A chlorophyll a oxygenase 1 gene ZmCAO1 contributes to grain yield and waterlogging tolerance in maize.

Chlorophylls function in photosynthesis, and are critical to plant developmental processes and responses to environmental stimuli. Chlorophyll b is synthesized from chlorophyll a by chlorophyll a oxygenase (CAO). Here, we characterize a yellow-green leaf (ygl) mutant and identify the causal gene which encodes a chlorophyll a oxygenase in maize (ZmCAO1). A 51 bp Popin transposon insertion in ZmCAO1 strongly disrupts its transcription. Low enzyme activity of ZmCAO1 leads to reduced concentrations of chlorophyll a and chlorophyll b, resulting in the yellow-green leaf phenotype of the ygl mutant. The net photosynthetic rate, stomatal conductance, and transpiration rate are decreased in the ygl mutant, while concentrations of δ-aminolevulinic acid (ALA), porphobilinogen (PBG) and protochlorophyllide (Pchlide) are increased. In addition, a ZmCAO1 mutation results in down-regulation of key photosynthetic genes, limits photosynthetic assimilation, and reduces plant height, ear size, kernel weight, and grain yield. Furthermore, the zmcao1 mutant shows enhanced reactive oxygen species production leading to sensitivity to waterlogging. These results demonstrate the pleiotropy of ZmCAO1 function in photosynthesis, grain yield, and waterlogging tolerance in maize.

CircWHSC1 serves as an oncogene to promote hepatocellular carcinoma progression.

BACKGROUND: Circular RNAs (circRNAs) function as vital regulators in multifarious cancers, including hepatocellular carcinoma (HCC). However, the roles of circRNA Wolf-Hirschhorn syndrome candidate gene-1 (circWHSC1) in HCC are barely known. METHODS: Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted for the levels of circWHSC1, miR-142-3p, miR-421, miR-665 and homeobox A1 (HOXA1) mRNA. Cell Counting Kit-8 (CCK-8) assay, colony formation assay and 5'-ethynyl-2'-deoxyuridine (EdU) assay were used to evaluate cell proliferation ability. Transwell assay was adopted for cell migration and invasion. Western blot assay was employed for protein levels. RNA pull-down assay, dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay were executed to verify the interaction between miR-142-3p and circWHSC1 or HOXA1. Murine xenograft model assay was conducted for the role of circWHSC1 in vivo. The morphology of exosomes was observed by transmission electron microscopy (TEM). RESULTS: CircWHSC1 was elevated in HCC tissues and cells, and high level of circWHSC1 was associated with worse overall survival of HCC patients. Knockdown of circWHSC1 suppressed HCC cell proliferation and metastasis in vitro and restrained tumorigenesis in vivo. CircWHSC1 functioned as the sponge for miR-142-3p, which directly targeted HOXA1. Inhibition of miR-142-3p ameliorated the effects of circWHSC1 knockdown on HCC cell proliferation and metastasis. Moreover, miR-142-3p overexpression restrained the growth and motility of HCC cells, with HOXA1 elevation reversing the impacts. Additionally, circWHSC1 was increased in HCC patients' serum and might be a diagnostic indicator for HCC. CONCLUSION: CircWHSC1 played a tumour-promoting role in HCC by elevating HOXA1 through sponging miR-142-3p.

Clinical significance and oncogene function of long noncoding RNA HAGLROS overexpression in ovarian cancer.

PURPOSE: To explore the clinical significance and mechanism of long noncoding RNA (lncRNA) HAGLROS in ovarian cancer. METHODS: The expression of HAGLROS in ovarian cancer was verified by online databases and quantitative reverse transcription polymerase chain reaction (qRT-PCR), and its relationship with clinicopathological parameters was analysed. Pearson correlation analysis was used to study the correlation between HAGLROS and miR-100 in ovarian cancer. Meta-analysis was used to explore the expression of miR-100 in ovarian cancer. In addition, we used bioinformatics to explore the target genes of miR-100 and perform functional analysis. RESULTS: HAGLROS was significantly upregulated in ovarian cancer (P < 0.001) and was closely related to disease stage (P = 0.033), tumour size (P = 0.032) and poor prognosis (P = 0.019). HAGLROS had a certain diagnostic value in ovarian cancer (area under the curve = 0.751). MiR-100 was negatively correlated with HAGLROS (r = 0.167, P = 0.001) and significantly downregulated in ovarian cancer. Bioinformatics analysis predicted a total of 31 potential target genes that interact with miR-100. These target genes were mainly involved in the regulation of cellular catabolic process, proteoglycan biosynthetic process and positive regulation of proteasomal ubiquitin-dependent protein catabolic process. Among them, mTOR and ZNRF2 are hub genes. CONCLUSION: HAGLROS is a potential biomarker for early diagnosis and prognosis evaluation of ovarian cancer. It can be used as a molecular sponge of miR-100 to regulate the expression of mTOR and ZNRF2 and affect the signal transduction of the mTOR pathway. HAGLROS is expected to be a new target for the treatment of ovarian cancer.

Single-Cell Non-coding RNA in Embryonic Development.

Non-coding RNAs (ncRNAs) have significant regulatory functions on the regulation of gene expression of various life activities after transcription, even though they do not encode proteins. During the development of embryos, ncRNAs, such as long non-coding RNAs (lncRNAs), microRNAs (miRNAs), circular RNAs (circRNAs), small nucleolar RNAs (snoRNAs), and Piwi-interacting RNAs (piRNAs), have been widely proven as key regulators. The emerging single-cell RNA sequencing technique is powerful for profiling "cell-to-cell" variability at the genomic level. It has been applied to detect the expression of ncRNAs during embryo development. In this chapter, we pay close attention to single-cell ncRNA expression and summarize their roles in embryo development.

Comprehensive analysis of a TPX2-related TRHDE-AS1/PKIA ceRNA network involving prognostic signatures in Hepatitis B virus-infected hepatocellular carcinoma.

Hepatitis B virus (HBV) infection is a main carcinogenic factor of hepatocellular carcinoma (HCC). TPX2 microtubule nucleation factor is recently recommended as a novel prognostic biomarker in HBV-infected HCC tissues. This study aimed to explore a TPX2-related ceRNA regulatory network in HBV-infected HCC and the potential impact on HCC prognosis. We comprehensively identified 541 differential expressed lncRNAs (DElncRNAs), 37 DEmiRNAs and 439 DEmRNAs from HBV-related TCGA-HCC cohorts in TPX2low and TPX2high groups. Based on their RNA-RNA interaction and expression analysis, four DElncRNAs (TRHDE-AS1, DLX6-AS1, SNHG14, HOXA11-AS), four DEmiRNAs (miR-23b, miR-320a, miR-589, miR-126) and five DEmRNAs (PKIA, PCDHA2, SHCBP1, PRSS16, KIF18A) in HCC tumor vs normal groups were subjected to the hub regulatory networks analysis and further prognostic value analysis. Importantly, the TRHDE-AS1/miR-23b/PKIA ceRNA network was associated with HCC prognosis. Furthermore, cellular location analysis and base-base interaction analysis indicated that the cytoplasmic lncRNA TRHDE-AS1 was regarded as a ceRNA to sponging miR-23b and then regulating PKIA. Interestingly, correlation analysis suggested the expression correlation between TRHDE-AS1 and PKIA in HCC. Finally, we further performed the methylation and immune infiltration analysis to explore the functional process of PKIA in HCC. We proposed a ceRNA regulatory network may help elucidate the mechanism by which TPX2 contributes to the prognosis of HBV-related HCC.

CNOT7 depletion reverses natural killer cell resistance by modulating the tumor immune microenvironment of hepatocellular carcinoma.

A major obstacle to effective cancer immunotherapy is the tumor immune microenvironment. Natural killer (NK) cell resistance has been suggested as a primary cause of poor prognosis in hepatocellular carcinoma (HCC), which seemingly correlates with CNOT7 overexpression. CNOT7, a cytoplasmic mRNA deadenylase that is highly expressed in HCC, may regulate cytokine transforming growth factor-ß1 (TGF-ß1) secretion by controlling nuclear factor-κB subunit p65 trafficking. CNOT7 depletion suppresses TGF-ß1 secretion in HCC and promotes interferon-γ (IFN-γ) secretion by NK cells, and we previously demonstrated that CNOT7 depletion reversed IFN-γ resistance in HCC cells. Therefore, we hypothesized that CNOT7 depletion might reverse NK cell resistance by influencing the tumor immune microenvironment of HCC. To test this hypothesis, we examined the correlation between CNOT7, STAT1, TGF-ß1 and IFN-γ expression with hepatitis B virus-related cirrhosis and HCC with hepatitis B virus-related cirrhosis. We found that modulation of CNOT7 expression alters TGF-ß1 secretion in HCC and IFN-γ secretion in NK cells. We also examined the effects of NK cells in HepG2 cells with CNOT7 knockdown, which showed that NK cell surface CD107a expression is up-regulated and caspase-3 expression is significantly enhanced in CNOT7-deficient HepG2 cells. Overall, our results show that knockdown of CNOT7 expression reverses NK cell resistance in HCC cells. Therefore, CNOT7 depletion has potential as a new adjuvant therapy in immunotherapy for HCC.

Long non-coding RNA FLJ33360 participates in ovarian cancer progression by sponging miR-30b-3p.

BACKGROUND: Long noncoding RNAs (lncRNAs) have been reported to play a key role in the development and progression of human malignancies. FLJ33360 is an lncRNA with unknown functions. This study was designed to determine the clinical significance and mechanism of FLJ33360 in ovarian cancer. MATERIALS AND METHODS: The clinical significance of FLJ33360 in ovarian cancer was determined using the Gene Expression Profiling Interactive Analysis (GEPIA) database, Kaplan-Meier Plotter database, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and statistical analysis. The regulatory relationships between FLJ33360 and miR-30b-3p were explored through bioinformatics, the Gene Expression Omnibus (GEO) database, the ArrayExpress database and meta-analysis. The possible pathways were predicted using Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis. In addition, the key target genes were identified using a protein-protein interaction (PPI) network, the Cancer Genome Atlas (TCGA) database, and correlation analysis. RESULTS: FLJ33360 expression was significantly downregulated in ovarian cancer tissue (P=0.0011) and was closely associated with International Federation of Gynecology and Obstetrics (FIGO) stage (P=0.027) and recurrence (P=0.002). FLJ33360 may have potential value in detecting ovarian cancer (area under the curve =0.793). Function analysis demonstrated that FLJ33360 can act as a molecular sponge of miR-30b-3p to regulate the expression of target genes that are mainly involved in positive regulation of smooth muscle cell migration, the unsaturated fatty acid metabolic process, and positive regulation of the epithelial to mesenchymal transition. Among these target genes, BCL2 is the hub gene. CONCLUSION: FLJ33360 is a potential biomarker for early diagnosis and prognostic assessment in ovarian cancer and may regulate the expression of genes by sponging miR-30b-3p and thus participate in the development of ovarian cancer.

Emerging Roles Of hsa-circ-0046600 Targeting The miR-640/HIF-1α Signalling Pathway In The Progression Of HCC.

PURPOSE: Circular RNAs (circRNAs) play important roles in the development and progression of various human cancers. hsa-circ-0046600 is a circRNA of unknown function. The purpose of this study was to investigate the biological function of hsa-circ-0046600 in hepatocellular carcinoma (HCC) and elucidate the possible molecular mechanisms of this circRNA. MATERIALS AND METHODS: GSE97332, quantitative reverse transcription polymerase chain reaction (qRT-PCR) and fluorescence in situ hybridization (FISH) were used to detect the expression of hsa-circ-0046600 in HCC tissues and cells. A dual-luciferase reporter assay was used to confirm the interaction between hsa-circ-0046600 and miR-640, and a meta-analysis confirmed the expression of miR-640 in HCC. Bioinformatics was used for the functional analysis of miR-640 target genes. N-cadherin and HIF-1α expression was measured by Western blot analysis. RESULTS: The expression level of hsa-circ-0046600 in HCC tissue was significantly higher than that in adjacent normal tissue (P < 0.05) and was associated with tumour size, TNM stage and pathological vascular invasion. Moreover, the downregulated expression of hsa-circ-0046600 significantly inhibited the migration of HepG2 and SK-HEP-1 cells. hsa-circ-0046600 is present mainly in the cytoplasm and promotes the expression of proteins such as HIF-1α by competitively binding to miR-640 in HCC, thereby affecting the malignant biological behaviour of liver cancer cells. CONCLUSION: hsa-circ-0046600 can be used as a new biomarker for HCC diagnosis and disease progression and provides a potential target for targeted therapy.

NLRP3 Deficiency Alleviates Severe Acute Pancreatitis and Pancreatitis-Associated Lung Injury in a Mouse Model.

The rapid production and release of a large number of inflammatory cytokines can cause excessive local and systemic inflammation in severe acute pancreatitis (SAP) and multiple organ dysfunction syndrome (MODS), especially pancreatitis-associated acute lung injury (P-ALI), which is the main cause of early death in patients with SAP. The NLRP3 inflammasome plays an important role in the maturation of IL-1ß and the inflammatory cascade. Here, we established a model of SAP using wild-type (NLRP3+/+) and NLRP3 knockout (NLRP3-/-) mice by intraperitoneal injections of caerulein (Cae) and lipopolysaccharide (LPS). Pathological injury to the pancreas and lungs, the inflammatory response, and neutrophil infiltration were significantly mitigated in NLRP3-/- mice. Furthermore, INF-39, an NLRP3 inflammasome inhibitor, could reduce the severity of SAP and P-ALI in a dose-dependent manner. Our results suggested that SAP and P-ALI were alleviated by NLRP3 deficiency in mice, and thus, reducing NLRP3 expression may mitigate SAP-associated inflammation and P-ALI.

Glucocorticoid receptor regulates expression of microRNA-22 and downstream signaling pathway in apoptosis of pancreatic acinar cells.

AIM: To elucidate the underlying mechanism that microRNA-22 (miR-22) promotes the apoptosis of rat pancreatic acinar cells (AR42J) and the elements that regulate the expression of miR-22. METHODS: One hundred nanomoles per liter of caerulein (Cae) was administrated to induce the apoptosis of AR42J cells and the apoptosis rate was detected by flow cytometry analysis. An amylase assay kit was used to measure the amylase expression level in the supernatant. Quantitative real-time PCR (qRT-PCR) was adopted to measure miR-22 expression. We used online tools to predict the potential transcription promoter of miR-22 and the binding sites, which was further identified by using luciferase reporter analysis, chromatin immunoprecipitation (ChIP) and ChIP-qPCR assays. Then, a mimic of miR-22, Nr3c1 plasmid encoding the glucocorticoid receptor (GR), and si-Nr3c1 were used to transfect AR42J cells, respectively. The mRNA expression of miR-22, Nr3c1, and Erb-b2 receptor tyrosine kinase 3 (ErbB3) was confirmed by qRT-PCR and the apoptosis rate of AR42J cells was detected by flow cytometry analysis. Western blot was used to detect the expression of ErbB3, GR, PI3k, PI3k-p85α, Akt, p-Akt, Bad, Bax, Bcl-xl, Bcl-2, and cleaved caspase3. RESULTS: After inducing apoptosis of AR42J cells in vitro, the expression of miR-22 was significantly increased by 2.20 ± 0.26 and 4.19 ± 0.54 times, respectively, at 3 h and 6 h in comparison with the control group. As revealed by qRT-PCR assay, the expression of miR-22 was 78.25 ± 6.61 times higher in the miR-22 mimic group relative to the miRNA control group, accompanied with an obviously increased acinar cell apoptosis rate (32.53 ± 1.15 vs 18.07 ± 0.89, P = 0.0006). The upregulation of miR-22 could suppress its target gene, ErbB3, and the phosphorylation of PI3k and Akt. Furthermore, we predicted the potential transcription promoter of miR-22 and the binding sites using online tools. Luciferase reporter analysis and site-directed mutagenesis indicated that the binding site (GACAGCCATGTACA) of the GR, which is encoded by the Nr3c1 gene. Downregulation of the expression of GR could upregulate the expression of miR-22, which further promoted the apoptosis of AR42J cells. CONCLUSION: GR transcriptionally represses the expression of miR-22, which further promotes the apoptosis of pancreatic acinar cells by downregulating the downstream signaling pathway.