Floxed Il1rl2 Locus with mCherry Reporter Element Reveals Distinct Expression Patterns of the IL-36 Receptor in Barrier Tissues.

IL-36 cytokines are emerging as beneficial in immunity against pathogens and cancers but can also be detrimental when dysregulated in autoimmune and autoinflammatory conditions. Interest in targeting IL-36 activity for therapeutic purposes is rapidly growing, yet many unknowns about the functions of these cytokines remain. Thus, the availability of robust research tools is essential for both fundamental basic science and pre-clinical studies to fully access outcomes of any manipulation of the system. For this purpose, a floxed Il1rl2, the gene encoding the IL-36 receptor, mouse strain was developed to facilitate the generation of conditional knockout mice. The targeted locus was engineered to contain an inverted mCherry reporter sequence that upon Cre-mediated recombination will be flipped and expressed under the control of the endogenous Il1rl2 promoter. This feature can be used to confirm knockout in individual cells but also as a reporter to determine which cells express the IL-36 receptor IL-1RL2. The locus was confirmed to function as intended and further used to demonstrate the expression of IL-1RL2 in barrier tissues. Il1rl2 expression was detected in leukocytes in all barrier tissues. Interestingly, strong expression was observed in epithelial cells at locations in direct contact with the environment such as the skin, oral mucosa, the esophagus, and the upper airways, but almost absent from epithelial cells at more inward facing sites, including lung alveoli, the small intestine, and the colon. These findings suggest specialized functions of IL-1RL2 in outward facing epithelial tissues and cells. The generated mouse model should prove valuable in defining such functions and may also facilitate basic and translational research.

A Florfenicol-Resistant Plasmid Shuttling Between Actinobacillus pleuropneumoniae and Glaesserella parasuis.

Porcine contagious pleuropneumonia, caused by Actinobacillus pleuropneumoniae, has resulted in significant economic losses to the swine industry. Although antibiotics are commonly employed to control this disease, their widespread use or misuse can lead to the development of antibiotic resistance in A. pleuropneumoniae. Consequently, it is crucial to conduct antimicrobial susceptibility testing on clinical isolates. In our study, we identified one strain of A. pleuropneumoniae with resistance to florfenicol and extracted a 5919 bp plasmid named pAPPJY, which confers florfenicol resistance. Sequence analysis revealed that the plasmid contains four open reading frames, namely rep, antioxin vbha family protein, floR, and a partial copy of lysr. Although a few variations in gene position were observed, the plasmid sequence exhibits a high degree of similarity to other florfenicol-resistant plasmids found in Glaesserella parasuis and A. pleuropneumoniae. Therefore, it is possible that the pAPPJY plasmid functions as a shuttle, facilitating the spread of florfenicol resistance between G. parasuis and A. pleuropneumoniae. In addition, partial recombination may occur during bacterial propagation. In conclusion, this study highlights the horizontal transmission of antibiotic resistance among different bacterial species through plasmids, underscoring the need for increased attention to antibiotic usage.

Outer Membrane Vesicles Associated with the Delivery of Amoxicillin Resistance in Glaesserella parasuis.

Glässer's disease is caused by Glaesserella parasuis, a common bacterium in the upper respiratory tract of pigs. Antibiotics are widely used to control this disease. A G. parasuis isolate with amoxicillin (AMX) resistance was identified in our previous study. Outer membrane vesicles (OMVs) are naturally released by G. parasuis and contain many compounds. To reveal the underlying mechanisms associated with AMX resistance delivery, OMVs from G. parasuis were successfully isolated and identified by transmission electron microscopy. In particular, we found that ß-lactamase existed in OMVs using label-free analysis, and further verified that OMVs carry ß-lactamase by Western blotting. The minimal inhibitory concentration and growth rate were determined to evaluate the ß-lactamase activity in G. parasuis OMVs. Moreover, the effect of different concentrations of OMVs from aHPS7 on the growth rate of AMX sensitive strains was examined. Our results further confirmed that OMVs isolated from aHPS7 contain ß-lactamase, which can prevent AMX-susceptible strains from killing by hydrolyzing AMX. Our results first showed that OMVs of G. parasuis play an important role in the spread of antibiotic resistance, which seriously hampered the prevention of this disease by the delivery of OMVs in different strains.

Pili Subunit PilA Contributes to the Cytoadhesion of Glaesserella Parasuis to Host Cells and Provides Immunoprotection.

Glaesserella parasuis (G. parasuis) is commonly located in the upper respiratory tract of pigs as an opportunistic pathogen. It can cause Glässer's disease, which leads to serious economic losses in the swine industry. The occurrence of the disease is often linked with the adhesion and colonization of the pathogen. The PilA pilus subunit is important for adhesion to the host, twitching motility, and biofilm formation in many bacteria. However, no research has focused on the function of PilA in G. parasuis. To further reveal the pathogenesis of G. parasuis and to search for subunit vaccine candidates, we investigated whether PilA could adhere to cells and provide immune protection. A bioinformatic analysis showed that the protein secondary structure of the G. parasuis PilA was similar to that of Haemophilus influenzae (HI). Cell adhesion, ELISA, and far-Western blotting showed that rPilA could bind porcine-derived, porcine kidney-15 (PK-15) cells, swine tracheal epithelial cells (STECs), and the extracellular matrix components fibronectin (FN) and laminin (LN). An immunogenicity analysis showed that recombinant PilA (rPilA) reacted specifically with convalescent and hyperimmune serum. Importantly, purified rPilA elicited a strong immune response and conferred robust protection against challenges with serovar 5 G. parasuis in mice. These results suggested that the PilA protein might help G. parasuis adhere to host cells by binding to FN and LN, and its immunogenicity establishes it as a promising, novel subunit vaccine candidate against infections with G. parasuis. IMPORTANCE G. parasuis is one of the most prevalent bacterial infections in swine production and can lead to huge economic losses around the world. A full understanding of colonization and immunity with G. parasuis infections will be essential in disease control. In this study, the PilA protein, which is a common virulence factor in other bacteria that mediates adherence to the host, was assessed. The results suggested that the PilA protein of G. parasuis can mediate adhesion to host cells through FN and LN, which provides a new idea for the study of the pathogenicity of G. parasuis. Furthermore, fimbriae usually have high immunogenicity. Immunogenicity and protective capacity results showed that the use of this recombinant PilA antigen might be a promising candidate vaccine antigen with which to prevent G. parasuis infections.

Combined effects of nanosized polystyrene and erythromycin on bacterial growth and resistance mutations in Escherichia coli.

Toxicological effects of nanoplastics have been demonstrated in a variety of organisms, yet their impacts on bacteria, especially on the antibiotic resistance evolution remain under explored. Herein, we report individual and combined effects of nano-polystyrene (nano-PS) and erythromycin (ERY) on growth and resistance mutations of Escherichia coli. The toxicity of nano-PS was dependent on size and functional modifications, with 30 nm and amino-modified PS (PS-NH2, 200 nm) showing the greatest toxicity. Adsorption of nano-PS onto bacterial surface and the subsequent increase of intracellular ROS or the probable mechanical damage were considered as the primary toxic mechanisms. Furthermore, nano-PS increased the bacterial resistance mutations, which was due to the oxidative damage to DNA and the SOS response. In addition, PS-NH2 presented synergistic effects with ERY while non-modified PS had no impact, although both of them showed adsorption capacity to ERY. This was likely because the positively charged PS-NH2 acted as a carrier of ERY and enhanced the interactions between ERY and the bacteria. Our findings raised the concerns about the risk of nanoplastics in accelerating the bacterial resistance evolution, and highlighted the necessity of including combined effects of nanoplastics and co-contaminants in risk assessment.

Characterization and protective activity of monoclonal antibodies directed against Fe (3+) ABC transporter substrate-binding protein of Glaesserella parasuis.

Glässer's disease is caused by the agent Glaesserella parasuis and is difficult to prevent and control. Candidate screening for subunit vaccines contributes to the prevention of this disease. Therefore, in this study, the inactivated G. parasuis reference serovar 5 strain (G. parasuis-5) was used to generate specific monoclonal antibodies (mAbs) to screen subunit vaccine candidates. Six mAbs (1A12, 3E3, 4C6, 2D1, 3E6, and 4B2) were screened, and they all reacted with the G. parasuis serovar 5 strain according to laser confocal microscopy and flow cytometry (FCM). Indirect enzyme-linked immunosorbent assay (ELISA) showed that one mAb 2D1, can react with all 15 reference serovars of G. parasuis. Protein mass spectrometry and Western blot analysis demonstrated that mAb 2D1 specifically reacts with Fe (3+) ABC transporter substrate-binding protein. A complement killing assay found that the colony numbers of bacteria were significantly reduced in the G. parasuis-5 group incubated with mAb 2D1 (p < 0.01) in comparison with the control group. Opsonophagocytic assays demonstrated that mAb 2D1 significantly enhanced the phagocytosis of 3D4/21 cells by G. parasuis (p < 0.05). RAW264.7 cells with stronger phagocytic ability were also used for the opsonophagocytic assay, and the difference was highly significant (p < 0.01). Passive immunization of mice revealed that mAb 2D1 can eliminate the bacteria in the blood and provide protection against G. parasuis-5. Our study found one mAb that can be used to prevent and control G. parasuis infection in vivo and in vitro, which may suggest that Fe (3+) ABC transporter substrate-binding protein is an immunodominant antigen and a promising candidate for subunit vaccine development.

ICEHpsaHPS7, a Novel Multiple Drug Resistance Integrative Conjugative Element in Glaesserella parasuis.

Integrative conjugative elements (ICEs) are a kind of novel self-transmissible mobile genetic element. In this study, a novel ICE was identified in Glaesserella (Haemophilus) parasuis We confirmed that it could mediate the migration of antimicrobial resistance genes in G. parasuis and found that there may have been a transferring potential between different serovar strains of G. parasuis These findings demonstrate that the ICE is crucial to the horizontal transfer of antimicrobial resistance among G. parasuis strains.

Trichostatin A increases BDNF protein expression by improving XBP-1s/ATF6/GRP78 axis in Schwann cells of diabetic peripheral neuropathy.

Diabetic peripheral neuropathy (DPN) is the common complication of diabetes mellitus. Histone deacetylase (HDAC) inhibitor trichostatin A (TSA) is reported to ameliorate the peripheral nerves degeneration of DPN. However, the exact mechanism is still not well elucidated. Here, we first revealed that TSA promoted nerve conduction and brain derived neurotrophic factor (BDNF) expression in the sciatic nerves of diabetic mice. In line, TSA also reversed high glucose-reduced mature BDNF expression in vitro cultured rat Schwann cells (RSC96). Then unexpectedly, the downstream targets of TSA HDAC1 and HDAC5 were not involved in TSA-improved BDNF expression. Furthermore, unfolded protein response (UPR) chaperone GRP78 was revealed to be downregulated with high glucose stimulation in RSC96 cells, which was avoided with TSA treatment. Also, GRP78 upregulation mediated TSA-improved mature BDNF expression in high glucose-cultured RSC96 cells by binding with BDNF. As well, TSA treatment enhanced the binding of GRP78 with BDNF in RSC96 cells. Again, UPR-associated transcription factors XBP-1s and ATF6 were involved in TSA-increased GRP78 expression in high glucose-stimulated RSC96 cells. Finally, conditioned medium from high glucose-cultured RSC96 cells delayed neuron SH-SY5Y differentiation and that from TSA-treated high glucose-cultured RSC96 cells promoted SH-SY5Y cell differentiation. Taken together, our findings suggested that TSA increased BDNF expression to ameliorate DPN by improving XBP-1s/ATF6/GRP78 axis in Schwann cells.

IFN-γ/mTORC1 decreased Rab11 in Schwann cells of diabetic peripheral neuropathy, inhibiting cell proliferation via GLUT1 downregulation.

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus. Rab11 is conserved gene-regulating vesicle traffic and reported to be involved in the pathogenesis of diabetes mellitus by affecting insulin sensitivity. We aimed to investigate the role of Rab11 in the pathogenesis of DPN. In this study, Rab11 expression decreased in the sciatic nerves of diabetic mice with impaired conduction function versus those of normal mice. In vitro experiment revealed interferon-γ (IFN-γ), not high glucose and interleukin 1ß was the main factor to lead to Rab11 downregulation in RSC96 cells. Again, both Rab11 knockdown and IFN-γ treatment caused cell viability inhibition and the decrease in BrdU-positive cells. In contrast, overexpression of Rab11 reversed IFN-γ-reduced cell proliferation. Furthermore, mTORC1 not mTORC2 was proven to be suppressed by IFN-γ treatment in RSC96 cells, indicated in decreased phospho-p70S6K. Inhibition of the mTORC1 pathway resulted in Rab11 expression downregulation in RSC96 cells. Activation of the mTORC1 pathway effectively prevented IFN-γ-reduced Rab11 expression in RSC96 cells. Also, glucose transporter 1 (GLUT1) was found to be downregulated in RSC96 cells with Rab11 silence and overexpression of GLUT1 reversed Rab11 blocking-caused proliferation inhibition. Taken together, our findings suggest that IFN-γ decreases Rab11 expression via the inhibition of the mTORC1 signaling pathway, causing reduced cell proliferation in Schwann cells of DPN by GLUT1 downregulation.

Haemophilus parasuis infection in 3D4/21 cells induces autophagy through the AMPK pathway.

Haemophilus parasuis (H. parasuis) is a common commensal in the upper respiratory tract of pigs, but causes Glässer's disease in stress conditions. To date, many studies focused on the immune evasion and virulence of H. parasuis; very few have focused on the role autophagy played in H. parasuis infection, particularly in porcine alveolar macrophages (PAMs). In this study, a PAM cell line, 3D4/21 cells were used to study the role of autophagy in H. parasuis infection. 3D4/21 cells tandemly expressing GFP, mCherry, and LC3 were infected with H. parasuis serovar 5 (Hps5). Western blot analysis and confocal and transmission electron microscopy showed that H. parasuis infection effectively induces autophagy. Using Hps strains of varying virulence (Hps4, Hps5, and Hps7) and UV-inactivated Hps5, we demonstrated that autophagy is associated with the internalisation of living virulent strains into cells. In 3D4/21 cells pretreated with rapamycin and 3-MA then infected by Hps4, Hps5, and Hps7, we demonstrated that autophagy affects invasion of H. parasuis in cells. AMPK signal results showed that Hps5 infection can upregulate the phosphorylation level of AMPK, which is consistent with the autophagy development. 3D4/21 cells pretreated with AICAR or Compound C then infected by Hps5 revealed that the autophagy induced by Hps5 infection is associated with the AMPK pathway. Our study contributes to the theoretical basis for the study of H. parasuis pathogenesis and development of novel drugs target for prevention Glässer's disease.

STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Schwann cells are the main supportive cells of the peripheral nerves. Schwann cells suffer inhibition of autophagy under hyperglycemia treatment in diabetic peripheral neuropathy (DPN). However, the exact mechanism is still not fully elucidated. We first observed the decrease of autophagy markers (LC3-II/LC3-I, P62) in the sciatic nerves of diabetic mice vs. normal mice, accompanied with the loss of myelinated nerve fibers and abnormal myelin sheath. In line with this, LC3-II/LC3-I and P62 were also significantly reduced in high glucose-treated rat Schwann cell 96 (RSC96) cells compared with normal glucose-treated cells. Furthermore, we found that trichostatin A [an inhibitor of histone deacetylase (HDAC)] evidently improved LC3-II/LC3-I in high glucose-treated RSC96 cells, without an effect on P62 expression. Again, HDAC1 and HDAC5 were revealed to be increased in RSC96 cells stimulated with high glucose. Inhibition of HDAC1 but not HDAC5 by small hairpin RNA vector enhanced LC3-II/LC3-I in high glucose-cultured RSC96 cells. In addition, LC3-II conversion regulators [autophagy-related protein (Atg)3, Atg5, and Atg7] were detected in high glucose-treated and HDAC1-knockdown RSC96 cells, and Atg3 was proven to be the key target of HDAC1. The presuppression of Atg3 offset the improvement of LC3-II/LC3-I resulting from HDAC1 inhibition in high glucose-treated RSC96 cells. The Janus kinase (JAK)-signal transducer and activator of transcription (STAT) signaling pathway was activated in RSC96 cells treated with high glucose, which was indicated by increased STAT3 phosphorylation. Blocking STAT3 phosphorylation by chemical inhibitor AG490 induced HDAC1 down-regulation followed by increases in Atg3 and LC3-II/LC3-I. Interestingly, we also found that AG490 treatment enhanced P62 expression in high glucose-stimulated RSC96 cells. Taken together, our findings demonstrate that hyperglycemia inhibits LC3-II/LC3-I in an HDAC1-Atg3-dependent manner and decreases P62 expression in an HDAC-independent manner via the JAK-STAT3 signaling pathway in the Schwann cells of DPN.-Du, W., Wang, N., Li, F. Jia, K., An, J., Liu, Y., Wang, Y., Zhu, L., Zhao, S. Hao, J. STAT3 phosphorylation mediates high glucose-impaired cell autophagy in an HDAC1-dependent and -independent manner in Schwann cells of diabetic peripheral neuropathy.

Detection and sequencing of porcine circovirus 3 in commercially sourced laboratory mice.

Porcine circovirus 3 (PCV3), a recently discovered virus, has spread widely in pigs throughout the world. In order to investigate the possibility of mice used to study the infection of PCV3, commercially sourced Balb/C and ICR mice were screened for PCV3 infection. Blood samples were collected from 20 mice (10 each of Balb/c and ICR), DNA was extracted, and subjected to PCR with PCV3 specific primers. We found all 20 serum samples tested positive for PCV3 DNA. From four mice, the complete genomes of PCV3 were amplified and sequenced, and a phylogenetic tree was constructed. The results showed that the amplified genome was 2000 bp, and sequence comparison showed that the homology of the complete genome and ORF2 gene with those of porcine PCV3 are 97.9%-98.8% and 96.9%-98.3%, respectively. Amino acids alignment results showed that the Cap protein of the mouse PCV3 isolates share 90.7%-96.3% amino acid homology with that of the references strains derived from pigs. Phylogenetic analysis based on ORF2 sequences showed that all PCV3 strains clustered together and were clearly separate from other circovirus species. We detected PCV3 in experimental mice in China for the first time, which is an opportunity to use mice to study the infection of PCV3 and a potential hazard to swine industry.

HistoneH3 demethylase JMJD2A promotes growth of liver cancer cells through up-regulating miR372.

Changes in histone lysine methylation status have been observed during cancer formation. JMJD2A protein is a demethylase that is overexpressed in several tumors. Herein, our results demonstrate that JMJD2A accelerates malignant progression of liver cancer cells in vitro and in vivo. Mechanistically, JMJD2A promoted the expression and mature of pre-miR372 epigenetically. Notably, miR372 blocks the editing of 13th exon-introns-14th exon and forms a novel transcript( JMJD2AΔ) of JMJD2A. In particular, JMJD2A inhibited P21(WAF1/Cip1) expression by decreasing H3K9me3 dependent on JMJD2AΔ. Thereby, JMJD2A could enhance Pim1 transcription by suppressing P21(WAF1/Cip1). Furthermore, through increasing the expression of Pim1, JMJD2A could facilitate the interaction among pRB, CDK2 and CyclinE which prompts the transcription and translation of oncogenic C-myc. Strikingly, JMJD2A may trigger the demethylation of Pim1. On the other hand, Pim1 knockdown and P21(WAF1/Cip1) overexpression fully abrogated the oncogenic function of JMJD2A. Our observations suggest that JMJD2A promotes liver cancer cell cycle progress through JMJD2A-miR372-JMJD2AΔ-P21WAF1/Cip1-Pim1-pRB-CDK2-CyclinE-C-myc axis. This study elucidates a novel mechanism for JMJD2A in liver cancer cells and suggests that JMJD2A can be used as a novel therapeutic targets of liver cancer.

Inflammatory cytokine IL6 cooperates with CUDR to aggravate hepatocyte-like stem cells malignant transformation through NF-κB signaling.

Inflammatory cytokines and lncRNAs are closely associated with tumorigenesis. Herein, we reveal inflammatory cytokines IL6 cooperates with long noncoding RNA CUDR to trigger the malignant transformation of human embryonic stem cells-derived hepatocyte-like stem cells. Mechanistically, IL6 cooperates with CUDR to cause MELLT3 to interact with SUV39h1 mRNA3'UTR and promote SUV39h1 expression. Moreover, the excessive SUV39h1 also increases tri-methylation of histone H3 on nineth lysine (H3K9me3). Intriguingly, under inflammatory conditions, H3K9me3 promotes the excessive expression and phosphorylation of NF-κB, and in turn, phorsphorylated NF-κB promotes the expression and phosphorylation of Stat3. Furthermore, that the phosphorylated Stat3 loads onto the promoter region of miRs and lncRNAs. Ultimately, the abnormal expression of miRs and lncRNAs increased telomerase activity, telomere length and microsatellite instability (MSI), leading to malignant transformation of hepatocyte-like stem cells.

HULC cooperates with MALAT1 to aggravate liver cancer stem cells growth through telomere repeat-binding factor 2.

The dysregulation of lncRNAs has increasingly been linked to many human diseases, especially in cancers. Our results demonstrate HULC, MALAT1 and TRF2 are highly expressed in human hepatocellular carcinoma tissues, and HULC plus MALAT1 overexpression drastically promotes the growth of liver cancer stem cells. Mechanistically, both HULC and MALAT1 overexpression enhanced RNA polII, P300, CREPT to load on the promoter region of telomere repeat-binding factor 2(TRF2), triggering the overexpression, phosphorylation and SUMOylation of TRF2. Strikingly, the excessive TRF2 interacts with HULC or MALAT1 to form the complex that loads on the telomeric region, replacing the CST/AAF and recruiting POT1, pPOT1, ExoI, SNM1B, HP1 α. Accordingly, the telomere is greatly protected and enlonged. Furthermore, the excessive HULC plus MALAT1 reduced the methylation of the TERC promoter dependent on TRF2, increasing the TERC expression that causes the increase of interplay between TRET and TERC. Ultimately, the interaction between RFC and PCNA or between CDK2 and CyclinE, the telomerase activity and the microsatellite instability (MSI) are significantly increased in the liver cancer stem cells. Our demonstrations suggest that haploinsufficiency of HULC/MALAT1 plays an important role in malignant growth of liver cancer stem cell.

Inflammatory related gene IKKα, IKKß, IKKγ cooperates to determine liver cancer stem cells progression by altering telomere via heterochromatin protein 1-HOTAIR axis.

Cancer stem cells are associated with tumor recurrence. IKK is a protein kinase that is composed of IKKα, IKKß, IKKγ. Herein, we demonstrate that IKKα plus IKKß promoted and IKKγ inhibited liver cancer stem cell growth in vitro and in vivo. Mechanistically, IKKα plus IKKß enhanced and IKKγ inhibited the interplay among HP1α, HP1ß and HP1γ that competes for the interaction among HP1α, SUZ12, HEZ2. Therefore, IKKα plus IKKß inhibited and IKKγ enhanced the activity of H3K27 methyltransferase SUZ12 and EZH2, which methylates H3K27 immediately sites on HOTAIR promoter region. Therefore, IKKα plus IKKß increased and IKKγ decreased the HOTAIR expression. Strikingly, IKKα plus IKKß decreases and IKKγ increases the HP1α interplays with DNA methyltransferase DNMT3b, which increases or decreases TERRA promoter DNA methylation. Thus IKKα plus IKKß reduces and IKKγ increases to recruit TRF1 and RNA polymerase II deposition and elongation on the TERRA promoter locus, which increases or decreases TERRA expression. Furthermore, IKKα plus IKKß decreases/increases and IKKγ increases/decreases the interplay between TERT and TRRRA/between TERT and TREC. Ultimately, IKKα plus IKKß increases and IKKγ decreases the telomerase activity. On the other hand, at the telomere locus, IKKα plus IKKß increases/drcreases and IKKγ decreases/increases TRF2, POT1, pPOT1, Exo1, pExo1, SNM1B, pSNM1B/CST-AAF binding, which keep active telomere regulatory genes and poised for telomere length. Strikingly, HOTAIR is required for IKKα plus IKKß and IKKγ to control telomerase activity and telomere length. These observations suggest that HOTAIR operates the action of IKKα, IKKß, IKKγ in liver cancer stem cells. This study provides a novel basis to elucidate the oncogenic action of IKKα, IKKß, IKKγ and prompts that IKKα, IKKß, IKKγ cooperate to HOTAR to be used as a novel therapeutic targets for liver cancer.

Double mutant P53 (N340Q/L344R) promotes hepatocarcinogenesis through upregulation of Pim1 mediated by PKM2 and LncRNA CUDR.

P53 is frequently mutated in human tumors as a novel gain-of-function to promote tumor development. Although dimeric (M340Q/L344R) influences on tetramerisation on site-specific post-translational modifications of p53, it is not clear how dimeric (M340Q/L344R) plays a role during hepatocarcinogenesis. Herein, we reveal that P53 (N340Q/L344R) promotes hepatocarcinogenesis through upregulation of PKM2. Mechanistically, P53 (N340Q/L344R) forms complex with CUDR and the complex binds to the promoter regions of PKM2 which enhances the expression, phosphorylation of PKM2 and its polymer formation. Thereby, the polymer PKM2 (tetramer) binds to the eleventh threonine on histone H3 that increases the phosphorylation of the eleventh threonine on histone H3 (pH3T11). Furthermore, pH3T11 blocks HDAC3 binding to H3K9Ac that prevents H3K9Ac from deacetylation and stabilizes the H3K9Ac modification. On the other hand, it also decreased tri-methylation of histone H3 on the ninth lysine (H3K9me3) and increases one methylation of histone H3 on the ninth lysine (H3K9me1). Moreover, the combination of H3K9me1 and HP1 α forms more H3K9me3-HP1α complex which binds to the promoter region of Pim1, enhancing the expression of Pim1 that enhances the expression of TERT, oncogenic lncRNA HOTAIR and reduces the TERRA expression. Ultimately, P53 (N340Q/L344R) accerlerates the growth of liver cancer cells Hep3B by activating telomerase and prolonging telomere through the cascade of P53 (N340Q/L344R)-CUDR-PKM2-pH3T11- (H3K9me1-HP1α)-Pim1- (TERT-HOTAIR-TERRA). Understanding the novel functions of P53 (N340Q/L344R) will help in the development of new liver cancer therapeutic approaches that may be useful in a broad range of cancer types.

RETRACTED: SET1A Cooperates With CUDR to Promote Liver Cancer Growth and Hepatocyte-like Stem Cell Malignant Transformation Epigenetically

Long noncoding RNA CUDR plays an important role during tumorigenesis. Herein, we demonstrate that SET1A cooperates with CUDR to accelerate hepatocarcinogenesis and promote malignant transformation of hepatocyte-like stem cells. Mechanistically, CUDR enhances the phosphorylation of RB1, C-myc expression, and the interplay between the SET1A and pRB1. Notably, CUDR acts as a sponge cushion that shows a link between SET1A and pRB1, producing a activated pRB1-SET1A complex. On the other hand, the pRB1-SET1A complex may carry methyls(me) to occupy the position of H3K4, resulting in specific tri-methylation of forth lysine of histone H3 (H3K4me3). Thereby, the H3K4me3 loads on the TRF2 promoter region which causes the TRF2 overexpression. Ultimately, the excessive TRF2 binds to telomere repeat DNA, prolonging the telomere length. These findings provide the first demonstration that SET1A cooperates with CUDR to play a positive potential role during hepatocarcinogenesis and hepatocyte-like stem cells' malignant transformation epigenetically.

CUDR promotes liver cancer stem cell growth through upregulating TERT and C-Myc.

Cancer up-regulated drug resistant (CUDR) is a novel non-coding RNA gene. Herein, we demonstrate excessive CUDR cooperates with excessive CyclinD1 or PTEN depletion to accelerate liver cancer stem cells growth and liver stem cell malignant transformation in vitro and in vivo. Mechanistically, we reveal the decrease of PTEN in cells may lead to increase binding capacity of CUDR to CyclinD1. Therefore, CUDR-CyclinD1 complex loads onto the long noncoding RNA H19 promoter region that may lead to reduce the DNA methylation on H19 promoter region and then to enhance the H19 expression. Strikingly, the overexpression of H19 increases the binding of TERT to TERC and reduces the interplay between TERT with TERRA, thus enhancing the cell telomerase activity and extending the telomere length. On the other hand, insulator CTCF recruits the CUDR-CyclinD1 complx to form the composite CUDR-CyclinD1-insulator CTCF complex which occupancied on the C-myc gene promoter region, increasing the outcome of oncogene C-myc. Ultimately, excessive TERT and C-myc lead to liver cancer stem cell and hepatocyte-like stem cell malignant proliferation. To understand the novel functions of long noncoding RNA CUDR will help in the development of new liver cancer therapeutic and diagnostic approaches.