymorphism of the TSSK6 gene associated with spermatogenic impairment in a Chinese population.

TSSK6 is a member of the testis-specific serine/threonine kinase family. Male Tssk6 knockout mice are infertile owing to spermatogenic impairment, including sperm count reduction, a decrease in motile sperm number and motility rates, and an increase in the number of sperms with abnormal morphology. We investigated the possible association between variations of the TSSK6 gene and spermatogenic impairment in humans. Mutation screening of TSSK6 was carried out in 519 patients with azoospermia (n = 273) or severe oligozoospermia (n = 246) and in 359 controls with normozoospermia by denaturing high-performance liquid chromatography and DNA sequencing. The frequencies of alleles and genotypes of gene polymorphism were compared between patients and controls. A novel triallelic polymorphism in TSSK6, c.822+126T>G/C, was identified. The frequencies of genotype TT and allele T were increased dramatically in infertile patients compared with controls, whereas genotype TG, allele G and allele C frequencies were significantly higher in controls than in patients. Further study revealed that the allele C frequency of controls was remarkably higher than that of patients with oligospermia. Our findings, for the first time, suggested an association of c.822+126T>G/C in TSSK6 with spermatogenic impairment in humans in which allele T may be a risk factor for male infertility, while alleles C and G may decrease susceptibility to male infertility.

[Clinical characteristics and spastin gene mutation analysis on an autosomal dominant kindred with hereditary spastic paraplegia].

OBJECTIVE: To investigate the clinical characteristics and analyze spastin gene mutation on a kindred with hereditary spastic paraplegia (HSP). METHODS: All family members were studied through clinical examinations. The proband and another two patients in this kindred were subjected to electromyography (EMG) examinations. The proband was subjected to thoracic MRI examination too. Mutation analysis of spastin gene was screened by polymerase chain reaction combined with DNA sequencing in the proband and his father. RESULTS: All patients in the kindred manifested as classical HSP. Thoracic MRI revealed atrophies of the spinal cord in the proband. No abnormal spastin gene mutation was detected in these two patients. CONCLUSION: This kindred has typical clinical manifestations of HSP. The pathogenesis has no association with mutation of the exons of spastin gene.

Association of Sst I polymorphism in apolipoprotein C3 gene with hypertriglyceridaemia in coronary atherosclerotic heart disease and type II diabetes mellitus in Chinese population.

Several independent population studies have reported that the apolipoprotein C3 (APOC3) Sst I polymorphism in apolipoprotein (apo) A1 /C3/A4/A5 gene cluster is associated with Hypertriglyceridaemia (HTG). HTG is a known risk factor for coronary atherosclerotic heart disease(CHD)and type II diabetes mellitus (non-insulin-dependent diabetes, NIDDM). The aim of this study is to investigate the association between the APOC3 gene Sst I polymorphism and the hypertriglyceridaemia in CHD and NIDDM in Chinese population. The genotype and allele frequencies of APOC3 Sst I polymorphism (S1/S2) were analyzed by PCR-restriction fragment length polymorphism in 267 CHD patients, 246 NIDDM patients and 491 unrelated healthy control individuals. The frequencies of minor allele 52 in CHD group, NIDDM group and control group were 0.301, 0.307 and 0.286, respectively. Compared with controls, there was no significant difference in distribution of genotype and allele frequencies of Sst I polymorphic site in CHD patients and NIDDM patients, respectively. However, the frequency of S1 S2 genotype in the HTG subgroup was significantly higher than that of the normal triglyceridaemia subgroup (NTG) in CHD patients (0.542 > 0.357, chi2 = 8.77, P = 0.0124). In NIDDM patients, the frequency of S2 S2 genotype in the HTG subgroup was significantly high, compared with that in the NTG subgroup (0.200 > 0.055, chi2 = 20.21, P = 0.0000), and there was significantly difference in the distribution of allele frequencies in subgroups of NTG and HTG (chi2 = 19.86, P = 0.0000). The level of triglyceride (TG) in S1 S2 genotype patients of CHD group were higher than that of S1 S1 genotype patients (P = 0.036). In NIDDM and controls groups, S2 S2 genotype individuals exhibited a significant increase in plasma TG concentrations, respectively compared with S1 S1 and S1 S2 genotype individuals of each group (P < 0.01). The minor allele S2, which was associated with both CHD with HTG and NIDDM with HTG and may contribute to the susceptibility of hypertriglyceridemia in CHD and NIDDM patients, may be one of the genetic predispositions to both CHD with HTG and NIDDM with HTG in Chinese population.

[Construction of recombinant ZNF230/GFP fused plasmids and their expression and cellular localization].

To use green fluorescent protein as a marker to study the localization of the fusion protein, the mutant full length cDNAs of human ZNF230 and mouse znf230 with their stop codon TGA changed to TGG were obtained by PCR amplification, and then cloned into pGEM-Teasy vector. After the double enzyme cutting, the mutated human and mouse ZNF230(znf230) were inserted into mammalian expression plasmid pEGFP-N1. Thus we constructed the plasmid with fusion gene of ZNF230 and green fluorescent protein(GFP). Then the Cos cell was transfected with the fused gene by liposome. Fluorescence microscopy showed that green fluorescence protein expressed over the whole cell when transfected with vector pEGFP-N1. While after the transfection with pEGFP-ZNF230, the fluorescence located mainly on the nuclei of the cells. We demonstrated that the transfected Cos cell line can express human ZNF230 and mouse znf230 with high efficiency. When transfected with the constructed recombinant pEGFP-ZNF230 vector, the ZNF230 protein localizes mainly on the nucleus.