Dual-luciferase Reporter Assay System Research Articles

Micro-RNA-338-3p Promotes the Development of Atherosclerosis by Targeting Desmin and Promoting Proliferation

Atherosclerosis (AS) is a dynamic and multi-stage process that involves various cells types, such as vascular smooth muscle cells (VSMCs) and molecules such as microRNAs. In this study, we investigated how miR-338-3p works in the process of AS. To determine how miR-338-3p was expressed in AS, an AS rat model was established and primary rat VSMCs were cultured. Real-time polymerase chain reaction was performed to detect miR-338-3p expression. Markers of different VSMC phenotypes were tested by Western blot. Immunofluorescent staining was employed to observe the morphologic changes of VSMCs transfected with miR-338-3p mimics. A dual luciferase reporter assay system was used to verify that desmin was a target of miR-338-3p. To further identify the role of miR-338-3p in the development of AS, VSMC proliferation and migration were evaluated by EdU incorporation assay, MTT assay, and wound healing assay. miR-338-3p expression was upregulated in the aortic tissues of an AS rat model and in primary rat VSMCs from a later passage. The transfection of miR-338-3p mimics in VSMCs promoted the synthetic cell phenotype. Bioinformatics analysis proposed desmin as a candidate target for miR-338-3p and the dual luciferase reporter assay confirmed in vivo that desmin was a direct target of miR-338-3p. The MTT and EdU incorporation assay revealed increased cell viability when miR-338-3p mimics were transfected. The increased expression of PCNA was a consistent observation, although a positive result was not obtained with respect to VSMC mobility. In AS, miR-338-3p expression was elevated. Elevated miR-338-3p inhibited the expression of desmin, thus promoting the contractile-to-synthetic VSMC phenotypic transition. In addition to morphologic changes, miR-338-3p enhanced the proliferative but not mobile ability of VSMCs. In summary, miR-338-3p promotes the development of AS.

Abnormal Expression of microRNA-296-3p in Type 2 Diabetes Patients and its Role in Pancreatic β-Cells Function by Targeting Tensin Homolog Deleted on Chromosome Ten.

Diabetes mellitus (DM), a familiar disease, is characterized by high blood glucose levels owing to insulin deficiency. Researches have suggested that the incidence rate of diabetes is increasing and it has become an important global epidemic. The type 2 diabetes mellitus (T2DM) is featured with pancreatic β-cell loss and lack of insulin release. Nevertheless, the therapeutic methods that was helpful to improve pancreatic β-cell damage still unclear. Previous report have revealed that tensin homolog deleted on chromosome ten (PTEN) was remarkably enhanced in serum of patients with T2DM, and the lack of PTEN may prevent function deficiency of pancreatic β-cells in DM. However, the underlying mechanisms are rarely illustrated. Our purpose in this report was to illustrated the roles and potential mechanism of microRNA-296-3p (miR-296-3p) in uric acid (UA)-induced pancreatic β-cell injury. The direct target of miR-296-3p was predicted and verified by dual-luciferase reporter system and TargetScan assay. Moreover, Min6 cells were induced by 5mg/dl UA and the cell proliferation, apoptosis, and insulin release were evaluated using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, flow cytometry and glucose-stimulated insulin secretion (GSIS), respectively. Quantitative reverse transcription PCR (qRT-PCR) and western blot assay were adopted to analyze the levels of miR-296-3p, PTEN and apoptosis-related proteins. TargetScan and Dual-luciferase reporter system confirmed that PTEN directly target miR-296-3p. MiR-296-3p was downregulated in UA-induced Min6 cells and the serum of type 2 diabetes patients, while PTEN was upregulated in UA-induced Min6 cells. Upregulation of miR-296-3p by mimic dramatically promoted miR-296-3p level and decreased PTEN level. Besides, PTEN was over-expressed after PTEN-plasmid transfection. UA treatment prominently decreased cell viability, promoted apoptotic cells, enhanced Bax levels, declined Bcl-2 level as well as decreased insulin release in Min6 cells. MiR-296-3p mimic significantly alleviated UA-induced pancreatic β-cells dysfunction, and PTEN-plasmid eliminated the protective effect of miR-296-3p on insulin release, cell viability, and apoptosis of pancreatic β-cells in UA-stimulated Min6 cells. In summary, our findings revealed that upregulation of miR-296-3p protected pancreatic β-cells functions against UA-induced dysfunction by targeting PTEN, which provides a novel agent for type 2 diabetes treatment.

Long Noncoding RNA MALAT1 Regulates the Progression of Atherosclerosis by miR-330-5p/NF-κB Signal Pathway.

Long non-coding RNA metastasis-associated lung adenocarcinoma transcript 1 (MALAT1) was reported to be related to atherosclerosis (AS) progression. However, the underlying mechanism of MALAT1 in AS remains unknown. Quantitative real-time polymerase chain reaction was performed to detect the expression of MALAT1 and miR-330-5p. Western blot was applied to assess the protein levels of cluster of differentiation 36, interleukin-1β, interleukin-6 and tumor necrosis factor-α, phosphorylation of nuclear factor kappa-B inhibitor alpha and phosphorylation of p65. Flow cytometry assay, cell counting kit 8 assay, triglyceride, and total cholesterol detection assays were used to detect the apoptosis, viability, and lipid indexes of THP-1 macrophages-derived foam cells. Online database starbasev2.0 was used to predict the binding sequences between MALAT1 and miR-330-5p and it was verified by dual-luciferase reporter system and RNA immunoprecipitation assay. Besides, an AS mice model was used to evaluate the effect of MALAT1 in vivo. As a result, MALAT1 was overexpressed, whereas miR-330-5p was downregulated in THP-1 macrophages-derived foam cells. MiR-330-5p was a target of MALAT1. MALAT1 depletion inhibited cell formation, apoptosis, and inflammation in THP-1 macrophages-derived foam cells. Besides, MALAT1 overexpression promoted the inflammation in AS mice model, which promoted the pathogenesis of AS. Furthermore, miR-330-5p regulated the nuclear factor kappa light chain enhancer of activated B cells (NF-κB) pathway in THP-1 macrophages-derived foam cells. Moreover, MALAT1 regulated NF-κB signal pathway to mediate the pathogenesis of AS by sponging miR-330-5p. MALAT1 sponges miR-330-5p to activate NF-κB signal pathway in THP-1 macrophages-derived foam cells. This finding may provide a novel biomarker for AS diagnosis.

SOX17 Loss-of-Function Mutation Underlying Familial Pulmonary Arterial Hypertension

Pulmonary arterial hypertension (PAH) refers to a rare, progressive disorder that is characterized by occlusive pulmonary vascular remodeling, resulting in increased pulmonary arterial pressure, right-sided heart failure, and eventual death. Emerging evidence from genetic investigations of pediatric-onset PAH highlights the strong genetic basis underpinning PAH, and deleterious variants in multiple genes have been found to cause PAH. Nevertheless, PAH is of substantial genetic heterogeneity, and the genetic defects underlying PAH in the overwhelming majority of cases remain elusive. In this investigation, a consanguineous family suffering from PAH transmitted as an autosomal-dominant trait was identified. Through whole-exome sequencing and bioinformatic analyses as well as Sanger sequencing analyses of the PAH family, a novel heterozygous SOX17 mutation, NM_022454.4: c.379C>T; p. (Gln127*), was found to co-segregate with the disease in the family, with complete penetrance. The nonsense mutation was neither observed in 612 unrelated healthy volunteers nor retrieved in the population genetic databases encompassing the Genome Aggregation Database, the Exome Aggregation Consortium database, and the Single Nucleotide Polymorphism database. Biological analyses using a dual-luciferase reporter assay system revealed that the Gln127*-mutant SOX17 protein lost the ability to transcriptionally activate its target gene NOTCH1. Moreover, the Gln127*-mutant SOX17 protein exhibited no inhibitory effect on the function of CTNNB1-encode β-catenin, which is a key player in vascular morphogenesis. This research firstly links SOX17 loss-of-function mutation to familial PAH, which provides novel insight into the molecular pathogenesis of PAH, suggesting potential implications for genetic and prognostic risk evaluation as well as personalized prophylaxis of the family members affected with PAH.

Diagnostic significance of circulating miR-485-5p in patients with lupus nephritis and its predictive value evaluation for the clinical outcomes.

Lupus nephritis (LN) is one of the main risk factors contributing to morbidity and mortality of systemic lupus erythematosus (SLE). This study aimed to investigate the potential role of miR-485-5p in human LN. Quantitative real-time polymerase chain reaction was used for the measurement of miR-485-5p levels. The levels of interleukin-1β (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α) in serum were determined by enzyme-linked immunosorbent assay. The diagnostic role of miR-485-5p in LN was evaluated by the receiver operating characteristic (ROC) curve. The impact of miR-485-5p on end-stage renal disease (ESRD) was compared by Kaplan-Meier analysis and Cox regression analysis. The target gene was determined by a dual-luciferase reporter assay system. MiR-485-5p was highly expressed in SLE and LN patients compared with the healthy controls, and LN patients had the highest level of miR-485-5p. The expression level of miR-485-5p in active LN patients was significantly increased compared with that in nonactive cases. MiR-485-5p expression showed a positive correlation with the levels of estimated glomerular filtration rate, serum creatinine, proteinuria, SLE disease activity index score, and inflammatory cytokines. The ROC analysis results indicated that serum miR-485-5p was a promising biomarker for the early diagnosis of LN, and it can distinguish active LN patients from nonactive ones. Phosphatase and tensin homolog was a direct target of miR-485-5p, and negatively associated with serum miR-485-5p levels. More ESRD events were observed in cases with high miR-485-5p expression, miR-485-5p was an independent factor for the risk of ESRD in LN patients. Serum miR-485-5p might be a novel promising diagnostic marker for LN and has potential predictive value for ESRD risk in LN patients.

MicroRNA-665 regulates the proliferation, apoptosis and adhesion of gastric cancer cells by binding to cadherin 3.

Numerous studies have reported that abnormal cadherin 3 (CDH3) and microRNA (miRNA/miR)-665 expression can induce the progression of gastric cancer (GC). However, the mechanism of interaction between miR-665 and CDH3 in GC requires further investigation. The present study aimed to investigate the influence of miR-665 and CDH3 in GC development. The effect of miR-665 and CDH3 on GC tissues and cell lines was examined using reverse transcription-quantitative PCR. Subsequently, CDH3 protein expression in GC cell lines was detected using western blotting. To confirm the association between miR-665 and CDH3, a dual-luciferase reporter assay system was employed. Cell proliferation and adhesion were analyzed using BrdU ELISA, MTT and cell adhesion assays. Finally, caspase-3 activity assay kit and FITC apoptosis detection kit were used for the determination of apoptosis of GC cells. The current findings confirmed the upregulation of CDH3 expression in GC cell lines and tissues. Experimental results indicated that CDH3 overexpression increased cell proliferation and adhesion, but decreased the apoptosis level of the cells. Similarly, the miR-665 inhibitor enhanced cell proliferation and adhesion, but inhibited apoptosis of GC cells. Additionally, it was observed that CDH3 was a direct target of miR-665 in GC cells and that miR-665 inhibited CDH3 expression, thereby repressing the progression of GC. In conclusion, the present study suggested that by targeting CDH3, miR-665 suppressed the progression of GC. These findings may provide a significant theoretical basis for future GC clinical therapy.

PLOD2-driven IL-6/STAT3 signaling promotes the invasion and metastasis of oral squamous cell carcinoma via activation of integrin β1.

We previously reported that high expression of procollagen-lysine 2-oxoglutarate 5-dioxygenase 2 (PLOD2) leads to stabilization and plasma membrane translocation of integrin β1 to promote the invasion and metastasis of oral squamous cell carcinoma (SCC). The present study aimed to further understand the relationship between PLOD2-integrin β1 signaling and the tumor microenvironment. This study provided further advanced insights indicating that elevated interleukin (IL)-6 in the tumor microenvironment acts as a key molecule that triggers PLOD2-integrin β1 axis-derived acceleration of tumor invasion and metastasis. It was found using the dual-luciferase reporter assay system that signal transducer and activator of transcription 3 (STAT3) activation by IL-6 was essential for increasing the expression levels of PLOD2 through direct activation of the PLOD2 promoter in oral SCC, whereas IL-6 stimulation did not contribute to integrin β1 expression or the subsequent maturation process towards a functional form on the plasma membrane. Furthermore, the expression of IL-6 in oral SCC tissues was mainly observed in the tumor stroma. Finally, with double immunofluorescence staining, it was found that IL-6 expression occurred in CD163-positive M2 macrophages distributed around the tumor nest. These results combined with our previous results indicate that as IL-6 significantly increases STAT3-mediated PLOD2 promoter activity, IL-6 released by M2-type tumor-associated macrophages is a crucial factor that promotes PLOD2-integrin β1 axis-enhanced invasion and metastasis of oral SCC cells.

Downregulation of TAF9B by miR-7-5p Inhibits the Progression of Osteosarcoma.

BackgroundOsteosarcoma (OS) is a malignant bone tumor with high metastatic potential. As a regulatory factor of apoptosis, TATA-box binding protein (TBP) associated factor 9B (TAF9B) is rarely studied in tumors.MethodsWe investigated the role and mechanism of TAF9B in OS cells by overexpression and knockdown. CCK8, colony formation, transwell, and flow cytometry analysis were performed to detect proliferation, migration, invasion, and apoptosis.ResultsTAF9B overexpression promotes the proliferation, migration, and invasion of OS cells, while TAF9B knockdown gives the opposite result. TAF9B inhibits apoptosis by upregulating Bcl-2 and downregulating Bax and Cleaved-caspase-3. Through starBase analysis, it was found that miR-7-5p can bind to the 3ʹUTR region of TAF9B, which is further confirmed by the dual luciferase reporter system assay. MiR-7-5p downregulates the expression of TAF9B in MG63 and U2OS cells. The proliferation and invasion of OS cells are inhibited after miR-7-5p mimics transfection and are promoted after miR-7-5p inhibitor transfection. TAF9B rescues the inhibitory effect of miR-7-5p on OS cells. TAF9B also activates the AKT/mTOR signaling pathway.ConclusionAccording to our results, miR-7-5p inhibits the translation of TAF9B and then suppresses growth and metastasis through the AKT/mTOR signaling pathway in OS cells, thereby indicating the potential value of miR-7-5p and TAF9B as therapeutic targets for human OS.

SOX17 loss-of-function variation underlying familial congenital heart disease

As the most prevalent form of human birth defect, congenital heart disease (CHD) contributes to substantial morbidity, mortality and socioeconomic burden worldwide. Aggregating evidence has convincingly demonstrated that genetic defects exert a pivotal role in the pathogenesis of CHD, and causative mutations in multiple genes have been causally linked to CHD. Nevertheless, CHD is of pronounced genetic heterogeneity, and the genetic components underpinning CHD in the overwhelming majority of patients remain obscure. In this research, a four-generation consanguineous family suffering from CHD transmitted in an autosomal dominant mode was recruited. By whole-exome sequencing and bioinformatics analyses as well as Sanger sequencing analyses of the family members, a new heterozygous SOX17 variation, NM_022454.4: c.553G > T; p.(Glu185*), was identified to co-segregate with CHD in the family, with complete penetrance. The nonsense variation was neither detected in 310 unrelated healthy volunteers used as controls nor retrieved in such population genetics databases as the Exome Aggregation Consortium database, Genome Aggregation Database, and the Single Nucleotide Polymorphism database. Functional assays by utilizing a dual-luciferase reporter assay system unveiled that the Glu185*-mutant SOX17 protein had no transcriptional activity on its two target genes NOTCH1 and GATA4, which have been reported to cause CHD. Furthermore, the mutation abrogated the synergistic transactivation between SOX17 and NKX2.5, another established CHD-causing transcription factor. These findings firstly indicate SOX17 loss-of-function mutation predisposes to familial CHD, which adds novel insight to the molecular mechanism of CHD, implying potential implications for genetic risk appraisal and individualized prophylaxis of the family members affected with CHD.

Long non-coding RNA THRIL inhibits miRNA-24-3p to upregulate neuropilin-1 to aggravate cerebral ischemia-reperfusion injury through regulating the nuclear factor κB p65 signaling.

Purpose: The aim of this study was to investigate the role of the tumor necrosis factor and HNRNPL related immunoregulatory long non-coding RNA (THRIL) in cerebral ischemia-reperfusion injury.Methods: A rat middle cerebral artery occlusion/ischemia-reperfusion (MCAO/IR) model and an oxygen glucose deprivation/reoxygenation (OGD/R) cell model were constructed. THRIL was knocked down using siTHRIL. Neurological deficit score was detected based on the criteria of Zea-Longa. Brain region 2,3,5-Triphenyltetrazolium (TTC) staining and quantitative analysis of cerebral infarction volume, RT-qPCR, and fluorescence immunostaining were performed for assessing THRIL expression. MTT assay was used to detect the cell proliferation ability after transfection, TUNEL assay was applied to detect apoptosis, and western blot and ELISA detected related protein expression. A dual luciferase reporter system and RIP assay were used to confirm the target relationship.Results: THRIL was upregulated in both in vitro and in vivo models of brain ischemia-reperfusion injury. Knockdown of THRIL attenuated OGD/R neuronal apoptosis and OGD/R-induced inflammation. THRIL targeted and regulated the expression of miR-24-3p/neuropilin-1 (NRP1) axis. THRIL silencing significantly improved the neurological functioning of rats in the MCAO/R model by miR-24-3p/NRP1/NF-κB p65 signaling pathway.Conclusion: THRIL could aggravate cerebral ischemia-reperfusion injury by competitively binding to miR-24-3p to promote the upregulation of NRP1 and further promoted the activation of the NF-κB p65 signaling pathway.

MiR-130b-3p is high-expressed in polycystic ovarian syndrome and promotes granulosa cell proliferation by targeting SMAD4

BackgroundBeing one of the most prevalent metabolic and endocrine disorders, Polycystic Ovary Syndrome (PCOS) has been proven to be associated with microRNA-130b-3p (miR-130b-3p). However, the exact role played by miR-130b-3p in the pathogenesis and progression of PCOS remains unknown. Thus, this article is focused on elucidating the function of miR-130b-3p in the pathogenesis of PCOS. MethodsThe expression levels of miR-130b-3p and SMAD4 in tissues and cells responsible for the development of PCOS were determined by RT-qPCR and western blot. A miR-130b-3p mimic/inhibitor or si-SMAD4 were transfected into KGN cells. The cell viability was detected by CCK-8 and EDU methods. The activity of caspase-3 was measured by caspase-3 analysis. Subsequently, apoptosis and the cell cycle were measured via flow cytometry. The correlation between SMAD4 and miR-130b-3p was confirmed using an RNA pull-down assay and a dual luciferase reporter system assay. ResultsMiR-130b-3p was upregulated in the KGN cells and ovarian granulosa cells (GCs) of PCOS patients. It was found that miR-130b-3p overexpression or SMAD4 silencing can promote KGN cell proliferation and positive EDU rates, induce S phase arrest, inhibit apoptosis and caspase-3 activity. On the other hand, miR-130b-3p inhibitors reduce KGN cell proliferation, inhibit apoptosis and reverse the effect of si-SMAD4. ConclusionMiR-130b-3p directly interacts with SMAD4 to induce KGN cell proliferation, inhibit apoptosis, suggesting that miR-130b-3p expression is positively correlated with the development of PCOS. This may serve as new evidence for the abnormal proliferation of GCs in PCOS.

MicroRNA-221 promotes tumor progression by targeting HHIP in human glioblastoma.

BackgroundMicroRNAs are found to be aberrantly expressed in multiple cancers, including glioblastoma (GBM), and microRNA-221 (miR-221) has been verified as an oncogene in various human cancers. Nevertheless, the role of miR-221 in GBM is unclear. This study aimed to investigate the miR-221 expression level in GBM and to evaluate its function and underlying mechanisms.MethodsWestern blotting and qPCR were used to determine the expression of human hedgehog-interacting protein (HHIP) and miR-221 levels. MiR-221-inhibited cell models were constructed, and siRNA was used for HHIP silencing. Cell proliferation was analyzed by MTT and colony formation assays and a subcutaneous xenograft model. Cell migration and invasion was analyzed by wound healing and Transwell invasion assays. A dual luciferase reporter assay system was used to clarify the relationship between miR-221 and HHIP.ResultsThe results of this study revealed that miR-221 expression was upregulated in GBM tissues and A172, U251, as well as T98G cells, as detected by real-time PCR analysis. MTT, Transwell, and colony formation assays revealed that miR-221 knockdown could suppress GBM cells from proliferating, migrating, and invading in vitro. Moreover, animal experiments showed that tumor growth in vivo was inhibited when miR-221 expression decreased. Furthermore, HHIP was predicted and verified to be a target of miR-221 by bioinformatics analysis, and luciferase and western blot assays. In addition, HHIP silencing rescued the suppressive effect of a miR-221 inhibitor on the proliferation, migration, and invasion of GBM cells.ConclusionsOur results indicated that miR-221 is upregulated in GBM and enhances tumor progression by targeting HHIP, which suggests this may be a potential therapeutic target for GBM.

LncRNA MEG8 promotes NSCLC progression by modulating the miR-15a-5p-miR-15b-5p/PSAT1 axis

BackgroundNon-small cell lung cancer (NSCLC) is the most common tumor with severe morbidity and high mortality. Long non-coding RNAs (lncRNAs) as crucial regulators participate in multiple cancer progressions. However, the role of lncRNA MEG8 in the development of NSCLC remains unclear. Here, we aimed to investigate the effect of lncRNA MEG8 on the progression of NSCLC and the underlying mechanism.MethodsCell proliferation was analyzed by EdU assays. The impacts of lncRNA MEG8, miR-15a-5p, and miR-15b-5p on cell invasion and migration of NSCLC were assessed by transwell assay. The luciferase reporter gene assay was performed using the Dual-luciferase Reporter Assay System. The effect of lncRNA MEG8, miR-15a-5p, and miR-15b-5p on tumor growth was evaluated in nude mice of Balb/c in vivo.ResultsWe revealed that the expression levels of MEG8 were elevated in the NSCLC patient tissues compared to that in adjacent normal tissues. The expression of MEG8 was negatively relative to that of miR-15a-5p and miR-15b-5p in the NSCLC patient tissues. The expression of MEG8 was upregulated, while miR-15a-5p and miR-15b-5p were downregulated in NSCLC cell lines. The depletion of MEG8 inhibited NSCLC cell proliferation, migration, and invasion in vitro. MEG8 contributed to NSCLC progression by targeting miR-15a-5p/miR-15b-5p in vitro. LncRNA MEG8 contributes to tumor growth of NSCLC via the miR-15a/b-5p/PSAT1 axis in vivo. Thus, we concluded that lncRNA MEG8 promotes NSCLC progression by modulating the miR-15a/b-5p/PSAT1 axis.ConclusionsOur findings demonstrated that lncRNA MEG8 plays a critical role in NSCLC development. LncRNA MEG8, miR-15a-5p, miR-15b-5p, and PSAT1 may serve as potential targets for NSCLC therapy.

TLR4 rs1927914 polymorphism contributes to serum TLR4 levels in patients with aortic aneurysm

Toll-like receptor 4 (TLR4) is a crucial regulator of inflammatory reactions and vascular remodeling. Elevated TLR4 expression has been proved to be correlated with an increased risk of aortic aneurysm (AA). This study aimed to explore the influence of TLR4 gene polymorphisms on TLR4 expression levels and its probable functional significance in AA disease. A total of 294 AA patients and 285 controls were enrolled in the study and serum TLR4 levels were detected by ELISA. All the participants were genotyped for two tag-SNPs in TLR4 (rs1927914 in the promoter region and rs11536889 in the 3′-untranslated region) using the KASP method. Relative luciferase activity was measured by the dual-luciferase reporter assay system. The rs1927914 TC, TC/CC genotypes and C allele showed associations with increased serum TLR4 levels in the total population and AA patients (all P<0.05). Further stratified analysis demonstrated that AA subjects with TC or TC/CC genotype of rs1927914 had significantly higher serum levels of TLR4 than those with TT genotype in male, age>60y, hypertension, diabetes, TAA type and size>5.0 cm subgroups (all P<0.05). In binary logistic analysis, rs1927914 TC genotype and dominant model presented significant associations with high TLR4 levels (OR = 1.579 and 1.431, P = 0.020 and 0.049, respectively) after adjusting age, hypertension and diabetes. However, rs11536889 polymorphism had no significant influence on serum TLR4 levels. Regarding rs1927914, luciferase activity of the C allele construct was significantly increased in comparison with the T allele construct (0.589 ± 0.004 vs. 0.340 ± 0.014, P<0.001). Our results provided evidence that rs1927914 polymorphism contributed to serum TLR4 levels, possibly by influencing promoter activity of TLR4, and could be a novel genetic factor in the formation of AA.

Long non-coding RNA COL4A2-AS1 facilitates cell proliferation and glycolysis of colorectal cancer cells via miR-20b-5p/hypoxia inducible factor 1 alpha subunit axis

ABSTRACT Long non-coding RNAs (lncRNAs) have critical functions in tumorigenesis and progression of colorectal cancer (CRC). The role of lncRNA COL4A2-AS1 (COL4A2-AS1) lacks system investigation. The current study comprehensively analyzed the expression, biological functions, and mechanism of COL4A2-AS1 in CRC through performing real-time quantitative PCR (RT-qPCR), Western blot, cell transfection, cell colony assay, MTT assay, flow cytometry and dual-luciferase reporter system assays. A xenograft model of CRC was constructed to further verify the function of COL4A2-AS1 in CRC progression in vivo. The data revealed an upregulated expression of COL4A2-AS1 in CRC tissues and cell lines than paired adjacent tissues and normal cell line. Silencing COL4A2-AS1 inhibited proliferation, aerobic glycolysis, and promoted apoptosis of CRC cells in vivo and in vitro. However, overexpression of COL4A2-AS1 significantly promoted CRC cell proliferation and aerobic glycolysis. In CRC cells, miR-20b-5p was sponged by COL4A2-AS1 and hypoxia-inducible factor 1 alpha subunit (HIF1A). Restoration of HIF1A expression reversed the inhibitory effects of silencing COL4A2-AS1 on aerobic glycolysis and proliferation of CRC cells. The current findings showed that COL4A2-AS1 promoted the proliferation, and aerobic glycolysis of CRC cells potentially through modulating the miR-20b-5p/HIF1A axis.

LncRNA MSTRG.29039.1 Promotes Proliferation by Sponging hsa-miR-12119 via JAK2/STAT3 Pathway in Multiple Myeloma.

Noncoding RNA (ncRNA) is involved in the occurrence, development, metastasis, and drug resistance of tumors and involves a variety of biological functions. In addition, miRNA can regulate proliferation and migration and even regulate epigenetics to promote the development of multiple myeloma (MM). However, the mechanism of ncRNA involved in MM is still unclear, and there are many unknown ncRNAs to be explored. This research is aimed at discovering the unknown lncRNA in MM through high-throughput sequencing and to study the mechanism and role of competitive endogenous RNA (ceRNA) involved in the pathogenesis of MM for the development of novel molecular markers and potential new targeted drugs. We screened out 262 new lncRNAs with statistical differences by RNA sequencing and selected the lncRNA MSTRG.29039.1 according to the expression and function of lncRNAs and their target genes in MM. We verified that MSTRG.29039.1 and its target gene OSMR were highly expressed in MM. After knockdown of MSTRG.29039.1 in MM cell lines, the expression of OSMR was decreased, and the expression of hsa-miR-12119 was upregulated which can also promote cell apoptosis and inhibit proliferation. Then, we knocked down hsa-miR-12119 and MSTRG.29039.1, we found that apoptosis of MM cells was reduced, and cell proliferation was increased compared with just knocking down hsa-miR-12119. We further verified the direct binding relationship between MSTRG.29039.1 and OSMR by the dual-luciferase reporter assay system. Thus, MSTRG.29039.1 can competitively bind with miRNA to counteract the inhibitory effect of miRNA on OSMR, which regulates cell proliferation and apoptosis through the JAK2/STAT3 pathway. In a conclusion, lncRNA MSTRG.29039.1 could promote proliferation by sponging hsa-miR-12119 via the JAK2/STAT3 pathway in multiple myeloma. This may be a molecular marker and a potential therapeutic target for MM.

Circular RNA_0062582 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via regulation of microRNA-145/CBFB axis

ABSTRACT Osteoporosis poses a threat to human health worldwide. To date, there have been few studies regarding targeted treatment of osteoporosis. We aimed to identify the possible molecular mechanism of circular RNA (circ)_0062582 in osteogenic differentiation, and the interactions among circ_0062582, microRNA-145 (miR-145) and core-binding factor subunit β (CBFB). The proliferation of human bone marrow mesenchymal stem cells (hBMSCs) was tested with a cell counting kit-8 assay. Circ_0062582, miR-145 and CBFB were overexpressed by transient transfection. Dual-luciferase reporter assay system was used to analyze the combination among circ_0062582, miR-145 and CBFB. Additionally, the levels of circ_0062582, miR-145, CBFB, osterix (OSX), osteocalcin (OCN) and collagen type 1 (COL1) were detected by means of RT-qPCR or western blot analysis. Alkaline phosphatase and Alizarin red stainings were performed to analyze the degree of osteogenic differentiation under the control of circ_0062582, miR-145 and CBFB. The results demonstrated that circ_0062582 level was notably elvated during osteogenic differentiation of hBMSCs. Circ_0062582 overexpression significantly promoted osteogenic differentiation and upregulated the levels of osteogenic differentiation-related proteins, including OSX, OCN and COL1. In addition, miR-145, which was identified as the target gene of circ_0062582, could specifically target CBFB 3′-UTR regions. Next, these changes caused by the overexpression of circ_0062582 were reversed following the addition of miR-145 mimic. Following overexpression of CBFB, osteogenic differentiation was increased. In summary, these results demonstrated that the role of circ_0062582 in osteoporosis is mediated through regulating the expression level of CBFB via miR-145.

MicroRNA-302a is involved in folate deficiency-induced apoptosis through the AKT-FOXO1-BIM pathway in mouse embryonic stem cells

BackgroundOur previous study had shown that microRNA (miR)-302a played a key role in folate deficiency-induced apoptosis in mouse embryonic stem cells. However, details regarding the mechanism remain unclear. Transcription factors (TFs) and miRNAs are two key elements in gene regulation. The aim of this study is to construct the TF-miRNA gene regulation network and demonstrate its possible mechanism.MethodsThe TF-miRNA gene regulation network was constructed via bioinformatics methods. Chromatin immuno-coprecipitation PCR was selected to confirm the binding between miR-302a and TF. mRNA and protein levels were detected by Real-time quantitative PCR and western blotting. TargetScan prediction and Dual-Luciferase Reporter Assay system were used to confirm whether the miRNA binded directly to the predicted target gene.ResultsFOXO1 and miR-302a were selected as the key TF and miRNA, respectively. FOXO1 was confirmed to bind directly to the upstream promoter region of miR-302a. Real-time quantitative PCR and immunoblotting showed that in folate-free conditions, miR-302a and AKT were down regulated, while FOXO1 and Bim were up-regulated significantly. Additionally, treatment with LY294002 inhibitor revealed the involvement of the Akt/FOXO1/Bim signaling pathway in folate deficiency-induced apoptosis, rather than the ERK pathway. Finally, TargetScan prediction and double luciferase reporting experiments illustrated the ability of miR-302a to target the Bim 3′UTR region.ConclusionThe involvement of miR-302a in folate deficiency-induced apoptosis through the AKT-FOXO1-BIM pathway in mESCs is a unique demonstration of the regulation mechanism of nutrient expression in embryonic development.

Down-regulation of miR-215 attenuates lipopolysaccharide-induced inflammatory injury in CCD-18co cells by targeting GDF11 through the TLR4/NF-kB and JNK/p38 signaling pathways.

Ulcerative colitis (UC) is a risk factor for carcinogenesis of colorectal cancer, which is associated with disruption of the epithelial barrier and disorder of the inflammatory response. It has been reported that the expression of microRNA (miR)-215 is upregulated in patients with long-term UC. The present study aimed to investigate the effects of miR-215 on lipopolysaccharide (LPS)-induced inflammatory injury in CCD-18Co cells, as well as to identify the underlying possible molecular mechanisms. CCD-18Co cells were treated with 1 µg/ml LPS to induce inflammatory injury. Reverse transcription-quantitative PCR was performed to determine the expression of miR-215 in LPS-treated CCD-18Co cells. Moreover, a dual luciferase reporter system assay was used to evaluate the interaction of miR-215 and growth differentiation factor 11 (GDF11) in CCD-18Co cells. The expression of miR-215 was significantly upregulated in LPS-treated CCD-18Co cells. Knockdown of miR-215 significantly alleviated the inflammatory response and oxidative stress in LPS-treated CCD-18Co cells. In addition, GDF11 was identified as a direct binding target of miR-215 in CCD-18Co cells. Knockdown of miR-215 significantly increased the expression of GDF11, but decreased the expression levels of Toll-like receptor (TLR)4, phosphorylated (p)-p65, iNOS, p-p38 and p-JNK in LPS-treated CCD-18Co cells. Collectively, the present findings indicated that knockdown of miR-215 alleviated oxidative stress and inflammatory response in LPS-treated CCD-18Co cells by upregulating GDF11 expression and inactivating the TLR4/NF-κB and JNK/p38 signaling pathways.

KLF13 loss-of-function variation contributes to familial congenital heart defects.

Congenital heart defect (CHD) represents the most common form of human developmental abnormality and contributes to substantial morbidity, mortality, and socioeconomic burden worldwide. Accumulating evidence underscores the strong genetic basis of CHD. Nevertheless, CHD is of pronounced genetic heterogeneity, and the genetic determinants underlying CHD in most patients are still unclear. This study was mainly sought to identify the causative gene for CHD in a consanguineous Chinese family. Whole-exosome sequencing and bioinformatics analyses were performed in a Chinese family with CHD (double-outlet right ventricle and ventricular septal defect), which was transmitted in an autosomal dominant pattern. A total of 312 unrelated healthy individuals were then genotyped for the identified genetic variation. The functional effect of the identified variation was characterized by utilizing a Dual-Luciferase reporter assay system. A novel heterozygous variation, NM_015995.3: c.370G>T; p.(Glu124*), was identified in the KLF13 gene, which encodes Kruppel-like factor 13 key to proper heart development. Genetic analysis of the pedigree unveiled that the variation co-segregated with CHD, with complete penetrance. The variation was absent from 624 control chromosomes. The biological analysis revealed that the Glu124*-mutant KLF13 protein failed to transactivate its cardiac target genes ACTC1 and ANP. Furthermore, the variation disrupted the synergistic transactivation between KLF13 and GATA4, as well as GATA6, two other genes that have been recognized to cause CHD. These findings firstly indicate that genetically defective KLF13 predisposes to familial CHD, implying potential implications for genetic counseling and an improved prophylactic strategy in a subset of CHD patients.