A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra-Tcrd locus.

The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra-Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.

Mutual interaction between YAP and CREB promotes tumorigenesis in liver cancer.

UNLABELLED: Yes-associated protein (YAP), the downstream effecter of the Hippo-signaling pathway as well as cyclic adenosine monophosphate response element-binding protein (CREB), has been linked to hepatocarcinogenesis. However, little is known about whether and how YAP and CREB interact with each other. In this study, we found that YAP-CREB interaction is critical for liver cancer cell survival and maintenance of transformative phenotypes, both in vitro and in vivo. Moreover, both CREB and YAP proteins are highly expressed in a subset of human liver cancer samples and are closely correlated. Mechanistically, CREB promotes YAP transcriptional output through binding to -608/-439, a novel region from the YAP promoter. By contrast, YAP promotes protein stabilization of CREB through interaction with mitogen-activated protein kinase 14 (MAPK14/p38) and beta-transducin repeat containing E3 ubiquitin protein ligase (BTRC). Gain-of-function and loss-of-function studies demonstrated that phosphorylation of CREB by MAPK14/p38 at ser133 ultimately leads to its degradation. Such effects can be enhanced by BTRC through phosphorylation of MAPK14/p38 at Thr180/Tyr182. However, YAP negatively controls phosphorylation of MAPK14/p38 through inhibition of BTRC expression. CONCLUSION: There is a novel positive autoregulatory feedback loop underlying the interaction between YAP and CREB in liver cancer, suggesting that YAP and CREB form a nexus to integrate the protein kinase A, Hippo/YAP, and MAPK14/p38 pathways in cancer cells and thus may be helpful in the development of effective diagnosis and treatment strategies against liver cancer.

O-GlcNAcylated LARP1 positively regulated by circCLNS1A facilitates hepatoblastoma progression through DKK4/ß-catenin signalling.

BACKGROUND: Accumulating studies have shown that La-related protein 1 (LARP1) is involved in the occurrence and development of various tumours. However, the expression pattern and biological role of LARP1 in hepatoblastoma (HB) remain unclear so far. METHODS: LARP1 expression level in HB and adjacent normal liver tissues was analysed by qRT-PCR, Western blotting and immunohistochemistry assays. The prognostic significance of LARP1 was evaluated by Kaplan-Meier method and multivariate Cox regression analysis. In vitro and in vivo functional assays were implemented to clarify the biological effects of LARP1 on HB cells. Mechanistically, the regulatory roles of O-GlcNAcylation and circCLNS1A in LARP1 expression were investigated by co-immunoprecipitation (co-IP), immunofluorescence, RNA immunoprecipitation (RIP), RNA pull-down and protein stability assays. Moreover, RNA-sequencing, co-IP, RIP, mRNA stability and poly(A)-tail length assays were performed to investigate the association between LARP1 and DKK4. The expression and diagnostic significance of plasma DKK4 protein in multi-centre cohorts were evaluated by ELISA and ROC curves. RESULTS: LARP1 mRNA and protein levels were remarkably elevated in HB tissues and associated with worse prognosis of HB patients. LARP1 knockdown abolished cell proliferation, triggered cell apoptosis in vitro as well as prohibited tumour growth in vivo, whereas LARP1 overexpression incited HB progression. Mechanistically, O-GlcNAcylation of LARP1 Ser672 by O-GlcNAc transferase strengthened its binding to circCLNS1A and then protected LARP1 from TRIM-25-mediated ubiquitination and proteolysis. LARP1 upregulation subsequently led to DKK4 mRNA stabilisation by competitively interacting with PABPC1 to prevent DKK4 mRNA from B-cell translocation gene 2-dependent deadenylation and degradation, thus facilitating ß-catenin protein expression and nuclear import. CONCLUSION: This study indicates that upregulated protein level of O-GlcNAcylated LARP1 mediated by circCLNS1A promotes the tumorigenesis and progression of HB through LARP1/DKK4/ß-catenin axis. Hence, LARP1 and DKK4 are promising therapeutical target and diagnostic/prognostic plasma biomarker for HB.

The N6-methyladenosine modification enhances ferroptosis resistance through inhibiting SLC7A11 mRNA deadenylation in hepatoblastoma.

BACKGROUND: Solute carrier family 7 member 11 (SLC7A11) is overexpressed in multiple human tumours and functions as a transporter importing cystine for glutathione biosynthesis. It promotes tumour development in part by suppressing ferroptosis, a newly identified form of cell death that plays a pivotal role in the suppression of tumorigenesis. However, the role and underlying mechanisms of SLC7A11-mediated ferroptosis in hepatoblastoma (HB) remain largely unknown. METHODS: Reverse transcription quantitative real-time PCR (RT-qPCR) and western blotting were used to measure SLC7A11 levels. Cell proliferation, colony formation, lipid reactive oxygen species (ROS), MDA concentration, 4-HNE, GSH/GSSG ratio and cell death assays as well as subcutaneous xenograft experiments were used to elucidate the effects of SLC7A11 in HB cell proliferation and ferroptosis. Furthermore, MeRIP-qPCR, dual luciferase reporter, RNA pulldown, RNA immunoprecipitation (RIP) and RACE-PAT assays were performed to elucidate the underlying mechanism through which SLC7A11 was regulated by the m6A modification in HB. RESULTS: SLC7A11 expression was highly upregulated in HB. SLC7A11 upregulation promoted HB cell proliferation in vitro and in vivo, inhibiting HB cell ferroptosis. Mechanistically, SLC7A11 mRNA exhibited abnormal METTL3-mediated m6A modification, which enhanced its stability and expression. IGF2 mRNA-binding protein 1 (IGF2BP1) was identified as the m6A reader of SLC7A11, enhancing SLC7A11 mRNA stability and expression by inhibiting SLC7A11 mRNA deadenylation in an m6A-dependent manner. Moreover, IGF2BP1 was found to block BTG2/CCR4-NOT complex recruitment via competitively binding to PABPC1, thereby suppressing SLC7A11 mRNA deadenylation. CONCLUSIONS: Our findings demonstrated that the METTL3-mediated SLC7A11 m6A modification enhances HB ferroptosis resistance. The METTL3/IGF2BP1/m6A modification promotes SLC7A11 mRNA stability and upregulates its expression by inhibiting the deadenylation process. Our study highlights a critical role of the m6A modification in SLC7A11-mediated ferroptosis, providing a potential strategy for HB therapy through blockade of the m6A-SLC7A11 axis.

miR-597 inhibits breast cancer cell proliferation, migration and invasion through FOSL2.

Many reports suggest that the discovery of microRNAs (miRNAs) might provide a novel therapeutical target for many diseases, even of human cancers; however, there are no reports on the role of miR-597 in human cancers. In the present study, by detecting mRNA expression with qRT-PCR, compared with the adjacent normal tissues we found that miR-597 was significantly downregulated in breast cancer tissues. By using the MTT assay, the cell wound-healing assay and the cell invasion assay, we demonstrated that miR-597 mimics were able to suppress breast cancer cell proliferation, migration and invasion. Additionally, with flow cytometry, we found that mir-597 influenced the growth of breast cancer cells through regulating the G1-S phase transition. Furthermore, we identified one binding site for miR-597 at the 3'UTR of the FOSL2 gene, using bioinformatics methods and the luciferase reporter assay, it was confirmed that FOSL2 was a direct target of miR-597. Moreover, overexpression of FOSL2 in MDA-MB­231 and SK-BR-3 cells can block the vast majority of the miR-597 roles, suggesting that miR-597 acts as a tumor suppressor in breast cancer cells by the downregulation of FOSL2. Additionally, we also found a negative correlation between the expression of FOSL2 and miR-597 in the tumor samples. This new regulatory mechanism in breast cancer may provide another method for diagnosis and therapy.

Distinct response of the hepatic transcriptome to Aflatoxin B1 induced hepatocellular carcinogenesis and resistance in rats.

Aflatoxin is a natural potent carcinogen and a major cause of liver cancer. However, the molecular mechanisms of hepatocellular carcinogenesis remain largely unexplored. In this study, we profiled global gene expression in liver tissues of rats that developed hepatocellular carcinoma (HCC) from aflatoxin B1 (AFB1) administration and those that were AFB1-resistant, as well as rats without AFB1 exposure as a control. AFB1 exposure resulted in extensive perturbation in gene expression with different functions in HCC and AFB1 resistance (AR) samples. The differentially expressed genes (DEGs) in HCC sample were enriched for cell proliferation, cell adhesion and vasculature development that largely contribute to carcinogenesis. Anti-apoptosis genes were up-regulated in HCC sample whereas apoptosis-induction genes were up-regulated in AR sample. AFB1 exposure also caused extensive alteration in expression level of lncRNAs. Among all the 4511 annotated lncRNAs, half of them were highly expressed only in HCC sample and up-regulated a group of protein-coding genes with cancer-related functions: apoptosis regulation, DNA repair, and cell cycle. Intriguingly, these genes were down-regulated by lncRNAs highly expressed in AR sample. Collectively, apoptosis is the critical biological process for carcinogenesis in response to AFB1 exposure through changes in expression level of both protein-coding and lncRNA genes.

miR-889 promotes proliferation of esophageal squamous cell carcinomas through DAB2IP.

MicroRNAs have been reported to play critical roles in various cancers, but there has been no study on the role of miR-889 in cancers. Here, we report that over-expression of miR-889 leads to rapid proliferation of EC109 and EC9706 cells in vitro and in vivo by inducing cells into S-phase. Using bioinformatics methods, DAB2IP was further confirmed to be a direct target of miR-889. In addition, the expression of DAB2IP, which was negatively correlated with that of miR-889, was significantly associated with clinicopathological features of ESCC patients. In conclusion, miR-889 is an important regulator in ESCC and both miR-889 and DAB2IP may serve as promising biomarkers and therapeutic targets in patients with ESCC.

microRNA-155 acts as an oncogene by targeting the tumor protein 53-induced nuclear protein 1 in esophageal squamous cell carcinoma.

MicroRNA-155 (miR-155) is overexpressed in many human cancers; however, the function of miR-155 is largely unknown in esophageal squamous cell carcinoma (ESCC). In the present study, we found that miR-155 is dramatically increased in ESCC tissues compared with the paired adjacent normal tissues, which suggested that miR-155 acts as an oncogene in ESCC. We predicted that tumor protein p53-induced nuclear protein 1 (TP53INP1) is a candidate target gene of miR-155 given that miR-155 expression decreased mRNA and protein levels of TP53INP1 as determined by RT-PCR and Western blot analysis. In addition, miR-155 and TP53INP1 showed a negative relation in ESCC tissues. Dual luciferase-based reporter assay indicated direct regulation of TP53INP1 by miR-155. Furthermore, we demonstrated that RNA interference of TP53INP1 increased the proliferation and colonies formation of EC-1 cells. Up-regulation of TP53INP1 abrogated miR-155 induced growth in EC-1 cells and mutation of TP53INP1 in 3'-UTR restored the effects when co-transfected with miR-155. We also indicated that overexpression of miR-155 significantly promoted the proliferation of EC-1 cells in vitro and the development of tumors in nude mice. Taken together, our study reveals that miR-155 acts as an oncogene by targeting TP53INP1 in ESCC.

Arene ruthenium(ii) complex, a potent inhibitor against proliferation, migration and invasion of breast cancer cells, reduces stress fibers, focal adhesions and invadopodia.

Effective chemotherapy drugs for cancer that would inhibit tumor growth and suppress metastasis are currently lacking. In this study, a series of arene ruthenium complexes, [(η6-arene)Ru(H2iip)Cl]Cl (arene = p-cymene, RAWQ03; CH3C6H5, RAWQ04; and C6H6, RAWQ11), were synthesized and their inhibitory activity against tumor cells were evaluated. The results showed that the complex RAWQ11 inhibited the growth of MDA-MB-231 breast cancer cells by inducing S-phase arrest, which is closely related to the inhibition of cell mitosis-mediated cell nucleus damage. Further studies showed that RAWQ11 can inhibit the invasion and metastasis of MDA-MB-231 cells. The morphology of MDA-MB-231 cells changed, the number of focal adhesions decreased, and the stress fibers de-polymerized upon dealing with the complex RAWQ11. The FITC-gelatin assay confirmed that the formation of invadopodia in MDA-MB-231 cells was significantly blocked by RAWQ11. Furthermore, RAWQ11 can block the AKT signal pathway by upregulating the PTEN expression through binding and downregulating miR-21. These results demonstrated that this type of arene ruthenium(ii) complex can block the invadopodia formation by regulating the PTEN/AKT signal pathway mediated by miR-21 to inhibit the invasion and metastasis of breast cancer cells. Therefore, this complex can be used as a potential dual functional agent to inhibit the growth and metastasis of tumor cells.

Effects and interactions of MiR-577 and TSGA10 in regulating esophageal squamous cell carcinoma.

Testis specific 10 (TSGA10) was originally identified as a testis-specific protein and tumor-associated antigen in a number of cancer types. In this study, we found that down-regulation of TSGA10 was associated with increased malignancy and clinical features of esophageal squamous cell carcinomas (ESCCs). Moreover, increased expression of TSGA10 inhibited, while its knockdown promoted, tumor formation in vivo in nude mice. At the 3'UTR of the TSGA10 gene we identified two binding sites for microRNA-577 (miR-577). Further investigation demonstrated that expression levels of miR-577 and TSGA10 were negatively correlated to each other in ESCC cell lines and tumor samples. Moreover, manipulation of miR-577 and TSGA10 expression confirmed that miR-577 can regulate TSGA10 and in turn affect cell proliferation in vitro. Additionally, with flow cytometry and manipulation of the mir-577/TSGA10 axis, it was found that mir-577/TSGA10 axis influenced the growth of ESCC through regulating the G1-S phase transition. We also obtained evidence to establish that mir-577/TSGA10 axis activation was always accompanied by inactivation of the p53 pathway or the Rb pathway or both, thus, the latter two pathways are obligatory for progression of ESCCs with mir-577/TSGA10 axis activation. In addition, we found that such an interactive pathway in regulating cancer cell proliferation was restricted to a few cancer types including ESCC, but not uniformly applicable to other cancer types. This newly discovered regulatory mechanism provides a new dimension for ESCC diagnosis and therapy.