SIRT7-Induced PHF5A Decrotonylation Regulates Aging Progress Through Alternative Splicing-Mediated Downregulation of CDK2.

Dysregulation of protein posttranslational modification (PTM) can lead to a variety of pathological processes, such as abnormal sperm development, malignant tumorigenesis, depression, and aging process. SIRT7 is a NAD+-dependent protein deacetylase. Besides known deacetylation, SIRT7 may also have the capacity to remove other acylation. However, the roles of SIRT7-induced other deacylation in aging are still largely unknown. Here, we found that the expression of SIRT7 was significantly increased in senescent fibroblasts and aged tissues. Knockdown or overexpression of SIRT7 can inhibit or promote fibroblast senescence. Knockdown of SIRT7 led to increased pan-lysine crotonylation (Kcr) levels in senescent fibroblasts. Using modern mass spectrometry (MS) technology, we identified 5,149 Kcr sites across 1,541 proteins in senescent fibroblasts, and providing the largest crotonylome dataset to date in senescent cells. Specifically, among the identified proteins, we found SIRT7 decrotonylated PHF5A, an alternative splicing (AS) factor, at K25. Decrotonylation of PHF5A K25 contributed to decreased CDK2 expression by retained intron (RI)-induced abnormal AS, thereby accelerating fibroblast senescence, and supporting a key role of PHF5A K25 decrotonylation in aging. Collectively, our data revealed the molecular mechanism of SIRT7-induced k25 decrotonylation of PHF5A regulating aging and provide new ideas and molecular targets for drug intervention in cellular aging and the treatment of aging-related diseases, and indicating that protein crotonylation has important implications in the regulation of aging progress.

miR-30c-5p inhibits glioma proliferation and invasion via targeting Bcl2.

BACKGROUND: Glioma is a highly malignant brain tumor, characterized by the poor prognosis and high recurrence rates. Previous studies have confirmed that miRNA-30c-5p is closely associated with tumor cell biological properties. The present study explored the biological role of miR-30c-5p in human glioma malignant behavior and underlying mechanisms. METHODS: Levels of miR-30c-5p were detected in glioma tissues and adjacent normal tissues. Two glioma cell lines including U87 and U251 were transfected with miR-30c-5p mimic or inhibitors. Cell proliferation was evaluated by MTT assay and colony formation assay. Cell apoptosis and invasive potential of glioma cells were assessed by flow cytometry and transwell assays, respectively. Luciferase reporter assay was performed to validate the target gene of miR-30c-5p. RESULTS: Levels of miR-30c-5p were dramatically decreased in glioma tissues as compared to the adjacent normal tissues. Upregulation of miR-30c-5p significantly suppressed cell growth and colony formation, and induced apoptosis in glioma cells. In contrast, inhibition of miR-30c-5p promoted the proliferation and inhibited apoptosis in tumor cells. Furthermore, miR-30c-5p strongly suppresses the invasion of glioma cells. Western blot showed that Bcl-2 was significantly decreased following treatment with miR-30c-5p mimics and increased after miR-30c-5p inhibitor treatment. Moreover, luciferase reporter assays indicated that transfection of miR-30c-5p led to a marked reduction of luciferase activity, but had no effect on Bcl-2 3'-UTR mutated fragment. Mechanically, miR-30c-5p promoted the activation of caspase 3 and caspase 9 in glioma cells. Furthermore, miR-30c-5p promoted apoptosis and inhibited colony formation and migration, and knockdown of Bcl2 further increased the number of apoptotic cells and suppressed colony formation and migration of glioma cells. By contrast, miR-30c-5p inhibitors decreased apoptosis and increased colony formation and migration, and restored Bcl2 expression further suppressed glioma cell apoptosis and enhanced colony formation and migration. CONCLUSIONS: These results demonstrated that miR-30c-5p regulated growth, apoptosis and migration in glioma cells by targeting Bcl2, suggesting that miR-30c-5p might serve as a novel target for glioma therapy.

Nuclear paraspeckle assembly transcript 1 promotes the metastasis and epithelial-mesenchymal transition of hepatoblastoma cells by inhibiting miR-129-5p.

The abnormal expression of nuclear paraspeckle assembly transcript 1 (NEAT1) may serve critical functions for the development and progression of various types of human tumor. However, the expression and biological function of NEAT1 in hepatoblastoma (HB) and the underlying mechanisms for the function of NEAT1 in HB remain largely uncharacterized. In the present study, the results of reverse transcription-quantitative polymerase chain reaction revealed that the expression of NEAT1 was significantly elevated in HB tissues. HB tissues with metastasis also exhibited significantly increased levels of NEAT1 compared with tissues without metastasis. The biological functions of NEAT1 were then assessed using gain-/loss-of-function studies. The results of in vitro assays revealed that inhibiting NEAT1 expression reduced the migration and invasion of HepG2 cells. By contrast, the induced expression of NEAT1 exhibited the opposite effect. The present study also demonstrated that the inhibition of NEAT1 expression prevented the epithelial-mesenchymal transition of HepG2 cells, whereas forced expression of NEAT1 exhibited the opposite effect. In addition, it was confirmed that NEAT1 could modulate the expression of microRNA (miR)-129-5p in HepG2 cells, and that NEAT1 may exert its effect on the metastatic behaviors and epithelial-mesenchymal transition of HepG2 cells by inhibiting miR-129-5p. In conclusion, the present study indicated that NEAT1 expression was aberrantly increased in HB and that it may promote the metastasis of HB cells by inhibiting miR-129-5p. Targeting NEAT1 may potentially be a novel therapeutic option for treating patients with HB.

Actein inhibits glioma growth via a mitochondria-mediated pathway.

Previous studies indicate that the triterpene glycoside Actein from the herb black cohosh inhibits growth of human breast cancer cells. This study sought to investigate the effects of Actein on glioma cell growth and explore the potential mechanisms. Our results showed that administration of Actein significantly inhibited glioma cell viability in a dose- and time-dependent manner. Actein also increasingly inhibited the colony formation processes in glioma U87 cells and U251 cells. Administration of Actein also induced mitochondria-related apoptosis by increasing expression of pro-apoptotic factors Bax, cleaved caspase-3, cleaved caspase-9 and cleaved poly (ADP-ribose) polymerase 1 (PARP1) as well as decreasing anti-apoptotic Bcl-2 expression in U87 cells and U251 cells. In a xenograft model of glioma, Actein suppressed tumor growth and consistently induced cell apoptosis with the same mechanisms observed in vitro. In all, this study is the first report to address the growth inhibitory effects of Actein on glioma growth and propose that mitochondria-mediated apoptosis pathway may underlie the biological activities of Actein in glioma. Our study suggests that administration of Actein may serve as a potent therapeutic strategy for treatment of glioma.

Histone deacetylase 5 promotes Wilms' tumor cell proliferation through the upregulation of c-Met.

The histone deacetylase (HDAC) family is comprised of enzymes, which are involved in modulating the majority of critical cellular processes, including transcriptional regulation, apoptosis, proliferation and cell cycle progression. However, the biological function of HDAC5 in Wilms' tumor remains to be fully elucidated. The present study aimed to investigate the expression and function of HDAC5 in Wilm's tumor. It was demonstrated that the mRNA and protein levels of HDAC5 were upregulated in human Wilms' tumor tissues. Overexpression of HDAC5 in G401 cells was observed to significantly promote cellular proliferation, as demonstrated by the results of an MTT assay and bromodeoxyuridine incorporation assay. By contrast, HDAC5 knockdown using small interfering RNA suppressed the proliferation of the G401 cells. At the molecular level, the present study demonstrated that HDAC5 promoted the expression of c­Met, which has been previously identified as an oncogene. In addition, downregulation of c­Met inhibited the proliferative effects of HDAC5 in human Wilms' tumor cells. Taken together, these results suggested that HDAC5 promotes cellular proliferation through the upregulation of c­Met, and may provide a novel therapeutic target for the treatment of patients with Wilms' tumor.

The viral ankyrin repeat protein (ORF124L) from infectious spleen and kidney necrosis virus attenuates nuclear factor-κB activation and interacts with IκB kinase ß.

The ankyrin (ANK) repeat is one of the most common protein-protein interaction motifs, found predominantly in eukaryotes and bacteria, but the functions of the ANK repeat are rarely researched in animal viruses, with the exception of poxviruses. Infectious spleen and kidney necrosis virus (ISKNV) is a typical member of the genus Megalocytivirus in the family Iridoviridae and is a causative agent of epizootics in fish. The genome of ISKNV contains four putative viral ANK (vANK) repeat proteins and their functions remain largely unknown. In the present study, it was found that ORF124L, a vANK repeat protein in ISKNV, encodes a protein of 274 aa with three ANK repeats. Transcription of ORF124L was detected at 12 h post-infection (p.i.) and reached a peak at 40 h p.i. ORF124L was found to localize to both the nucleus and the cytoplasm in mandarin fish fry cells. ISKNV ORF124L interacted with the mandarin fish IκB kinase ß protein (scIKKß), and attenuated tumour necrosis factor alpha (TNF-α)- or phorbol myristate acetate (PMA)-induced activity of a nuclear factor κB (NF-κB)-luciferase reporter but did not interfere with the activity of an activator protein 1 (AP-1)-luciferase reporter. Phosphorylation of IκBα and nuclear translocation of NF-κB were also impaired by ISKNV ORF124L. In summary, ORF124L was identified as a vANK repeat protein and its role in inhibition of TNF-α-induced NF-κB signalling was investigated through interaction with the mandarin fish IKKß. This work may help to improve our understanding of the function of fish iridovirus ANK repeat proteins.

Molecular cloning of IKKß from the mandarin fish Siniperca chuatsi and its up-regulation in cells by ISKNV infection.

The IκB kinase ß (IKKß) plays crucial roles in regulating activation of nuclear factor-kappa B (NF-κB) in response to proinflammatory factors and microbial and viral infections. Here, we report the cloning of an IKKß cDNA (named SicIKKß) from the mandarin fish Siniperca chuatsi. The full-length cDNA is 4052bp and contains an ORF that encodes a predicted protein of 743-amino acid residues. The deduced amino acid sequence of SicIKKß has the same domain organization as human IKKß, which consists of a serine/threonine kinase domain, a leucine zipper motif and a putative helix-loop-helix motif. Quantitative RT-PCR showed that SicIKKß was ubiquitously expressed in tissues of mandarin fish, and its expression in mandarin fish fry (MFF-1) cells was up-regulated during the course of ISKNV infection.