mcPGK1-dependent mitochondrial import of PGK1 promotes metabolic reprogramming and self-renewal of liver TICs.

Liver tumour-initiating cells (TICs) contribute to tumour initiation, metastasis, progression and drug resistance. Metabolic reprogramming is a cancer hallmark and plays vital roles in liver tumorigenesis. However, the role of metabolic reprogramming in TICs remains poorly explored. Here, we identify a mitochondria-encoded circular RNA, termed mcPGK1 (mitochondrial circRNA for translocating phosphoglycerate kinase 1), which is highly expressed in liver TICs. mcPGK1 knockdown impairs liver TIC self-renewal, whereas its overexpression drives liver TIC self-renewal. Mechanistically, mcPGK1 regulates metabolic reprogramming by inhibiting mitochondrial oxidative phosphorylation (OXPHOS) and promoting glycolysis. This alters the intracellular levels of α-ketoglutarate and lactate, which are modulators in Wnt/ß-catenin activation and liver TIC self-renewal. In addition, mcPGK1 promotes PGK1 mitochondrial import via TOM40 interactions, reprogramming metabolism from oxidative phosphorylation to glycolysis through PGK1-PDK1-PDH axis. Our work suggests that mitochondria-encoded circRNAs represent an additional regulatory layer controlling mitochondrial function, metabolic reprogramming and liver TIC self-renewal.

PRC1 and RACGAP1 are Diagnostic Biomarkers of Early HCC and PRC1 Drives Self-Renewal of Liver Cancer Stem Cells.

Hepatocellular carcinoma (HCC) is the fourth leading cause of cancer-related deaths across the world. Due to the lack of reliable markers for early HCC detection, most HCC patients are diagnosed in middle/late stages. Liver cancer stem cells (CSCs), which are drivers of liver tumorigenesis, usually emerge in the early HCC stage and are also termed as liver tumor initiation cells (TIC). Liver CSCs contribute to initiation, propagation, and metastasis of HCC and also play a key role in tumor therapy. Taking advantage of online-available data sets, bioinformatic analyses, and experimental confirmation, here we have screened out PRC1 and RACGAP1 as reliable markers for early HCC detection. PRC1 or RACGAP1 knockdown dramatically inhibited the proliferation, migration, and invasion capacities of HCC cells, conferring PRC1 and RACGAP1 as predominant modulators for HCC propagation and metastasis. Moreover, the sphere formation capacity of HCC cells was impaired after PRC1 knockdown, revealing the function of PRC1 as a modulator for liver CSC self-renewal. Furthermore, the inhibitor of PRC1 had same phenotypes as PRC1 knockdown in HCC cells. Altogether, PRC1 and RACGAP1 are identified both as prognosis markers for early HCC detection and therapeutic targets for liver cancer and liver CSCs, adding additional layers for the early prognosis and therapy of HCC.

Screening and predicted value of potential biomarkers for breast cancer using bioinformatics analysis.

Breast cancer is the most common cancer and the leading cause of cancer-related deaths in women. Increasing molecular targets have been discovered for breast cancer prognosis and therapy. However, there is still an urgent need to identify new biomarkers. Therefore, we evaluated biomarkers that may aid the diagnosis and treatment of breast cancer. We searched three mRNA microarray datasets (GSE134359, GSE31448 and GSE42568) and identified differentially expressed genes (DEGs) by comparing tumor and non-tumor tissues using GEO2R. Functional and pathway enrichment analyses of the DEGs were performed using the DAVID database. The protein-protein interaction (PPI) network was plotted with STRING and visualized using Cytoscape. Module analysis of the PPI network was done using MCODE. The associations between the identified genes and overall survival (OS) were analyzed using an online Kaplan-Meier tool. The redundancy analysis was conducted by DepMap. Finally, we verified the screened HUB gene at the protein level. A total of 268 DEGs were identified, which were mostly enriched in cell division, cell proliferation, and signal transduction. The PPI network comprised 236 nodes and 2132 edges. Two significant modules were identified in the PPI network. Elevated expression of the genes Discs large-associated protein 5 (DLGAP5), aurora kinase A (AURKA), ubiquitin-conjugating enzyme E2 C (UBE2C), ribonucleotide reductase regulatory subunit M2(RRM2), kinesin family member 23(KIF23), kinesin family member 11(KIF11), non-structural maintenance of chromosome condensin 1 complex subunit G (NCAPG), ZW10 interactor (ZWINT), and denticleless E3 ubiquitin protein ligase homolog(DTL) are associated with poor OS of breast cancer patients. The enriched functions and pathways included cell cycle, oocyte meiosis and the p53 signaling pathway. The DEGs in breast cancer have the potential to become useful targets for the diagnosis and treatment of breast cancer.

Long Non-coding RNA H19 Regulates Porcine Satellite Cell Differentiation Through miR-140-5p/SOX4 and DBN1.

The H19 gene promotes skeletal muscle differentiation in mice, but the regulatory models and mechanisms of myogenesis regulated by H19 are largely unknown in pigs. Therefore, the regulatory modes of H19 in the differentiation of porcine skeletal muscle satellite cells (PSCs) need to be determined. We observed that H19 gene silencing could decrease the expressions of the myogenin (MYOG) gene, myogenic differentiation (MYOD), and myosin heavy chain (MYHC) in PSCs. Therefore, we constructed and sequenced 12 cDNA libraries of PSCs after knockdown of H19 at two differentiation time points to analyze the transcriptome differences. A total of 11,419 differentially expressed genes (DEGs) were identified. Among these DEGs, we found through bioinformatics analysis and protein interaction experiment that SRY-box transcription factor 4 (SOX4) and Drebrin 1 (DBN1) were the key genes in H19-regulated PSC differentiation. Functional analysis shows that SOX4 and DBN1 promote PSC differentiation. Mechanistically, H19 regulates PSC differentiation through two different pathways. On the one hand, H19 functions as a molecular sponge of miR-140-5p, which inhibits the differentiation of PSCs, thereby modulating the derepression of SOX4. On the other hand, H19 regulates PSC differentiation through directly binding with DBN1. Furthermore, MYOD binds to the promoters of H19 and DBN1. The knockdown of MYOD inhibits the expression of H19 and DBN1. We determined the function of H19 and provided a molecular model to elucidate H19's role in regulating PSC differentiation.

Long Non-Coding RNA H19 Promotes Porcine Satellite Cell Differentiation by Interacting with TDP43.

Long non-coding RNAs (lncRNAs) have been implicated in fundamental and diverse biological processes, including myogenesis. However, the molecular mechanisms involved in this process remain largely unexplored. This study found that H19 affected the differentiation of porcine satellite cells (PSCs) by directly binding to the DNA/RNA-binding protein TDP43. Functional analyses showed that TDP43 knockdown decreased PSC differentiation, whereas TDP43 overexpression exerted opposite effects in vitro. Furthermore, rescue experiments demonstrated that TDP43 can rescue the decrease in PSC differentiation caused by H19 knockdown. Mechanistically, H19 may act as a scaffold to recruit TDP43 to the promoters of MYOD and thereby activate the transcription of MYOD, leading to PSC differentiation. In summary, we elucidate the molecular mechanism by which H19 and TDP43 regulate myogenesis.

MEG3 Promotes Differentiation of Porcine Satellite Cells by Sponging miR-423-5p to Relieve Inhibiting Effect on SRF.

Although thousands of long noncoding RNAs (lncRNAs) have been identified in porcine growth and development, the regulation mechanisms of functional lncRNAs have not been well explored. In this study, using 5'- and 3'-rapid amplification of cDNA ends (RACE) assays, we obtained two different variants of lncRNA maternally expressed gene 3 (MEG3), namely, MEG3 v1 and MEG3 v2, that were both highly expressed in porcine skeletal muscle and in the early stage of the differentiation of porcine satellite cells. Moreover, we identified the core transcript MEG3 v2. Functional analyses showed that MEG3 overexpression could effectively arrest myoblasts in the G1 phase, inhibit DNA replication, and promote myoblast differentiation, whereas MEG3 knockdown resulted in the opposite effects. Interestingly, the expression of serum response factor (SRF), a crucial transcription factor for myogenesis process, remarkably increased and decreased in mRNA and protein levels with the respective overexpression and knockdown of MEG3. Dual luciferase reporter assay showed that MEG3 could attenuate the decrease of luciferase activity of SRF induced by miR-423-5p in a dose-dependent manner. MEG3 overexpression could relieve the inhibitory effect on SRF and myoblast differentiation induced by miR-423-5p. In addition, results of RNA immunoprecipitation analysis suggested that MEG3 could act as a ceRNA for miR-423-5p. Our findings initially established a novel connection among MEG3, miR-423-5p, and SRF in porcine satellite cell differentiation. This novel role of MEG3 may shed new light on understanding of molecular regulation of lncRNA in porcine myogenesis.

Transcriptome Analysis Reveals the Effect of Long Intergenic Noncoding RNAs on Pig Muscle Growth and Fat Deposition.

Muscle growth and fat deposition are the two important biological processes in the development of pigs which are closely related to the pig production performance. Long intergenic noncoding RNAs (lincRNAs), with lack of coding potential and the length of at least 200nt, have been extensively studied to play important roles in many biological processes. However, the importance and molecular regulation mechanism of lincRNAs in the process of muscle growth and fat deposition in pigs are still to be further studied comprehensively. In our study, we used the data, including liver, abdominal fat, and longissimus dorsi muscle of 240 days' age of two F2 full-sib female individuals from the white Duroc and Erhualian crossbreed, to identify 581 putative lincRNAs associated with pig muscle growth and fat deposition. The 581 putative lincRNAs shared many common features with other mammalian lincRNAs, such as fewer exons, lower expression levels, and shorter transcript lengths. Cross-tissue comparisons showed that many transcripts were tissue-specific and were involved in the important biological processes in their corresponding tissues. Gene ontology and pathway analysis revealed that many potential target genes (PTGs) of putative lincRNAs were involved in pig muscle growth and fat deposition-related processes, including muscle cell proliferation, lipid metabolism, and fatty acid degradation. In Quantitative Trait Locus (QTLs) analysis, some PTGs were screened from putative lincRNAs, MRPL12 is associated with muscle growth, GCGR and SLC25A10 were associated with fat deposition, and PPP3CA, DPYD, and FGGY were related not only to muscle growth but also to fat deposition. Therefore, it implied that these lincRNAs might participate in the biological processes related to muscle growth or fat deposition through homeostatic regulation of PTGs, but the detailed molecular regulatory mechanisms still needed to be further explored. This study lays the molecular foundation for the in-depth study of the role of lincRNAs in the pig muscle growth and fat deposition and further provides the new molecular markers for understanding the complex biological mechanisms of pig muscle growth and fat deposition.

Transcriptome Analysis Suggests the Roles of Long Intergenic Non-coding RNAs in the Growth Performance of Weaned Piglets.

Long intergenic non-coding RNAs (lincRNAs) have been considered to play a key regulatory role in various biological processes. An increasing number of studies have utilized transcriptome analysis to obtain lincRNAs with functions related to cancer, but lincRNAs affecting growth rates in weaned piglets are rarely described. Although lincRNAs have been systematically identified in various mouse tissues and cell lines, studies of lincRNA in pigs remain rare. Therefore, identifying and characterizing novel lincRNAs affecting the growth performance of weaned piglets is of great importance. Here, we reconstructed 101,988 lincRNA transcripts and identified 1,078 lincRNAs in two groups of longissimus dorsi muscle (LDM) and subcutaneous fat (SF) based on published RNA-seq datasets. These lincRNAs exhibit typical characteristics, such as shorter lengths and lower expression relative to protein-encoding genes. Gene ontology analysis revealed that some lincRNAs could be involved in weaned piglet related processes, such as insulin resistance and the AMPK signaling pathway. We also compared the positional relationship between differentially expressed lincRNAs (DELs) and quantitative trait loci (QTL) and found that some of DELs may play an important role in piglet growth and development. Our work details part of the lincRNAs that may affect the growth performance of weaned piglets and promotes future studies of lincRNAs for molecular-assisted development in weaned piglets.

Identification and Functional Prediction of Long Intergenic Non-coding RNAs Related to Subcutaneous Adipose Development in Pigs.

An increasing number of studies have shown that long intergenic non-coding RNAs (lincRNAs) are a very important class of non-coding RNAs that plays a vital role in many biological processes. Adipose tissue is an important place for storing energy, but few studies on lincRNAs were related to pig subcutaneous fat development. Here, we used published RNA-seq data from subcutaneous adipose tissue of Italian Large White pigs and identified 252 putative lincRNAs, wherein 34 were unannotated. These lincRNAs had relatively shorter length, lower number of exons, and lower expression level compared with protein-coding transcripts. Gene ontology and pathway analysis indicated that the adjacent genes of lincRNAs were involved in lipid metabolism. In addition, differentially expressed lincRNAs (DELs) between low and high backfat thickness pigs were identified. Through the detection of quantitative trait locus (QTL), DELs were mainly located in QTLs related to adipose development. Based on the expression correlation of DEL genes and their differentially expressed potential target genes, we constructed a co-expression network and a potential pathway of DEL's effect on lipid metabolism. Our study identified and analyzed lincRNAs in subcutaneous adipose tissue, and results suggested that lincRNAs may be involved in the regulation of subcutaneous fat development. Our findings provided new insights into the biological function of porcine lincRNAs.