PRMT1 promotes the proliferation and metastasis of gastric cancer cells by recruiting MLXIP for the transcriptional activation of the ß-catenin pathway.

Protein arginine methyltransferase 1 (PRMT1), a type I PRMT, is overexpressed in gastric cancer (GC) cells. To elucidate the function of PRMT1 in GC, PRMT1 expression in HGC-27 and MKN-45 cells was knocked down by short hairpin RNA (shRNA) or inhibited by PRMT1 inhibitors (AMI-1 or DCLX069), which resulted in inhibition of GC cell proliferation, migration, invasion, and tumorigenesis in vitro and in vivo. MLX-interacting protein (MLXIP) and Kinectin 1 (KTN1) were identified as PRMT1-binding proteins. PRMT1 recruited MLXIP to the promoter of ß-catenin, which induced ß-catenin transcription and activated the ß-catenin signaling pathway, promoting GC cell migration and metastasis. Furthermore, KTN1 inhibited the K48-linked ubiquitination of PRMT1 by decreasing the interaction between TRIM48 and PRMT1. Collectively, our findings reveal a mechanism by which PRMT1 promotes cell proliferation and metastasis mediated by the ß-catenin signaling pathway.

Erratum: Lycorine hydrochloride inhibits melanoma cell proliferation, migration and invasion via down-regulating p21Cip1/WAF1.

[This corrects the article on p. 1391 in vol. 11, PMID: 33948364.].

ARIH2 regulates the proliferation, DNA damage and chemosensitivity of gastric cancer cells by reducing the stability of p21 via ubiquitination.

Ariadne homolog 2 (ARIH2) is a key member of the RING-between-RING (RBR) E3 ligase family, which is characterized by an RBR domain involved in the polyubiquitination process. However, the molecular mechanism and biological function of ARIH2 in the pathogenesis of gastric cancer remain unclear. In this paper, we found that high ARIH2 expression is correlated with poor prognosis in gastric cancer patients and that ARIH2 can significantly promote the proliferation of gastric cancer cells. The effect of ARIH2 knockdown on colony formation and tumorigenesis of gastric cancer cells was also shown both in vivo and in vitro. Further mechanistic investigations revealed that ARIH2 interacts with p21 and induces p21 ubiquitination, and that the K48 residue of ubiquitin and the K161 residue of p21 play key roles in ARIH2-mediated p21 ubiquitination. We identified ARIH2 as an E3 ligase of p21 by an in vitro ubiquitination assay. In addition, ARIH2 knockdown induced DNA damage, and then induced cell apoptosis and regulated the chemosensitivity of gastric cancer cells after combined treatment with 5-fluorouracil. Generally, our results indicated that ARIH2 promotes the proliferation of gastric cancer cells and regulates p21 expression. These data demonstrate the need to further evaluate the potential therapeutic implications of ARIH2 in gastric cancer.

CBX3 accelerates the malignant progression of glioblastoma multiforme by stabilizing EGFR expression.

CBX3, also known as HP1γ, is a major isoform of heterochromatin protein 1, whose deregulation has been reported to promote the development of human cancers. However, the molecular mechanism of CBX3 in glioblastoma multiforme (GBM) are unclear. Our study reported the identification of CBX3 as a potential therapeutic target for GBM. Briefly, we found that, CBX3 is significantly upregulated in GBM and reduces patient survival. In addition, functional assays demonstrated that CBX3 significantly promote the proliferation, invasion and tumorigenesis of GBM cells in vitro and in vivo. Mechanistically, Erlotinib, a small molecule targeting epidermal growth factor receptor (EGFR) tyrosine kinase, was used to demonstrate that CBX3 direct the malignant progression of GBM are EGFR dependent. Previous studies have shown that PARK2(Parkin) and STUB1(Carboxy Terminus of Hsp70-Interacting Protein) are EGFR-specific E3 ligases. Notably, we verified that CBX3 directly suppressed PARK2 and STUB1 at the transcriptional level through its CD domain to reduce the ubiquitination of EGFR. Moreover, the CSD domain of CBX3 interacted with PARK2 and regulated its ubiquitination to further reduce its protein level. Collectively, these results revealed an unknown mechanism underlying the pathogenesis of GBM and confirmed that CBX3 is a promising therapeutic target.

Lycorine hydrochloride inhibits melanoma cell proliferation, migration and invasion via down-regulating p21Cip1/WAF1.

Lycorine hydrochloride (LH) is an active ingredient sourced from the medicinal herb Lycoris radiata. Previous studies have suggested that LH exerts tumor suppression activity in several human cancers. However, the anti-cancer effect of LH in melanoma and the potential molecular mechanisms still need to be further studied. p21Cip1/WAF1, unlike its traditional cyclin-dependent kinase (CDK) inhibitor role, is believed to act as an oncogene under certain cellular conditions. In this research, an increased expression of p21Cip1/WAF1 was found in human melanoma tissues and positively related to the tumor invasion depth. High level of p21Cip1/WAF1 was found to correlate with bad outcomes of melanoma patients by Kaplan-Meier survival analysis. Functional experiments demonstrated that the proliferation, migration and invasion ability of A375 and MV3 melanoma cells was powerfully inhibited by LH through inducing S phase cell cycle arrest and regulating epithelial-mesenchymal transition (EMT). In NOD/SCID mice model, LH effectively inhibited the xenograft tumor growth and lung metastasis of A375 cells. Further research revealed that LH reduced p21Cip1/WAF1 protein by accelerating its ubiquitination. Importantly, the LH-induced suppression of cell proliferation and metastasis was rescued by p21Cip1/WAF1 overexpression, both in vitro an in vivo. Taken together, LH, which suppresses the proliferation and metastasis of melanoma cells via down-regulating p21Cip1/WAF1, is expected to be developed as an effective medicine for melanoma therapy.

Hedgehog promotes cell proliferation in the midgut of silkworm, Bombyx mori.

The Hedgehog (Hh) signaling pathway is one of the major regulators of embryonic development and tissue homeostasis in multicellular organisms. However, the role of this pathway in the silkworm, especially in the silkworm midgut, remains poorly understood. Here, we report that Bombyx mori Hedgehog (BmHh) is expressed in most tissues of silkworm larvae and that its functions are well-conserved throughout evolution. We further demonstrate that the messenger RNA of four Hh signaling components, BmHh ligand, BmPtch receptor, signal transducer BmSmo and transcription factor BmCi, are all upregulated following Escherichia coli or Bacillus thuringiensis infection, indicating the activation of the Hh pathway. Simultaneously, midgut cell proliferation is strongly promoted. Conversely, the repression of Hh signal transduction with double-stranded RNA or cyclopamine inhibits the expression of BmHh and BmCi and reduces cell proliferation. Overall, these findings provide new insights into the Hh signaling pathway in the silkworm, B. mori.

Zinc finger protein RP-8, the Bombyx mori ortholog of programmed cell death 2, regulates cell proliferation.

Programmed cell death 2 (PDCD2) is a highly conserved eukaryotic protein indispensable for various physiological processes such as cell proliferation, development, and apoptosis. In the present study, we identified a Zinc finger protein RP-8 from the silkworm, Bombyx mori (BmZfrp8), the ortholog of PDCD2 protein. The quantitative real-time PCR analysis revealed the ubiquitous distribution of BmZfrp8 in the different tissues; however, the gene's transcription level was highest in those of the silk gland, testis, and ovary. Additionally, the expression levels of BmZfrp8 were unequal on different days of embryonic development, and it reached the highest level on the 5th day of early development. The challenge with pathogens influenced the expression level of BmZfrp8 in both hemocyte and fat body when compared with the control. Administration of 20-hydroxyecdysone significantly enhanced the BmZfrp8 expression in hemocyte. The knock-down of BmZfrp8 by double-stranded RNA suppressed the expression of developmental pathway associated genes as well as cell cycle-associated genes. Furthermore, the RNAi treated cells also showed cell cycle arrest compared to the control group. Taken together, BmZfrp8 may have a critical biological role in of B. mori, since it regulates the expression of the developmental pathway and cell cycle-associated genes.

20-Hydroxyecdysone regulates the transcription of the lysozyme via Broad-Complex Z2 gene in silkworm, Bombyx mori.

Broad-Complex Z2 (Br-C Z2) is an ecdysone inducible transcription factor that regulates physiological, innate immune and developmental events in insects. Here, we identified an orthologue of Br-C Z2 from silkworm, Bombyx mori (BmBr-C Z2) to study its involvement in immune responses. The quantitative real-time PCR analysis revealed that BmBr-C Z2 was expressed ubiquitously in all tested tissues under normal physiological conditions. Further, developmental profile displayed that BmBr-C Z2 expression was detectable in different developmental stages, however the gene's expression was highest in the molting and pre-pupal stages. Administration of 20-hydroxyecdysone (20E) enhanced the expression levels of BmBr-C Z2 in hemocytes. The challenge with pathogens and pathogen associated molecular patterns (PAMPs) also upregulated the mRNA levels of BmBr-C Z2 in hemocytes when compared with the control. By contrast, the ectopic expression of BmBr-C Z2 remarkably increased the production of antimicrobial peptides, while the knock-down of this gene by double stranded RNA decreased their production. Dual-luciferase assay exhibited that BmBr-C Z2 induced the expression of lysozyme by directly binding to its promoter region. The treatment of Escherichia coli following the knock-down of BmBr-C Z2 strongly reduced the survival rate of silkworm larvae. These results suggest that BmBr-C Z2 plays an important biological role in the innate immune responses of silkworm by regulating immune-related genes.

PHF19 promotes the proliferation, migration, and chemosensitivity of glioblastoma to doxorubicin through modulation of the SIAH1/ß-catenin axis.

PHD finger protein 19 (PHF19), a critical component of the polycomb repressive complex 2 (PRC2), is crucial for maintaining the repressive transcriptional activity of several developmental regulatory genes and plays essential roles in various biological processes. Abnormal expression of PHF19 causes dysplasia or serious diseases, including chronic myeloid disorders and tumors. However, the biological functions and molecular mechanisms of PHF19 in glioblastoma (GBM) remain unclear. Here, we demonstrated that PHF19 expression was positively associated with GBM progression, including cell proliferation, migration, invasion, chemosensitivity, and tumorigenesis. Using XAV-939, a Wnt/ß-catenin inhibitor, we found that the effects of PHF19 on GBM cells were ß-catenin-dependent. We also demonstrated that PHF19 expression was positively correlated with cytoplasmic ß-catenin expression. PHF19 stabilized ß-catenin by inhibiting the transcription of seven in absentia homolog 1 (SIAH1), an E3 ubiquitin ligase of ß-catenin, through direct binding to the SIAH1 promoter region. Taken together, our results revealed the novel PHF19-SIAH1-ß-catenin axis as a potential and promising therapeutic target.

Integrin ß3 plays a novel role in innate immunity in silkworm, Bombyx mori.

Integrins are transmembrane receptors that play essential roles in many physiological and pathological processes through cell-to-cell and cell-to-extracellular matrix (ECM) interactions. In the current study, a 2653-bp full-length cDNA of a novel integrin ß subunit (designated Bmintegrin ß3) was obtained from silkworm hemocytes. Bmintegrin ß3 has the typical conserved structure of the integrin ß family. The qRT-PCR results showed that Bmintegrin ß3 was specifically expressed in the hematological system and that its expression was significantly increased after challenge with different types of PAMPs and bacteria. The recombinant Bmintegrin ß3 protein displayed increased aggregation with S. aureus, suggesting that Bmintegrin ß3 might directly bind to PAMPs. Interestingly, Bmintegrin ß3 knockdown promoted PPO1, PPO2, BAEE, SPH78, SPH125, and SPH127 expression and accelerated the melanization process. Unexpectedly, the expression of genes related to phagocytosis, the Toll pathway, and the IMD pathway was also up-regulated after Bmintegrin ß3 knockdown. Thus, Bmintegrin ß3 might be a pattern recognition protein (PRP) for PAMPs and might directly bind to bacteria and enhance the phagocytosis activity of hemocytes. Moreover, Bmintegrin ß3 and its ligand might negatively regulate the expression of immune-related genes through an unknown mechanism. In summary, our studies provide new insights into the immune functions of Bmintegrin ß3 from the silkworm, Bombyx mori.

[Cloning and characterization of BmBrat in silkworm, Bombyx mori].

NHL proteins, which play important roles in regulation of cell proliferation and differentiation, have been extensively studied on mammals. Here, we cloned a member of NHL protein family namely BmBrat in silkworm. The full-length cDNA sequence of BmB rat was obtained by means of the rapid amplification of cDNA ends (RACE), including 3 614 bp. The ORF is 2 580 bp long, encoding a protein with 859 amino acid residues. The molecular weight is 94.3 kDa and the isoeledtric point (pI) is 6.65. The BmBrat expression profile was detected by RT-PCR at L5D3 larval stage, and it was expressed in all tissues, including silk gland, midgut, fat body and malpighian tubule. However, it was highly expressed in ovary and head. The expression profile was also detected at different stage of embryo development, and reached a peak at the 4th and 5th days of the embryonic period. Anti-BmBrat polyclonal antibody was generated f6llowing prokaryotic expression, protein purification and mice immunization, which is highly specific and effective for recognizing BmBrat protein through Western blotting and immunofluorescence staining. Subcellular localization of BmBrat in hemocytes revealed that it was specifically expressed in cytoplasm. This study provides a foundation for further research of the biological function of BmBrat gene.

Molecular cloning, characterization and expression analysis of cathepsin O in silkworm Bombyx mori related to bacterial response.

Cathepsins are the main members of the cysteine family and play important roles in immune response in vertebrates. The Cathepsin O of Bombyx mori (BmCathepsin O) was cloned from the hemocytes by the rapid amplification of cDNA ends (RACE). The genomic DNA was 6131bp long with a total of six exons and five introns. Its pre-mRNA was spliced to generate two spliceosomes. By comparisons with other reported cathepsins O, it was concluded that the identity between them ranged from 29 to 39%. Expression analysis indicated that BmCathepsin O was specific-expressed in hemocytes, and highly expressed at the 4th molting and metamorphosis stages. Immunofluorescence assay and qRT-PCR showed that BmCathepsin O was expressed in granulocytes and plasmatocytes. Interestingly, BmCathepsin O was significantly up-regulated after stimulated by 20-hydroxyecdysone (20-E) in vivo, which suggested that BmCathepsin O may be regulated by 20E. Moreover, activation of BmCathepsin O was also observed in hemocytes challenged by Escherichia coli, indicating its potential involvement in the innate immune system of silkworm, B. mori. In summary, our studies provide a new insight into the functional features of Cathepsin O.