Dual Luciferase Activity Assay Research Articles

Characterization of pectin methylesterase gene family and its possible role in juice sac granulation in navel orange (Citrus sinensis Osbeck)

BackgroundCitrus is one of the most important fresh fruit crops worldwide. Juice sac granulation is a physiological disorder, which leads to a reduction in soluble solid concentration, total sugar, and titratable acidity of citrus fruits. Pectin methylesterase (PME) catalyzes the de-methylesterification of homogalacturonans and plays crucial roles in cell wall modification during plant development and fruit ripening. Although PME family has been well investigated in various model plants, little is known regarding the evolutionary property and biological function of PME family genes in citrus.ResultsIn this study, 53 non-redundant PME genes were identified from Citrus sinensis genome, and these PME genes were divided into four clades based on the phylogenetic relationship. Subsequently, bioinformatics analyses of gene structure, conserved domain, chromosome localization, gene duplication, and collinearity were performed on CsPME genes, providing important clues for further research on the functions of CsPME genes. The expression profiles of CsPME genes in response to juice sac granulation and low-temperature stress revealed that CsPME genes were involved in the low temperature-induced juice sac granulation in navel orange fruits. Subcellular localization analysis suggested that CsPME genes were localized on the apoplast, endoplasmic reticulum, plasma membrane, and vacuole membrane. Moreover, yeast one-hybrid screening and dual luciferase activity assay revealed that the transcription factor CsRVE1 directly bound to the promoter of CsPME3 and activated its activity.ConclusionIn summary, this study conducts a comprehensive analysis of the PME gene family in citrus, and provides a novel insight into the biological functions and regulation patterns of CsPME genes during juice sac granulation of citrus.

The Circ-CYP24A1-miR-224-PRLR Axis Impairs Cell Proliferation and Apoptosis in Recurrent Miscarriage.

AimRecurrent miscarriage (RM) is associated with numerous clinical factors. However, some RM occurred without specific factors. It has been revealed that some molecules such as hormones, miRNAs, and transcription factors are involved in RM by regulating proliferation, apoptosis, etc. However, the mechanism of RM has yet to be identified clearly. Circular RNAs (circRNAs) are a class of endogenous non-coding RNAs that often act as sponges for miRNAs or binds to proteins involved in biological processes. However, the functional role of circRNAs in the uterine decidua of patients with early RM is still unclear. In this study, we aimed to investigate the mechanisms of circ-CYP24A1 in RM.MethodsThe Dual-Luciferase Activity Assay was designed to analyze the bonding between circ-CYP24A1 and miR-224, and miR-224 and prolactin receptor (PRLR) mRNA 3′UTR. In situ hybridization (ISH) and immunohistochemistry (IHC) were used to observe the expression of circ-CYP24A1 and PRLR in the decidua. Rescue experiments were performed to investigate the regulating effects of circ-CYP24A1, miR-224, and PRLR. Western blotting was conducted to test the expression level of PRLR. The proliferation and apoptosis-related markers in Ishikawa cells were analyzed using CCK8, immunofluorescence staining, and the terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay.ResultsIn this study, based on the microarray analysis data, we identified a high level of circ-CYP24A1 and PRLR in the decidua of patients with early RM. Based on the bioinformatics prediction, the binding relationship between circ-CYP24A1 and miR-224, as well as miR-224 and PRLR, were verified. Functional experiments demonstrated that circ-CYP24A1 regulated proliferation and apoptosis by binding to and inhibiting miR-224, resulting in increased PRLR expression. Taken together, this study provides new insights into the mechanism of RM.ConclusionIn this study, we found that circ-CYP24A1 plays a role in RM by impairing the balance of cell proliferation and apoptosis by sponging miR-224, thereby regulating PRLR.

The Competitive Endogenous RNA (ceRNA) Regulation in Porcine Alveolar Macrophages (3D4/21) Infected by Swine Influenza Virus (H1N1 and H3N2).

H1N1 and H3N2 are the two most common subtypes of swine influenza virus (SIV). They not only endanger the pig industry, but are also a huge risk of zoonotic diseases. However, the molecular mechanism and regulatory network of pigs (hosts) against influenza virus infection are still unclear. In this study, porcine alveolar macrophage cell (3D4/21) models infected by swine influenza virus (H1N1 and H3N2) were constructed. The expression profiles of miRNAs, mRNAs, lncRNAs and circRNAs after H1N1 and H3N2 infected 3D4/21 cells were revealed in this study. Then, two ceRNAs (TCONS_00166432-miR10391-MAN2A1 and novel_circ_0004733-miR10391-MAN2A1) that regulated H1N1 and H3N2 infection in 3D4/21 cells were verified by the methods of bioinformatics analysis, gene overexpression, gene interference, real-time quantitative PCR (qPCR), dual luciferase activity assay and RNA immunoprecipitation (RIP). In addition, the important candidate molecules (miR-10391, TCONS_00166432, and novel_circ_0004733) were identified by qPCR and enzyme linked immunosorbent assay (ELISA). Finally, the regulatory effect and possible molecular mechanism of the target gene MAN2A1 were identified by the methods of gene interference, qPCR, Western blot and ELISA. The results of this study suggested that TCONS_00166432 and novel_circ_0004733 could competitively bind miR-10391 to target the MAN2A1 gene to regulate swine influenza virus infecting 3D4/21 cells. This study reported for the first time the ceRNA networks involved in the regulation of the swine influenza virus infecting 3D4/21 cells, which provided a new insight into the molecular mechanism of 3D4/21 cells against swine influenza virus infection.

LINC 01436 is overexpressed in colorectal cancer and promotes cancer cell proliferation by suppressing tumor-suppressive miR-466 maturation.

LINC 01436 (lncRNA) promotes lung and gastric cancers. However, it is unclear whether it participates in colorectal cancer (CRC) progression. Therefore, the study was carried out to analyze the role of LINC 01436 in CRC. LINC 01436 expressions in CRC tissues were analyzed by RT-qPCR, and its prognostic value was investigated in a follow-up study. Correlation between LINC 01436 and mature miR-466 or miR-466 precursor was analyzed by linear regression. Mature miR-466 and miR-466 precursor levels in CRC cells with LINC 01436 overexpression were studied using RT-qPCR. CRC cell proliferation was evaluated using CCK-8 assay. LINC 01436 was upregulated in CRC and predicted poor survival. LINC 01436 was inversely correlated with mature miR-466, but not miR-466 precursor. LINC 01436 was predicted to bind with miR-466 precursor. Their interaction was further verified by dual-luciferase activity assay. In CRC cells, LINC 01436 overexpression downregulated mature miR-466 but not miR-466 precursor. Cell proliferation analysis showed that LINC 01436 overexpression rescued cell proliferation reduced by miR-466. LINC 01436 is overexpressed in CRC and promotes cancer cell proliferation by suppressing miR-466 maturation.

Epigenetic axis of SNHG19/miR-137/TNFAIP1 modulates amyloid beta peptide 25-35-induced SH-SY5Y cytotoxicity.

Aims: In this study, the authors hypothesized that, in an in vitro Alzheimer's disease model, the epigenetic axis of SNHG19/hsa-miR-137 functionally regulates amyloid beta peptide 25-35 (Aβ25-35)-induced SH-SY5Y cytotoxicity. Methods: Dual luciferase activity assay demonstrated that SNHG19 could directly bind hsa-miR-137. In Aβ25-35-treated SH-SY5Y cells, SNHG19 was upregulated and hsa-miR-137 downregulated. Results: SNHG19 knockdown ameliorated Aβ25-35-induced SH-SY5Y cytotoxicity, then reversed by secondary hsa-miR-137 downregulation. TNFAIP1 was dynamically regulated by Aβ25-35 and gene modifications in SH-SY5Y cells. Finally, upregulation of TNFAIP1 reversed the protective effect of SNHG19 knockdown on Aβ25-35-induced cytotoxicity. Conclusions: The authors concluded that the epigenetic axis of SNHG19/hsa-miR-137/TNFAIP1 may functionally regulate Aβ25-35-induced SH-SY5Y cytotoxicity, thus making it a potential molecular target for Alzheimer's disease treatment.

The R2R3-MYB transcription factor FaMYB63 participates in regulation of eugenol production in strawberry.

The biosynthetic pathway of volatile phenylpropanoids, including 4-allyl-2-methoxyphenol (eugenol), has been investigated in petunia (Petunia hybrida). However, the regulatory network for eugenol accumulation in strawberry (Fragaria × ananassa Duch.) fruit remains unclear. Here, an R2R3-type MYB transcription factor (TF; FaMYB63) was isolated from strawberry by yeast one-hybrid (Y1H) screening using the promoter of the FaEGS1 (eugenol synthase 1 [EGS 1]) gene, which encodes the enzyme responsible for the last step in eugenol biosynthesis. FaMYB63 is phylogenetically distinct from other R2R3-MYB TFs, including FaEOBІІ (EMISSION OF BENZENOID II [EOBII]), which also participates in regulating eugenol biosynthesis in strawberry receptacles. Reverse transcription quantitative PCR (RT-qPCR) assays showed that the expression of FaMYB63 was tissue-specific and consistent with eugenol content through strawberry fruit development, was repressed by abscisic acid, and was activated by auxins (indole-3-acetic acid). Overexpression and RNA interference-mediated silencing of FaMYB63 resulted in marked changes in the transcript levels of the biosynthetic genes FaEGS1, FaEGS2, and FaCAD1 (cinnamyl alcohol dehydrogenase 1 [CAD1]) and, thereby, the accumulation of eugenol. Electrophoretic mobility shift, Y1H, GUS activity, and dual-luciferase activity assays demonstrated that the transcript levels of FaEOBІІ and FaMYB10 were regulated by FaMYB63, but not the other way around. Together, these results demonstrate that FaMYB63 directly activates FaEGS1, FaEGS2, FaCAD1, FaEOBІІ, and FaMYB10 to induce eugenol biosynthesis during strawberry fruit development. These findings deepen the understanding of the regulatory network that influences eugenol metabolism in an edible fruit crop.

MiR-124-3p targeting PDE4B attenuates LPS-induced ALI through the TLR4/NF-κB signaling pathway

BackgroundA large number of studies have found that microRNAs (miRNAs) and phosphodiesterase 4 (PDE4) are crucial regulators of inflammatory responses in acute lung injury (ALI). ObjectiveThis study will explore the protective effect of miR-124-3p on ALI and its related mechanism. MethodsThe ALI mouse model was established by intratracheal administration of lipopolysaccharide (LPS) and evaluated by haematoxylin and eosin (HE) staining, lung injury score, inflammation factors, polymorphonuclear leukocyte (PMN) count, total protein and lung wet weight/dry weight (W/D) ratio. MiR-124-3p was overexpressed in vivo by intratracheal administration of miR-agomir, and PDE4B was expressed at low level in vivo by intratracheal administration of a PDE4B inhibitor. The mRNA expression level was detected by qRT–PCR, and the protein expression level was detected by Western blot. The relationship between miR-124-3p and PDE4B was detected by dual-luciferase activity assay. ResultsWe found that miR-124-3p was downregulated in LPS-induced ALI. Overexpression of miR-124-3p alleviated lung injury by inhibiting the Toll-like receptor 4 (TLR4)/nuclear factor-kappa B (NF-κB) signaling pathway. Furthermore, we confirmed that miR-124-3p suppressed the TLR4/NF-κB signaling pathway by directly targeting PDE4B. ConclusionmiR-124-3p targeting PDE4B had a protective effect on LPS-induced ALI by inhibiting the TLR4/NF-κB signaling pathway.

STAT1 Mediates the Transcription of CircIFI30 and Promotes the Progression of Triple-Negative Breast Cancer by Up-Regulating CDCA4.

Signal transducers and activators of transcription 1 (STAT1) is an important transcription factor that regulates the growth, survival, differentiation and apoptosis of various tumor cells. However, the biological roles of STAT1 and potential mechanisms in triple-negative breast cancer (TNBC) remain largely unknown. The expression levels of STAT1, CircIFI30, CDCA4, and epithelial-mesenchymal transition (EMT)-associated molecules (MM2, MMP9, E-cadherin, and N-cadherin) were evaluated using quantitative reverse transcription polymerase chain reaction (RT-qPCR). Furthermore, cell-counting kit-8 assay, Transwell assay, flow cytometry, and immunofluorescence staining were performed to investigate the biological functions of STAT1 and CircIFI30 in TNBC cells. In addition, Dual luciferase activity assay and chromatin immunoprecipitation qPCR were used to predict the interaction between STAT1 and CircIFI30 promoter. The effects of CircIFI30 on the stability of CDCA4 mRNA were also confirmed in further function study. Up-regulation of STAT1 was detected in TNBC tissues and cells, which were positively correlated with tumor metastasis, advanced clinical stage and poor survival rate. Up-regulated STAT1 could promote the proliferation, invasion, migration, EMT and inhibit the apoptosis of TNBC cells. RNA-seq indicated has_circ_0005571 (CircIFI30) was significantly down-regulated in TNBC cells after knockdown of STAT1. Moreover, STAT1 could be novel transcription factor that binds to CircIFI30 promoter to enhance its transcription. Additionally, knockdown of CirclFl30 down regulated the expression of cell division cycleassociated protein 4 (CDCA4) through reducing the stability of its mRNA. Our data revealed the STAT1/CircIFI30/CDCA4 axis could regulate the proliferation, invasion, migration, EMT and apoptosis of TNBC cells. Therefore, STAT1 may be a putative therapeutic candidate for targeted treatment of TNBC.

Upregulating microRNA-373-3p promotes apoptosis and inhibits metastasis of hepatocellular carcinoma cells

ABSTRACT Hepatocellular carcinoma (HCC) is one of the most prevalent malignancies in the digestive system. Abnormal miR-373-3p and TFAP4 expressions are critical in many malignant tumors, but it is unclear whether they work in the context of HCC. qRT-PCR measured miR-373-3p expression in HCC tissues and adjacent normal tissues. Flow cytometry and Western blot analyzed cell apoptosis. EMT, Transwell, and wound healing assay examined HCC cell migration and EMT, respectively. Western blot determined the profile of TFAP4/PI3K/AKT. IHC detected Ki67, E-cadherin, and vimentin in the tumor tissues. Moreover, the downstream target of miR-373-3p was predicted using the database. Dual luciferase activity assay and RIP verified the binding correlation between TFAP4 and miR-373-3p. In HCC tissues and cell lines, miR-373-3p was downregulated, and its overexpression stepped up HCC cell apoptosis and suppressed migration and EMT. Furthermore, miR-373-3p overexpression elevated Bax and caspase 3 expressions and attenuated Bcl2’s level. A xenograft tumor experiment in nude mice unveiled that miR-373-3p overexpression dampened tumor growth and proliferation. miR-373-3p cramped PI3K/AKT pathway activation. miR-373-3p negatively modulated TFAP4, and TFAP4 overexpression inverted miR-373-3p-mediated anti-tumor effects. Additionally, TFAP4 enhanced IGF1 expression, and promoted IGF1R-PI3K/AKT pathway activation. Collectively, miR-373-3p functions as an anti-tumor gene in HCC by inhibiting TFAP4/PI3K/AKT pathway.

Lnc-AC145676.2.1-6-3 can influence STX3-induced abnormal autophagy by sponging hsa-miR-1292-3p in intestinal aGVHD.

Intestinal acute graft-versus-host disease (aGVHD) is a serious complication of allogeneic hematopoietic stem cell transplantation (allo-HSCT). Abnormal autophagy levels in intestinal aGVHD have been confirmed in many studies. LncRNAs exert coregulatory functions and participate in a variety of intracellular regulatory processes. In this study, we investigated how lnc-AC145676.2.1-6-3 regulates dysregulated STX3-related autophagy in aGVHD. First, we established a mouse model of aGVHD by transplanting a mononuclear cell suspension from Balb/c donor mice treated with 60Co X-rays into CB6F1 recipient mice. STX3-related indicators were analyzed by Western blotting (WB) and immunohistochemistry which confirmed that STX3 plays an important role in dysregulating autophagy in intestinal aGVHD. TNF-αinduced Caco-2 cells, which is an in vitro model of intestinal barrier dysfunction, were established to verify the effect of STX3. The direct interaction between the partners of lnc-AC145676.2.1-6-3-mediated hsa-miR-1292-3p and STX3 axis was evaluated by the Dual-Luciferase activity assay. We performed PCR, WB, and immunofluorescence in Caco-2 cells to determine whether the abnormal autophagy levels were influenced by lnc-AC145676.2.1-6-3. The results showed that lnc-AC145676.2.1-6-3 could significantly suppress the number of autophagic vacuoles, the LC3-II/I ratio, and beclin1 levels by increasing STX3 levels. Lnc-AC145676.2.1-6-3 may play an important role in intestinal aGVHD by targeting STX3.

MiR-3571 modulates the proliferation and migration of vascular smooth muscle cells by targeting claudin 1.

Background and aims: The miRNA-based post-transcription modification has been extensively studied in hypertension. It however remains elusive how miRNA expression is regulated in this pathological process. We hypothesize that hydroxymethylation in the promoter regions tightly controls the levels of key miRNAs, which in turn affects the development of hypertension.Methods: The levels of hydroxymethylation in the promoter regions from thoracic aortic tissues were compared between spontaneously hypertensive rats (SHRs) and normotensive Wistar-Kyoto rats (WKYs), using hydroxymethylcytosine DNA immunoprecipitation (hMeDIP) sequencing. The altered hydroxymethylation level of miR-3571 was confirmed by glucosylation-coupled hydroxymethylation-sensitive qPCR. We further identified claudin 1(CLDN1) as a key target of miR-3571 via bioinformatic prediction (targetscan) and dual-luciferase activity assays. Finally, we analyzed the contribution of miR-3571/CLDN1 axis in the proliferation and migration of vascular smooth muscle cells (VSMCs).Results: The hydroxymethylation level of miR-3571 promoter region in thoracic aortic tissue from spontaneously hypertensive rats was lower than that from normotensive Wistar-Kyoto rats. Accordingly, the expression of miR-3571 was lower during hypertension, with up-regulated CLDN1 protein levels. More importantly, we found that miR3571 overexpression led to phenotypic changes of VSMCs, and inhibited the proliferation and migration of muscle cells via suppressing CLDN1 as well. Our findings further suggested that CLDN1 up-regulation increase the activity of ERK1/2 in VSMCs.Conclusions: Our study suggested that hydroxymethylation in the promoter regions controlled the level of miR-3571 and revealed the important roles of miR-3571 and CLDN1 in VSMCs during the development of hypertension. In addition, our results also indicated that miR-3571/CLDN1 axis regulated the functions of VSMCs via the ERK1/2 pathway. Taken together, our findings support miR-3571 as a novel biomarker for the diagnosis and prevention of hypertension.

Exosomal miR-455-3p from BMMSCs prevents cardiac ischemia-reperfusion injury

Bone marrow mesenchymal stem cells (BMMSCs) exert protective effects against myocardial infarction (MI). Here, we focused on the function and mechanism of miR-455-3p from BMMSCs-derived exosomes (BMMSCs-Exo) in myocardial infarction. BMMSCs were isolated from rat bone marrow, and the exosomes from the culture medium of BMMSCs were separated, and administered to H9C2 cells under hypoxia-reperfusion (H/R) stimulation. MTT and TUNEL staining analyzed cell viability and apoptosis, respectively. RT-qPCR determined miR-455-3p expression. Apoptosis-related proteins, autophagy-associated proteins, and the MEKK1-MKK4-JNK signaling pathway were detected. The interaction between miR-455-3p and MEKK1 was confirmed through dual luciferase activity and RIP assay. An in vivo ischemia reperfusion (I/R) model was established in rats. 2, 3, 5 triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (H&E) staining, Masson staining, and TUNEL staining evaluated the infarct volume and histopathological changes. miR-455-3p's expression was down-regulated in BMMSCs-derived exosomes, I/R myocardial tissues, and H/R myocardial cells. miR-455-3p enriched by BMMSC exosomes reduced H/R-mediated cardiomyocyte damage and death-related autophagy. miR-455-3p upregulation suppressed MEKK1-MKK4-JNK. MEKK1 overexpression notably mitigated cell apoptosis, cramped cell viability, suppressed autophagy expansion, and attenuated Exo-miR-455-3p's protection on H/R myocardial cells. In-vivo trials reflected that BMMSC exosomes enriched with miR-455-3p repressed ischemia reperfusion-induced myocardial damage and myocardial cell function. miR-455-3p, shuttled by exosomes from MSCs, targets the MEKK1-MKK4-JNK signaling pathway to guard against myocardial ischemia-reperfusion damage.

CiRS-7 Enhances the Progression of Hepatocellular Carcinoma through miR-944/NOX4 Pathway.

Recently, increasing numbers of non-coding RNA have been uncovered in research. As a new class of non-coding RNA, circular RNA has been identified to be involved in various diseases including many cancers. The circular RNA ciRS-7 is reported to play critical roles in tumorigenesis. However, the role of ciRS-7 in hepatocellular carcinoma (HCC) remains unclear. In this study, we investigated the expression and function of ciRS-7 in HCC cells and cancer tissues. CCK8 was applied to detect the influence of ciRS-7 on proliferation. Wound heal assay and invasion assay were used to identify the effects on migration and invasion. Quantitative reverse transcription polymerase chain reaction (qRT-PCR), Western blot, RNA pull-down, and luciferase reporter assay were used to investigate the downstream targets of ciRS-7. The results showed that ciRS-7 was highly expressed in both hepatocellular carcinoma cells and tissues. Overexpression of ciRS-7 could promote the proliferation, migration, and invasion of HCC. Further study showed that ciRS-7 regulated the miR-944 level through acting as a microRNA sponge. q-RT-PCR, Western blot, RNA pull-down and dual luciferase activity assays showed that miR-944 targeted and regulated the expression of NOX4. Furthermore, the tumor-promoting effect of ciRS-7 could be blocked by inhibition of miR-944/NOX4. Our study demonstrated that ciRS-7 enhanced the proliferation, migration, and invasion of HCC through miR-944/NOX4 pathway. ciRS-7 could be a promising therapeutic target for HCC.

Long non-coding RNA placenta‑specific protein 2 regulates micorRNA-19a/tumor necrosis factor α to participate in polycystic ovary syndrome

ABSTRACT Polycystic ovary syndrome (PCOS) is a type of hormonal disorder that affects about 5–20% of females at their reproductive age worldwide. MicorRNA-19a (miR-19a) is a well-characterized miRNA in cancer biology and its function is mainly mediated by targeting tumor necrosis factor α (TNF-α), which plays critical roles in PCOS. Our preliminary analysis predicted the potential interaction between miR-19a and long non-coding RNA (lncRNA) placenta‑specific protein 2 (PLAC2). Therefore, this study aimed to explore the role of PLAC2 in PCOS. Ovarian tissues were collected from 62 PCOS patients and 62 healthy females. Granulosa-like tumor cells (KGN) was prepared, and transient transfections was conducted. Dual-luciferase activity assay was used to investigate the interaction between PLAC2 and miR-19a. qPCR assays were performed for the expression analysis of miR-19a/TNF-α. In addition, Western blot analysis and cell apoptosis assay were conducted. The results showed that PLAC2 was upregulated in PCOS. PLAC2 and miR-19a showed a direct interaction, while overexpression of PLAC2 and miR-19a did not affect the expression of each other in KGN cells. Instead, overexpression of PLAC2 led to upregulated TNF-α, which is a target of miR-19a. Cell apoptosis analysis showed that PLAC2 and TNF-α promoted the apoptosis of KGN cells. Overexpression of miR-19a played an opposite role. In addition, the overexpression of PLAC2 reduced the effects of overexpression of miR-19a. Therefore, PLAC2 may regulate miR-19a/TNF-α to participate in PCOS.

Hsa_circ_0001879 promotes the progression of atherosclerosis by regulating the proliferation and migration of oxidation of low density lipoprotein (ox-LDL)-induced vascular endothelial cells via the miR-6873-5p-HDAC9 axis

ABSTRACT Atherosclerosis (AS) is a typical vascular disease. Emerging evidence has shown that circRNAs play key roles in the progression of AS, but the potential function and underlying mechanism of hsa_circ_0001879 remains unknown. We detected the expression level of hsa_circ_0001879 was determined by qRT-PCR, and the proliferation rate and migration ability of HUVECs were measured by CCK-8 assay and Transwell assay, respectively. Proliferative markers and epithelium mesenchymal transition (EMT) markers were measured through immunoblotting. A dual luciferase activity assay was performed to detect the interaction between circRNAs, miRNAs, and mRNAs. Hsa_circ_0001879 was upregulated in AS patients. Hsa_circ_0001879 inhibited the proliferation and migration ability of Human umbilical vein endothelial cells (HUVECs). Hsa_circ_0001879 directly bound to miR-6873-5p and acted as a sponge. miR-6873-5p-induced HDAC9 mRNA degradation was inhibited by hsa_circ_0001879. Hsa_circ_0001879 decreased the proliferation and migration of HUVECs by inhibiting miR-6873-5p-induced HDAC9 degradation.

Hsa_circ_0008434 regulates USP9X expression by sponging miR-6838-5p to promote gastric cancer growth, migration and invasion

BackgroundThe role of circular RNAs (circRNAs) in the occurrence and development of gastric cancer (GC) has recently attracted increasing interest. The following study investigates the role of a newly discovered hsa_circ_0008434, which has been confirmed to be highly expressed in GC tissues, in regulating GC biological behaviour.MethodsHigh-throughput RNA sequencing was used to identify differentially expressed genes between normal gastric tissues and GC tissues; actinomycin D and RNase R assays were used to determine the stability and loop structure of hsa_circ_0008434; and the miRanda database was used to predict the target genes of hsa_circ_0008434. The role of hsa_circ_0008434 in cell proliferation, migration, and invasion was examined using CCK-8, wound healing, Transwell and colony formation assays. The regulatory relationships among hsa_circ_0008434, microRNA-6838 (miR-6838), and ubiquitin-specific peptidase 9X (USP9X) were determined by dual-luciferase activity assays. The expression of hsa_circ_0008434 and miR-6838 was measured by qPCR; the expression of USP9X was detected by immunohistochemistry and Western blotting. The effects of hsa_circ_0008434 on in vivo tumour growth were assessed in xenograft models.ResultsWe found that hsa_circ_0008434 was one of the most upregulated circRNAs in GC tissue versus normal tissue. Further in vitro testing indicated that by acting as a miRNA sponge for miR-6838-5p, hsa_circ_0008434 promotes the expression of USP9X and further increases the proliferation, migration, and invasion of GC cells. In addition, animal studies indicated that hsa_circ_0008434 could promote tumour growth in vivo.ConclusionsHsa_circ_0008434 may promote GC proliferation, invasion and migration by regulating the expression of miR-6838 and USP9X.

CircRNAome profiling reveals circFgfr2 regulates myogenesis and muscle regeneration via a feedback loop.

BackgroundCircular RNAs (circRNAs) represent a novel class of non‐coding RNAs formed by a covalently closed loop and play crucial roles in many biological processes. Several circRNAs associated with myogenesis have been reported. However, the dynamic expression, function, and mechanism of circRNAs during myogenesis and skeletal muscle development are largely unknown.MethodsStrand‐specific RNA‐sequencing (RNA‐seq) and microarray datasets were used to profile the dynamic circRNAome landscape during skeletal muscle development and myogenic differentiation. Bioinformatics analyses were used to characterize the circRNAome and identify candidate circRNAs associated with myogenesis. Bulk and single‐cell RNA‐seq were performed to identify the downstream genes and pathways of circFgfr2. The primary myoblast cells, C2C12 cells, and animal model were used to assess the function and mechanism of circFgfr2 in myogenesis and muscle regeneration in vitro or in vivo by RT‐qPCR, western blotting, dual‐luciferase activity assay, RNA immunoprecipitation, RNA fluorescence in situ hybridization, and chromatin immunoprecipitation.ResultsWe profiled the dynamic circRNAome in pig skeletal muscle across 27 developmental stages and detected 52 918 high‐confidence circRNAs. A total of 2916 of these circRNAs are conserved across human, mouse, and pig, including four circRNAs (circFgfr2, circQrich1, circMettl9, and circCamta1) that were differentially expressed (|log2 fold change| > 1 and adjusted P value < 0.05) in various myogenesis systems. We further focused on a conserved circRNA produced from the fibroblast growth factor receptor 2 (Fgfr2) gene, termed circFgfr2, which was found to inhibit myoblast proliferation and promote differentiation and skeletal muscle regeneration. Mechanistically, circFgfr2 acted as a sponge for miR‐133 to regulate the mitogen‐activated protein kinase kinase kinase 20 (Map3k20) gene and JNK/MAPK pathway. Importantly, transcription factor Kruppel like factor 4 (Klf4), the downstream target of the JNK/MAPK pathway, directly bound to the promoter of circFgfr2 and affected its expression via an miR‐133/Map3k20/JNK/Klf4 auto‐regulatory feedback loop. RNA binding protein G3BP stress granule assembly factor 1 (G3bp1) inhibited the biogenesis of circFgfr2.ConclusionsThe present study provides a comprehensive circRNA resource for skeletal muscle study. The functional and mechanistic analysis of circFgfr2 uncovered a circRNA‐mediated auto‐regulatory feedback loop regulating myogenesis and muscle regeneration, which provides new insight to further understand the regulatory mechanism of circRNAs.

Integrated Analysis Reveals a lncRNA-miRNA-mRNA Network Associated with Pigeon Skeletal Muscle Development.

Growing evidence has demonstrated the emerging role of long non-coding RNA as competitive endogenous RNA (ceRNA) in regulating skeletal muscle development. However, the mechanism of ceRNA regulated by lncRNA in pigeon skeletal muscle development remains unclear. To reveal the function and regulatory mechanisms of lncRNA, we first analyzed the expression profiles of lncRNA, microRNA (miRNA), and mRNA during the development of pigeon skeletal muscle using high-throughput sequencing. We then constructed a lncRNA–miRNA–mRNA ceRNA network based on differentially expressed (DE) lncRNAs, miRNAs, and mRNAs according to the ceRNA hypothesis. Functional enrichment and short time-series expression miner (STEM) analysis were performed to explore the function of the ceRNA network. Hub lncRNA–miRNA–mRNA interactions were identified by connectivity degree and validated using dual-luciferase activity assay. The results showed that a total of 1625 DE lncRNAs, 11,311 DE mRNAs, and 573 DE miRNAs were identified. A ceRNA network containing 9120 lncRNA–miRNA–mRNA interactions was constructed. STEM analysis indicated that the function of the lncRNA-associated ceRNA network might be developmental specific. Functional enrichment analysis identified potential pathways regulating pigeon skeletal muscle development, such as cell cycle and MAPK signaling. Based on the connectivity degree, lncRNAs TCONS_00066712, TCONS_00026594, TCONS_00001557, TCONS_00001553, and TCONS_00003307 were identified as hub genes in the ceRNA network. lncRNA TCONS_00026594 might regulate the FSHD region gene 1 (FRG1)/ SRC proto-oncogene, non-receptor tyrosine kinase (SRC) by sponge adsorption of cli-miR-1a-3p to affect the development of pigeon skeletal muscle. Our findings provide a data basis for in-depth elucidation of the lncRNA-associated ceRNA mechanism underlying pigeon skeletal muscle development.

Investigation of the Heterogeneity Evolution and Drug Resistance Mechanism of Diffuse Large B-Cell Lymphoma Under the Treatment of a Novel HDAC Inhibitor LAQ824

Aims:Diffuse large B-cell lymphoma (DLBCL), the most frequent malignant lymphoma subtype, is a group of highly invasive diseases with great heterogeneity in genomic alterations, clinical characteristics, morphological manifestations, treatment response and prognosis. Although most DLBCL patients can be cured by immunochemotherapy, nearly 40% of DLBCL patients still develop drug resistance and relapse. For relapsed/ refractory (R/R) DLBCL patients, there is still no optimal treatment. The heterogeneity and clonal evolution of tumor cells are the core driving forces for the occurrence and development of DLBCL, and the root causes for their refractory, recurrence and drug resistance. In this study, we screened out a novel small molecule compound effectively killing DLBCL cells, and analyzed its potential mechanism of anti-tumor. Meanwhile, by using single cell sequencing technology, we try to further investigate the heterogeneity and clona evolution and drug resistance mechanism of DLBCL under different drug pressure, explore core driver factors of drug resistance, evaluate and develop new treatment strategies.Methods:In this study, GEXSCOPE microfluidic platform was used for single-cell transcriptome sequencing. Seruat software was used for cell type recognition and clustering analysis. In order to further investigate the molecular mechanism of LAQ824 inducing the apoptosis of DLBCL cells and explore the target of LAQ824, antibody chip was performed to detect the phosphorylation of related signaling pathway. Gene expression was detected by real-time qPCR and Western blot. ChIP, RNA interfering (RNAi) and dual-luciferase activity assay were performed to validate the potential drug resistance targets for LAQ824. Moreover, WES of 21 DLBCL cell lines were performed to map mutations and analyze the correlation between related mutations and LAQ824 resistance. In this study, we established DLBCL animal models using NOD SCID mice transplanted with DLBCL cell lines, by which we could evaluate the tumor inhibition efficiency of LAQ824 alone and/or combination with other small molecular inhibitors.Results:Using GDSC database, we screened out Dacinostat (LAQ824), a novel HDAC inhibitor, was highly sensitive that could effectively induce the apoptosis of most DLBCL cells at low concentrations. Functional assay showed that LAQ824 could inhibit cell proliferation and promote apoptosis of tumor cells. LAQ824 treatment could significantly upregulate the acetylation level of histone H3 within a certain concentration range, and the DNA damage repair function of DLBCL cells was supressed by inhibiting Chk2 expression, thus significantly inducing cell apoptosis and effectively killing DLBCL cells. Meanwhile, through single-cell sequencing analysis, it was found that c-Fos could be activated under certain drug pressure of LAQ824. As a potential drug-resistant core driver gene, the expression level of c-Fos is highly correlated with IC50 of LAQ824 and the prognosis of patients with DLBCL, which can be used as a sensitivity indicator of LAQ824. Treatment with c-Fos inhibitor combined with LAQ824 can significantly improve the tumor inhibition rate, validated both in vitro and in vivo, which is expected to alleviate the recurrence and drug resistance of DLBCL patients.Conclusions:In general, we explores potential therapeutic drugs for DLBCL parients, adjusts and explores new clinical treatment strategies on this basis, and provides theoretical basis and data support for the realization of individualized precise treatment and the solution of DLBCL recurrence and drug resistance. DisclosuresNo relevant conflicts of interest to declare.

Quercetin improves atrial fibrillation through inhibiting TGF-β/Smads pathway via promoting MiR-135b expression

PurposeTo investigate the role and mechanism of quercetin in isoprenaline (ISO)-induced atrial fibrillation (AF). Study designRat cardiac fibroblasts (RCFs) models and RCFs were used to explore the effect and underlying mechanism of quercetin in isoprenaline (ISO)-induced atrial fibrillation (AF) in vivo and in vitro by a series of experiments. MethodsDifferentially expressed microRNAs were screened from human AF tissues using the GEO2R and RT-qPCR. The expressions of TGF-β/Smads pathway molecules (TGFβ1, TGFBR1, Tgfbr1, Tgfbr2, Smad2, Smad3, Smad4) in AF tissues were detected by RT-qPCR and Western blot. The relationships between miR-135b and genes (Tgfbr1, Tgfbr2, Smad2) were analyzed by Pearson correlation, TargetScan and dual-luciferase activity assay. RCFs induced by ISO were treated with quercetin (20 or 50 μM), miR-135b mimic and inhibitor, siTgfbr1 and their corresponding controls, then the cell viability was determined by MTT and the expressions of cyclin D1, α-SMA, collagen-related molecules, TGF-β/Smads pathway molecules, and miR-135b were measured by RT-qPCR and Western blot. ISO-induced rats were treated with quercetin (25 mg/kg/day) via gavage, miR-135b antagomir, agomir and their corresponding controls. The treated rats were used for the detection of miR-135b expression by RT-qPCR, histopathological observation by HE and Masson staining, and the detection of Col1A1 and fibronectin contents by immunohistochemical technique. ResultsThe expression of miR-135b was downregulated, and those of TGFBR1, TGFBR2, target genes of miR-135b were upregulated in human AF tissues and negatively regulated by miR-135b in RCFs. Through inhibiting TGF-β/Smads pathway via promoting miR-135b expression, quercetin treatment inhibited proliferation, myofibroblast differentiation and collagen deposition in ISO-treated RCFs, as evidenced by reduced expressions of cyclin D1, α-SMA, collagen-related genes and proteins, and alleviated fibrosis and collagen deposition of atrial tissues in ISO-treated rats. ConclusionQuercetin may alleviate AF by inhibiting fibrosis of atrial tissues through inhibiting TGF-β/Smads pathway via promoting miR-135b expression.