Regulation of Hepatocytes in G0 and G1 Phases by NOTCH3 mRNA, miR-369-3p, and rno-Rmdn2_0006 during the Initial Stage of Rat Liver Regeneration.

The key event of liver regeneration initiation (LRI) is the switch of hepatocytes from the G0 phase to the G1 phase. This study aimed to use the data from large-scale quantitatively detecting and analyzing (LQDA) to reveal the regulation of hepatocytes in the G0 or G1 phase by competing endogenous RNAs (ceRNAs) during LRI. The hepatocytes of the rat liver right lobe were isolated 0, 6, and 24 h after partial hepatectomy. Their ceRNA expression level was measured using LQDA, and the correlation among their expression, interaction, and role was revealed by ceRNA comprehensive analysis. The expression of neurogenic loci notch homologous protein 3 (NOTCH3) mRNA was upregulated in 0 h, but the expression of miR-369-3p and rno-Rmdn2_0006 of hepatocytes did not change significantly. Meanwhile, the expression of the G0 phase-related gene CDKN1c was promoted by NOTCH3 upregulation, and the expression of the G1 phase-related gene PSEN2 was inhibited by NOTCH3 downregulation. On the contrary, the expression of NOTCH3 mRNA and rno-Rmdn2_0006 was upregulated at 6 h, but the expression of miR-136-3p was downregulated. The expression of the G1 phase-related genes CHUK, DDX24, HES1, NET1, and STAT3 was promoted by NOTCH3 upregulation, and the expression of the G0 phase-related gene CDKN1a was inhibited by NOTCH3 downregulation. These results suggested that the ceRNAs and the NOTCH3-regulated G0 phase- and G1 phase-related genes showed a correlation in expression, interaction, and role. They together regulated the hepatocytes in the G0 phase at 0 h and in the G1 phase at 6 h. These findings might help understand the mechanism by which ceRNA together regulated the hepatocytes in the G0 or G1 phase.

Fat storage-inducing transmembrane proteins: beyond mediating lipid droplet formation.

Fat storage-inducing transmembrane proteins (FITMs) were initially identified in 2007 as members of a conserved endoplasmic reticulum (ER) resident transmembrane protein gene family, and were found to be involved in lipid droplet (LD) formation. Recently, several studies have further demonstrated that the ability of FITMs to directly bind to triglyceride and diacylglycerol, and the diphosphatase activity of hydrolyzing fatty acyl-CoA, might enable FITMs to maintain the formation of lipid droplets, engage in lipid metabolism, and protect against cellular stress. Based on the distribution of FITMs in tissues and their important roles in lipid droplet biology and lipid metabolism, it was discovered that FITMs were closely related to muscle development, adipocyte differentiation, and energy metabolism. Accordingly, the abnormal expression of FITMs was not only associated with type 2 diabetes and lipodystrophy, but also with cardiac disease and several types of cancer. This study reviews the structure, distribution, expression regulation, and functionality of FITMs and their potential relationships with various metabolic diseases, hoping to provide inspiration for fruitful research directions and applications of FITM proteins. Moreover, this review will provide an important theoretical basis for the application of FITMs in the diagnosis and treatment of related diseases.

circRNA-14723 promotes hepatocytes proliferation in rat liver regeneration by sponging rno-miR-16-5p.

Circular RNA (circRNA) is a subclass of noncoding RNA (ncRNA) detected within mammalian tissues and cells. However, its regulatory role during the proliferation phase of rat liver regeneration (LR) remains unreported. This study was designed to explore their regulatory mechanisms in cell proliferation of LR. The circRNA expression profile was detected by high-throughput sequencing. It was indicated that 260 circRNAs were differentially expressed during the proliferation phase of rat LR. Among them, circ-14723 displayed a significantly differential expression. We further explored its regulatory mechanism in rat hepatocytes (BRL-3A cells). First, EdU, flow cytometry and western blot (WB) indicated that knocking down circ-14723 inhibited BRL-3A cells proliferation. Second, RNA-Pulldown and dual-luciferase report assay showed that circ-14723 could sponge rno-miR-16-5p. At last, WB showed that the reported target genes of rno-miR-16-5p, CCND1, and CCNE1 were downregulated after knocking down circ-14723. In conclusion, we found that circ-14723 exerted a critical role in G1/S arrest to promote cell proliferation via rno-miR-16-5p/CCND1 and CCNE1 axis in rat LR. This finding further revealed the regulatory mechanisms of circRNA on cell proliferation of LR, and might provide a potential target for clinical problems.

Serine peptidase inhibitor Kazal type III (SPINK3) promotes BRL-3A cell proliferation by targeting the PI3K-AKT signaling pathway.

The serine protease inhibitor, Kazal type III (SPINK3), is a trypsin inhibitor associated with liver disease, which highly overexpresses in a variety of cancers. In one of our previous studies of our laboratory, Spink3 was observed to be significantly upregulated in rat liver regeneration (LR) via a gene expression profile. For the current study, rat hepatocyte BRL-3A cells were treated by gene addition/interference, and the addition of the exogenous rat recombinant protein SPINK3. It was revealed that both the overexpression of endogenous Spink3 and addition of exogenous rat recombinant SPINK3 (rrSPINK3) significantly promoted the cell proliferation of BRL-3A cells, whereas cell proliferation was inhibited when Spink3 was interfered. Furthermore, quantitative reverse transcription polymerase chain reaction and western blot results revealed that three signaling pathways, including extracellular-signal-regulated kinase 1/2 (ERK1/2), Janus kinase (JAK)-signal transducer and activator of transcription (STAT), and phosphatidylinositol-3-kinase (PI3K)-protein kinase B (AKT), as well as their related genes, were altered following endogenous Spink3 addition/interference. Also, the PI3K-AKT and SRC-p38 pathways and their related genes were modified following exogenous SPINK3 treatment. Among them, the common signaling pathway was PI3K-AKT pathway. We concluded that SPINK3 could activate the PI3K-AKT pathway by enhancing the expression of AKT1 to regulate the proliferation of BRL-3A cells. This study may contribute to shedding light on the potential mechanisms of SPINK3 that regulate the proliferation of BRL-3A cells.

Comparative transcriptomics analysis revealing flower trichome development during flower development in two Lonicera japonica Thunb. cultivars using RNA-seq.

BACKGROUND: Lonicera japonica Thunb. (L. japonica) has the functions of clearing away heat and detoxifying, broad-spectrum antibacterial and anti-virus, etc. More than 70% of anti-inflammatory and cold Chinese patent medicines contain L. japonica. Trichomes comprise specialized multicellular structures that have the capacity to synthesize and secrete secondary metabolites and protect plants from biotic and abiotic stresses. The extraction of trichome secretions has great commercial value. However, little is known about the trichome formation mechanism in L. japonica. Therefore, the study of trichome development between different varieties provides a basis for selecting suitable planting resources. RESULTS: Here, we present a genome-wide comparative transcriptome analysis between two L. japonica cultivars, toward the identification of biological processes and functional gene activities that occur during flowering stage trichome development. In this study, the density and average lengths of flower trichomes were at their highest during three-green periods (S2). Using the Illumina RNA-Seq method, we obtained 134,304 unigenes, 33,733 of which were differentially expressed. In an analysis of 40 differentially expressed unigenes (DEGs) involved in trichome development, 29 of these were transcription factors. The DEGs analysis of plant hormone signal transduction indicated that plant growth and development may be independent of gibberellin (GA) and cytokinine (CTK) signaling pathways, and plant stress may be independent of jasmonic acid (JA) and ethylene (ET) signaling pathways. We screened several genes involved in the floral biosynthesis of odors, tastes, colors, and plant hormones, and proposed biosynthetic pathways for sesquiterpenoid, triterpenoid, monoterpenoid, flavonoid, and plant hormones. Furthermore, 82 DEGs were assigned to cell cycles and 2616 were predicted as plant resistance genes (PRGs). CONCLUSIONS: This study provides a comprehensive characterization of the expression profiles of flower development during the seven developmental stages of L. japonica, thereby offering valuable insights into the molecular networks that underly flower development in L. japonica.

A combined proteomic and metabolomic analyses of the priming phase during rat liver regeneration.

By comparing differentially abundant proteins and metabolites, the protein expression, metabolic changes and metabolic regulation mechanisms during the priming phase of liver regeneration (LR) were investigated. We combined proteomic analysis via isobaric tags for relative and absolute quantification (iTRAQ) with metabolomic analysis via nontargeted liquid chromatography-mass spectrometry (LC-MS). LC-MS was used to examine 29 energy metabolites expression alterations in targeted metabolomics. A total number of 441 differentially expressed proteins and 65 metabolites were identified. PSMB10, PSMB5, RCG_63409, PSME4 and PSMB7 were key node proteins, these proteins are involved in the proteasome pathway. The most strongly enriched transcription factor motif was TP63. These results point out a critical role of the proteasome pathway (defense mechanisms) and of TP63 (metabolic regulator) as the key transcription factor during the priming phase of LR. Metabolomic and metabolite analysis showed that profiling indicates upregulation of arginine biosynthesis and glycolysis as the main ATP-delivering pathway. Integrative proteomic and metabolomic analysis showed that biomolecular changes were primarily related to the neurological disease, cell death and survival and cell morphology. What's more, neurotransmitters may play an important role in the regulation of LR.

Comprehensive analysis of lncRNA-miRNA-mRNA during proliferative phase of rat liver regeneration.

This study aims to reveal the regulatory mechanism of lncRNAs-miRNAs-mRNAs network during the proliferative phase of liver regeneration (LR). High-throughput sequencing technology was performed, and a total of 1,738 differentially expressed lncRNAs (DE lncRNAs), 167 known differentially expressed miRNAs (DE miRNAs), and 2,727 differentially expressed mRNAs were identified. Then, the target DE lncRNAs and DE mRNAs regulated by the same miRNAs were screened and a ceRNA regulatory network containing 32 miRNAs, 107 lncRNAs, and 270 mRNAs was constructed. Insulin signaling pathway, pyrimidine metabolism, axon guidance, carbohydrate digestion and absorption, and pyruvate metabolism were significantly enriched in the network. Through literature review and the regulatory relationship between lncRNAs and miRNAs, nine core lncRNAs were identified, which might play important roles during the proliferative phase of rat LR. This study analyzed lncRNA-miRNA-mRNA regulatory network for the first time during the proliferative phase of rat LR, providing clues for exploring the mechanism of LR and the treatment of liver diseases.

Genome-wide RAD sequencing to identify a sex-specific marker in Chinese giant salamander Andrias davidianus.

BACKGROUND: Chinese giant salamander Andrias davidianus is an endangered species. The success of artificial breeding provides a useful way to protect this species. However, the method to identify the sex and mechanism of sex determination were unclear which hinder the improvement of the artificial breeding. Detection of a sex specific marker provides an effective approach to identify genetic sex and investigate the sex determination mechanism. RESULTS: We used restriction-site-associated DNA (RAD) sequencing to isolate a sex-specific genetic marker in A. davidianus to expand knowledge of the sex determination mechanism. Four male and four female specimens were subjected to RAD sequencing, which generated 934,072,989 reads containing approximately 134.4 Gb of sequences. The first round of comparison of the assembled sequence against the opposite sex raw reads revealed 19,097 female and 17,994 male unmatched sequences. Subsequently, 19,097 female sequences were subjected to a BLAST search against male genomic data, which revealed 308 sequences unmapped to the male genome. One hundred of these were randomly selected and validated by PCR in five male and five female specimens, and four putative sex-specific sequences were produced. Further validation was performed by PCR in another 24 females and 24 males, and all female individuals exhibited the expected specific bands, while the males did not. To apply the sex-specific marker, three specimens reversed from genetic female to physiological male were found in a group exposed to elevated temperature, and 13 individuals reversed from genetic male to physiological female were obtained in a 17ß-estradiol exposed group. CONCLUSION: This is the first report of a sex-specific marker in A. davidianus and may have potential for elucidation of its sex determination mechanism and, hence, its conservation.

Comprehensive CircRNA expression profile and selection of key CircRNAs during priming phase of rat liver regeneration.

BACKGROUND: Rat liver regeneration (LR) proceeds along a process of highly organized and ordered tissue growth in response to the loss or injury of liver tissue, during which many physiological processes may play important roles. The molecular mechanism of hepatocyte proliferation, energy metabolism and substance metabolism during rat LR had been elucidated. Further, the correlation of circular RNA (circRNA) abundance with proliferation has recently been clarified. However, the regulatory capacity of circRNA in rat LR remains a fascinating topic. RESULTS: To investigate the regulatory mechanism of circRNA during priming phase of rat LR, high-throughput RNA sequencing technology was performed to unbiasedly profile the expression of circRNA during priming phase of rat LR. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) biological pathway analysis was conducted to predict the functions of differentially expressed circRNAs and their host linear transcripts. Co-expression networks of circRNA-miRNA were constructed based on the correlation analysis between the differentially expressed LR-related circRNAs and the condition of their miRNA binding sites. To excavate the key circRNAs in the early phase of rat LR, we comprehensively evaluated and integrated the relationship of expression level between the circRNAs and the linear transcripts as well as the distribution of miRNA binding sites in circRNA sequences. CONCLUSIONS: This paper is the first to employ the comprehensive circRNA expression profile and to investigate circRNA-miRNA interactions during priming phase of rat LR. Two thousand four hundred twelve circRNAs were detected, and 159 circRNAs deriving from 116 host linear transcripts differentially expressed (p < 0.05). Six significantly changed circRNAs during priming phase of rat LR were screened as key circle molecules, and then were validated by qRT-PCR. This study will lay the foundation for revealing the functional roles of circRNAs during rat LR and help solve the remaining clinical problems.

Serine peptidase inhibitor Kazal type I (SPINK1) promotes BRL-3A cell proliferation via p38, ERK, and JNK pathways.

Serine peptidase inhibitor Kazal type I (SPINK1) has the similar spatial structure as epidermal growth factor (EGF); EGF can interact with epidermal growth factor receptor (EGFR) to promote proliferation in different cell types. However, whether SPINK1 can interact with EGFR and further regulate the proliferation of hepatocytes in liver regeneration remains largely unknown. In this study, we investigated the role of SPINK1 in a rat liver hepatocyte line of BRL-3A in vitro. The results showed the upregulation of endogenous Spink1 (gene addition) significantly increased not only the cell viability, cell numbers in S and G2 /M phase, but also upregulated the genes/proteins expression related to cell proliferation and anti-apoptosis in BRL-3A. In contrast, the cell number in G1 phase and the expression of pro-apoptosis-related genes/proteins were significantly decreased. The similar results were observed when the cells were treated with exogenous rat recombinant SPINK1. Immunoblotting suggested SPINK1 can interact with EGFR. By Ingenuity Pathway Analysis software, the SPINK1 signalling pathway was built; the predicted read outs were validated by qRT-PCR and western blot; and the results showed that p38, ERK, and JNK pathways-related genes/proteins were involved in the cell proliferation upon the treatment of endogenous Spink1 and exogenous SPINK1. Collectively, SPINK1 can associate with EGFR to promote the expression of cell proliferation-related and anti-apoptosis-related genes/proteins; inhibit the expression of pro-apoptosis-related genes/proteins via p38, ERK, and JNK pathways; and consequently promote the proliferation of BRL-3A cells. For the first time, we demonstrated that SPINK1 can associate with EGFR to promote the proliferation of BRL-3A cells via p38, ERK, and JNK pathways. This work has direct implications on the underlying mechanism of SPINK1 in regulating hepatocytes proliferation in vivo and liver regeneration after partial hepatectomy.

The novel protein C3orf43 accelerates hepatocyte proliferation.

BACKGROUND: Our previous study found that single-pass membrane protein with coiled-coil domains 1 (C3orf43; XM_006248472.3) was significantly upregulated in the proliferative phase during liver regeneration. This indicates that C3orf43 plays a vital role in liver cell proliferation. However, its physiological functions remains unclear. METHODS: The expressions of C3orf43 in BRL-3A cells transfected with C3orf43-siRNA (C3-siRNA) or overexpressing the vector plasmid pCDH-C3orf43 (pCDH-C3) were measured via RT-qPCR and western blot. Cell growth and proliferation were determined using MTT and flow cytometry. Cell proliferation-related gene expression was measured using RT-qPCR and western blot. RESULTS: It was found that upregulation of C3orf43 by pCDH-C3 promoted hepatocyte proliferation, and inhibition of C3orf43 by C3-siRNA led to the reduction of cell proliferation. The results of qRT-PCR and western blot assay showed that the C3-siRNA group downregulated the expression of cell proliferation-related genes like JUN, MYC, CCND1 and CCNA2, and the pCDH-C3 group upregulated the expression of those genes. CONCLUSION: These findings reveal that C3orf43 may contribute to hepatocyte proliferation and may have the potential to promote liver repair and regeneration.

A preliminary in vivo study of the effects of OPN on rat liver regeneration induced by partial hepatectomy.

Osteopontin (OPN) is a member of Th1 cytokine secreted by activated lymphocytes and macrophages. However, it deserves to be studied whether OPN could promote cell activation or proliferation, and then facilitate hepatic self-repair during liver regeneration (LR). This study is designed to further reveal the effects of OPN on LR in vivo. Firstly, quantitative reverse transcription-PCR (qRT-PCR) and western blot (WB) were utilized to validate the expression profile of endogenous OPN in rat regenerating livers after partial hepatectomy (PH). Then OPN expression vector, two shRNA expression vectors and their respective test vectors were successfully constructed. Afterwards, test vectors were administrated into mouse livers via tail vein to find the more efficient shRNA. Furthermore, OPN expression vector and the more efficient shRNA expression vector were injected into rat regenerating livers, and then the changes in liver regeneration and hepatic microstructure were respectively detected by liver regeneration rate and HE staining, while the expressions of several marker genes were detected by qRT-PCR and WB. Endogenous OPN was strikingly up-regulated in both mRNA and protein level during LR, especially at 12 and 72 h after PH. The shRNA expression vector Opn(313) was found to be more efficient than Opn(887) in silencing the expression of Opn. Then OPN expression vector and Opn(313) were injected into rat remnant livers, and it showed that OPN overexpression aggravated hepatic necrosis and leukocytes infiltration, while OPN silencing inhibited liver regeneration rate and the expressions of PCNA and CCL2, but augmented that of BAX. In conclusion, OPN might enhance inflammation and cell proliferation, attenuate cell apoptosis, and ultimately facilitate liver regeneration at the termination stage of liver regeneration.

Analysis of the ways and methods of signaling pathways in regulating cell cycle of NIH3T3 at transcriptional level.

BACKGROUND: To analyze the ways and methods of signaling pathways in regulating cell cycle progression of NIH3T3 at transcriptional level, we modeled cell cycle of NIH3T3 and found that G1 phase of NIH3T3 cell cycle was at 5-15 h after synchronization, S phase at 15-21 h, G2 phase at 21-22 h, M phase at 22-25 h. RESULTS: Mouse Genome 430 2.0 microarray was used to detect the gene expression profiles of the model, and results showed remarkable changes in the expressions of 64 cell cycle genes and 960 genes associated with other physiological activity during the cell cycle of NIH3T3. For the next step, IPA software was used to analyze the physiological activities, cell cycle genes-associated signal transduction activities and their regulatory roles of these genes in cell cycle progression, and our results indicated that the reported genes were involved in 17 signaling pathways in the regulation of cell cycle progression. Newfound genes such as PKC, RAS, PP2A, NGR and PI3K etc. belong to the functional category of molecular mechanism of cancer, cyclins and cell cycle regulation HER-2 signaling in breast cancer signaling pathways. These newfound genes could promote DNA damage repairment and DNA replication progress, regulate the metabolism of protein, and maintain the cell cycle progression of NIH3T3 modulating the reported genes CCND1 and C-FOS. CONCLUSION: All of the aforementioned signaling pathways interacted with the cell cycle network, indicating that NIH3T3 cell cycle was regulated by a number of signaling pathways.

Expression profiles uncover the correlation of OPN signaling pathways with rat liver regeneration at cellular level.

Osteopontin (OPN) could participate in the occurrence of multiple liver diseases via promoting inflammation, cell activation, proliferation, and migration. However, the correlation of OPN with liver regeneration (LR) is poorly defined. Previous studies from us and others revealed that OPN was probably involved in the activation and proliferation of various hepatic cell types during LR. In this study, to further investigate the underlined mechanism of OPN in regulating LR, eight hepatic cell types were isolated and purified from rat regenerative livers at 10 time points. The gene expression profiles of above hepatic cells were assayed by Rat Genome 230 2.0 chips, and then IPA software was used to uncover the correlations of gene expression changes with physiological activities. The findings demonstrated that the majority of the OPN pathway-related genes were up-regulated in hepatocytes (HCs), pit cells (PCs), oval cells (OCs), and biliary epithelial cells (BECs) but down-regulated in other four cell types including sinusoidal endothelial cells (SECs), Kupffer cells (KCs), dendritic cells (DCs), and hepatic stellate cells (HSCs). Thereafter, functional enriched analysis by IPA indicated that OPN signaling pathway might promote cell proliferation, activation, migration, and inflammation in HCs, OCs, BECs, and PCs, and slightly boost proliferation and migration of SECs and KCs but inhibit inflammation response and chemotaxis in SECs and KCs and almost all physiological processes in DCs and HSCs. Morever, apoptosis, cell death, and necrosis were remarkably inhibited through JAK/STAT, ERK1/2, and NF-kB branches in almost every cell type. These above results suggest that OPN signaling pathway is closely related to HCs, OCs, BECs, and PCs but has less regulatory effect on SECs, KCs, HSCs, and DCs during rat LR.

Thrombopoietin Signaling Pathway Regulates Hepatocyte Activation in Rat Liver Regeneration.

Thrombopoietin (THPO) signaling pathway regulates cell activation and many other physiological activities. To study its role in liver regeneration (LR), hepatocytes were isolated from rat regenerating livers and gene expression profile was detected using the Rat Genome 230 2.0 Array. Spectral function (E t ) and information correlation coefficient (ICC) were used to analyze gene synergy based on gene expression changes. The results showed that 35 genes related to THPO signaling pathway were significantly changed during rat LR. Functional analysis with ICC showed that five genes, STAT3, PLSCR1, CTGF, PRLR, and LCP1, played a key role in hepatocyte activation. Fourteen channels of THPO signaling pathway participated in regulating hepatocyte activation during rat LR.

[Regulation of autophagy on dendritic cells during rat liver regeneration by IPA].

To understand the mechanism underlying autophagy in regulating dendritic cells during rat liver regeneration, we used the method of percoll density gradient centrifugation combined with immunomagnetic bead to isolate dendritic cells, the Rat Genome 230 2.0 Array to determine the expression changes of autophagy-related genes, and Ingenuity Pathway Analysis 9.0 (IPA) to determine the autophagy activities. The results indicated that LC3, BECN1, ATG7 and SQSTM1 genes had significant expression changes during rat liver regeneration. There were 593 genes related to autophagy, among which 210 genes were identified as significant. We also showed that the activity of autophagy was enhanced in the priming phase and teminal phase of liver regeneration, weakened in the proliferative stage by comparative analysis method of IPA. The autophagy-related physiological activities mainly included RNA expression, RNA transcription, cell differentiation and proliferation, involving in PPARα/RXRα activation, acute phase response signaling, TREM1 signaling, IL-6 signaling, IL-8 signaling and IL-1 signaling, whose activities were increased or decreased in liver regeneration. Cluster analysis found that P53 and AMPK signaling participated in the regulation of dendritic cells autophagy, with AMPK signaling in the priming phase of liver regeneration, and both signaling pathways in the terminal phase. We conclude that dendritic cells autophagy played an important role in initiation of the immune response in priming phase and depletion of dendritic cells in late phase during rat liver regeneration.

[Effect of autophagy on liver regeneration].

Autophagy is a lysosome-mediated degradation pathway, which plays an important role in hepatic physiological and pathological processes, in eukaryotic cells. The liver has a remarkable regenerative capacity. After acute or chronic injury, the residual hepatic cells can be activated to enter the cell-cycle for proliferation, in order to compensate for lost liver tissue and recover liver function. In this review, we summarize the relationship between liver regeneration (LR) after various types of injury and autophagy. For example, autophagy is activated to accelerate LR after physically, alcohol and food borne induced liver injury, while the role of autophagy in animal models of LR after chemical injury remains controversial. Autophagy can also be used to promote the replication of virus particles by some hepatotropic viruses (e.g., HBV, HCV) and inhibit LR after viral infection. Studies on mechanisms of autophagy and LR will contribute to clarify the regenerative process and provide new methods for the treatment of liver disease.

High expression of GPR50 promotes the proliferation, migration and autophagy of hepatocellular carcinoma cells in vitro.

G protein-coupled receptors (GPCRs) play important roles in tumorigenesis and the development of hepatocellular carcinoma (HCC). GPR50 is an orphan GPCR. Previous studies have indicated that GPR50 could protect against breast cancer development and decrease tumor growth in a xenograft mouse model. However, its role in HCC remains indistinct. To detect the role and the regulation mechanism of GPR50 in HCC, GPR50 expression was analyzed in HCC patients (gene expression omnibus database (GEO) (GSE45436)) and detected in HCC cell line CBRH-7919, and the results showed that GPR50 was significantly up-regulated in HCC patients and CBRH-7919 cell line compared to the corresponding normal control. Gpr50 cDNA was transfected into HCC cell line CBRH-7919, and we found that Gpr50 promoted the proliferation, migration, and autophagy of CBRH-7919. The regulation mechanism of GPR50 in HCC was detected by isobaric tags for relative and absolute quantification (iTRAQ) analysis, and we found that GPR50 promoted HCC was closely related to CCT6A and PGK1. Taken together, GPR50 may promote HCC progression via CCT6A-induced proliferation and PGK1-induced migration and autophagy, and GPR50 could be an important target for HCC.

Comparative Transcriptome and Metabolome Profiling Reveal Mechanisms of Red Leaf Color Fading in Populus × euramericana cv. 'Zhonghuahongye'.

Anthocyanins are among the flavonoids that serve as the principal pigments affecting the color of plants. During leaf growth, the leaf color of 'Zhonghuahongye' gradually changes from copper-brown to yellow-green. At present, the mechanism of color change at different stages has not yet been discovered. To find this, we compared the color phenotype, metabolome, and transcriptome of the three leaf stages. The results showed that the anthocyanin content of leaves decreased by 62.5% and the chlorophyll content increased by 204.35%, 69.23%, 155.56% and 60%, respectively. Differential metabolites and genes were enriched in the pathway related to the synthesis of 'Zhonghuahongye' flavonoids and anthocyanins and to the biosynthesis of secondary metabolites. Furthermore, 273 flavonoid metabolites were detected, with a total of eight classes. DFR, FLS and ANS downstream of anthocyanin synthesis may be the key structural genes in reducing anthocyanin synthesis and accumulation in the green leaf of 'Zhonghuahongye'. The results of multi-omics analysis showed that the formation of color was primarily affected by anthocyanin regulation and its related synthesis-affected genes. This study preliminarily analyzed the green regression gene and metabolic changes in 'Zhonghuahongye' red leaves and constitutes a reference for the molecular breeding of 'Zhonghuahongye' red leaves.

Metabolome analysis reveals flavonoid changes during the leaf color transition in Populus × euramericana 'Zhonghuahongye'.

Introduction: To investigate the mechanism of leaf color change at different stages in Populus × euramericana 'Zhonghuahongye' ('Zhonghong' poplar). Methods: Leaf color phenotypes were determined and a metabolomic analysis was performed on leaves at three stages (R1, R2 and R3). Results: The a*, C* and chromatic light values of the leaves decreased by 108.91%, 52.08% and 113.34%, while the brightness L values and chromatic b* values gradually increased by 36.01% and 13.94%, respectively. In the differential metabolite assay, 81 differentially expressed metabolites were detected in the R1 vs. R3 comparison, 45 were detected in the R1 vs. R2 comparison, and 75 were detected in the R2 vs. R3 comparison. Ten metabolites showed significant differences in all comparisons, which were mostly flavonoid metabolites. The metabolites that were upregulated in the three periods were cyanidin 3,5-O-diglucoside, delphinidin, and gallocatechin, with flavonoid metabolites accounting for the largest proportion and malvidin 3- O-galactoside as the primary downregulated metabolite. The color shift of red leaves from a bright purplish red to a brownish green was associated with the downregulation of malvidin 3-O-glucoside, cyanidin, naringenin, and dihydromyricetin. Discussion: Here, we analyzed the expression of flavonoid metabolites in the leaves of 'Zhonghong' poplar at three stages and identified key metabolites closely related to leaf color change, providing an important genetic basis for the genetic improvement of this cultivar.