Pseudouridine synthase 1 promotes hepatocellular carcinoma through mRNA pseudouridylation to enhance the translation of oncogenic mRNAs.

BACKGROUND AND AIMS: Pseudouridine is a prevalent RNA modification and is highly present in the serum and urine of patients with HCC. However, the role of pseudouridylation and its modifiers in HCC remains unknown. We investigated the function and underlying mechanism of pseudouridine synthase 1 (PUS1) in HCC. APPROACH AND RESULTS: By analyzing the TCGA data set, PUS1 was found to be significantly upregulated in human HCC specimens and positively correlated with tumor grade and poor prognosis of HCC. Knockdown of PUS1 inhibited cell proliferation and the growth of tumors in a subcutaneous xenograft mouse model. Accordingly, increased cell proliferation and tumor growth were observed in PUS1-overexpressing cells. Furthermore, overexpression of PUS1 significantly accelerates tumor formation in a mouse HCC model established by hydrodynamic tail vein injection, while knockout of PUS1 decreases it. Additionally, PUS1 catalytic activity is required for HCC tumorigenesis. Mechanistically, we profiled the mRNA targets of PUS1 by utilizing surveying targets by apolipoprotein B mRNA-editing enzyme 1 (APOBEC1)-mediated profiling and found that PUS1 incorporated pseudouridine into mRNAs of a set of oncogenes, thereby endowing them with greater translation capacity. CONCLUSIONS: Our study highlights the critical role of PUS1 and pseudouridylation in HCC development, and provides new insight that PUS1 enhances the protein levels of a set of oncogenes, including insulin receptor substrate 1 (IRS1) and c-MYC, by means of pseudouridylation-mediated mRNA translation.

Chemical and metabolic profiling of Codonopsis Radix extract with an integrated strategy using ultra-high-performance liquid chromatography coupled with mass spectrometry.

Codonopsis radix was commonly used as food materials or herbal medicines in many countries. However, the comprehensive analysis of chemical constituents, and in vivo xenobiotics of Codonopsis radix remain unclear. In the present study, an integrated strategy with feature-based molecular networking using ultra-high-performance liquid chromatography coupled with mass spectrometry was established to systematically screen the chemical constituents and the in vivo xenobiotics of Codonopsis radix. A step-by-step manner based on a composition database, visual structure classification, discriminant ions, and metabolite software prediction was proposed to overcome the complexities due to the similar structure of chemical constituents and metabolites of Codonopsis radix. As a result, 103 compounds were tentatively characterized, 20 of which were identified by reference standards. Besides, a total of 50 xenobiotics were detected in vivo, including 26 prototypes and 24 metabolites, while the metabolic features of the pyrrolidine alkaloids were elucidated for the first time. The metabolism reactions of pyrrolidine alkaloids and sesquiterpene lactones included oxidation, methylation, hydration, hydrogenation, demethylation, glucuronidation, and sulfation. This study provided a generally applicable approach to the comprehensive investigation of the chemical and metabolic profile of traditional Chinese medicine and offered reasonable guidelines for further screening of quality control indicators and pharmacodynamics mechanism of Codonopsis radix.

LncRNA 4930581F22Rik promotes myogenic differentiation by regulating the ERK/MAPK signaling pathway.

The skeletal muscle is the largest organ in mammals and is the primary motor function organ of the body. Our previous research has shown that long non-coding RNAs (lncRNAs) are significant in the epigenetic control of skeletal muscle development. Here, we observed progressive upregulation of lncRNA 4930581F22Rik expression during skeletal muscle differentiation. Knockdown of lncRNA 4930581F22Rik hindered skeletal muscle differentiation and resulted in the inhibition of the myogenic markers MyHC and MEF2C. Furthermore, we found that lncRNA 4930581F22Rik regulates myogenesis via the ERK/MAPK signaling pathway, and this effect could be attenuated by the ERK-specific inhibitor PD0325901. Additionally, in vivo mice injury model results revealed that lncRNA 4930581F22Rik is involved in skeletal muscle regeneration. These results establish a theoretical basis for understanding the contribution of lncRNAs in skeletal muscle development and regeneration.

RNA-binding protein CCDC137 activates AKT signaling and promotes hepatocellular carcinoma through a novel non-canonical role of DGCR8 in mRNA localization.

BACKGROUND: RNA binding proteins (RBPs)-regulated gene expression play a vital role in various pathological processes, including the progression of cancer. However, the role of RBP in hepatocellular carcinoma (HCC) remains much unknown. In this study, we aimed to explore the contribution of RBP CCDC137 in HCC development. METHODS: We analyzed the altered expression level and clinical significance of CCDC137 in database and HCC specimens. In vitro cell assays and in vivo spontaneous mouse models were used to assess the function of CCDC137. Finally, the molecular mechanisms of how CCDC137 regulates gene expression and promotes HCC was explored. RESULTS: CCDC137 is aberrantly upregulated in HCC and correlates with poor clinical outcomes in HCC patients. CCDC137 markedly promoted HCC proliferation and progression in vitro and in vivo. Mechanistically, CCDC137 binds with FOXM1, JTV1, LASP1 and FLOT2 mRNAs, which was revealed by APOBEC1-mediated profiling, to increase their cytoplasmic localization and thus enhance their protein expressions. Upregulation of FOXM1, JTV1, LASP1 and FLOT2 subsequently synergistically activate AKT signaling and promote HCC. Interestingly, we found that CCDC137 binds with the microprocessor protein DGCR8 and DGCR8 has a novel non-canonical function in mRNA subcellular localization, which mediates the cytoplasmic distribution of mRNAs regulated by CCDC137. CONCLUSIONS: Our results identify a critical proliferation-related role of CCDC137 and reveal a novel CCDC137/DGCR8/mRNA localization/AKT axis in HCC progression, which provide a potential target for HCC therapy.

Simultaneous determination of multiple constituents of Qi-Lin pill by UPLC-MS/MS: Applications to pharmacokinetics and testicular tissue distribution in rats.

Qi-Lin pill (QLP) is an effective traditional Chinese medicine prescription (TCMP) that has been used for the treatment of the oligoasthenozoospermia in China. Recently, some articles described the pharmacological effects of QLP and multiple ingredients in QLP contribute to its effects. However, the pharmacokinetic and target tissue distribution data of QLP are still unknown. In the present study, according to the Bioanalytical Method Validation Guidance of FDA, a sensitive and selective UPLC-MS/MS method was developed and validated for simultaneous determination of multiple constituents in rat plasma and testicular tissue, including morusimic acid A, codonopyrridium B, magnoflorine, emodin, 2,3,5,4'-tetrahydroxystilbene-2-O-ß-D-glucoside (THSG), ecliptasaponin A, paeoniflorin, albiflorin, gallic acid, danshensu, salvianolic acid A, catechin, isosinensetin, nobiletin, formononetin, calycosin, icariside II, icariin and epimedin C. For 19 analytes, the LLOQs reached 0.01-4 ng/mL. And all calibration curves showed favorable linearity (r ≥ 0.9903) in linear ranges. The intra-day and inter-day precision (relative standard deviation) for all analytes was less than 14.92 %, and the accuracies (as relative error) were in the range of - 6.44 % to 6.22 %. Extraction recoveries and matrix effects of analytes and IS were acceptable. All analytes were stable during the assay and storage in plasma samples. The method was successfully applied for the pharmacokinetics and testis distribution of multiple chemical constituents in QLP after a single oral dose. As a result, high exposure of danshensu, gallic acid, paeoniflorin and albiflorin were observed in rat plasma and testicular tissue. Among the flavonoids, isosinensetin and nobiletin had high exposure in testicular tissue. Moreover, alleviation of progesterone reduction was evaluated in H2O2-induced R2C leydig cells, and danshensu, gallic acid, paeoniflorin, albiflorin and nobiletin showed potent activity. Therefore, these five components were considered to be the effective components of QLP due to their relatively high exposure in vivo and biological activity. This finding also provided relevant information on action mechanism of QLP in the treatment of oligoasthenozoospermia.

The Conserved LncRNA DIO3OS Restricts Hepatocellular Carcinoma Stemness by Interfering with NONO-Mediated Nuclear Export of ZEB1 mRNA.

Hepatocellular carcinoma (HCC) is an aggressive and fatal disease caused by a subset of cancer stem cells (CSCs). It is estimated that there are approximately 100 000 long noncoding RNAs (lncRNAs) in humans. However, the mechanisms by which lncRNAs affect tumor stemness remain poorly understood. In the present study, it is found that DIO3OS is a conserved lncRNA that is generally downregulated in multiple cancers, including HCC, and its low expression correlates with poor clinical outcomes in HCC. In in vitro cancer cell lines and an in vivo spontaneous HCC mouse model, DIO3OS markedly represses tumor development via its suppressive role in CSCs through downregulation of zinc finger E-box binding homeobox 1 (ZEB1). Interestingly, DIO3OS represses ZEB1 post-transcriptionally without affecting its mRNA levels. Subsequent experiments show that DIO3OS interacts with the NONO protein and restricts NONO-mediated nuclear export of ZEB1 mRNA. Overall, these findings demonstrate that the DIO3OS-NONO-ZEB1 axis restricts HCC development and offers a valuable candidate for CSC-targeted therapeutics for HCC.

Characterization of Long Non-coding RNAs Modified by m6A RNA Methylation in Skeletal Myogenesis.

Proper development of mammalian skeletal muscle relies on precise gene expression regulation. Our previous studies revealed that muscle development is regulated by both mRNA and long non-coding RNAs (lncRNAs). Accumulating evidence has demonstrated that N6-methyladenosine (m6A) plays important roles in various biological processes, making it essential to profile m6A modification on a transcriptome-wide scale in developing muscle. Patterns of m6A methylation in lncRNAs in developing muscle have not been uncovered. Here, we reveal differentially expressed lncRNAs and report temporal m6A methylation patterns in lncRNAs expressed in mouse myoblasts and myotubes by RNA-seq and methylated RNA immunoprecipitation (MeRIP) sequencing. Many lncRNAs exhibit temporal differential expression, and m6A-lncRNAs harbor the consensus m6A motif "DRACH" along lncRNA transcripts. Interestingly, we found that m6A methylation levels of lncRNAs are positively correlated with the transcript abundance of lncRNAs. Overexpression or knockdown of m6A methyltransferase METTL3 alters the expression levels of these lncRNAs. Furthermore, we highlight that the function of m6A genic lncRNAs might correlate to their nearby mRNAs. Our work reveals a fundamental expression reference of m6A-mediated epitranscriptomic modifications in lncRNAs that are temporally expressed in developing muscle.