Luciferase Reporter Gene Experiment Research Articles

MiR-137 mediated high expression of TIGD1 promotes migration, invasion, and suppresses apoptosis of lung adenocarcinoma

ObjectivesTigger transposable element-derived 1 (TIGD1) expression and its underlying functions and regulatory mechanisms in lung adenocarcinoma (LUAD) remain unknown. Therefore, we intended to explore the expression, potential functions, and regulatory mechanisms of TIGD1 in LUAD. Materials and methodsTIGD1 expression in LUAD tissues was determined by immunohistochemistry analysis of a tissue microarray. Functional experiments were conducted to determine how TIGD1 affects LUAD tumorigenesis and metastasis. The molecular mechanisms by which TIGD1 induces LUAD progression were determined. ResultsTIGD1 was upregulated in LUAD tissues and was related to lymph node metastases. TIGD1 knockdown suppressed LUAD cell proliferation, migration, and invasion, while promoted cell apoptosis. Furthermore, decreased metastatic nodules were observed in the TIGD1 knockdown mouse metastasis model. Moreover, microarray analysis was performed to determine the potential downstream genes of TIGD1 in LUAD. Hallmark pathway analysis revealed that the downstream genes of TIGD1 were involved in epithelial-mesenchymal transition (EMT). Western blotting confirmed that vimentin and TWIST was downregulated in TIGD1 knockdown cells, while E-cadherin was upregulated. Ingenuity pathway and hallmark pathway analyses revealed that TIGD1 regulated the interleukin-6 signaling pathway and related gene members. Western blotting, quantitative real-time polymerase chain reaction, enzyme-linked immunosorbent assay indicated that downregulation of TIGD1 decreased interleukin-6 and CXCL1 expression. TIGD1 expression was negatively correlated with immune infiltration in LUAD. The upstream microRNA of TIGD1 was predicted, and subsequent luciferase reporter gene experiments confirmed the interactions between microRNA-137 and TIGD1. The expression of miR-137 was significantly downregulated in LUAD tissues and miR-137 suppressed the proliferation, migration, and invasion of LUAD cells, partially through negatively regulating the expression of TIGD1. ConclusionOur findings suggest that TIGD1, which was regulated by microRNA-137, contributed to LUAD progression by promoting cell proliferation, migration, invasion, and EMT and suppressing cell apoptosis.

The mechanism of miR-665 targeting GNB3 in aluminum-induced neuronal apoptosis

BackgroundAluminum exerts neurotoxic effects through various mechanisms, mainly manifested as impaired learning and memory function. MethodsForty SD rats were divided into 0, 10, 20, and 40 mM maltol aluminum [Al(mal)3] groups. Cell experiments are divided into 0, 100, 200, and 400 μM Al(mal)3 dose group and control, Al(mal)3, Al(mal)3+inhibitor NC, Al(mal)3+miR-665 inhibitor intervention group. Water maze was used to detect the learning and memory function of rats, HE staining was used to observe the morphology and number of neurons in the CA1 area of the rat hippocampus, Flow cytometry was used to detect the apoptosis of PC12 cells, PCR and Western blotting were used to detect the expression of Caspase3, miR-665 and GNB3/PI3K/AKT proteins. The target binding relationship between miR-665 and GNB3 was verified by double luciferase reporter gene experiment. ResultsIn vivo experimental results showed that with the increase of Al(mal)3 concentration, the escape latency of rats was prolonged, the target quadrant dwell time was shortened, and the number of crossing platform was reduced. Moreover, the arrangement of neurons was loose and the number decreased; the expression of Caspase3 and miR-665 increased, while the expression of GNB3/PI3K/AKT proteins decreased. In vitro experiments, with the increase of Al(mal)3 concentration, apoptosis rate of PC12 cells increased, the expression of Caspase3, miR-665 and GNB3/PI3K/AKT proteins were consistent with rat results. After inhibiting miR-665 in the intervention group experiment, apoptosis rate of PC12 cells in the aluminum exposure group decreased, the expression of Caspase3 and miR-665 decreased, and the expression of GNB3/PI3K/AKT proteins increased. ConclusionMiR-665 plays an important role in aluminum induced neuronal apoptosis by targeting GNB3 and regulating the PI3K/AKT pathway.

The role and mechanism of circadian clock gene BMAL1 in the proliferation and metastasis of nasopharyngeal carcinoma by inhibition TGF-β1/Smads pathway.

6027 Background: To explore the molecular mechanism of biological clock gene BMAL1 inhibiting the proliferation and metastasis of nasopharyngeal carcinoma(NPC). Methods: 1. IHC detected the expression of BMAL1 in 41 cases of nasopharyngeal chronic inflammatory tissue and 71 cases of NPCtissue. PCR and Western blot measured the expression levels of BMAL1 in NP69 in NPC cells and human immortalized nasopharyngeal epithelial cells, respectively. 2. To detect the effect of BMAL1 on the proliferation capacity of NPCcells in vitro and in vivo; Scratch healing and Transwell invasion migration experiments detected the effect of BMAL1 on the invasion and migration ability of NPCcells in vitro. Fluorescence in vivo imaging and HE staining to determine the transfer of nude mouse tail intravenous injection model. PCR and Western blot detect the effect of BMAL1 on the expression of EMT-related markers at the transcriptional and protein levels in NPC cells. 3. A series of molecular biological means such as bioinformatics technology, ChIP experiment, double luciferase reporter gene experiment, immunofluorescence colocalization experiment and Co-IP were used to verify its mechanism. Results: 1. The expression of BMAL1 in NPCtissues and cells is reduced. The expression of BMAL1 in NPC tissues was associated with the M stage of nasopharyngeal carcinoma patients (p=0.006). 2. Overexpression of BMAL1 can inhibit the proliferation, invasion and migration ability of nasopharyngeal cancer cells, while knocking down BMAL1 is the opposite. Overexpression of BMAL1 inhibits EMT of NPCcells. 3. There are 5 BMAL1 binding sites in the TGF-β1promoter region, and BMAL1 can directly bind to the TGF-β1promoter region, which can inhibit the transcriptional activity of TGF-β1. Overexpression of BMAL1 inhibits TGF-β1/Smads pathway activity. Under the induction of recombinant human TGF-β1, the proliferation ability of NPC cells was enhanced, the activity of TGF-β1/Smads signaling pathway was enhanced, and nasopharyngeal cancer cells underwent EMT, and cell invasion and migration capacity increased. After overexpression of BMAL1, TGF-β1 no longer promotes TGF-β1/Smads signaling pathway activity, EMT is inhibited, and cell invasion and migration ability is weakened. BMAL1 combined with TGF-β1 has certain diagnostic value in predicting whether nasopharyngeal carcinoma metastasis. Conclusions: BMAL1 expression is downregulated in nasopharyngeal carcinoma, and its expression level is related to the development and metastasis of nasopharyngeal carcinoma. BMAL1 can inhibit the proliferation, epithelial-mesenchymal transformation and invasion and metastasis ability of NPC cells, and its mechanism is related to the inhibition of TGF-β1/Smads signaling pathway activity. It is speculated that it may become a new prognostic marker and therapeutic target for nasopharyngeal carcinoma.

Schwann cell-derived extracellular vesicles promote memory impairment associated with chronic neuropathic pain.

The pathogenesis of memory impairment, a common complication of chronic neuropathic pain (CNP), has not been fully elucidated. Schwann cell (SC)-derived extracellular vesicles (EVs) contribute to remote organ injury. Here, we showed that SC-EVs may mediate pathological communication between SCs and hippocampal neurons in the context of CNP. We used an adeno-associated virus harboring the SC-specific promoter Mpz and expressing the CD63-GFP gene to track SC-EVs transport. microRNA (miRNA) expression profiles of EVs and gain-of-function and loss-of-function regulatory experiments revealed that miR-142-5p was the main cargo of SC-EVs. Next, luciferase reporter gene and phenotyping experiments confirmed the direct targets of miR-142-5p. The contents and granule sizes of plasma EVs were significantly greater in rats with chronic sciatic nerve constriction injury (CCI)than in sham rats. Administration of the EV biogenesis inhibitor GW4869 ameliorated memory impairment in CCI rats and reversed CCI-associated dendritic spine damage. Notably, during CCI stress, SC-EVs could be transferred into the brain through the circulation and accumulate in the hippocampal CA1-CA3 regions. miR-142-5p was the main cargo wrapped in SC-EVs and mediated the development of CCI-associated memory impairment. Furthermore, α-actinin-4 (ACTN4), ELAV-like protein 4 (ELAVL4) and ubiquitin-specific peptidase 9 X-linked (USP9X) were demonstrated to be important downstream target genes for miR-142-5p-mediated regulation of dendritic spine damage in hippocampal neurons from CCI rats. Together, these findings suggest that SCs-EVs and/or their cargo miR-142-5p may be potential therapeutic targets for memory impairment associated with CNP.

Differentially expressed miR-4310 functions as a tumor suppressor in colorectal cancer.

Colorectal cancer (CRC) is a digestive tract malignancy microRNAs (miRNAs) have attracted much attention as biomarkers in tumor studies. This work focused on the predictive potential and mechanism of miR-4310 in CRC. The miRNA expression profile sets were obtained from the Gene Expression Omnibus (GEO) database, and the appropriate miRNA was screened by GEO2R. The CRC tissues and control tissues of 88 patients with CRC were collected, and the expression of miR-4310 was detected by quantitative real-time PCR, and the efficacy of miR-4310 in diagnosing CRC was evaluated by the receiver operating characteristic curve (ROC). The effects of miR-4310 on the proliferation, migration, and invasion of CRC cells were explored by Cell Counting Kit-8 (CCK-8) and Transwell experiments. Predicting the potential binding sites of miR-4310 and Runt-related transcription factor 1 (RUNX1) by four predictive websites. The relationship between miR-4310 and RUNX1 was confirmed by a double luciferase reporter gene experiment. The bioinformatics analysis found that miR-4310 was differentially expressed in CRC tissues and this finding was certified by the expression of miR-4310 in CRC tissues of collected patients and cultured CRC cell lines. The expression of miR-4310 had a predictive possibility for CRC patients. MiR-4310/RUNX1 pathway had effects on CRC viability, migration, and invasion. MiR-4310 had the potential to be a biomarker for early screening of CRC. MiR-4310 and RUNX1 participated in the regulation of CRC cells.

DNA methylation of microRNA-365-1 induces apoptosis of hair follicle stem cells by targeting DAP3

BackgroundDNA methylation is a crucial epigenetic alteration involved in diverse biological processes and diseases. Nevertheless, the precise role of DNA methylation in chemotherapeutic drug-induced alopecia remains unclear. This study examined the role and novel processes of DNA methylation in regulating of chemotherapeutic drug-induced alopecia. MethodsA mouse model of cyclophosphamide (CTX)-induced alopecia was established. Hematoxylin-eosin staining and immunohistochemical staining for the Ki67 proportion and a mitochondrial membrane potential assay (JC-1) were performed to assess the structural integrity and proliferative efficiency of the hair follicle stem cells (HFSCs). Immunofluorescence staining and real-time fluorescence quantitative PCR (RT-qPCR) were performed to determine the expression levels of key HFSC markers, namely Lgr5, CD49f, Sox9, CD200, and FZD10. Differential DNA methylation levels between the normal and CTX-induced model groups were determined through simple methylation sequencing and analyzed using bioinformatics tools. The expression levels of miR-365-1, apoptosis markers, and DAP3 were detected through RT-qPCR and western blotting. In parallel, primary mouse HFSCs were extracted and used as a cell model, which was constructed using 4-hydroperoxycyclophosphamide. The luciferase reporter gene assay was conducted to confirm miR-365-1 binding to DAP3. To measure the expression of relevant indicators, superoxide dismutase (SOD) and malondialdehyde (MDA) kits were used. Methylation-specific PCR (MS-PCR) was performed to determine DNA methylation levels. The regulatory relationship within HFSCs was confirmed through plasmid overexpression of miR-365-1 and DAP3. ResultIn the alopecia areata model, a substantial number of apoptotic cells were observed within the hair follicles on the mouse backs. Immunofluorescence staining revealed that the expression of HFSC markers significantly reduced in the CTX group. Both RT-qPCR and western blotting demonstrated a noteworthy difference in DNA methyltransferase expression. Simple methylation sequencing unveiled that DNA methylation substantially increased within the dorsal skin of the CTX group. Subsequent screening identified miR-365-1 as the most differentially expressed miRNA. miR-365-1 was predicted and confirmed to bind to the target gene DAP3. In the CTX group, SOD and ATP expression markedly reduced, whereas MDA levels were significantly elevated. Cellular investigations revealed 4-HC-induced cell cycle arrest and decreased expression of HFSC markers. MS-PCR indicated hypermethylation modification of miR-365-1 in the 4-HC-induced HFSCs. The luciferase reporter gene experiment confirmed the binding of miR-365-1 to the DAP3 promoter region. miR-365-1 overexpression dramatically reduced apoptotic protein expression in the HFSCs. However, this effect was slightly reversed after DAP3 overexpression in lentivirus. ConclusionThis study explored the occurrence of miR-365-1 DNA methylation in chemotherapeutic drug-induced alopecia. The results unveiled that miR-365-1 reduces cell apoptosis by targeting DAP3 in HFSCs, thereby revealing the role of DNA methylation of the miR-365-1 promoter in chemotherapeutic drug-induced alopecia.

N6-methyladenosine-induced miR-182-5p promotes multiple myeloma tumorigenesis by regulating CAMK2N1.

Methyltransferase like 3 (METTL3) has been reported to promote tumorigenesis of multiple myeloma (MM), however, the molecular mechanism still needs further research. The N6-methyladenosine (m6A) level in tissues or cells was measured by m6A kit and dot blot assay. The mRNA and protein expression were detected by quantitative real-time PCR (RT-qPCR) and Western blot, respectively. The cell counting kit-8 and colony formation assay were used to detect the cell proliferation. Coimmunoprecipitation (Co-IP) experiment verified the binding of two proteins. The luciferase reporter experiment demonstrated the targeted binding of miR-182-5p and CaMKII inhibitor 1 (CAMK2N1). More importantly, tumor growth was measured in xenograft mice. Our data showed that the expression of METTL3 was significantly increased in MM patients' samples and MM cells. METTL3 overexpression promoted MM cells proliferation, and METTL3 knockdown inhibited MM cells proliferation. Mechanically, METTL3-dependent m6A participated in DiGeorge syndrome critical region 8 (DGCR8)-mediated maturation of pri-miR-182. Upregulation of miR-182-5p further enhanced the promoting proliferation effect of METTL3 overexpression on MM cells. Moreover, the luciferase reporter gene experiment proved that miR-182-5p targetedly regulated CAMK2N1 expression. Xenograft tumor in nude mice further verified that METTL3 promoted MM tumor growth through miR-182/CAMK2N1 signal axis. In summary, the METTL3/miR-182-5p/CAMK2N1 axis plays an important role in MM tumorigenesis, which may provide a new target for MM therapy.

MicroRNA-34c-5p Reduces Malignant Properties of Lung Cancer Cells through Regulation of TBL1XR1/Wnt/β-catenin Signaling.

Lung cancer is common cancer with high mortality. A growing number of studies have focused on investigating the regulatory effects of microRNAs (miRs/miRNAs) during cancer progression. Nevertheless, the biological function of miR- 34c-5p in lung cancer and the underlying mechanism have not been determined. This study explored the effect of miR-34c-5p on the malignant behaviors of lung cancer cells. In this study, we utilized diverse public databases to obtain differentially expressed miRNAs. Then, qRT-PCR and western blot were conducted to determine miR-34c-5p and transducin β-like 1 X-linked receptor 1 (TBL1XR1) expression. Next, H1299 and H460 cells were transfected with miR-34c-5p-mimic and pcDNA3.1- TBL1XR1. To examine the anticancer effects of miR-34c-5p, CCK-8, scratch, and Matrigel-Transwell assays were conducted to test cell viability, migration, and invasion, respectively. The StarBase database and dual-luciferase reporter gene assay were used to predict and verify the relationship between miR-34c-5p and TBL1XR1. Finally, Wnt/β-catenin signaling- and epithelial-mesenchymal transition (EMT)- related protein levels were detected using western blot. The results demonstrated that miR-34c-5p was poorly expressed in lung cancer cells, while TBL1XR1 was highly expressed. The findings also confirmed the direct interaction between miR-34c-5p and TBL1XR1. In H1299 and H460 cells, miR-34c-5p overexpression inhibited cell proliferation, migration, and invasion, Wnt/β-catenin signaling activity, and EMT, while TBL1XR1 upregulation reversed these effects of miR-34c-5p overexpression. These findings illustrated that miR-34c-5p might repress the malignant behaviors of lung cancer cells via TBL1XR1, providing evidence for miR-34c-5p-based lung cancer therapy.

The role of HMGA2 in activating the IGFBP2 expression to promote angiogenesis and LUAD metastasis via the PI3K/AKT/VEGFA signaling pathway.

This study investigates the molecular mechanism of HMGA2-mediated regulation of IGFBP2 expression in the PI3K/AKT/VEGFA signaling pathway, which is involved in angiogenesis and LUAD metastasis. Target genes with prognostic implications for LUAD patients were selected using bioinformatics, and previously published literature was referenced to predict the molecular regulatory mechanisms. A549 cells were used for in vitro validation. Cell proliferation and viability were assessed using CCK-8 and EdU assays, while cell migration ability was evaluated using Transwell and wound healing assays. Changes in angiogenesis were examined using an angiogenesis assay. The targeted binding of HMGA2 with the IGFBP2 promoter was confirmed through dual luciferase reporter gene experiments and ChIP assays. In vivo validation was performed using a xenograft mouse model, and changes in angiogenesis and tumor metastasis were observed using western blot, immunofluorescence, and H&E staining. Bioinformatics analysis revealed that HMGA2 was one of the AAGs that differed between normal individuals and LUAD patients and could serve as a critical mRNA for predicting LUAD prognosis. Results from in vitro experiments demonstrated that the expression of the HMGA2 gene was significantly upregulated in LUAD cell lines. Through mediating the expression of IGFBP2, the HMGA2 gene activated the PI3K/AKT/VEGFA signaling pathway, promoting the proliferation, migration, and angiogenesis of A549 cells. In vivo, animal experiments further confirmed that HMGA2 facilitated angiogenesis and the development and metastasis of LUAD through mediating IGFBP2 expression and activating the PI3K/AKT/VEGFA signaling pathway. HMGA2 promotes angiogenesis and healthy growth and metastasis of LUAD by activating the PI3K/AKT/VEGFA signaling pathway by mediating IGFBP2 expression.

HDAC2-miRNA183 Epigenetic Circuit Contributes to the Growth of Philadelphia-Positive Acute Lymphoblastic Leukemia Via BCR-ABL/PTEN/p-AKT and c-MYC Signaling Pathways

Purpose: To characterize the expression and function of leukemia related/specific microRNAs (miRNAs) in Philadelphia-positive Acute Lymphoblastic Leukemia (Ph-positive ALL), and to identify the mechanistic signaling pathways in Ph-positive ALL. Method: Differentially expressed miRNAs in Ph-positive ALL patient samples and cell line were identified using RNA-seq and qPCR. Cell proliferation and apoptosis were detected with CCK-8 and flow cytometry assays, respectively. Bioinformatics software predicted the upstream and downstream regulatory pathway of miRNA183; the dual luciferase reporter gene analysis verified the regulatory effect of miRNA183 on the target gene BCR-ABL; ChIP experiment detected the recruitment of HDACs in the miRNA183 promoter region, and western blot detected the expression of related protein in the miRNA183 related pathway. Results: We identified thirty-one differentially expressed miRNAs in three Ph-positive ALL samples vs. normal karyotype B-ALL samples. Among these alternately expressed miRNAs, we found miRNA183 was significantly downregulated in Ph-positive ALL. qPCR verified the down-expression of miRNA183 in Ph-positive ALLcasesamples and Ph-positive ALL cell line SUP-B15. Forced over-expression of miRNA183 inhibited proliferation of SUP-B15 cells, but had no effect on NALM-6 cells (normal karyotype B-ALL). Furthermore, either forced over-expression of miRNA183 or silence down of BCR-ABL1 promoted SUP-B15 cell apoptosis.TargetScan software prediction found that miRNA183 and BCR-ABL (e1a2) had potential binding sites, dual luciferase reporter gene experiments further proved that miRNA183 could direct bind to BCR-ABL gene; qPCR and western blot further showed that miRNA183 inhibited the expression of P190. Our results showed that BCR-ABL (e1a2) is the target gene of miRNA183. In addition, forced over-expression of miRNA183 and silence down of BCR-ABL1 regulated the downstream proteins including PTEN, PARP, p-AKT and c-MYC with the same change trend. In complementary loss-of-function studies, inhibition of miRNA183 restored the expression of these proteins.Treatment of SUP-B15 cells with decitabine (DAC) and histone deacetylase inhibitor (HDACi), respectively, resulted in decrease of cell viability in a dose-dependent manner. Surprisingly, miRNA183 levels were significantly increased in HDACi exposure but not in DAC exposure. Furthermore, miRNA183 inhibitor reduced HDACi-induced upregulation of miRNA183 expression and attenuated HDACi-mediated apoptosis in SUP-B15 cells. Biology software prediction showed the presence of a transcription start site (TSS) 5 kb upstream of miRNA183. ChIP assay further demonstrated that HDAC2 but not HDAC1 was specifically recruited to the miRNA183 promoter region. Additionally, after HDACi treatment, HDACi-induced H3K27ac was recruited and accumulated at the miRNA183 promoter region. Western blotassay further proved the changes of the related proteins. Conclusion :Our results indicate that miRNA183 is a potential biomarker for Ph-positive ALL and suggest that a novel “HDAC2-miRNA183 Epigenetic Circuit” may be involved in the growth and progression of Ph-positive ALL by PTEN/p-AKT and c-MYC signaling pathways. Taken together, this study may provide novel approaches to the design of targeted drugs for Ph-positive ALL.

MiR-590-5p/Tiam1-mediated glucose metabolism promotes malignant evolution of pancreatic cancer by regulating SLC2A3 stability

BackgroundT lymphoma invasion and metastasis 1 (Tiam1) is a tumor related gene that specifically activates Rho-like GTPases Rac1 and plays a critical role in the progression of various malignancies. Glycolysis plays an important role in cancer progression, it is crucial for supplying energy and producing metabolic end products, which can maintain the survival of tumor cells. As yet, however, the mechanism of Tiam1 in glycolysis reprogramming of pancreatic cancer (PC) remains to be clarified. Here, we investigated the functional role of Tiam1 in PC cell proliferation, metastasis and glycolysis reprogramming. It is expected to provide a new direction for clinical treatment.MethodsThe clinical relevance of Tiam1 was evaluated in 66 patients with PC, the effect of Tiam1 on cell proliferation was detected via 5-Ethynyl-2′-deoxyuridine (EdU) and colony formation. The ability of cell migration was detected by the wound healing and Transwell. Quantitative real time polymerase chain reaction (qRT-PCR) and luciferase reporter gene experiments clarify the regulatory relationship of miR-590-5p inhibiting Tiam1. Detection of the molecular mechanism of Tiam1 regulating glucose metabolism reprogramming in PC by glucose metabolism kit. RNA sequencing and Co-Immunoprecipitation (CoIP) have identified glucose transporter protein 3 (SLC2A3) as a key downstream target gene for miR-590-5p/Tiam1.ResultsWe found that Tiam1 expression increased in PC tissues and was associated with lymph node metastasis. The silencing or exogenous overexpression of Tiam1 significantly altered the proliferation, invasion, and angiogenesis of PC cells through glucose metabolism pathway. In addition, Tiam1 could interact with the crucial SLC2A3 and promote the evolution of PC in a SLC2A3-dependent manner. Moreover, miR-590-5p was found to exacerbate the PC cell proliferation, migration and invasion by targeting Tiam1. Furthermore, the reversing effects on proliferation, migration and invasion were found in PC cells with miR-590-5p/Tiam1 overexpression after applying glucose metabolism inhibition.ConclusionsOur findings demonstrate the critical role of Tiam1 in PC development and the miR-590-5p/Tiam1/SLC2A3 signaling pathway may serve as a target for new PC therapeutic strategies.Graphical

Circ_0082878 contributes to prostate cancer progression via the miR-455-3p/WTAP axis.

Circ_0082878 has been found to be strongly expressed in prostate cancer (PCa). However, its roles and potential mechanism in PCa have not been investigated. This study aims to clarify it. RNase R digestion method was adopted for verifying the circular structure of circ_0082878. RT-qPCR assay is aimed to detect the expressions of circ_0082878, miR-455-3p and WTAP in PCa tissues and cells. For identifying cell proliferation, migration and invasion abilities, CCK-8 and transwell assay were used. To show the correlation between miR-455-3p and WTAP or circ_0082878, the luciferase reporter gene, RNA RIP and RNA pull-down experiments were employed. We employed western blot to detect protein level of WTAP. In addition, the impact of circ_0082878 on PCa cells in vivo was also studied. It was found that circ_0082878 and WTAP were highly expressed in PCa tissues and cells, whereas miR-455-3p was lowly expressed. Inhibition of circ_0082878 restrained the growth of PCa in vitro and in vivo. Regarding mechanism, miR-455-3p was the target of circ_0082878, and WTAP was the target of miR-455-3p. Circ_0082878 could downregulate the level of miR-455-3p, and inhibiting of miR-455-3p expression could partially eliminate the inhibitory impact of low expression of circ_0082878 on the proliferation and migration of PCa cells. Additionally, over-expression of miR-455-3p resulted in the reduced level of WTAP, and WTAP over-expression counteracted the tumor suppressive impact of miR-455-3p in PCa cells. Moreover, the obtained findings indicated that circ_0082878 may exert tumor-promoting activity in PCa via sponging miR-455-3p and then upregulating WTAP. This indicates that the circ_0082878/miR-455-3p/WTAP axis can probably become the possible therapeutic target for PCa.

RBM12 regulates the progression of hepatocellular cancer via miR-497–5p/CPNE1 Axis

BackgroundHepatocellular Carcinoma (HCC), also called hepatocellular cancer, has emerged as a highly prevalent malignancy globally. By binding to specific RNA via one or more spherical RNA Domains (RBDs) or RNA Motifs (RBMs), RNA Binding Proteins (RBPs) can affect RNA modification, splicing, localization, translation, and stability. MethodsThis paper builds on previous research by further investigating the impact of RBM12 on LC progression. In order to determine the effect of RBM12 expression on the prognosis of patients with hepatocellular cancer, we first investigated its expression in liver cancer cells (LCC) and tissues. The effect of RBM12 on the malignant biological behavior of LCC was subsequently detected using cytological experiments. To explore the upstream mechanism affecting RBM12, we predicted the miRNA targeting RBM12. According to the database, miR-497–5p was the best candidate gene. The double Luciferase reporter gene experiment was executed to validate the bounding of miR-497–5p with RBM12. ResultsAccording to the cytological experiments, a high RBM12 expression promoted the propagation, migration, and invasion of LCC and impeded liver cancer cell apoptosis. By secreting TGF-β1, RBM12 could induce the EMT process. The miR-497–5p expression is suppressed in hepatocellular cancer. As shown by the CCK8, plate cloning, Transwell, EDU, and other experiments, miR-497–5p suppressed RBM12 expression and tumor growth. The double Luciferase reporter gene system was utilized to verify the combination of miR-497–5p and RBM12. The CPNE1 is a downstream gene regulated by RBM12. A high CPNE1 expression was exhibited in LCC and tissues. The CPNE1 is essential in the process where RBM12 promotes the incidence and progression of liver cancer. ConclusionsBy elucidating the exact molecular mechanism through which RBM12 promotes the initiation and progression of LC, thus, the current investigation provides some reference for the clinical management of LC.

LncRNA HOTAIR regulates the PI3K/AKT pathway via the miR-126-3p/PIK3R2 axis to participate in synovial angiogenesis in rheumatoid arthritis.

The abnormal expression of long noncoding RNA (LncRNA) HOTAIR has been associated with synovial angiogenesis in rheumatoid arthritis (RA). The aim of this study is to investigate whether LncRNA HOTAIR plays a role in synovial angiogenesis in RA by regulating the phosphoinositide 3-kinase/protein kinase B (PI3K/AKT) pathway through the miR-126-3p/PIK3R2 axis. In this study, we conducted in vitro experiments by designing overexpression plasmids and small interfering RNAs targeting LncRNA HOTAIR and then transfected them into rheumatoid arthritis fibroblast-like synoviocytes (RA-FLS). We then co-cultured the RA-FLS with human umbilical vein endothelial cells (HUVEC) to establish a RA-FLS-induced HUVEC model. We investigated the effects of LncRNA HOTAIR on the proliferation, migration, lumen forming ability of HUVEC, as well as the expression of synovial endothelial cell markers, angiogenic factors, and the PI3K/AKT pathway. To validate the interactions between LncRNA HOTAIR, miR-126-3p, and PIK3R2, we used bioinformatics and luciferase reporter experiments. We also employed real-time fluorescence quantitative, Western blotanalysis, and immunofluorescence techniques to analyze the target genes and proteins. The expression of LncRNA HOTAIR was upregulated in HUVEC induced by RA-FLS. The overexpression of LncRNA HOTAIR significantly increased the expression of vascular endothelial growth factor, basic fibroblast growth factor, CD34, and CD105 in HUVEC, promoting their proliferation, migration, and lumen formation. At the same time, the overexpression of LncRNA HOTAIR inhibited the expression of miR-126-3p, promoted the expression of PIK3R2, activated the PI3K/AKT pathway, and promoted the expression of PI3K, AKT and phosphorylated-AKT, while the silence of LncRNA HOTAIR reversed these expressions. Bioinformatics and double luciferase reporter gene experiments confirmed the targeting relationship among LncRNA HOTAIR, miR-126-3p, and PIK3R2. Finally, the rescue experiments showed that PI3K agonists could reverse the inhibitory effect of silent LncRNA HOTAIR on HUVEC. LncRNA HOTAIR has the potential to activate the PI3K/AKT pathway, likely through the regulatory axis involving miR-126-3p/PIK3R2, consequently contributing to synovial angiogenesis in RA.

Hsa-miR-148a-3p promotes malignant behavior of breast cancer cells by downregulating DUSP1

To investigate the role of Hsa-miR-148a-3p in regulating biological behaviors of breast cancer cells and explore the mechanism. TCGA database was used to identify the differential miRNAs and mRNAs in breast cancer, and the protein-protein interaction (PPI) network was constructed using String and Cytoscape to screen the top 10 hub genes and construct the miRNA-TOP10hub network. RT-qPCR was used to detect the expressions of Hsa-miR-148a-3p and DUSP1 in breast cancer tissues and cell lines. The effects of Hsa-miR-148a-3p mimic and inhibitor on proliferation, migration, invasion and apoptosis of MCF-7 cells were analyzed, and luciferase reporter gene experiment was performed to verify the binding of Hsa-miR-148a-3p to DUSP1. The effect of Hsa-miR-148a-3p overexpression on breast cancer cell xenograft growth was evaluated in nude mice. Kaplan-Meier survival curve analysis was used to analyze the survival of the tumor-bearing mice, and the expression level of DUSP1 in the xenografts was detected using immunohistochemistry. A total of 54 differential miRNAs and 799 differential mRNAs were identified in breast cancer; 3716 target genes were intersected with the differential mRNA, resulting in 150 intersected genes. The top 10 hub genes were downregulated in breast cancer tissues in the PPI network. Double luciferase reporter gene experiment confirmed that Hsa-miR-148a-3p was capable of binding to DUSP1. Hsa-miR-148a-3p was up-regulated and DUSP1 was down-regulated significantly in breast cancer tissues and cells (P<0.01). In breast cancer cells, Hsa-miR-148a-3p mimic strongly promoted cell proliferation, migration and invasion and inhibited cell apoptosis (P<0.01). Hsa-miR-148a-3p overexpression obviously promoted xenograft growth in nude mice (P<0.01), shortened survival time of the mice (P<0.01), and reduced the expression of DUSP1 in the xenografts (P<0.01). Hsa-miR-148a-3p promotes malignant behavior of breast cancer cells by inhibiting the expression of DUSP1.

Interferon-gamma and tumor necrosis factor-alpha synergistically enhance the immunosuppressive capacity of human umbilical-cord-derived mesenchymal stem cells by increasing PD-L1 expression.

The immunosuppressive capacity of mesenchymal stem cells (MSCs) is dependent on the "license" of several proinflammatory factors to express immunosuppressive factors such as programmed cell death 1 ligand 1 (PD-L1), which determines the clinical therapeutic efficacy of MSCs for inflammatory or immune diseases. In MSCs, interferon-gamma (IFN-γ) is a key inducer of PD-L1 expression, which is synergistically enhanced by tumor necrosis factor-alpha (TNF-α); however, the underlying mechanism is unclear. To reveal the mechanism of pretreated MSCs express high PD-L1 and explore the application of pretreated MSCs in ulcerative colitis. We assessed PD-L1 expression in human umbilical-cord-derived MSCs (hUC-MSCs) induced by IFN-γ and TNF-α, alone or in combination. Additionally, we performed signal pathway inhibitor experiments as well as RNA interference experiments to elucidate the molecular mechanism by which IFN-γ alone or in combination with TNF-α induces PD-L1 expression. Moreover, we used luciferase reporter gene experiments to verify the binding sites of the transcription factors of each signal transduction pathway to the targeted gene promoters. Finally, we evaluated the immunosuppressive capacity of hUC-MSCs treated with IFN-γ and TNF-α in both an in vitro mixed lymphocyte culture assay, and in vivo in mice with dextran sulfate sodium-induced acute colitis. Our results suggest that IFN-γ induction alone upregulates PD-L1 expression in hUC-MSCs while TNF-α alone does not, and that the co-induction of IFN-γ and TNF-α promotes higher expression of PD-L1. IFN-γ induces hUC-MSCs to express PD-L1, in which IFN-γ activates the JAK/STAT1 signaling pathway, up-regulates the expression of the interferon regulatory factor 1 (IRF1) transcription factor, promotes the binding of IRF1 and the PD-L1 gene promoter, and finally promotes PD-L1 mRNA. Although TNF-α alone did not induce PD-L1 expression in hUC-MSCs, the addition of TNF-α significantly enhanced IFN-γ-induced JAK/STAT1/IRF1 activation. TNF-α up-regulated IFN-γ receptor expression through activation of the nuclear factor kappa-B signaling pathway, which significantly enhanced IFN-γ signaling. Finally, co-induced hUC-MSCs have a stronger inhibitory effect on lymphocyte proliferation, and significantly ameliorate weight loss, mucosal damage, inflammatory cell infiltration, and up-regulation of inflammatory factors in colitis mice. Overall, our results suggest that IFN-γ and TNF-α enhance both the immunosuppressive ability of hUC-MSCs and their efficacy in ulcerative colitis by synergistically inducing high expression of PD-L1.

MiR-601-induced BMSCs senescence accelerates steroid-induced osteonecrosis of the femoral head progression by targeting SIRT1.

The imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) is not only the primary pathological feature but also a major contributor to the pathogenesis of steroid-induced osteonecrosis of the femoral head (SONFH). Cellular senescence is one of the main causes of imbalanced BMSCs differentiation. The purpose of this study was to reveal whether cellular senescence could participate in the progression of SONFH and the related mechanisms. The rat SONFH model was constructed, and rat BMSCs were extracted. Aging-related indicators were detected by SA-β-Gal staining, qRT-PCR and Western Blot experiments. Using H2O2 to construct a senescent cell model, and overexpressing and knocking down miR-601 and SIRT1 in hBMSCs, the effect on BMSCs differentiation was explored by qRT-PCR, Western Blot experiment, oil red O staining (ORO), alizarin red staining (ARS), and luciferase reporter gene experiment. A rat SONFH model was established to test the effects of miR-601 and metformin in vivo. The current study showed that glucocorticoids (GCs)-induced BMSCs senescence, which caused imbalanced osteogenesis and adipogenesis of BMSCs, was responsible for the SONFH progression. Further, elevated miR-601 caused by GCs was demonstrated to contribute to BMSCs senescence through targeting SIRT1. In addition, the anti-aging drug metformin was shown to be able to alleviate GCs-induced BMSCs senescence and SONFH progression. Considering the role of BMSCs aging in the progression of SONFH, this provides a new idea for the prevention and treatment of SONFH.

YY1 regulates the proliferation and invasion of triple-negative breast cancer via activating PLAUR.

It is well-established that breast cancer is a highly prevalent malignancy among women, emphasizing the need to investigate mechanisms underlying its pathogenesis and metastasis. In this study, the Gene Expression Omnibus (GEO) database was utilized to conduct differential expression analysis in breast cancer and adjacent tissues. Upregulated genes were selected for prognostic analysis of breast cancer. The expression of urokinase plasminogen activator receptor (uPAR), also known as PLAUR, was assessed using RT-qPCR and western blot. Immunofluorescence staining was employed to determine PLAUR localization. Various cellular processes were analyzed, including proliferation, migration, invasion, apoptosis, and cell cycle. Bioinformatics analysis was used to predict transcription factors of PLAUR, which were subsequently validated in a double luciferase reporter gene experiment. Rescue experiments confirmed the impact of PLAUR on the proliferation, apoptosis, and migration of MDA-MB-231 cells. Furthermore, the effects of PLAUR were evaluated in an orthotopic tumor transplantation and lung metastasis nude mouse model. Our findings substantiated the critical involvement of PLAUR in the progression of triple-negative breast cancer (TNBC) in vitro and among TNBC patients with a poor prognosis. Additionally, we demonstrated Yin Yang-1 (YY1) as a notable transcriptional regulator of PLAUR, whose activation could transcriptionally enhance the proliferation and invasion capabilities of TNBC cells. We also identified the downstream mechanism of PLAUR associated with PLAU, focal adhesion kinase (FAK), and AKT. Overall, these findings offer a novel perspective on PLAUR as a potential therapeutic target for TNBC.

Effect of exosomes from nasopharyngeal carcinoma on endothelium pyroptosis and metastasis.

93 Background: Nasopharyngeal carcinoma (NPC) is a malignant tumor occurring in the epithelium of nasopharyngeal mucosa. Radiotherapy can achieve a good therapeutic effect for NPC in situ. However, there is no effective treatment once distant metastasis occurring. Epstein-Barr virus (EBV) is an important pathogenic factor of NPC and also promotes tumor metastasis. The passage of circulating tumor cells through the vascular barrier is one of the rate-limiting steps of tumor distant metastasis, and vascular endothelial cells are an important component. Exosomes are an important way of information transmission between cells. And some small molecules in exosomes involve in tumor metastasis. How the exosomes of NPC cells affect vascular endothelial cells and promote metastasis has not been studied. Therefore, it is of great significance to explore the effect of the NPC exosomes with EBV component on the vascular endothelial cells of metastatic foci and the mechanism of promoting tumor metastasis, in order to find effective therapeutic targets and molecular markers indicating distant metastasis. Methods: 1. Immunohistochemical analysis was used to analyze the vascular endothelium of liver and lung tissues with NPC metastasis compared with that of non-tumor liver and lung in pathological sections; 2. Using transmission electron microscope, cells and dye molecules through vascular endothelial cell monolayer experiment, immunofluorescence and animal tumorigenesis experiment to detect the effect of NPC exosomes on vascular endothelial cells and NPC metastasis; 3. Using CRISPR-Cas9 knockout EBNA1, cell function test and miRNA sequencing technology to analyze to analyze molecular types that induce vascular endothelial cell pyroptosis in NPC exosomes; 4. Using bioinformatics prediction, luciferase reporter gene, immunofluorescence and animal tumorigenesis experiment to further clarify the molecular pathway of molecules in the NPC exosomes that induce endothelial cell pyroptosis. Results: 1. Immunohistochemistry showed that P20 and NT-GSDMD were significantly higher in vascular endothelial cells of liver and lung tissues with NPC metastasis than in non-tumor liver and lung; 2. NPC exosomes can promote the increase of molecules related to vascular endothelial cell pyroptosis (P20, NT-GSDMD and IL-1 β), the formation of vesicular protrusion of cell membrane and the destruction of vascular endothelial monolayer barrier. And vascular endothelial cell pyroptosis can promote the migration of circulating tumor cells out of blood vessels to form metastatic foci; 3. BART2-5p in NPC exosomes is the main effector molecule that induces vascular endothelial cell pyroptosis; 4. MRE11A is the target molecule of BART2-5p to induce vascular endothelial cell pyroptosis. Conclusions: 1. It was found for the first time that the vascular endothelial cell pyroptosis in liver and lung metastasis of NPC; 2. It is proved that BART2-5p in NPC exosomes is involved in regulating the vascular endothelial cell pyroptosis, and promoting NPC cells to go through blood vessels and form metastatic foci; 3. MRE11A mediates BART2-5p to induce the vascular endothelial cell pyroptosis.

MiR-320a upregulation improves IL-1β-induced osteoarthritis via targeting the DAZAP1 and MAPK pathways

PurposeOsteoarthritis (OA), a constant illness described by articular cartilage degeneration, usually manifested by joint pain and helpless development. Numerous literatures suggest that microRNAs play an important regulatory role in OA, yet the role of miR-320a in OA remains largely obscure.Materials and methodsTo evaluate the expression of miR-320a mRNA, quantitative real-time polymerase chain reaction was used. Cell counting kit-8 assay, Edu staining, Annexin V-FITC/PI apoptosis detection assay, Caspases 3 staining, and trypan staining were conducted to monitor cell proliferation and apoptosis. Western blot was applied to examine DAZAP1 and ERK/JNK/MAPK associated protein expression. Luciferase reporter gene experiments were performed to confirm the relationships between miR-320a and DAZAP1. ELISA assay was adopted to analyze the secretion of inflammation cytokines IL-6, IL-8, and TNF-α.ResultsIn an in vitro osteoarthritis model caused by IL-1β, miR-320a expression was markedly reduced. Overexpression of miR-320a restored IL-1β-inhibited chondrocyte proliferation, induced apoptosis and inflammatory response. Mechanistically, miR-320a affected HC-A cell proliferation, apoptosis and inflammatory response by regulating DAZAPI. Meanwhile, the ERK/JNK/MAPK pathway is also involved in the regulatory role of miR-320a on OA.ConclusionOur results show an important role for miR-320a and provide new therapeutic targets for avoiding and treating osteoarthritis.