Dual Luciferase Activity Assay Research Articles

Clinical Value of lncRNA CASC19 in Myocardial Infarction and its Role in Myocardial Infarction-Induced Cardiomyocyte Apoptosis.

To explore the effect of long non-coding RNA cancer susceptibility 19 (lncRNA CASC19) on the activity, apoptosis, and oxidative stress response of cardiomyocytes, so as to assess the clinical relevance and molecular mechanism of CASC19 in myocardial infarction (MI). CASC19 level was determined by using real-time quantitative polymerase chain reaction (RT-qPCR). MI model was constructed using hypoxia induction, and rat cardiomyocytes H9c2 were divided into control group, MI group, MI small interference negative control (MI-si-NC) group, MI-si-CASC19 group, MI-si-CASC19+microRNA-NC (miR-NC) group, and MI-si-CASC19+miR-218-5p inhibitor group. Tetramethylazolium salt colorimetric method and flow cytometry were used to evaluate cell activity and apoptotic capacity. Cellular oxidative stress was evaluated using malondialdehyde and superoxide dismutase kits. The relationship between CASC19 and miR-218-5p was confirmed by using dual-luciferase activity assay. CASC19 levels were enhanced in MI patients and hypoxia-induced cardiomyocytes. Downregulating CASC19 promoted the proliferation, while suppressed apoptosis and oxidative stress in the MI cell model. Moreover, low expression of miR-218-5p reversed the promotion of proliferation and inhibition of apoptosis and oxidative stress in MI cell models by silencing CASC19. Briefly, CASC19 may serve as a diagnostic marker for MI by sponging miR-218-5p to inhibit apoptosis and oxidative stress in cardiomyocytes and promote cell survival.

Eurycomanone inhibits osteosarcoma growth and metastasis by suppressing GRP78 expression

Ethnopharmacological relevanceOsteosarcoma (OS) is characterized by rapid growth and frequent pulmonary metastasis. Eurycoma longifolia Jack, a flowering plant primarily found in Southeast Asian countries, is commonly used in traditional herbal medicine. Its root extract is mainly used for against cancer, malaria, parasites and other conditions. The active compound in its root extract, eurycomanone (EUR), has been proven to inhibit lung and liver cancer proliferation. Aim of the studyOur research aimed to investigate the inhibitory effect and underlying molecular mechanism of EUR on OS growth and metastasis. Materials and methodsIn vitro experiments: western blotting (WB) screened 41 compounds that inhibited GRP78 expression and evaluated the protein levels of GRP78, PARP, cleaved-PARP, MMP2, and MMP9. Cell proliferation was evaluated using CCK-8, EdU, colony formation assay, and cell apoptosis was assessed by flow cytometry. Transwell, wound healing, and tube formation assays were performed to determine the effect of EUR on tumor invasion, migration, and angiogenesis, respectively. Quantitative real-time polymerase chain (qRT-PCR) and dual-luciferase activity assays detected GRP78 mRNA stability and transcription levels post-EUR and thapsigargin treatment. RNA-Seq identified signaling pathways inhibited by EUR. In vivo experiments: effects of EUR in mice were evaluated by H&E staining to detect lung metastasis and potential toxic effects in tissues. Immunohistochemical (IHC) staining detected the expression of Ki-67, CD31, and cleaved caspase-3 in tumors. ResultsGRP78 is highly expressed in OS and correlated with poor prognosis. In vitro, eurycomanone (EUR) significantly downregulated GRP78 expression, inhibited cell proliferation, migration, invasion, tube formation, and induced apoptosis. Moreover, it enhanced trichostatin A (TSA) sensitivity and exhibited inhibitory effects on other cancer types. Mechanistically, EUR decreased GRP78 mRNA stability and transcription. In vivo, EUR inhibited proliferation and invasion in tibial and PDX models. ConclusionsOur study demonstrated that EUR inhibits the growth and metastasis of OS by reducing GRP78 mRNA stability and inhibiting its transcription, which offers a novel approach for clinical treatment of OS.

MTHFR as a Novel Candidate Marker for Litter Size in Rabbits.

Litter size is a significant economic trait during animal reproduction. This current study attempted to decipher whether MTHFR promotes the apoptosis of granulosa cells (GCs) and inhibits their proliferation by investigating the effects of the MTHFR gene using flow cytometry and a Cell Counting Kit-8 (CCK-8) assay. MTHFR is linked with ovarian follicle development in the reproductive performance of 104 female New Zealand rabbits. We observed that MTHFR could regulate the mRNA of follicular development-related genes (TIMP1, CITED1, FSHR, GHR, HSD17B1, and STAR) with a qRT-PCR, and we observed the protein expression of CITED1 and GHR using a western blot (WB) analysis. The dual luciferase activity assays helped identify the core promoter region of the MTHFR gene, and the polymorphism of the MTHFR promoter region was studied using Sanger sequencing. The results indicated four single nucleotide polymorphisms (SNPs) within the core promoter region, among which the g.-680C>A locus was significantly associated with both the total and alive litter sizes. Additionally, the CC genotype was associated with the largest total and alive litter sizes, compared to the CA and AA genotypes (p < 0.05). In conclusion, this study investigated the effects of MTHFR on ovarian granulosa cells and its association with selected reproductive parameters in rabbits. The results provide a theoretical foundation for the use of MTHFR as a molecular marker in rabbits.

CircPDSS1 (hsa_circ_0017998) silencing induces ferroptosis in non-small-cell lung cancer cells by modulating the miR-137/SLC7A11/GPX4/GCLC axis

BackgroundCircular RNAs (circRNAs) regulate the tumorigenesis of non-small-cell lung cancer (NSCLC). CircPDSS1 (hsa_circ_0017998) has been newly discovered, and its role in NSCLC remains elusive. We aimed to investigate the functional roles and downstream targets of circPDSS1 in NSCLC cells. Materials and methodsCellular viabilities were measured through the Cell Counting Kit-8 (CCK-8) assay, whereas cell death was assessed through flow cytometry. The lactate dehydrogenase activity, malondialdehyde levels, ferrous iron, and reactive oxygen species were measured using commercial assay kits. The interaction between circPDSSA/ microRNA-137 (miR-137) and miR-137/solute carrier family 7 member 11 (SLC7A11) was assayed through a dual luciferase activity assay. Finally, the mRNA and protein levels were measured using real-time reverse transcriptase-polymerase chain reaction and western blots, respectively. ResultsCircPDSS1 expression was upregulated in NSCLC cells, compared with healthy lung cells. CircPDSS1 silencing suppressed the viability of NSCLC cells. Additionally, circPDSS1 knockdown induced ferroptosis rather than other types of cell death in NSCLC cells. Mechanically, circPDSS1 functions as a “sponge” to inversely control miR-137 expression, which directly targets SLC7A11. Moreover, circPDSS1 silencing causes the downregulation of glutathione peroxidase 4 (GPX4) and glutamate-cysteine ligase catalytic subunit (GCLC). ConclusionsTargeting the circPDSS1/miR-137/SLC7A11/GPX4/GCLC axis may be a promising strategy to kill NSCLC cells.

Long Non-coding RNA PCED1B Antisense RNA 1 Promotes Cell Proliferation and Invasion in Hepatocellular Carcinoma by Regulating the MicroRNA-34a/CD44 Axis.

This study aimed to examine the role of long non-coding RNA PCED1B antisense RNA 1 (PCED1B-AS1) in the development of hepatocellular carcinoma (HCC). A total of 62 pairs of HCC tissues and adjacent non-tumor tissues were obtained from 62 HCC patients. The interactions of PCED1B-AS1 and microRNA-34a (miR-34a) were detected by dual luciferase activity assay and RNA pull-down assay. The RNA expression levels of PCED1B-AS1, miR-34a and CD44 were detected by RT-qPCR, and the protein expression level of CD44 was determined by Western blotting. The cell proliferation was detected by cell proliferation assay, and the cell invasion and migration by transwell invasion assay. The HCC tumor growth after PCED1B-AS1 was downregulated was determined by in vivo animal study. PCED1B-AS1 was highly expressed in HCC tissues, which was associated with poor survival of HCC patients. Furthermore, PCED1B-AS1 interacted with miR-34a in HCC cells, but they did not regulate the expression of each other. Additionally, PCED1B-AS1 increased the expression level of CD44, which was targeted by miR-34a. The cell proliferation and invasion assay revealed that miR-34a inhibited the proliferation and invasion of HCC in vitro, while CD44 exhibited the opposite effects. Furthermore, PCED1B-AS1 suppressed the role of miR-34a. Moreover, the knockdown of PCED1B-AS1 repressed the HCC tumor growth in nude mice in vivo. PCED1B-AS1 may play an oncogenic role by regulating the miR-34a/CD44 axis in HCC.

Circ_TMCO3 Inhibits the Progression of Cervical Cancer by Activating FRMD6 Expression by Restraining miR-1291.

Cervical cancer (CC) is one of the most common cancers that threaten the life of women. More and more circular RNAs (circRNAs) have been found to be maladjusted in tumor tissues. However, the mechanism of circ_TMCO3 in CC needs to be studied. In this study, quantitative real-time polymerase chain reaction (qRT-PCR), western blot, and immunohistochemistry (IHC) were used to detect the expressions of circ_TMCO3, miR-1291, and FERM domain-containing protein 6 (FRMD6). Cell viability, proliferation, apoptosis, migration, invasion, and protein level were detected via 3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT), 5-Ethynyl-2'-deoxyuridine (EdU), flow cytometry, transwell and western blot, respectively. The glycolysis level was detected via specific kits. Dual-luciferase activity assay was used to analyze the targeted relationship between miR-1291 and circ_TMCO3 or FRMD6. Xenograft models were used to analyze the effect of circ_TMCO3 on the growth of CC tumors in vivo. Circ_TMCO3 and FRMD6 were low expressed in tumor tissues, and miR-1291 was conspicuously upregulated in tumor tissues. Upregulation of circ_TMCO3 dramatically curbed cell viability, proliferation, migration, and invasion, and enhanced cell apoptosis, while those effects were attenuated after the overexpression of miR-1291. MiR-1291 could directly target FRMD6, and knockdown of FRMD6 could restore the inhibitory effect of miR-1291 silencing on tumor cell growth. In terms of mechanism, circ_TMCO3 was confirmed as a miR-1291 sponge to regulate the expression of FRMD6. Tumor growth was markedly retarded with the overexpression of circ_TMCO3. In conclusion, circ_TMCO3 inhibited tumorigenicity of CC via miR-1291/FRMD6 axis, providing a potential therapeutic strategy for CC patients.

Novel indel variation of LTBP4 gene associates with risk of sudden cardiac death in Chinese populations with coronary artery disease

The objective of this study is to investigate whether common genetic variants of the LTBP4 gene are linked to the susceptibility of sudden cardiac death in individuals who have atherosclerotic coronary artery disease (SCD-CAD) in Chinese populations. A total of 208 SCD-CAD cases and 638 controls were included in the analysis, and logistic regression was employed to assess the association between a 4-bp insertion/deletion polymorphism (rs34005443) within LTBP4 and the susceptibility to SCD-CAD among Chinese individuals. Logistic regression analysis demonstrated a notable association between the insertion allele of rs34005443 and an escalated susceptibility to SCD-CAD [odds ratio (OR) = 1.434; 95 % confidence interval:1.14–1.80; P = 1.79 × 10−3]. Genotype-phenotype correlation analysis was performed using Genotype-Tissue expression (GTEx) database and further validated by human myocardium using qPCR. Correlation analysis revealed that LTBP4 expression level was lower in samples with the insertion allele. Furthermore, the dual-luciferase activity assays indicated that rs34005443 may play a regulatory role. Additionally, we predicted 30 transcription factors that are likely to bind to rs34005443 and its highly linked genetic variants via 3DSNP database. Subsequent GO and KEGG analysis indicated that these transcription factors have a significant function in regulating gene expression. Finally, PPI network analysis suggested a tight connection between LTBP4 proteins and TGFβs, highlighting these genes as potential hub genes in the context of SCD-CAD. In summary, our study revealed that rs34005443 might contribute to SCD-CAD susceptibility by regulating LTBP4 expression. These findings revealed that this indel could be a potentially functional marker for molecular diagnosis and risk stratification of SCD-CAD.

Pigeon novel-miR-741 targets OTUD1 to inhibit proliferation and promote apoptosis of crop fibroblasts

Trichomonas gallinae (T. gallinae) is a globally distributed protozoan parasite and could cause serious damage to the pigeon industry. MiRNAs have important roles in regulating parasite infection, but its impacts on T. gallinae resistance have rarely been reported. In the present study, we identified a new miRNA (novel-miR-741) and its predicted target OTU deubiquitinase 1 (OTUD1) that might be associated with immunity to T. gallinae in pigeon. Novel-miR-741 and OTUD1 over-expression vectors and interference vectors were constructed. Results from dual luciferase activity assay demonstrated that OTUD1 was a downstream target of novel-miR-741. The Cell Counting Kit-8 and apoptosis assays showed that novel-miR-741 inhibited the proliferation and promoted apoptosis of pigeon crop fibroblasts. Meanwhile, mRNA levels of OTUD1 were significantly reduced in novel-miR-741 mimic-transfected fibroblasts, while mRNA levels of OTUD1 were significantly increased in the novel-miR-741 inhibitor-transfected fibroblasts. The regulatory roles of si-OTUD1 on fibroblasts proliferation, apoptosis, and migration were similar to novel-miR-741 mimic. Our findings demonstrated that novel-miR-741 inhibited the proliferation, and migration of crop fibroblasts, while OTUD1 promoted the proliferation and migration of crop fibroblasts. Therefore, the regulation of OTUD1 by novel-miR-741 was proposed as a potential therapeutic strategy for T. gallinae.

Stereotactic body radiation therapy suppresses myeloid-derived suppressor cells by regulating miR-21/Sorbin and SH3 Domain-containing Protein 1 axis.

Stereotactic body radiation therapy (SBRT) is a targeted form of radiotherapy used to treat early-stage cancers. Despite its effectiveness, the impact of SBRT on myeloid-derived suppressor cells (MDSCs) is not well understood. In this study, we examined how SBRT affects the differentiation and survival of MDSCs, as well as delved into the molecular mechanisms involved. SBRT was utilized on bone marrow (BM)-derived MDSCs to investigate its impact on the differentiation and survival of MDSCs using flow cytometry. An animal model of lung cancer was created to assess the anti-cancer properties of SBRT and the role of miR-21 expression in MDSCs. The interplay of miR-21 and Sorbin and SH3 domain-containing protein 1 (SORBS1) in MDSC differentiation was explored through dual luciferase activity assay, RT-qPCR, and Western blot analysis. The findings suggest that SBRT led to an increase in miR-21 levels, inhibited MDSC differentiation, and triggered cell apoptosis in BM cells. Inhibition of miR-21 reversed the effects of SBRT on MDSC differentiation and apoptosis. Additionally, it was revealed that SORBS1 was a downstream target of miR-21 in BM cells, and the miR-21/SORBS1 axis played a role in regulating MDSC differentiation and apoptosis induced by SBRT. Modulating miR-21 levels in vivo impinged on the response to SBRT treatment and the quantity of MDSCs in a mouse model of lung cancer. Our data indicate that the upregulation of miR-21 induced by SBRT may contribute to the inhibition of MDSC expansion in a lung cancer model.

MiRNA-449c-5p regulates the JAK-STAT pathway in inhibiting cell proliferation and invasion in human breast cancer cells by targeting ERBB2.

Breast cancer is a highly prevalent disease worldwide, and early diagnosis and treatment could reduce the mortality rate of breast cancer patients. microRNAs (miRNA) have been shown to regulate the occurrences and progression of many types of cancers. Thus, it is crucial to find novel biomarkers in breast cancer. miR-449c-5p acted as a biomarker in non-small cell lung cancer, gastric carcinoma, and so forth. ERBB2 is an ideal target for breast cancer therapy. However, the molecular mechanisms between miR-449c-5p and ERBB2 in breast cancer remain poorly understood. Our study focused on the regulatory role of miR-449c-5p in breast cancer and its targeting relationship with ERBB2. The miR-449c-5p expression in breast cancer tissue and normal tissue was searched from the online database (Starbase). The clinical prognosis of miR-449c-5p and ERBB2 was predicted by using the Kaplan-Meier analysis method. The expression of miR-449c-5p mimics and inhibitors was measured by qRT-PCR. T47D cells were transfected with miR-449c-5p mimics and miR-449c-5p inhibitors. After that, CCK-8, colony formation assays and Transwell assays were used to evaluate the cell proliferation ability, migration and invasion. Whether ERBB2 was the target gene of the miR-449c-5p was predicted by Starbase and verified by dual-luciferase activity assay. In addition, protein levels and the relationship between signalling pathways were measured and validated using western blotting analysis. We confirmed that miR-449c-5p was highly expressed in breast cancer tissue, and its downregulation was linked with poor prognosis. Overexpression of miR-449c-5p inhibited the proliferation, migration and invasion of breast cancer cells. ERBB2 was a target of miR-449c-5p. The invasion, migration, and proliferation of breast cancer cells were inhibited by miR-449c-5p/ERBB2 through JAK-STAT. This study demonstrated that miR-449c-5p inhibits breast cancer cell proliferation, migration and invasion by targeting ERBB2 via JAK/STAT, which means miR-449c-5p, is a potential biomarker for breast cancer and provides a novel insight for diagnosis.

Long noncoding RNA MCM3AP-AS1 attenuates sepsis-induced cardiomyopathy by improving inflammation, oxidative stress, and mitochondrial function through mediating the miR-501-3p/CADM1/STAT3 axis

Oxidative stress and inflammation are highly important for sepsis-mediated myocardial damage. The long noncoding RNA (lncRNA) MCM3AP-AS1 is involved in inflammatory diseases, but its function in acute myocardial injury during sepsis has not been fully elucidated. LPS and cecal ligation and puncture (CLP) were used to construct in vitro and in vivo sepsis-induced myocardial damage models, respectively. qRT-PCR was used to evaluate alterations in MCM3AP-AS1 and miR-501-3p alterations. After the MCM3AP-AS1 and miR-501-3p knockdown or overexpression models were established, the viability, apoptosis, inflammation, oxidative stress, and mitochondrial function of the myocardial cells were examined. Dual luciferase activity assay, RNA immunoprecipitation, and fluorescence in situ hybridization (FISH) confirmed the correlation among MCM3AP-AS1, miR-501-3p, and CADM1. Previous studies revealed that MCM3AP-AS1 was downregulated in sepsis patients, myocardial cells treated with LPS, and in the CLP mouse sepsis model, whereas miR-501-3p expression was increased. MCM3AP-AS1 overexpression hampered myocardial damage mediated by LPS and abated inflammation, oxidative stress, and mitochondrial dysfunction in myocardial cells and THP-1 cells. In contrast, MCM3AP-AS1 knockdown or miR-501-3p overexpression promoted all the effects of LPS. In vivo, MCM3AP-AS1 overexpression increased the survival rate of CLP mice; ameliorated myocardial injury; decreased the levels of TNF-α, IL-1β, IL-6, iNOS, COX2, ICAM1, VCAM1, PGE2, and MDA; and increased the levels of SOD, GSH-PX, Nrf2, and HO-1. Mechanistic studies demonstrated that MCM3AP-AS1 acted as a competitive endogenous RNA to repress miR-501-3p, enhance CADM1 expression, and dampen STAT3/nuclear factor-kappaB (NF-κB) activation. MCM3AP-AS1 suppresses myocardial injury elicited by sepsis by mediating the miR-501-3p/CADM1/STAT3/NF-κB axis.

MiRNA-146a-5p Inhibits Hypoxia-Induced Myocardial Fibrosis Through EndMT.

Cardiac Vascular disease particularly myocardial infarction (MI) is a threat to health worldwide. microRNAs (miRNAs) have been shown to regulate myocardial fibrosis. Therefore, it is potential to investigate the mechanism of miRNA and fibrosis following myocardial infarction. Hypoxia human cardiac microvascular endothelial cells (HCMECs) were selected for the vitro experimental model. The miR-146a-5p expression was tested via RT-qPCR. The level of endothelial-to-mesenchymal transition (EndMT) and fibrosis markers were detected by Western blotting and immunofluorescence. Then, the inflammation, cell viability and apoptosis were investigated. The target was predicted by an online database and verified by a dual-luciferase activity assay. An MI mouse model was created to validate that miR-146a-5p regulates cardiac fibrosis in vivo. MI mouse was transfected with miR-146a-5p lentivirus. Subsequently, its effect on cardiac fibrosis of infarcted hearts was assessed by In situ hybridization (ISH), Immunohistochemistry (IHC), Triphenylterazolium chloride (TTC) staining and Masson staining. Herein, we confirmed that miR-146a-5p was down-regulated in hypoxia HCMECs. Overexpression of miR-146a-5p inhibited hypoxia-induced cardiac fibrosis following myocardial infarction by inhibiting EndMT in HCMECs. Thioredoxin-interacting protein (TXNIP) was a target that was negatively regulated by miR-146a-5p. Up-regulation of miR-146a-5p inhibited cardiac fibrosis via regulating EndMT by targeting TXNIP, and it also regulated EndMT to inhibit cardiac fibrosis in vivo.

METTL3/IGF2BP3-regulated m6A modification of HYOU1 confers doxorubicin resistance in breast cancer

Chemoresistance is a main reason for therapeutic failure and poor prognosis for breast cancer (BC) patients, especially for triple-negative BC patients. How the molecular mechanisms underlying the chemoresistance to doxorubicin (Dox) in BC is not well understood. Here, we revealed that METTL3/IGF2BP3-regulated m6A modification of HYOU1 increased Dox resistance in BC cells. CCK-8 and Annexin V-FITC/PI staining assays were employed to measure viability and cell death. Western blotting and qRT–PCR assays were applied to assay the expression of genes. Knockdown and rescue experiments were used to assay the role of METTL3, IGF2BP3 and HYOU1 in regulating BC cell responses to Dox. RIP, MeRIP and dual-luciferase activity assays were applied to examine the function of METTL3/IGF2BP3 in the m6A modification of HYOU1 mRNA. It was found that global mRNA m6A methylation levels were upregulated in Dox-resistant BC cell lines. The methyltransferase METTL3 was upregulated in Dox-resistant BC cell lines, and downregulation of METTL3 could overcome this resistance. Furthermore, HYOU1 was identified as a downstream target of METTL3-mediated m6A modification. Downregulation of HYOU1 could overcome Dox resistance, while forced expression of HYOU1 resulted in Dox resistance in BC cells. METTL3 cooperated with IGF2BP3 to modulate the m6A modification of HYOU1 mRNA and increase its stability. Collectively, our findings unveiled the key roles of the METTL3/IGF2BP3/HYOU1 axis in modulating Dox sensitivity in BC cells; thus, targeting this axis might be a potential strategy to increase Dox efficacy in the treatment of BC.

Hsa_circ_0001707 regulates endothelial-mesenchymal transition in esophageal squamous cell carcinoma via miR-203a-3p/Snail2 pathway.

Esophageal squamous cell carcinoma (ESCC) is a malignant tumor with high mortality and poor prognosis. Despite intensive research focused on tumor suppression, the 5-year survival rate of ESCC is lower than 15%. Therefore, investigate fundamental mechanisms involved in ESCC is on-demand crucial for diagnostics and developing targeted therapeutic drugs. Circular RNAs (circRNAs), as an emerging class of non-coding RNA, have been elucidated that circRNAs participated in regulating a variety of pathological processes and tumorigenesis. Nevertheless, the functional role of circRNAs in the occurrence and development of ESCC remains unclear. We identify a novel circRNA (hsa_circ_0001707), which was highly expressed in ESCC patients' tissues and cell lines. Furthermore, gain- and loss-of-function assays were performed and found that overexpression of hsa_circ_0001707 significantly promote tumor proliferation, metastasis, and invasion. By functioning as a competing endogenous RNA (ceRNA), the dual-luciferase activity assay verified that hsa_circ_0001707 can endogenously bind with miR-203a-3p and regulate its downstream gene Snail2. Rescue assay further confirms that hsa_circ_0001707 downregulation could partially attenuate the facilitation effect of miR-203a-3p, thereby inhibiting the endothelial-mesenchymal transition (EMT) process of ESCC. Our results suggested that hsa_circ_0001707 play an oncogenic role in the pathogenesis of ESCC, which might be a potential biomarker for diagnostics and targeting therapy.

LRRC75A-AS1 Inhibits Chondrogenic Differentiation of Bmscs via Targeting the Mir-140-3p/Wnt/Β-Catenin Pathway.

Bone marrow mesenchymal stem cells (BMSCs) are pluripotent cells with the ability to differentiate into adipocytes, chondrocytes, and osteoblasts. BMSCs are widely used in regenerative medicine and cartilage tissue engineering. Role of lncRNA LRRC75A-AS1 (leucine-rich repeat containing 75A antisense RNA 1) in the chondrogenic differentiation of BMSCs was investigated in this study. BMSCs were isolated from rat bone marrow and then identified using flow cytometry. Alcian blue staining was used to detect chondrogenic differentiation. The effect of LRRC75A-AS1 on chondrogenic differentiation was assessed by western blot. The downstream target of LRRC75A-AS1 was determined by dual luciferase activity assay. BMSCs were identified with positive CD29 and CD44 staining and negative staining of CD34 and CD45. LRRC75A-AS1 was decreased during the chondrogenic differentiation of BMSCs. Silencing of LRRC75A-AS1 increased collagen II (COL II), aggrecan and SOX9 and promoted chondrogenic differentiation. However, over-expression of LRRC75A-AS1 inhibited chondrogenic differentiation. miR- 140-3p was increased during chondrogenic differentiation and interacted with LRRC75A-AS1. miR-140- 3p bind to wnt3a, and inhibition of miR-140-3p up-regulated wnt3a and nuclear β- catenin expression. Wnt3a and nuclear β-catenin were decreased during chondrogenic differentiation. Inhibition of miR-140- 3p attenuated LRRC75A-AS1 deficiency-induced up-regulation of COL II, aggrecan and SOX9. LRRC75A-AS1 suppressed chondrogenic differentiation of BMSCs through down-regulation of miR-140-3p and up-regulation of the wnt/β-catenin pathway.

Upregulation of miR-21-5p rescues the inhibition of cardiomyocyte proliferation induced by high glucose through negative regulation of Rhob.

Increasing evidence shows that maternal hyperglycemia inhibits cardiomyocyte (CM) proliferation and promotes cell apoptosis during fetal heart development, which leads to cardiac dysplasia. Accumulating evidence suggests that the overexpression of miR-21 in CMs has a protective role in cardiac function. Therefore, we investigated whether miR-21 can rescue CM injury caused by high glucose. First, we performed biological function analysis of miR-21-5p overexpression in H9c2 cells treated with high glucose. We found that the proliferation of H9c2 cells treated with high glucose decreased significantly and was rescued after overexpression of miR-21-5p. CCK-8 and EdU incorporation assays were performed to assess cell proliferation. The cell proliferation of the miR-21-5p mimic transfection group was improved compared with that of the NC mimic group (*p < 0.05, miR-21-5p mimics vs. NC mimics) when the proliferation of H9c2 cells was reduced by high glucose (****p < 0.0001, high glucose (HG) vs. normal glucose (NG)). Then, we verified the targeted and negative regulation of miR-21-5p on Rhob using a dual-luciferase activity assay and RT-qPCR, respectively. We further demonstrated that miR-21-5p regulates Rhob to rescue the inhibition of CM proliferation induced by high glucose. The CCK-8 results showed that the cell proliferation of the siRNA-Rhob group was higher than that of the NC mimic group (***p < 0.001) and that of the cotransfection group with Up-Rhob plasmids and miR-21-5p mimics was lower than that of the miR-21-5p mimic group (*p < 0.05). Conclusion: Overexpression of miR-21-5p rescues the inhibition of high glucose-induced CM proliferation through regulation of Rhob.

Kaempferol ameliorated alcoholic liver disease through inhibiting hepatic bile acid synthesis by targeting intestinal FXR-FGF15 signaling

BackgroundAlcoholic liver disease (ALD) is characterized by the disturbance of bile acids homeostasis, which further deteriorates ALD. Bile acid metabolism and its related signal molecules have become new therapeutic targets for alcoholic liver disease. This study aimed to investigate the impact of kaempferol (KAE) on ALD and elucidate its underlying mechanisms. MethodsC57BL/6 N mice were utilized to establish Binge-on-Chronic alcohol exposure mice model. KAE was administered as an interventional drug to chronic alcohol-fed mice for four weeks to assess its effects on liver damage and bile acid metabolism. And Z-Guggulsterone (Z-Gu), a global FXR inhibitor, was used to investigate the impact of intestinal FXR-FGF15 signal in ALD mice. Additionally, intestinal epithelial cells were exposed to alcohol or specific bile acid to induce the damage of FXR activity in vitro. The dual luciferase activity assay was employed to ascertain the interplay between KAE and FXR activity. ResultsThe results indicated that KAE treatment exhibited a significant hepatoprotective effect against chronic alcohol-fed mice. Accompanied by the intestinal FXR activation, the administration of KAE suppressed hepatic bile acid synthesis and promoted intestinal bile acid excretion in chronic ALD mice. And the notable alterations in total bile acid levels and composition were observed in mice after chronic alcohol feeding, which were reversed by KAE supplementation. And more, the protective effects of KAE on ALD mice were deprived by the inhibition of intestinal FXR activation. In vitro experiments demonstrated that KAE effectively activated FXR-FGF15 signaling, mitigated the damage to FXR activity in intestinal epithelial cells caused by alcohol or specific bile acids. Additionally, luciferase activity assays revealed that KAE directly promoted FXR expression, thereby enhancing FXR activity. ConclusionKAE treatment inhibited hepatic bile acids synthesis, maintained bile acids homeostasis in ALD mice by directly activating intestinal FXR-FGF15 signaling, which effectively alleviated liver injury induced by chronic alcohol consumption.

Functional Study of Amorpha fruticosa WRKY20 Gene in Response to Drought Stress.

The WRKY gene family in plants regulates the plant's response to drought through regulatory networks and hormone signaling. AfWRKY20 (MT859405) was cloned from Amorpha fruticosa (A. fruticosa) seedlings using RT-PCR. The binding properties of the AfWRKY20 protein and the W-box (a DNA cis-acting element) were verified both in vivo and in vitro using EMSA and Dual-Luciferase activity assays. RT-qPCR detected that the total expression level of AfWRKY20 in leaves and roots was 22 times higher in the 30% PEG6000 simulated drought treatment compared to the untreated group. Under the simulated drought stress treatments of sorbitol and abscisic acid (ABA), the transgenic tobacco with the AfWRKY20 gene showed enhanced drought resistance at the germination stage, with significantly increased germination rate, green leaf rate, fresh weight, and root length compared to the wild-type (WT) tobacco. In addition, the superoxide dismutase (SOD) activity, chlorophyll content, and Fv/Fm ratio of AfWRKY20 transgenic tobacco were significantly higher than those of the WT tobacco under natural drought stress, while the malondialdehyde (MDA) content and 3,3'-diaminobenzidine (DAB) and nitroblue tetrazolium (NBT) staining levels were lower. The expression levels of oxidation kinase genes (NbSOD, NbPOD, and NbCAT) in transgenic tobacco under drought stress were significantly higher than those in WT tobacco. This enhancement in gene expression improved the ability of transgenic tobacco to detoxify reactive oxygen species (ROS). The survival rate of transgenic tobacco after natural drought rehydration was four times higher than that of WT tobacco. In summary, this study revealed the regulatory mechanism of AfWRKY20 in response to drought stress-induced ABA signaling, particularly in relation to ROS. This finding provides a theoretical basis for understanding the pathways of WRKY20 involved in drought stress, and offers genetic resources for molecular plant breeding aimed at enhancing drought resistance.

LncRNA UCA1 regulates miR-132/Lrrfip1 axis to promote vascular smooth muscle cell proliferation.

UCA1 is predicted to bind to miR-132, which is a key player in the proliferation of vascular smooth muscle cells (VSMCs). This research studied the role of lncRNA UCA1 in atherosclerosis. The binding of UCA1 to miR-132 was proved by dual luciferase activity assay and RNA immunoprecipitation. UCA1 and miR-132 failed to affect each other's expression in VSMCs. UCA1 was upregulated and miR-132 was decreased in atherosclerosis plasma. However, they are not closely correlated across atherosclerosis and control plasma sample. Interestingly, UCA1 suppressed the role of miR-132 in downregulating Lrrfip1 expression and promoting VSMC proliferation. Therefore, UCA1 is downregulated in atherosclerosis and may regulate miR-132/Lrrfip1 axis to promote VSMC proliferation.

Mutation of the ETS1 3′UTR interacts with miR-216a-3p to regulate granulosa cell apoptosis in sheep

ETS1, an important member of the ETS transcription factor family, is involved in a variety of physiological processes in living organisms, such as cell development, differentiation, proliferation and apoptosis, and is thought to be associated with embryonic development and reproduction. However, the polymorphism of ETS1 has been rarely studied, and its potential impact on the formation of reproductive traits in sheep remains unclear. Here, we first analyzed polymorphisms of ETS1 in a population of 382 small-tailed Han sheep with a lambing number record using the Kompetitive Allele Specific PCR (KASP) technique. The results showed the presence of a SNP locus rs161611767 (T > C) in the 3′UTR of ETS1. The association analysis showed the lambing number of first, second and third parity in the individuals with the CC genotype (2.51 ± 0.108, 2.51 ± 0.179, 1.27 ± 0.196) was higher than that of individuals with the TT genotype (1.79 ± 0.086, 1.56 ± 0.102, 0.56 ± 0.100) (P < 0.05). Then, molecular biotechnologies were used to investigate the effects of the EST1 rs161611767 mutant locus on host gene expression in sheep and the underlying mechanism of its effect on sheep reproduction. The RT‒qPCR results showed that the expression of ETS1 was higher in individuals with the CC genotype than in those with the TT genotype (P < 0.05). The dual luciferase reporter assay showed that the luciferase activity of ETS1 in sheep with the TT genotype was decreased compared to CC genotype (P < 0.05), confirming the existence of EST1 rs161611767 in the 3′UTR as a functional SNP. Given that the 3′UTR is an important regulatory region of gene transcription and translation, we performed bioinformatics prediction and confirmed that the SNP rs161611767 of ETS1 was a direct functional target of miR-216a-3p using dual luciferase activity assay, and the binding capacity of allele T was stronger than that of allele C. Subsequently, the cell transfection results showed that miR-216a-3p suppressed the endogenous expression of ETS1 in sheep primary granulosa cells (GCs). Finally, CCK-8, EdU, WB detection of marker proteins and flow cytometry were used to detect the effects of miR-216a-3p on GCs viability and proliferation/apoptosis, respectively. The results showed that miR-216a-3p inhibited the proliferation of GCs while promoting apoptosis of GCs. In conclusion, these results demonstrate that the SNP rs161611767 of ETS1 is associated with lambing number in small-tailed Han sheep, and miR-216a-3p can act as a regulatory element binding to the T mutation in rs161611767 to regulate ETS1 expression and affect GCs development, which may indirectly affect the number of lambs in sheep. These studies provide evidence for the involvement of ETS1 polymorphisms in sheep reproduction and are expected to provide new insights to elucidate the molecular genetic mechanisms of lambing traits in sheep.