A novel polypeptide CAPG-171aa encoded by circCAPG plays a critical role in triple-negative breast cancer.

BACKGROUND: The treatment of Triple-negative breast cancer (TNBC) has always been challenging due to its heterogeneity and the absence of well-defined molecular targets. The present study aims to elucidate the role of protein-coding circRNAs in the etiology and carcinogenesis of TNBC. METHODS: CircRNA expression data in TNBC (GEO: GSE113230, GSE101123) were reanalyzed and then circCAPG was selected for further study. To identify the polypeptide-coding function of circCAPG, a series of experiments, such as Mass spectrometry and dual-luciferase reporter assays were conducted. Cell proliferation, apoptosis and metastasis parameters were determined to investigate the cancerous functions CAPG-171aa plays in both TNBC organoids and nude mice. Mechanistically, the relation between CAPG-171aa and STK38 in TNBC was verified by immunoprecipitation analyses and mass spectrometry. The interactions between SLU7 and its binding site on circCAPG were validated by RIP-qPCR experiments. RESULTS: In both TNBC clinical samples and cell lines, the expression level of circCAPG was identified to be higher compared with normal ones and positively correlated with the overall survival (n = 132) in a 10-year follow-up study, in which the area under the curve of receiver operating characteristic was 0.8723 with 100% specificity and 80% sensitivity. In addition, we found that circCAPG knockdown (KD) significantly inhibited the growth of TNBC organoids. Intriguingly, circCAPG can be translated into a polypeptide named CAPG-171aa which promotes tumor growh by disrupting the binding of serine/threonine kinase 38 (STK38) to SMAD-specific E3 ubiquitin protein ligase 1 (SMURF1) and thereby preventing MEKK2 ubiquitination and proteasomal degradation. Furthermore, we found that SLU7 Homolog- Splicing Factor (SLU7) can regulate the bio-generation of circCAPG through binding to the flanking Alu sequences of circRNA transcripts. CONCLUSIONS: circCAPG significantly enhances the proliferation and metastasis of TNBC cells by encoding a novel polypeptide CAPG-171aa and afterwards activates MEKK2-MEK1/2-ERK1/2 pathway. Additionally, the formation of circCAPG is found to be mediated by SLU7. The present study provides innovative insight into the role of protein-coding circRNAs CAPG-171aa in TNBC, and its capacity to serve as a promising prognostic biomarker and potential therapeutic target in TNBC.

A novel polypeptide encoded by the circular RNA ZKSCAN1 suppresses HCC via degradation of mTOR.

BACKGROUND: hsa_circ_0001727 (circZKSCAN1) has been reported to be a tumor-associated circRNA by sponging microRNAs. Intriguingly, we found that circZKSCAN1 encoded a secretory peptide (circZKSaa) in the liver. The present study aims to elucidate the potential role and molecular mechanism of circZKSaa in the regulation of hepatocellular carcinoma (HCC) progression. METHODS: The circRNA profiling datasets (RNA-seq data GSE143233 and GSE140202) were reanalyzed and circZKSCAN1 was selected for further study. Mass spectrometry, polysome fractionation assay, dual-luciferase reporter, and a series of experiments showed that circZKSCAN1 encodes circZKSaa. Cell proliferation, apoptosis, and tumorigenesis in nude mice were examined to investigate the functions of circZKSaa. Mechanistically, the relationship between the circZKSaa and mTOR in HCC was verified by immunoprecipitation analyses, mass spectrometry, and immunofluorescence staining analyses. RESULTS: Receiver operating characteristic (ROC) analysis demonstrated that the secretory peptide circZKSaa encoded by circZKSCAN1 might be the potential biomarker for HCC tissues. Through a series of experiments, we found that circZKSaa inhibited HCC progression and sensitize HCC cells to sorafenib. Mechanistically, we found that the sponge function of circZKSCAN1 to microRNA is weak in HCC, while overexpression of circZKSaa promoted the interaction of FBXW7 with the mammalian target of rapamycin (mTOR) to promote the ubiquitination of mTOR, thereby inhibiting the PI3K/AKT/mTOR pathway. Furthermore, we found that the high expression of cicZKSCAN1 in sorafenib-treated HCC cells was regulated by QKI-5. CONCLUSIONS: These results reveal that a novel circZKSCAN1-encoded peptide acts as a tumor suppressor on PI3K/AKT/mTOR pathway, and sensitizes HCC cells to sorafenib via ubiquitination of mTOR. These findings demonstrated that circZKSaa has the potential to serve as a therapeutic target and biomarker for HCC treatment.

Nucleolar protein NOC4L inhibits tumorigenesis and progression by attenuating SIRT1-mediated p53 deacetylation.

SIRT1 is an NAD+-dependent deacetylase and plays an important role in the deacetylation of both histone and non-histone proteins. Many studies revealed that SIRT1 is upregulated in a variety of tumors and tightly associated with tumorigenesis and cancer progression, but the detailed underlying mechanism of the biological processes remains unclarified. In the present study, we found a nucleolar protein NOC4L, human ortholog of yeast Noc4p, which is essential for the nuclear export of the ribosomal 40S subunit and could bind to SIRT1 to inhibit SIRT1 mediated deacetylation of p53. NOC4L interacts with SIRT1 in variety of cells under nucleolar stress and directly interacts with SIRT1 in vitro. Furthermore, we determined the C-terminal of NOC4L and the catalytic domain of SIRT1 were required for their interaction. Overexpression of NOC4L did not change the protein levels of SIRT1 or p53, but increased the acetylation of p53 and promoted cell apoptosis. Additionally, NOC4L inhibited tumor cell proliferation in a p53-dependent manner and restrained tumor growth in a nude mice xenograft model. Clinically, colorectal cancer patients with the high expression of NOC4L had a better prognosis as TP53 was normally expressed, but no significant difference was observed in survival with mutant TP53. Taken together, our results identified a novel SIRT1 regulatory protein and broaden our understanding of the molecular mechanism of how nucleolar protein NOC4L regulates p53 under nucleolar stress. This research provides an insight into tumorigenesis and cell self-protection in the early stage of DNA damage.

Adtrp regulates thermogenic activity of adipose tissue via mediating the secretion of S100b.

Brown and beige adipose tissues dissipate chemical energy in the form of heat to maintain your body temperature in cold conditions. The impaired function of these tissues results in various metabolic diseases in humans and mice. By bioinformatical analyses, we identified a functional thermogenic regulator of adipose tissue, Androgen-dependent tissue factor pathway inhibitor [TFPI]-regulating protein (Adtrp), which was significantly overexpressed in and functionally activated the mature brown/beige adipocytes. Hereby, we knocked out Adtrp in mice which led to multiple abnormalities in thermogenesis, metabolism, and maturation of brown/beige adipocytes causing excess lipid accumulation in brown adipose tissue (BAT) and cold intolerance. The capability of thermogenesis in brown/beige adipose tissues could be recovered in Adtrp KO mice upon direct ß3-adrenergic receptor (ß3-AR) stimulation by CL316,243 treatment. Our mechanistic studies revealed that Adtrp by binding to S100 calcium-binding protein b (S100b) indirectly mediated the secretion of S100b, which in turn promoted the ß3-AR mediated thermogenesis via sympathetic innervation. These results may provide a novel insight into Adtrp in metabolism via regulating the differentiation and thermogenesis of adipose tissues in mice.

Deletion of Letmd1 leads to the disruption of mitochondrial function in brown adipose tissue.

Human cervical cancer oncogene (HCCR-1), also named as LETMD1, is an LETM-domain containing outer mitochondrial membrane protein which plays an important role in carcinogenesis. The present study found that the loss of Letmd1 in mice led to severe abnormities, such as brown adipose tissue (BAT) whitening, impaired thermogenesis of both BAT and beige fat, cold intolerance, diet-induced obesity, glucose intolerance and insulin resistance. Mechanically, the deletion of Letmd1 in BAT caused decreased level of both mitochondrial and intracellular Ca2+. The reduced intracellular Ca2+ could suppress the fission of mitochondria and ultimately lead to the disruption of BAT thermogenesis by regulating mitochondrial structures and functions. This study indicates that LETMD1 played a crucial role in BAT thermogenesis and energy homeostasis through regulating mitochondrial structures and functions, which provides a novel insight into therapeutic target exploration from oncogenes for metabolic disorders.

circMRPS35 promotes malignant progression and cisplatin resistance in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer-related death worldwide. Circular RNAs (circRNAs), a novel class of non-coding RNA, have been reported to be involved in the etiology of various malignancies. However, the underlying cellular mechanisms of circRNAs implicated in the pathogenesis of HCC remain unknown. In this study, we identified a functional RNA, hsa_circ_0000384 (circMRPS35), from public tumor databases using a set of computational analyses, and we further identified that circMRPS35 was highly expressed in 35 pairs of HCC from patients. Moreover, knockdown of the expression of circMRPS35 in Huh-7 and HCC-LM3 cells suppressed their proliferation, migration, invasion, clone formation, and cell cycle in vitro, and it suppressed tumor growth in vivo as well. Mechanically, circMRPS35 sponged microRNA-148a-3p (miR-148a), regulating the expression of Syntaxin 3 (STX3), which modulated the ubiquitination and degradation of phosphatase and tensin homolog (PTEN). Unexpectedly, we detected a peptide encoded by circMRPS35 (circMRPS35-168aa), which was significantly induced by chemotherapeutic drugs and promoted cisplatin resistance in HCC. These results demonstrated that circMRPS35 might be a novel mediator in HCC progress, and they raise the potential of a new biomarker for HCC diagnosis and prognosis, as well as a novel therapeutic target for HCC patients.

CRISPR/Cas9 genome editing technology in filamentous fungi: progress and perspective.

Filamentous fungi play an important role in human health and industrial/agricultural production. With the increasing number of full genomes available for fungal species, the study of filamentous fungi has brought about a wider range of genetic manipulation opportunities. However, the utilization of traditional methods to study fungi is time consuming and laborious. Recent rapid progress and wide application of a versatile genome editing technology, i.e., the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 (CRISPR-related nuclease 9) system, has revolutionized biological research and has many innovative applications in a wide range of fields showing great promise in research and application of filamentous fungi. In this review, we introduce the CRISPR/Cas9 genome editing technology focusing on its application in research of filamentous fungi and we discuss the general considerations of genome editing using CRISPR/Cas9 system illustrating vector construction, multiple editing strategies, technical consideration of different sizes of homology arms on genome editing efficiency, off-target effects, and different transformation methodologies. In addition, we discuss the challenges encountered using CRISPR/Cas9 technology and give the perspectives of future applications of CRISPR/Cas9 technology for basic research and practical application of filamentous fungi.

Swainsonine induces autophagy via PI3K/AKT/mTOR signaling pathway to injure the renal tubular epithelial cells.

Swainsonine is a major toxic ingredients of locoweed plants, ingestion of these plants may cause locoism in livestock characterized by extensive cellular vacuolar degeneration of multiple tissues. However, so far, the mechanisms responsible for vacuolar degeneration induced by SW are not known. In this study, we investigated the role of autophagy in SW-induced TCMK-1 cells using Western blotting, transmission electron microscopy, immunofluorescent microscopy and qRT-PCR. The results showed that SW treatment increased the levels of LC3-II. The co-localization of LC3-II and lysosomal protein LAMP-2 results suggested that SW treatment does not interfere with fusion between autophagosome and lysosome. TEM results indicated that SW induced aggregation of the lysosome around the autophagosome. In addition, SW treatment suppressed p-PI3K, p-Akt, p-mTOR, p-p70S6K and p-4EBP1 level. In conclusion, SW induced autophagy via pI3K/AKT/mTOR signaling pathway and revealed the role of autophagy in causing the SW toxicity characterized by the vacuolar degeneration.

The study of metabolites from fermentation culture of Alternaria oxytropis.

BACKGROUND: The indolizidine alkaloid-swainsonine is produced by an endophytic fungus Alternaria oxytropis, which was isolated from locoweeds. Swainsonine has many biological activities such as anti-tumorigenic, anti-viral and bacteriostatic. However, the full complement of metabolites produced by Alternaria oxytropis is not known. This study is a chemical analysis of Alternaria oxytropis metabolites, which not only unravels the potential compounds from the fermentation broth but also in which solvent are they extracted, facilitating industrial application. RESULTS: Alternaria oxytropis isolated from Oxytropis gansuensis was cultured in Czapek's medium for 30d to collect the fermentation broth. The fermentation broth is treated with methanol and then evaporated to dryness to obtain a concentrate of the fermentation broth. The concentrate is added with water for the subsequent fractional extraction with petroleum ether, chloroform, ethyl acetate and n-butanol. Different fractions of the extract were eluted by wet packing and dry loading. The obtained eluate was combined by TLC to detect the same fraction, and then characterized by GC-MS and LC-MS. The results of GC-MS showed that 105 different compounds existed in the petroleum ether, chloroform, and ethyl acetate phases of Alternaria oxytropis fermentation broth. Moreover, the results of LC-MS indicated that the fermentation broth of Alternaria oxytropis contained five alkaloids, 2-hydroxy-indolizidine, retronecine, lentiginosine, swainsonine and swainsonine N-oxide. CONCLUSIONS: In addition to swainsonine and swainsonine N-oxide, 2-hydroxy-indolizidine, retronecine and lentiginosine were identified as the secondary metabolites of Alternaria oxytropis. Other compounds were also detected including 5,6-dihydroergosterol, eburicol, lanosterol, and L-phenylalanyl-L-proline lactam, which have potential applications as drugs.

A reactive oxygen species activation mechanism contributes to Sophoridine-induced apoptosis in rat liver BRL-3A cells.

ETHNOPHARMACOLOGICAL RELEVANCE: Sophora alopecuroides L., a traditional Chinese herb, has been widely used to treat numerous diseases throughout China. Quinolizidine alkaloids were identified as active components in Sophora alopecuroides L., and Sophoridine (SRI) is the major component in the Quinolizidine alkaloids. AIM OF THE STUDY: To investigate the toxic effects of SRI in rat liver BRL-3A cells and to explore potential ROS-related mechanisms. MATERIALS AND METHODS: Cell viability, cytotoxicity, apoptosis, intracellular generation of ROS, GSH/GSSG ratio and levels of proteins in mitochondria apoptosis pathway were analyzed. RESULTS: Our data indicated that SRI could suppress BRL-3A cells viability in a concentration- and time-dependent manner and increase cytotoxicity, ROS accumulation and cell apoptosis in a concentration-dependent manner. Expressions and activities of apoptotic related proteins were upregulated, whereas expression of Bcl-2 was downregulated after treatment. Furthermore, level of H2O2 was increased, whereas level of Superoxide was not changed after treatment. Moreover, the antioxidant N-acetylcysteine reversed SRI-induced apoptosis and ROS accumulation. CONCLUSION: Our data suggest that SRI promotes rat liver BRL-3A cells apoptosis by increasing intracellular ROS accumulation.

The Biosynthesis Pathway of Swainsonine, a New Anticancer Drug from Three Endophytic Fungi.

Swainsonine (SW) is the principal toxic ingredient of locoweed plants that causes locoism characterized by a disorder of the nervous system. It has also received widespread attention in the medical field for its beneficial anticancer and antitumor activities. Endophytic fungi, Alternaria sect. Undifilum oxytropis isolated from locoweeds, the plant pathogen Slafractonia leguminicola, and the insect pathogen Metarhizium anisopliae, produce swainsonine. Acquired SW by biofermentation has a certain foreground and research value. This paper mainly summarizes the local and foreign literature published thus far on the swainsonine biosynthesis pathway, and speculates on the possible regulatory enzymes involved in the synthesis pathway within these three fungi in order to provide a new reference for research on swainsonine biosynthesis by endophytic fungi.