Reporter Assay Research Articles

Targeted mutagenesis of specific genomic DNA sequences in animals for the in vivo generation of variant libraries.

Understanding how the number, placement and affinity of transcription factor binding sites dictates gene regulatory programs remains a major unsolved challenge in biology, particularly in the context of multicellular organisms. To uncover these rules, it is first necessary to find the binding sites within a regulatory region with high precision, and then to systematically modulate this binding site arrangement while simultaneously measuring the effect of this modulation on output gene expression. Massively parallel reporter assays (MPRAs), where the gene expression stemming from 10,000s of in vitro-generated regulatory sequences is measured, have made this feat possible in high-throughput in single cells in culture. However, because of lack of technologies to incorporate DNA libraries, MPRAs are limited in whole organisms. To enable MPRAs in multicellular organisms, we generated tools to create a high degree of mutagenesis in specific genomic loci in vivo using base editing. Targeting GFP integrated in genome of Drosophila cell culture and whole animals as a case study, we show that the base editor AID evoCDA1 stemming from sea lamprey fused to nCas9 is highly mutagenic. Surprisingly, longer gRNAs increase mutation efficiency and expand the mutating window, which can allow the introduction of mutations in previously untargetable sequences. Finally, we demonstrate arrays of >20 gRNAs that can efficiently introduce mutations along a 200bp sequence, making it a promising tool to test enhancer function in vivo in a high throughput manner.

CRISPR/Cas9-induced knockout of an amino acid permease gene (AAP6) reduced Arabidopsis thaliana susceptibility to Meloidogyne incognita

BackgroundPlant-parasitic root-knot nematode (Meloidogyne incognita) causes global yield loss in agri- and horticultural crops. Nematode management options rely on chemical method. However, only a handful of nematicides are commercially available. Resistance breeding efforts are not sustainable because R gene sources are limited and nematodes have developed resistance-breaking populations against the commercially available Mi-1.2 gene-expressing tomatoes. RNAi crops that manage nematode infection are yet to be commercialized because of the regulatory hurdles associated with transgenic crops. The deployment of the CRISPR/Cas9 system to improve nematode tolerance (by knocking out the susceptibility factors) in plants has emerged as a feasible alternative lately.ResultsIn the present study, a M. incognita-responsive susceptibility (S) gene, amino acid permease (AAP6), was characterized from the model plant Arabidodpsis thaliana by generating the AtAAP6 overexpression line, followed by performing the GUS reporter assay by fusing the promoter of AtAAP6 with the β-glucuronidase (GUS) gene. Upon challenge inoculation with M. incognita, overexpression lines supported greater nematode multiplication, and AtAAP6 expression was inducible to the early stage of nematode infection. Next, using CRISPR/Cas9, AtAAP6 was selectively knocked out without incurring any growth penalty in the host plant. The ‘Cas9-free’ homozygous T3 line was challenge inoculated with M. incognita, and CRISPR-edited A. thaliana plants exhibited considerably reduced susceptibility to nematode infection compared to the non-edited plants. Additionally, host defense response genes were unaltered between edited and non-edited plants, implicating the direct role of AtAAP6 towards nematode susceptibility.ConclusionThe present findings enrich the existing literature on CRISPR/Cas9 research in plant-nematode interactions, which is quite limited currently while compared with the other plant-pathogen interaction systems.

Oxidative modification of miR-30c promotes cardiac fibroblast proliferation via CDKN2C mismatch

The response of cardiac fibroblast proliferation to detrimental stimuli is one of the main pathological factors causing heart remodeling. Reactive oxygen species (ROS) mediate the proliferation of cardiac fibroblasts. However, the exact molecular mechanism remains unclear. In vivo, we examined the oxidative modification of miRNAs with miRNA immunoprecipitation with O8G in animal models of cardiac fibrosis induced by Ang II injection or ischemia‒reperfusion injury. Furthermore, in vitro, we constructed oxidation-modified miR-30c and investigated its effects on the proliferation of cardiac fibroblasts. Additionally, luciferase reporter assays were used to identify the target of oxidized miR-30c. We found that miR-30c oxidation was modified by Ang II and PDGF treatment and mediated by excess ROS. We demonstrated that oxidative modification of G to O8G occurred at positions 4 and 5 of the 5′ end of miR-30c (4,5-oxo-miR-30c), and this modification promoted cardiac fibroblast proliferation. Furthermore, CDKN2C is a negative regulator of cardiac fibroblast proliferation. 4,5-oxo-miR-30c misrecognizes CDKN2C mRNA, resulting in a reduction in protein expression. Oxidized miR-30c promotes cardiac fibroblast proliferation by mismatch mRNA of CDKN2C.

Characterization of NR1J1 Paralog Responses of Marine Mussels: Insights from Toxins and Natural Activators.

The pregnane X receptor (PXR) is a nuclear hormone receptor that plays a pivotal role in regulating gene expression in response to various ligands, particularly xenobiotics. In this context, the aim of this study was to shed light on the ligand affinity and functions of four NR1J1 paralogs identified in the marine mussel Mytilus galloprovincialis, employing a dual-luciferase reporter assay. To achieve this, the activation patterns of these paralogs in response to various toxins, including freshwater cyanotoxins (Anatoxin-a, Cylindrospermopsin, and Microcystin-LR, -RR, and -YR) and marine algal toxins (Nodularin, Saxitoxin, and Tetrodotoxin), alongside natural compounds (Saint John's Wort, Ursolic Acid, and 8-Methoxypsoralene) and microalgal extracts (Tetraselmis, Isochrysis, LEGE 95046, and LEGE 91351 extracts), were studied. The investigation revealed nuanced differences in paralog response patterns, highlighting the remarkable sensitivity of MgaNR1J1γ and MgaNR1J1δ paralogs to several toxins. In conclusion, this study sheds light on the intricate mechanisms of xenobiotic metabolism and detoxification, particularly focusing on the role of marine mussel NR1J1 in responding to a diverse array of compounds. Furthermore, comparative analysis with human PXR revealed potential species-specific adaptations in detoxification mechanisms, suggesting evolutionary implications. These findings deepen our understanding of PXR-mediated metabolism mechanisms, offering insights into environmental monitoring and evolutionary biology research.

Hypoxia-mediated repression of pyruvate carboxylase drives immunosuppression

BackgroundMetabolic plasticity mediates breast cancer survival, growth, and immune evasion during metastasis. However, how tumor cell metabolism is influenced by and feeds back to regulate breast cancer progression are not fully understood. We identify hypoxia-mediated suppression of pyruvate carboxylase (PC), and subsequent induction of lactate production, as a metabolic regulator of immunosuppression.MethodsWe used qPCR, immunoblot, and reporter assays to characterize repression of PC in hypoxic primary tumors. Steady state metabolomics were used to identify changes in metabolite pools upon PC depletion. In vivo tumor growth and metastasis assays were used to evaluate the impact of PC manipulation and pharmacologic inhibition of lactate transporters. Immunohistochemistry, flow cytometry, and global gene expression analyzes of tumor tissue were employed to characterize the impact of PC depletion on tumor immunity.ResultsPC is essential for metastatic colonization of the lungs. In contrast, depletion of PC in tumor cells promotes primary tumor growth. This effect was only observed in immune competent animals, supporting the hypothesis that repression of PC can suppress anti-tumor immunity. Exploring key differences between the pulmonary and mammary environments, we demonstrate that hypoxia potently downregulated PC. In the absence of PC, tumor cells produce more lactate and undergo less oxidative phosphorylation. Inhibition of lactate metabolism was sufficient to restore T cell populations to PC-depleted mammary tumors.ConclusionsWe present a dimorphic role for PC in primary mammary tumors vs. pulmonary metastases. These findings highlight a key contextual role for PC-directed lactate production as a metabolic nexus connecting hypoxia and antitumor immunity.Graphical abstract

Qiju Dihuang Pill protects the lens epithelial cells via alleviating cuproptosis in diabetic cataract

Ethnopharmacological relevanceQiju Dihuang Pill (QDP) is a traditional Chinese medicine prescription for the treatment of eye diseases. Novel literature reports that copper-induced cell death, called as cuproptosis, is a copper-dependent and differs distinctly from other types of cell death. Aim of the studyThe present study aims to investigate whether QDP could protect lens epithelial cells via alleviating copper-induced death in diabetic cataract. Materials and methodsThe different concentration of QDP medicated serum was administrated on high glucose (HG)-induced human lens epithelial cells (HLECs). The copper concentration was tested using Elabscience Copper Assay kit. The proliferation was detected using CCK-8 and EdU assays. The molecular binding was identified using RIP-PCR and luciferase reporter assay. ResultsResults indicated that HG culture condition triggered the copper concentration and repressed the proliferation of HLECs. Then, the elesclomol-Cu (Es–Cu) administration up-regulated the copper concentration and inhibited the proliferation, and cuproptosis inhibitor tetrathiomolybdate (TTM) could specifically reverse the consequence. QDP treatment reduced the copper concentration and cuproptosis-related genes (SLC31A1, FDX1). MeRIP-Seq and RIP-PCR confirmed that QDP reduced the stability of SLC31A1 mRNA through m6A modified site, and copper actually synergized the molecular binding efficiency. Rescue assay verified the role of QDP and SLC31A1 on HLECs’ cuproptosis characteristic. ConclusionThis research identified the protective role of QDP on HG-induced HLECs in DC through decreasing m6A/SLC31A1-mediated cuproptosis in DC. This finding provides novel insights into mechanisms for QDP and sheds light on the multifaceted role of traditional prescription on DC.

WRKY47 transcription factor modulates leaf senescence through regulating PCD-associated genes in Arabidopsis

Transcription factors play crucial roles in almost all physiological processes including leaf senescence. Cell death is a typical symptom appearing in senescing leaves, which is also classified as developmental programmed cell death (PCD). However, the link between PCD and leaf senescence still remains unclear. Here, we found a WRKY transcription factor WRKY47 positively modulates age-dependent leaf senescence in Arabidopsis (Arabidopsis thaliana). WRKY47 was expressed preferentially in senescing leaves. A subcellular localization assay indicated that WRKY47 was exclusively localized in nuclei. Overexpression of WRKY47 showed precocious leaf senescence, with less chlorophyll content and higher electrolyte leakage, but loss-of-function mutants of WRKY47 delayed this biological process. Through qRT-PCR and dual luciferase reporter assays, we found that WRKY47 could activate the expression of senescence-associated genes (SAGs) and PCD-associated genes to regulate leaf senescence. Furthermore, through electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP)-qPCR, WRKY47 was found to bind to W-box fragments in promoter regions of BFN1 (Bifunctional Nuclease 1) and MC6 (Metacaspase 6) directly. In general, our research revealed that WRKY47 regulates age-dependent leaf senescence by activating the transcription of two PCD-associated genes.

LncRNA MIR181A2HG inhibits keratinocytes proliferation through miR-223-3p/SOX6 axis.

Psoriasis is a complex and recurrent chronic inflammatory skin disease, and the abnormal proliferation of keratinocytes plays a crucial role in the pathogenesis of psoriasis. Long non-coding RNAs (lncRNAs) play an indispensable role in regulating cellular functions. This research aims to explore the potential impact of lncRNA MIR181A2HG on the regulation of keratinocyte proliferation. The expression level of MIR181A2HG and the mRNA level of KRT6, KRT16, and SOX6 were assessed using qRT-PCR. The viability and proliferation of keratinocytes were evaluated using CCK-8 and EdU assays. Cell cycle analysis was performed using flow cytometry. Dual-luciferase reporter assays were applied to test the interaction among MIR181A2HG/miR-223-3p/SOX6. Protein level was detected by Western blotting analysis. The findings indicated that psoriasis lesions tissue exhibited lower levels of MIR181A2HG expression compared to normal tissue. The overexpression of MIR181A2HG resulted in the inhibition of HaCaT keratinocytes proliferation. The knockdown of MIR181A2HG promoted cell proliferation. The dual-luciferase reporter assay and rescue experiments provided evidence of the interaction among MIR181A2HG, SOX6, and miR-223-3p. The lncRNA MIR181A2HG functions as a miR-223-3p sponge targeting SOX6 to regulate the proliferation of keratinocytes, which suggested that MIR181A2HG/miR-223-3p/SOX6 might be potential diagnostic and therapeutic targets for psoriasis.

Inhibitor of apoptosis stimulating protein of p53 protects against MPP+-induced neurotoxicity of dopaminergic neurons.

Inhibitor of apoptosis stimulating protein of p53 (iASPP) is related to the pathogenesis of several neurological disorders by affecting the oxidative stress and survival of neurons. However, whether iASPP has a role in Parkinson disease (PD) remains to be determined. This work explored the potential regulatory effect of iASPP in an in vitro model of PD based on 1-methyl-4-phenylpyridinium (MPP+)-evoked neurotoxicity of dopaminergic neurons in culture. MN9D neurons were treated with MPP+ at 200 µM in the culture media for 24h to induce neurotoxicity. Overexpression and silencing of iASPP in neurons were achieved by infecting recombinant adenovirus expressing iASPP and sh-iASPP, respectively. Protein expression was examined by immunoblotting. MPP+-evoked neurotoxicity of dopaminergic neurons was determined by cell viability, TUNEL, and flow cytometric assays. The transcriptional activity of nuclear erythroid factor 2-like 2 (Nrf2) was assessed by luciferase reporter assay. Kelch-like ECH-associated protein 1 (Keap1)-knockout neurons were generated by lentiCRISPR/Cas9-Keap1 constructs. Expression levels of iASPP declined in MPP+-stimulated neurons. Overexpression of iASPP in neurons exhibited inhibitory effects on MPP+-evoked apoptosis, α-synuclein accumulation, and oxidative stress, while iASPP-deficient neurons were more sensitive to MPP+-induced neurotoxicity. Overexpression of iASPP led to an enhancing effect on Nrf2 activation in MPP+-stimulated neurons. Mechanism research revealed that iASPP may contribute to the activation of Nrf2 by competing with Nrf2 in binding with Keap1. Notably, the regulatory effect of iASPP on Nrf2 was diminished in Keap1-knockout neurons. The chemical inhibition of Nrf2 or knockdown of Nrf2 abrogated the protective effects of iASPP on MPP+-induced neurotoxicity. To conclude, iASPP protects dopaminergic neurons against MPP+-induced neurotoxicity through modulation of the Keap1/Nrf2 axis. Therefore, iASPP may play a crucial role in mediating the loss of dopaminergic neurons in PD, and targeting the iASPP-Nrf2 axis could be a promising strategy for treating PD.

Mapping putative enhancers in mouse oocytes and early embryos reveals TCF3/12 as key folliculogenesis regulators.

Dynamic epigenomic reprogramming occurs during mammalian oocyte maturation and early development. However, the underlying transcription circuitry remains poorly characterized. By mapping cis-regulatory elements using H3K27ac, we identified putative enhancers in mouse oocytes and early embryos distinct from those in adult tissues, enabling global transitions of regulatory landscapes around fertilization and implantation. Gene deserts harbour prevalent putative enhancers in fully grown oocytes linked to oocyte-specific genes and repeat activation. Embryo-specific enhancers are primed before zygotic genome activation and are restricted by oocyte-inherited H3K27me3. Putative enhancers in oocytes often manifest H3K4me3, bidirectional transcription, Pol II binding and can drive transcription in STARR-seq and a reporter assay. Finally, motif analysis of these elements identified crucial regulators of oogenesis, TCF3 and TCF12, the deficiency of which impairs activation of key oocyte genes and folliculogenesis. These data reveal distinctive regulatory landscapes and their interacting transcription factors that underpin the development of mammalian oocytes and early embryos.

LINC01133 contributes to the malignant phenotypes of non-small cell lung cancer by targeting miR-30b-5p/FOXA1 pathway.

This study aimed to explore the mechanism of action of LINC01133 in non-small cell lung cancer. LINC01133 expression in NSCLC patient tissues and cells was detected by qRT-PCR. After transfecting siRNA-LINC01133 in NSCLC cells, the proliferation and invasive migration ability of the cells were assessed via CCK-8 and Transwell assay, respectively. The sublocalization of LINC01133 in NSCLC cells was analyzed by bioinformatics prediction and nucleoplasm separation assay and RNA-FISH assay. Analysis of the binding relationship between LINC01133, FOXA1 and miR-30b-5p was all through bioinformatics website analysis, dual-luciferase reporter and RNA Pulldown assay. Functional rescue experiments confirmed the character of miR-30b-5p and FOXA1 in LINC01133 regulating the NSCLC cells biological behavior. LINC01133 high expressions were found in NSCLC tissues and cells. siRNA-LINC01133 treatment inhibited NSCLC cells malignant behavior. Mechanistically: LINC01133 promoted FOXA1 expression through adsorption binding of miR-30b-5p. Knocking down miR-30b-5p expression or up-regulating FOXA1 expression was able to reverse siRNA-LINC01133 inhibitory effect of tumor cell malignant behavior. LINC01133 promoted FOX1 expression by competitively binding miR-30b-5p, which attenuated the targeting inhibitory effect of miR-30b-5p on FOXA1 and ultimately promoted proliferation and invasive migration of NSCLC cells.

Genetic variability of aquaporin expression in maize: from eQTLs to a MITE insertion regulating PIP2; 5 expression.

Plant aquaporins are involved in numerous physiological processes, such as cellular homeostasis, tissue hydraulics, transpiration, and nutrient supply, and are key players of the response to environmental cues. While varying expression patterns of aquaporin genes have been described across organs, developmental stages and stress conditions, the underlying regulation mechanisms remain elusive. Hence, this work aimed to shed light on the expression variability of four plasma membrane intrinsic protein (PIP) genes in maize (Zea mays) leaves, and its genetic causes, through eQTL (expression quantitative trait locus) mapping across a 252-hybrid diversity panel. Significant genetic variability in PIP transcript abundance was observed to different extents depending on the isoforms. The genome-wide association study mapped numerous eQTLs, both local and distant, thus emphasizing the existing natural diversity of PIP gene expression across the studied panel and the potential to reveal regulatory actors and mechanisms. One eQTL associated with PIP2; 5 expression variation was characterized. Genomic sequence comparison and in vivo reporter assay attributed, at least partly, the local eQTL to a transposon-containing polymorphism in the PIP2; 5 promoter. This work paves the way to the molecular understanding of PIP gene regulation and its possible integration into larger networks regulating physiological and stress-adaptation processes.

MicroR-1199-5p targeting SRD5A2 promotes the biological behavior and EMT of hypospadias cells.

Hypospadias, an oft-occurring penis anomaly, ranks among neonatal's foremost birth defects. The SRD5A2 can affect male reproductive system development and is abnormally expressed in its epithelial cells. This study exploration aimed at understanding the role of SRD5A2 in the development of hypospadias from a molecular perspective. SRD5A2 levels in hypospadias primary cells were analyzed by Western blot, while targeted interaction with miR-1199-5p was ascertained by dual-luciferase gene reporter assay. In vitro biological experiments were used to confirm the biological function of SRD5A2 in hypospadias. SRD5A2 expression was significantly upregulated, and miR-1199-5p expression was significantly downregulated in hypospadias primary cells. Intervention of SRD5A2 expression can affect cell proliferation, migration, invasion, EMT, and the expression of cell cycle-related proteins. Additionally, we found that SRD5A2 is regulated by upstream miR-1199-5p and can enhance the effect of SRD5A2 on hypospadias cells. Conclusions Silencing SRD5A2 promotes cell proliferation, invasion, and migration blocks the cell cycle at the G1 phase, and simultaneously promotes EMT, cell cycle, and cell proliferation-related protein expression. The biological function of SRD5A2 in hypospadias cells is regulated by miR-1199-5p. SRD5A2 may be an effective therapeutic target for hypospadias.

M2 macrophages regulate KDM6B/PFKFB2 metabolic reprogramming of cervical squamous cell carcinoma through CXCL1.

Macrophages in the tumor microenvironment can polarize into M1 or M2 forms, with M2 macrophages (M2φ) promoting tumor growth and metastasis in cervical squamous cell carcinoma (CESC). This study explored the effects of M2φ on CESC metabolic reprogramming both in vitro and in vivo. Results showed that M2φ secreted CXCL1, which significantly increased CESC migration and metabolic regulation. Further experiments revealed that CXCL1 upregulated KDM6B to enhance PFKFB2 transcriptional activity, thus regulating CESC glucose metabolism. Transcriptome sequencing screened 5 upregulated genes related to glycolysis, with PFKFB2 showing the most significant increase in cells treated with rCXCL1. Dual-luciferase reporter assay confirmed that rCXCL1 enhances PFKFB2 transcriptional activity. Bioinformatics analysis revealed a high correlation between expressions of KDM6B and PFKFB2 in CESC. Mechanistic experiments demonstrated that KDM6B inhibited H3K27me3 modification to activate PFKFB2 transcriptional expression. In conclusion, M2φ secreted CXCL1 to promote CESC cell migration and invasion, and CXCL1 activated KDM6B expression in CESC cells, inhibiting H3K27 protein methylation modification, and enhanced PFKFB2 transcriptional activity to regulate CESC glucose metabolism. These results provided new insights into the complex interplay between the immune system and cancer metabolism, which may have broader implications for understanding and treating other types of cancer.

LINC00472 suppresses non-small cell lung cancer progression via regulating miR-23a-3p/CCL22 axis.

Long non-coding RNA (lncRNA) LINC00472 has a close connection with the development of tumors. The aim was to explore the role of LINC00472 on NSCLC cell biological function in vivo and its potential mechanisms. The mRNA levels of LncRNA 00472 and microRNA-23a-3p, were determined by RT-qPCR. Cell Counting Kit-8, cell scratches and western blot assays were used to analyze the proliferation, migration and level of apoptosis-associated proteins. Luciferase reporter assay validates the binding between LINC00472/CCL22 and miR-23a-3p. LINC00472 and CCL22 were lowly expressed in NSCLC tissues and cells, while miR-23a-3p expression was upregulated. LINC00472 overexpression significantly depressed NSCLC cell cellular behavior, whereas promoting cell death. MiR-23a-3p could reverse these above-mentioned biological behavior changes caused by LINC00472 overexpression. Additionally, LINC00472 increased CCL22 expression through sponging miR-23a-3p. Knocking down CCL22 antagonized the inhibitory effect of LINC00472 on NSCLC cell survival. LINC00472 may reduce the cellular growth, and accelerate death of NSCLC through increasing CCL22 expression by targeting miR-23a-3p.

METTL3 promotes the osteogenic differentiation of human periodontal ligament cells by increasing YAP activity via IGF2BP1 and YTHDF1-mediated m6A modification.

N6-Methyladenosine (m6A) has been confirmed to play a dynamic role in osteoporosis and bone metabolism. However, whether m6A is involved in the osteogenic differentiation of human periodontal ligament cells (hPDLCs) remains unclear. The present study aimed to verify the role of methyltransferase-like 3 (METTL3)-mediated m6A modification in the osteogenic differentiation of hPDLCs. The METTL3, Runx2, Osx, and YAP mRNA expression was determined by qPCR. METTL3, RUNX2, OSX, YTHDF1, YAP, IGF2BP1, and eIF3a protein expression was measured by Western blotting and immunofluorescence assays. The levels of m6A modification were evaluated by methylated RNA immunoprecipitation (MeRIP) and dot blot analyses. MeRIP-seq and RNA-seq were used to screen potential candidate genes. Nucleic acid and protein interactions were detected by immunoprecipitation. Alizarin red staining was used to evaluate the osteogenic differentiation of hPDLCs. Gene transcription and promoter activities were assessed by luciferase reporter assays (n ≥ 3). The expression of METTL3 and m6A modifications increased synchronously with the osteogenic differentiation of hPDLCs (p = .0016). YAP was a candidate gene identified by MeRIP-seq and RNA-seq, and its mRNA and protein expression levels were simultaneously increased. METTL3 increased the m6A methylated IGF2BP1-mediated stability of YAP mRNA (p = .0037), which in turn promoted osteogenic differentiation (p = .0147). Furthermore, METTL3 increased the translation efficiency of YAP by recruiting YTHDF1 and eIF3a to the translation initiation complex (p = .0154), thereby promoting the osteogenic differentiation of hPDLCs (p = .0012). Our study revealed that METTL3-initiated m6A mRNA methylation promotes osteogenic differentiation of hPDLCs by increasing IGF2BP1-mediated YAP mRNA stability and recruiting YTHDF1 and eIF3a to the translation initiation complex to increase YAP mRNA translation. Our findings reveal the mechanism of METTL3-mediated m6A modification during hPDLC osteogenesis, providing a potential therapeutic target for periodontitis and alveolar bone defects.

Circ_0084615 promotes epithelial‐mesenchymal transition‐mediated tumor progression in hepatocellular carcinoma

AbstractAimCircRNAs have been identified as crucial regulators in tumorigenesis and progression. This study aimed to explore the biological role and underlying mechanism of circ_0084615 in hepatocellular carcinoma (HCC).MethodsThe expression of RNAs was detected by quantitative reverse transcription‐polymerase chain reaction (qRT‐PCR). The effects of circ_0084615 silencing on malignant behaviors of HCC cells were assessed by CCK‐8, colony formation, wound healing, and Transwell assays in vitro and tumor transplantation experiment in vivo. The expression of proteins was detected by Western blotting. Dual‐luciferase reporter assay and RNA‐binding protein immunoprecipitation were performed to explore the mechanism of circ_0084615.ResultsA significant upregulation of circ_0084615 was observed in HCC tissues, and positively correlated with the TNM staging. Silencing of circ_0084615 impeded HCC cell viability, colony formation, migration, invasion, epithelial‐mesenchymal transition, and xenograft tumor growth. Mechanistically, circ_0084615 could bind to miR‐1200 and eliminate its ability to destroy actin‐like 6A (ACTL6A) mRNA, thereby increasing ACTL6A expression and facilitating the malignant behaviors of HCC cells.ConclusionsThis study clarified the oncogenic activity and mechanism of circ_0084615, thereby providing potential diagnostic biomarker and therapeutic target for inhibiting HCC progression.

Circular RNA_015343 sponges microRNA-25 to regulate viability, proliferation, and milk fat synthesis of ovine mammary epithelial cells via INSIG1.

In our previous study, circ_015343 was found to inhibit the viability and proliferation of ovine mammary epithelial cells (OMECs) and the expression levels of milk fat synthesis marker genes, but the regulatory mechanism underlying the processes is still unclear. Accordingly in this study, the target relationships between circ_015343 with miR-25 and between miR-25 with insulin induced gene 1 (INSIG1) were verified, and the functions of miR-25 and INSIG1 were investigated in OMECs. The dual-luciferase reporter assay revealed that miR-25 mimic remarkably decreased the luciferase activity of circ_015343 in HEK293T cells cotransfected with a wild-type vector, while it did not change the activity of circ_015343 in HEK293T cells cotransfected with a mutant vector. These suggest that cic_015343 can adsorb and bind miR-25. The miR-25 increased the viability and proliferation of OMECs, and the content of triglycerides in OMECs. In addition, INSIG1 was found to be a target gene of miR-25 using a dual-luciferase reporter assay. Overexpression of INSIG1 decreased the viability, proliferation, and level of triglycerides of OMECs. In contrast, the inhibition of INSIG1 in expression had the opposite effect on activities and triglycerides of OMECs with overexpressed INSIG1. A rescue experiment revealed that circ_015343 alleviated the inhibitory effect of miR-25 on the mRNA and protein abundance of INSIG1. These results indicate that circ_015343 sponges miR-25 to inhibit the activities and content of triglycerides of OMECs by upregulating the expression of INSIG1 in OMECs. This study provided new insights for understanding the genetic molecular mechanism of lactation traits in sheep.

Extracellular vesicles activated cancer-associated fibroblasts promote lung cancer metastasis through mitophagy and mtDNA transfer.

Studies have shown that oxidative stress and its resistance plays important roles in the process of tumor metastasis, and mitochondrial dysfunction caused by mitochondrial DNA (mtDNA) damage is an important molecular event in oxidative stress. In lung cancer, the normal fibroblasts (NFs) are activated as cancer-associated fibroblasts (CAFs), and act in the realms of the tumor microenvironment (TME) with consequences for tumor growth and metastasis. However, its activation mechanism and whether it participates in tumor metastasis through antioxidative stress remain unclear. The role and signaling pathways of tumor cell derived extracellular vesicles (EVs) activating NFs and the characteristic of induced CAFs (iCAFs) were measured by the transmission electron microscopy, nanoparticle tracking analysis, immunofluorescence, collagen contraction assay, quantitative PCR, immunoblotting, luciferase reporter assay and mitochondrial membrane potential detection. Mitochondrial genome and single nucleotide polymorphism sequencing were used to investigate the transport of mtDNA from iCAFs to ρ0 cells, which were tumor cells with mitochondrial dysfunction caused by depletion of mtDNA. Further, the effects of iCAFs on mitochondrial function, growth and metastasis of tumor cells were analysed in co-culture models both in vitro and in vivo, using succinate dehydrogenase, glutathione and oxygen consumption rate measurements, CCK-8 assay, transwell assay, xenotransplantation and metastasis experiments as well as in situ hybridization and immunohistochemistry. Our findings revealed that EVs derived from high-metastatic lung cancer cells packaged miR-1290 that directly targets MT1G, leading to activation of AKT signaling in NFs and inducing NFs conversion to CAFs. The iCAFs exhibit higher levels of autophagy and mitophagy and more mtDNA release, and reactive oxygen species (ROS) could further promote this process. After cocultured with the conditioned medium (CM) of iCAFs, the ρ0 cells may restore its mitochondrial function by acquisition of mtDNA from CAFs, and further promotes tumor metastasis. These results elucidate a novel mechanism that CAFs activated by tumor-derived EVs can promote metastasis by transferring mtDNA and restoring mitochondrial function of tumor cells which result in resistance of oxidative stress, and provide a new therapeutic target for lung cancer metastasis.

MicroRNA-3061 downregulates the expression of PAX7/Wnt/Ca2+ signalling axis genes to induce premature ovarian failure in mice.

The in-depth mechanisms of microRNA regulation of premature ovarian failure (POF) remain unclear. Crispr-cas9 technology was used to construct transgenic mice. The qPCR and Western blot was used to detect the expression level of genes. H&E staining were used to detect ovarian pathological phenotypes. We found that the expression levels of microRNA-3061 were significantly higher in ovarian granulosa cells (OGCs) of POF mouse models than in controls. The miR-3061+/-/AMH-Cre+/- transgenic mice manifested symptoms of POF. RNA-Seq and luciferase reporter assay confirmed that the PAX7 was one of the target genes negatively regulated by microRNA-3061 (miR-3061-5p). Moreover, PAX7 mediated the expression of non-canonical Wnt/Ca2+ signalling pathway by binding to the motifs of promoters to stimulate the transcriptional activation of Wnt5a and CamK2a. In contrast, specific knock-in of microRNA-3061 in OGCs significantly downregulated the expression levels of PAX7 and inhibited the expression of downstream Wnt/Ca2+ signalling pathway. We also discerned a correlation between the expression levels of mRNAs of the Wnt/Ca2+ signalling pathway and the levels of E2 and FSH in POF patients by examining gene expression in the follicular fluid-derived exosomes of women. We confirmed that overexpression of microRNA-3061 induced proliferative inhibition of OGCs and ultimately induced POF in mice by suppressing the transcription factor PAX7 and downregulating expression levels of its downstream Wnt/Ca2+ signalling pathway genes.