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.

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.

Long noncoding RNA HULC is highly up-regulation in human hepatocellular carcinoma (HCC). However, the functions of HULC in hepatocarcinogenesis remains unclear.

Inflammatory and autophagy-related gene P62 is highly expressed in most human tumor tissues. Herein, we demonstrate that P62 promotes human mesenchymal stem cells' malignant transformation via the cascade of P62-tumor necrosis factor alpha (TNF-α)-CUDR-CTCF-insulin growth factor II (IGFII)-H-Ras signaling. Mechanistically, we reveal P62 enhances IGFII transcriptional activity through forming IGFII promoter-enhancer chromatin loop and increasing METTL3 occupancy on IGFII 3' UTR and enhances H-Ras overexpression by harboring inflammation-related factors, e.g., TNFR1, CLYD, EGR1, NFκB, TLR4, and PPARγ. Furthermore, the P62 cooperates with TNF-α to promote malignant transformation of mesenchymal stem cells. These findings, for the first time, provide insight into the positive role that P62 plays in malignant transformation of mesenchymal stem cells and reveal a novel link between P62 and the inflammation factors in mesenchymal stem cells.

Maternally expressed gene 3 (MEG3) encodes an lncRNA which is suggested to function as a tumor suppressor and has been showed to involve in a variety of cancers. Herein, our findings demonstrate that MEG3 inhibits the malignant progression of liver cancer cells in vitro and in vivo. Mechanistically, MEG3 promotes the expression and maturition of miR122 which targets PKM2. Therefore, MEG3 decreases the expression and nuclear location of PKM2 dependent on miR122. Furthermore, MEG3 also inhibits CyclinD1 and C-Myc via PKM2 in liver cancer cells. On the other hand, MEG3 promotes β-catenin degradation through ubiquitin-proteasome system dependent on PTEN. Strikingly, MEG3 inhibits β-catenin activity through PKM2 reduction and PTEN increase. Significantly, we also found that excessive β-catenin abrogated the effect of MEG3 in liver cancer. In conclusion, our study for the first time demonstrates that MEG3 acts as a tumor suppressor by negatively regulating the activity of the PKM2 and β-catenin signaling pathway in hepatocarcinogenesis and could provide potential therapeutic targets for the treatment of liver cancer.

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.

Several microRNAs are associated with carcinogenesis and tumour progression. Herein, our observations suggest both miR24-2 and Pim1 are up-regulated in human liver cancers, and miR24-2 accelerates growth of liver cancer cells in vitro and in vivo. Mechanistically, miR24-2 increases the expression of N6-adenosine-methyltransferase METTL3 and thereafter promotes the expression of miR6079 via RNA methylation modification. Furthermore, miR6079 targets JMJD2A and then increased the tri-methylation of histone H3 on the ninth lysine (H3K9me3). Therefore, miR24-2 inhibits JMJD2A by increasing miR6079 and then increases H3K9me3. Strikingly, miR24-2 increases the expression of Pim1 dependent on H3K9me3 and METTL3. Notably, our findings suggest that miR24-2 alters several related genes (pHistone H3, SUZ12, SUV39H1, Nanog, MEKK4, pTyr) and accelerates progression of liver cancer cells through Pim1 activation. In particular, Pim1 is required for the oncogenic action of miR24-2 in liver cancer. This study elucidates a novel mechanism for miR24-2 in liver cancer and suggests that miR24-2 may be used as novel therapeutic targets of liver cancer.

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.

MicroRNA24-2 (miR24-2) is associated with human tumorigenesis; however, its molecular mechanisms are poorly understood. Herein, our findings demonstrate that miR24-2 promotes the proliferation ability in vitro and the tumorigenic ability in vivo in human liver cancer stem cells (hLCSCs). Mechanically, the miR24-2 targets for 3' UTR (2,627-2,648) of protein arginine methyltransferase 7 (PRMT7) inhibit the translational ability of prmt7 gene. Moreover, miR24-2 inhibits the di-/tri-methylation of histone H4 arginine 3 by reducing PRMT7 and then promotes the expression of Nanog via long noncoding RNA HULC. Notably, miR24-2 inhibits histone deacetylase HDAC3 through miR675, which promotes the acetylation of histone H4 at lysine 16. Subsequently, miR24-2 enhances the interaction between LC3 and ATG4 dependent on PI3K and triggers cellular autophagy. Strikingly, miR24-2 inhibits the degradation of pyruvate kinase M1 via autophagosome-P62 in hLCSCs. Furthermore, miR24-2 enhances the activity of Src by promoting the binding of PKM1 to the Src promoter regions in hLCSCs. In particular, our results also indicate that src gene determines the oncogenic functions of miR24-2. These results provided a valuable theoretical basis for the discovery of liver cancer therapeutic targets and diagnosis markers based on miR24-2.

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.

microRNAs (miRNAs) are short non-coding RNAs that are involved in post-transcriptional regulation of gene expression in multicellular organisms by affecting both the stability and translation of mRNAs. miR675, embedded in H19's first exon, had been linked to the development of human cancers. Herein, we demonstrate miR675 overexpression promotes and silencing miR675 attenuated liver cancer cell growth in vitro and in vivo. Mechanistically, miR675 inhibits the heterochromatin1 isoform HP1α expression in human liver cancer cells which causes a dramatically decrease of the total histone H3 lysine 9 trimethylation (H3K9me3) , histone H3 lysine 27 trimethylation (H3K27me3) and a increase of histone H3 lysine 27 acetylation(H3K27Ac).Notably, a significant reduction of the H3K9me3 and H3K27me3 and the increment of H3K27Ac occupancy on the promoter region of EGR1 triggers EGR1 transcription, translation, sumoylation and activation which upregulates lincRNA H19. Strikingly, H19 may induce and activate tumor-specific pyruvate kinase M2 (PKM2) which is essential for the Warburg effect in its dimer and for gene expression in its teramer during tumorigenesis. Our results imply that miR675 is involved in the epigenetic regulation of H3K9me3, H3k27me3 and H3K27Ac for gene expression and function during hepatocarcinogenesis (e.g.C-myc,Pim1,Ras,CyclinD1,RB1).These findings sheds light on the significance of miR675-HP1α-EGR1-H19-PKM2 cascade signaling pathway in liver cancer.

Circular RNA (CircRNA) is a newly identified special class of non-coding RNA (ncRNA) that plays an important regulatory role in the progression of certain diseases. Herein, our results indicate that CircMEG3 is downregulated expression and negatively correlated with the expression of telomerase-related gene Cbf5 in human liver cancer. Moreover, CircMEG3 inhibits the growth of human liver cancer stem cells in vivo and in vitro. CircMEG3 inhibits the expression of m6A methyltransferase METTL3 dependent on HULC. Moreover, CircMEG3 inhibits the expression of Cbf5, a component of telomere synthetase H/ACA ribonucleoprotein (RNP; catalyst RNA pseudouracil modification) through METTL3 dependent on HULC. Thereby, CircMEG3 inhibits telomerase activity and shortens telomere lifespan dependent on HULC and Cbf5 in human liver cancer stem cell. Strikingly, increased Cbf5 abrogates the ability of CircMEG3 to inhibit malignant differentiation of human liver cancer stem cells. In summary, these observations provide important basic information for finding effective liver cancer therapeutic targets.

MicroRNAs are known to be involved in carcinogenesis. Recently, microRNA-372 (miR372) has been proven to play a substantial role in several human cancers, but its functions in liver cancer remain unclear. Herein, our results demonstrate that miR372 accelerates growth of liver cancer cells in vitro and in vivo. Mechanistically, miR372 enhances expression of Y-box-binding protein 1 (YB-1) by targeting for phosphatase and tensin homolog (PTEN) directly and consequently promotes phosphorylation of YB-1 via HULC looping dependent on ERK1/2 and PTEN. In particular, HULC knockdown or PTEN overexpression abrogated this miR372 action. Moreover, miR372 inhibits the degradation of β-catenin dependent on phosphorylation of YB-1 and then enhances the expression and activity of pyruvate kinase M2 isoform (PKM2) by β-catenin-LEF/TCF4 pathway. Furthermore, the loading of LEF/TCF4 on PKM2 promoter region was significantly increased in miR372 overexpressing Hep3B, and thus, glycolytic proton efflux rate (glycoPER) was significantly increased in rLV-miR372 group compared to the rLV group. Moreover, β-catenin knockdown abrogates this function of miR372. Ultimately, miR372 promotes the expression of erbB-2 through PKM2-pH3T11-acetylation on histone H3 lysine 9 (H3K9Ac) pathway. Of significance, both YB-1 knockdown and erbB-2 knockdown abrogate oncogenic action of miR372. Our observations suggest that miR372 promotes liver cancer cell cycle progress by activating cyclin-dependent kinase 2 (CDK2)-cyclin E-P21/Cip1 complex through miR372-YB-1-β-catenin-LEF/TCF4-PKM2-erbB-2 axis. This study elucidates a novel mechanism for miR372 in liver cancer cells and suggests that miR372 can be used as a novel therapeutic target of liver cancer.

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.

Cancer stem cells are associated with tumor recurrence. IKK is a protein kinase that is composed of IKKα, IKKβ, IKKγ. Herein, we demonstrate that IKKα plus IKKβ promoted and IKKγ inhibited liver cancer stem cell growth in vitro and in vivo. Mechanistically, IKKα plus IKKβ enhanced and IKKγ inhibited the interplay among HP1α, HP1β and HP1γ that competes for the interaction among HP1α, SUZ12, HEZ2. Therefore, IKKα plus IKKβ inhibited and IKKγ enhanced the activity of H3K27 methyltransferase SUZ12 and EZH2, which methylates H3K27 immediately sites on HOTAIR promoter region. Therefore, IKKα plus IKKβ increased and IKKγ decreased the HOTAIR expression. Strikingly, IKKα plus IKKβ decreases and IKKγ increases the HP1α interplays with DNA methyltransferase DNMT3b, which increases or decreases TERRA promoter DNA methylation. Thus IKKα plus IKKβ 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 IKKβ decreases/increases and IKKγ increases/decreases the interplay between TERT and TRRRA/between TERT and TREC. Ultimately, IKKα plus IKKβ increases and IKKγ decreases the telomerase activity. On the other hand, at the telomere locus, IKKα plus IKKβ 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 IKKβ and IKKγ to control telomerase activity and telomere length. These observations suggest that HOTAIR operates the action of IKKα, IKKβ, IKKγ in liver cancer stem cells. This study provides a novel basis to elucidate the oncogenic action of IKKα, IKKβ, IKKγ and prompts that IKKα, IKKβ, IKKγ cooperate to HOTAR to be used as a novel therapeutic targets for liver cancer.

Long non-coding RNA HOTAIR predicts negative tumor prognosis and exhibits oncogenic activity. Herein, we demonstrate HOTAIR promotes human liver cancer stem cell malignant growth through downregulation of SETD2. Mechanistically, HOTAIR reduces the recuritment of the CREB, P300, RNA polII onto the SETD2 promoter region that inhibits SETD2 expression and its phosphorylation. Thereby, the SETD2 binding capacity to substrate histone H3 is weakened, triggering a reduction of trimethylation on histone H3 thirty-sixth lysine, and thereby the H3K36me3-hMSH2-hMSH6-SKP2 complex is also decreased. Strikingly, the complex occupancy on chromosome is depressed, preventing from mismatch DNA repair. While reducing the degradation capacity of Skp2 for aging histone H3 bound to damaged DNA, the aging histone repair is impaired. Furthermore, that the damaged DNA escaped to repair can causes microsatellite instability(MSI) and abnormal expression of cell cycle related genes that may trigger the hepatocarcinogenesis. This study provides evidence for HOTAIR to promote tumorigenesis via downregulating SETD2 in liver cancer stem cells.

miR675 is highly expressed in several human tumor tissues and positively regulates cell progression. Herein, we demonstrate that miR675 promotes malignant transformation of human mesenchymal stem cells. Mechanistically, we reveal that miR675 enhances the expression of the polyubiquitin-binding protein p62. Intriguingly, P62 competes with SETD2 to bind histone H3 and then significantly reduces SETD2-binding capacity to substrate histone H3, triggering drastically the reduction of three methylation on histone H3 36th lysine (H3K36me3). Thereby, the H3K36me3-hMSH6-SKP2 triplex complex is significantly decreased. Notably, the ternary complex's occupancy capacity on chromosome is absolutely reduced, preventing it from DNA damage repair. By virtue of the reductive degradation ability of SKP2 for aging histone H3.3 bound to mismatch DNA, the aging histone H3.3 repair is delayed. Therefore, the mismatch DNA escapes from repair, triggering the abnormal expression of several cell cycle-related genes and causing the malignant transformation of mesenchymal stem cells. These observations strongly suggest understanding the novel functions of miR675 will help in the development of novel therapeutic approaches in a broad range of cancer types.

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.

miR-155 is associated with the promotion of tumorigenesis. Herein, we indicate that abnormal miR-155 was negatively correlated with the expression of P21WAF1/Cip1. Our results suggest that miR-155 alters the transcriptome and inhibits the expression of H3F3A in liver cancer cells. Therefore, miR-155 inhibits the methylation modification of histone H3 on the 27th lysine. Notably, on the one hand, miR-155-dependent CTCF loops cause the CDK2 interacting with cyclin E in liver cancer cells; on the other hand, miR-155 promotes the phosphorylation modification of CDK2 by inhibiting H3F3A. Subsequently, miR-155 competitively blocks the binding of RNA polymerase II (RNA Pol II) to the P21WAF1/CIP1 promoter by increasing the phosphorylation of CDK2, inhibiting the transcription and translation of P21WAF1/CIP1. Strikingly, excessive P21WAF1/CIP1 abolishes the cancerous function of miR-155. In conclusion, miR-155 can play a positive role in the development of liver cancer and influence a series of gene expression through epigenetic regulation.

The functions of HULC have been demonstrated in several cancers. However, its mechanism has not been elucidated in human liver cancer stem cells.