Nicotine promotes tumor progression and epithelial-mesenchymal transition by regulating the miR-155-5p/NDFIP1 axis in pancreatic ductal adenocarcinoma

BackgroundNicotine, the major component of cigarette smoke, has been reported to promote pancreatic ductal adenocarcinoma (PDAC) growth and invasion. Deregulation of microRNA (miRNA) expression is found in many cancers, including PDAC. The effects of nicotine on miRNAs change in PDAC progression remain unknown.MethodsThe effects of cigarette smoking/nicotine exposure on PDAC cell lines and tissues were evaluated. Quantitative real-time PCR and in situ hybridization assays were used to determine miR-155-5p expression in human PDAC tissue and cell lines upon cigarette smoking/nicotine exposure. Bioinformatics, loss-of-function experiments, luciferase reporter assay were performed to validate Nedd4 family interacting protein 1 (NDFIP1) as a direct target of miR-155-5p. The potentials of systemic miR-155-5p inhibitor-based therapy in overcoming nicotine exposure were evaluated in tumor xenograft model.ResultsNicotine promoted PDAC cells proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) in a dose-response manner. MiR-155-5p was found to be highly expressed in PDAC cell lines and tissues upon cigarette smoking/nicotine exposure. Functional studies showed that miR-155-5p knockdown could override the enhancement of oncogenic activity due to nicotine exposure in vitro and in vivo by directly interacting with the 3′ untranslated regions (UTRs) of NDFIP1.ConclusionsThese data demonstrate that nicotine-regulated miR-155-5p/NDFIP1 promotes tumor progression and EMT of PDAC.     

MiR-21-5p-expressing bone marrow mesenchymal stem cells alleviate myocardial ischemia/reperfusion injury by regulating the circRNA_0031672/miR-21-5p/programmed cell death protein 4 pathway

BACKGROUNDFor patients with coronary heart disease, reperfusion treatment strategies are often complicated by ischemia/reperfusion (I/R) injury (IRI), leading to serious organ damage and malfunction. The miR-21/programmed cell death protein 4 (PDCD4) pathway is involved in the IRI of cardiomyocytes; however, the aberrant miR-21 expression remains unexplained. Therefore, this study aimed to explore whether circRNA_0031672 downregulates miR-21-5p expression during I/R and to determine whether miR-21-5p-expressing bone marrow mesenchymal stem cells (BMSCs) reduce myocardial IRI.METHODSCircRNA_0031672, miR-21-5p, and PDCD4 expressions were evaluated in the I/R rat model and hypoxia/re-oxygenation (H/R)-treated H9C2 cells. Their interactions were subsequently investigated using luciferase reporter and RNA pulldown assays. Methyltransferase-like 3, a methyltransferase catalyzing N6-methyladenosine (m6A), was overexpressed in H9C2 cells to determine whether m6A modification influences miR-21-5p targeting PDCD4. BMSCs stably expressing miR-21 were co-cultured with H9C2 cells to investigate the protective effect of BMSCs on H9C2 cells upon H/R.RESULTSI/R downregulated miR-21-5p expression and upregulated circRNA_0031672 and PDCD4 expressions. CircRNA_0031672 knockdown increased miR-21-5p expression, but repressed PDCD4 expression, indicating that circRNA_0031672 competitively bound to miR-21-5p and prevented it from targeting PDCD4 mRNA. The m6A modification regulated PDCD4 expression, but had no effect on miR-21-5p targeting PDCD4. The circRNA_0031672/miR-21-5p/PDCD4 axis regulated myocardial cells viability and apoptosis after H/R treatment; co-culture with miR-21-5p-expressing BMSCs restored miR-21-5p abundance in H9C2 cells and further reduced H9C2 cells apoptosis induced by H/R.CONCLUSIONSWe identified a novel circRNA_0031672/miR-21-5p/PDCD4 signaling pathway that mediates the apoptosis of cardiomyocytes and successfully alleviates IRI in myocardial cells by co-culture with miR-21-5p-expressing BMSCs, offering novel insights into the IRI pathogenesis in cardiovascular diseases.

Coronary heart disease (CHD) is a major cause of death globally. According to the data released by the World Health Organization, the mortality rate due to CHD in China is the second highest in the world. Current therapeutic approaches for acute myocardial infarction include reperfusion strategies such as drug thrombolysis, coronary intervention, and vascular bypass grafting.^[1] However, the accompanying effect of ischemia/reperfusion (I/R) injury (IRI) remains unsolved. The IRI is a pathophysiologic process which can cause tissue damage and deterioration of multiple organs, ultimately leading to organ malfunction.^[2,3] Although multiple proposals have been made regarding the pathogenesis of IRI,^[1,4,5] further evidence is needed to understand the precise mechanisms. Therefore, delineating the detailed mechanisms underlying IRI is of vital importance for realizing the goal of early restoration of blood flow in ischemic tissues with minimal reperfusion injuries. MicroRNAs (miRNAs), 20−25 nucleotide-long non-coding RNAs, have been extensively studied and reported to modulate multiple cellular processes and cell signaling pathways by regulating the expression of messenger RNAs (mRNAs). MiRNAs also participate in regulating the response of cardiomyocytes to IRI by regulating various genes related to myocardial cell survival and apoptosis.^[6–8] Circular RNA (circRNA) is a less studied non-coding RNA. CircRNAs are mainly located in the cytoplasm and contain miRNA response elements that facilitate circRNAs to act as miRNA sponges through competitive interaction and inhibition of miRNA functions.^[9] Multiple studies have validated the circRNA-miRNA regulatory machinery and established the role of circRNA as a competing endogenous RNA.^[10–12]Among multiple types of RNA methylation modifications, including N1-methyladenosine, 5-methylcytosine, 5-hydroxymethylcytosine, N6-methyladenosine (m6A), and 7-methylguanine,^[13] the m6A modification occurs abundantly in mammals, accounting for almost half of the total RNA methylation events and playing crucial roles in RNA regulation.^[14,15] The m6A modification is often enriched near 3’ untranslated region (3’UTR), where miRNA binding sites may be present, raising the possibility of m6A and miRNA cooperatively or competitively targeting mRNAs.^[16]Bone marrow mesenchymal stem cells (BMSCs), the first type of mesenchymal stem cells identified, have attracted substantial attention for their pluripotency and ability to secrete extracellular vesicles. Large quantities of RNA or protein products generated by genetically modified BMSCs can be subsequently secreted extracellularly and internalized by surrounding cells, enabling BMSCs to be applied in clinical settings as potential vectors for gene therapy.^[17] In addition, BMSCs are reported to be a promising candidate in alleviating myocardial infarction or even heart failure.^[18,19]Apoptosis plays a major role in IRI and is considered a potential indicator of IRI severity. In particular, miR-21 has been shown to affect the apoptosis of myocardial cells by regulating the expression of multiple target genes.^[20,21] Expression of miR-21 has previously been shown to be downregulated in infarcted areas, whereas it is upregulated in the border regions of infarcted areas, and ischemia preconditioning prevented the downregulation of miR-21 in infarcted areas.^[22] The anti-apoptotic role of miR-21-5p in myocardial ischemia has been shown to be associated with pathways involving programmed cell death protein 4 (PDCD4) and activated caspase-1.^[20]Our previous study revealed the global alteration of circRNAs in hypoxic cardiomyocytes compared to that in normal controls, while the roles of these circRNAs in cardiomyocyte apoptosis remained elusive. Here, we identified circRNA_0031672, transcribed from the locus of RALGAPA1, and found that circRNA_0031672 modulated the apoptosis of cardiomyocytes under hypoxia by acting as a sponge of miR-21-5p. Methyltransferase-like 3 (METTL3), a methyltransferase catalyzing m6A, was overexpressed in H9C2 cells to determine whether the m6A modification influences miR-21-5p targeting PDCD4 mRNA. After confirming that m6A modification had no effect on miR-21-5p targeting PDCD4 mRNA, we co-cultured H9C2 cells with miR-21-5p-expressing BMSCs to reduce H9C2 injury after hypoxia/re-oxygenation (H/R). Thus, we propose a novel molecular mechanism of apoptosis during I/R and provide a potential therapeutic strategy for alleviating IRI in CHD patients by utilizing BMSCs.     

Circ_0000291 contributes to hepatocellular carcinoma tumorigenesis by binding to miR-1322 to up-regulate UBE2T

Background and objectivesCircular RNAs (circRNAs) are identified to show important regulatory functions in cancer biology. We attempted to analyze the role of circ_0000291 in hepatocellular carcinoma (HCC) progression and its related mechanism.MethodsThe circular characteristic of circ_0000291 was tested using exonuclease RNase R. Cell proliferation was analyzed by 5-Ethynyl-2’-deoxyuridine (EdU) incorporation and colony formation assays. Cell apoptosis was measured by flow cytometry and a caspase 3 activity assay kit. Transwell assays were performed to analyze cell migration and invasion abilities. Sphere formation assay was conducted to analyze cell stemness. Dual-luciferase reporter and RNA-pull down assays were conducted to verify the interaction between microRNA-1322 (miR-1322) and circ_0000291 or ubiquitin conjugating enzyme E2 T (UBE2T).ResultsCirc_0000291 was markedly up-regulated in HCC tissues and cell lines. HCC patients with high expression of circ_0000291 displayed a low survival rate. Circ_0000291 knockdown restrained the proliferation, migration, invasion, and stemness and induced the apoptosis of HCC cells. Circ_0000291 directly interacted with miR-1322 and negatively regulated miR-1322 expression. Circ_0000291 knockdown-mediated anti-tumor impacts in HCC cells were largely overturned by the interference of miR-1322. miR-1322 directly paired with the 3’ untranslated region (3’UTR) of UBE2T, and UBE2T was negatively regulated by miR-1322. UBE2T overexpression largely reversed circ_0000291 silencing-induced effects in HCC cells. Circ_0000291 positively regulated UBE2T expression by absorbing miR-1322 in HCC cells. Circ_0000291 silencing notably reduced the tumorigenic potential in vivo.ConclusionCirc_0000291 facilitated HCC progression by targeting miR-1322/UBE2T axis, which provided novel potential biomarkers and targets for HCC patients.     

LncRNA NEAT1 promotes proliferation of ovarian cancer cells and angiogenesis of co-incubated human umbilical vein endothelial cells by regulating FGF9 through sponging miR-365: An experimental study

Objective:To uncover the function of lncRNA NEAT1 in ovarian cancer (OC) cells and its mechanism.Methods:The expression patterns of lncRNA NEAT1 and FGF9 in human OC cells and human ovarian epithelial cells was determined. OC cells were transfected with sh-NEAT1, pcDNA3.1-NEAT1, miR-365 mimic, miR-365 inhibitor or pcDNA3.1-NEAT1 + sh-NEAT1 before cell proliferation rate and cell clone formation rate were measured. After the transfected OC cells were co-cultivated with human umbilical vein endothelial cells (HUVECs), Matrigel angiogenesis assay tested angiogenesis of HUVECs; qRT-PCR and Western blot tested the expressions of vascular endothelial growth factor (VEGF), angiogenin 1 (Ang-1) and matrix metalloproteinase 2 (MMP2). Dual-luciferase reporter assay determined the targeted binding of NEAT1 and FGF9 to miR-365.Results:LncRNA NEAT1 and FGF9 are over-expressed in OC cells. Knockdown of NEAT1 or FGF9, or over-expression of miR-365 results in decreased proliferation rate and cell clones as well as inhibited angiogenesis and down-regulated expressions of VEGF, Ang-1 and MMP2. Over-expression of NEAT1 or knockdown of miR-365 can reverse the effect caused by FGF9 knockdown. NEAT1 can down-regulate the expression of miR-365 while up-regulating that of FGF9. Dual-luciferase reporter assay determined that NEAT1 competes with FGF9 for binding to miR-365.Conclusion:LncRNA NEAT1 up-regulates FGF9 by sponging miR-365, thus promoting OC cell proliferation and angiogenesis of HUVECs.     

GPR120 promotes metastasis but inhibits tumor growth in pancreatic ductal adenocarcinoma

ObjectivesG-protein-coupled receptor 120 (GPR120) is a long-chain unsaturated fatty acid receptor, which regulates glucose metabolism and lipid. To date, there are disputes on the roles of GPR120 in the pathogenesis of cancer. Besides, little is known about its roles in the pathogenesis of pancreatic ductal adenocarcinoma (PDAC). This study was designed to investigate the roles of GPR120 in the pathogenesis of PDAC.MethodsImmunohistochemical staining (IHC) was used for detecting the level of GPR120, epithelial-mesenchymal transformation (EMT) markers, Ki-67 and CD31 in ninety-one PDAC patients. Western blot, CCK8, flow cytometry and transwell assays were performed to determine proliferation, apoptosis, and motility in vitro. Subcutaneous tumor model was established to validate the roles of GPR120 in vivo.ResultsGPR120 was highly expressed in PDAC tissues, which was associated with free fatty acids (FFAs), lymph node metastasis (LNM), and poor prognosis. Moreover, GPR120 activation led to down-regulation of E-cadherin and up-regulation of Snail, Vimentin, N-cadherin, MMP2, MMP9, and CD31. Additionally, GPR120 decreased the expression of P-PI3K, P-AKT and CMYC and increased the level of P-JAK2, P-STAT3, Wnt5a, total β-catenin and β-catenin in nucleus.ConclusionsGPR120 promoted proliferation inhibition and apoptosis of PDAC, and contributed to PDAC metastasis via inducing EMT and angiogenesis. GPR120 served as a double-edged sword in the pathogenesis of PDAC.     

环状RNA _005647通过结合miR-99b-5p抑制心肌细胞中肥厚相关基因的表达

目的研究环状RNA circRNA_005647抑制心肌肥厚相关基因表达的作用机制。方法建立血管紧张素Ⅱ(AngⅡ)灌注诱导的心肌肥厚小鼠模型。原代分离获得C57BL/6乳小鼠心肌细胞(NMVC),AngⅡ处理NMVC建立心肌细胞肥大模型。利用NMVC,分别感染circRNA_005647重组腺病毒(rAd-circRNA_005647)和转染miR-99b-5p模拟物来研究对NMVC肥大的影响。通过双荧光素酶报告基因实验和RNA Pull-down实验验证circRNA_005647与miR-99b-5p的结合作用。实时荧光定量PCR和Western blot检测NMVC中心肌肥厚相关基因的mRNA和蛋白表达水平。结果在AngⅡ诱导的小鼠心肌肥厚模型和心肌细胞肥大模型中,circRNA_005647及其宿主基因肌球蛋白IXA(Myo9a)表达升高。过表达circRNA_005647可抑制AngⅡ诱导的NMVC中肥厚相关基因心房钠尿肽(Anp)和肌球蛋白重链7(Myh7)基因表达升高。circRNA_005647与miR-99b-5p间存在结合作用。过表达circRNA_005647可抑制miR-99b-5p促心肌细胞肥大的作用。结论circRNA_005647通过结合miR-99b-5p发挥抑制心肌细胞肥大的作用。     

circZFR promotes cell proliferation and migration by regulating miR-511/AKT1 axis in hepatocellular carcinoma

BackgroundEmerging data suggest the crucial regulatory roles of circular RNAs (circRNAs) in hepatocellular carcinoma (HCC). However, the pathophysiology role of circZFR in HCC remains largely unknown.AimsThis study aims to disclose the functions of circZFR in HCC progression and its potential molecular mechanism.MethodscircZFR and miR-511 were identified by qRT-PCR. Colony formation assay, wound-healing assay, transwell assay, and flow cytometry assay were performed to determine the cell proliferation, migration, invasion and apoptosis. Western blotting and immunohistochemistry (IHC) were utilized to evaluate the expression level of AKT1, GSK3β, β-catenin and cascades of proliferation-related proteins both in vitro and in vivo. Dual luciferase reporter assay was conducted to evaluate the interactions among circZFR, miR-511 and AKT1.ResultsThe expression of circZFR was enhanced and the expression of miR-511 was down-regulated in HCC tissues and cells. Functionally, circZFR silencing or miR-511 overexpression suppressed cell proliferation, migration and invasion, and induced apoptosis of HCC cells. Mechanistically, circZFR acted as a miR-511 sponge to up-regulate its target gene AKT1, which activated cascades of proliferation-related proteins (c-Myc, cyclin D1, Survivin and Bcl-2). Furthermore, depletion of circZFR inhibited tumorigenesis and decreased the expression level of AKT1 in xenograft models.ConclusioncircZFR promotes HCC progression by directly down-regulating miR-511 to activate AKT1 signaling, suggesting that circZFR is a potential target in HCC treatment. Targeting circZFR may provide therapeutic benefits for HCC.     

MicroRNA-145 promotes esophageal cancer cells proliferation and metastasis by targeting SMAD5

Objective: To clarify the relative expression and molecular function of microRNA (miR)-145 in esophageal cancer and understand its mechanistic involvement in this disease.

Material and methods: The relative expression of miR-145 in clinical samples was analyzed using the public GSE43732 dataset. The prognostic analysis with respect to miR-145 expression was performed with Kaplan–Meier plot. Cell viability was measured by MTT assay and the anchorage-independent growth was evaluated by soft agar assay. The migration and invasion of esophageal cancer cells were measured using transwell chamber. The regulatory effect of miR-145 on SMAD5 was determined by dual-luciferase reporter assay. The endogenous SMAD5 protein was measured by Western blot.

Results: We demonstrated high expression of miR-145 associated with late stage and unfavorable prognosis of esophageal cancer. Ectopic expression of miR-145 mimic significantly stimulated cell proliferation and anchorage-independent growth. Furthermore, high level of miR-145 significantly promoted both migration and invasion in vitro. Notably, we identified SMAD5 as direct target of miR-145, the suppressed expression of which consequently led to increased cell proliferation and migration/invasion.

Conclusion: Our study uncovered the crucial role of miR-145/SMAD5 in esophageal cancer and highlighted its target potential for diagnostic and therapeutic purpose.     

The role of microRNA-145 in human embryonic stem cell differentiation into vascular cells

Background

Recent studies have reported that microRNA-145 (miR-145) is a critical mediator in the regulation of proliferation, differentiation, and phenotype expression of smooth muscle cells (SMCs). Previously, we established a system for differentiating human ESCs into vascular cells including endothelial cells (ECs) and vascular smooth muscle cells (SMCs). In the present study, we investigated the role of miR-145 in the differentiation process from human ESCs into ECs and SMCs.

Methods and results

Undifferentiated human ESCs were induced to differentiate into vascular lineage according to our established method. Quantitative RT-PCR analysis revealed that human ESC-derived precursor of SMCs (ES-pre-SMCs), similar to human aortic SMCs, expressed a significant amount of miR-145 as well as smooth muscle-specific proteins, compared to undifferentiated human ESCs, adult ECs, or ESC-derived ECs (ES-ECs). However, morphological analysis revealed that human ES-pre-SMCs appeared round and flattened in shape, though human aortic SMCs exhibited the typical spindle-like morphology of SMCs. In addition, Krüppel-like factor 4 and 5 (KLF4 and 5), direct targets of miR-145 and suppressors of smooth muscle differentiation, were upregulated in ES-pre-SMCs compared to aortic SMCs, indicating ES-pre-SMCs were not fully differentiated SMCs. Overexpression of miR-145 in ES-pre-SMCs upregulated the expression of smooth muscle markers, repressed KLF4 and 5 expressions, and changed their morphology into a differentiated spindle-like shape. Furthermore, by introduction of miR-145, ES-pre-SMC proliferation was significantly inhibited and carbachol-stimulated contraction of ES-pre-SMCs was significantly increased. In contrast, downregulation of miR-145 in ES-pre-SMCs upregulated KLF4 and 5 expressions, suppressed the expression of smooth muscle markers, and left unchanged their proliferation and contractility. In ES-ECs, miR-145 overexpression did not induce the synthesis of smooth muscle-related proteins nor suppress the expression of endothelial nitric oxide synthase.

Conclusion

We showed that miR-145 can regulate the fate and phenotype of human ES-pre-SMCs as they become fully differentiated SMCs. Overexpression of miR-145 on human ES-pre-SMCs is a promising method to obtain functional mature SMCs from human ESCs, which are required for reliable experimental research in the fields of atherosclerosis, hypertension and other vascular diseases.     

Circ_0006768 upregulation attenuates oxygen–glucose deprivation/reoxygenation-induced human brain microvascular endothelial cell injuries by upregulating VEZF1 via miR-222-3p inhibition

Circular RNAs (circRNAs) have been widely implicated in multiple diseases, including ischemic stroke. This study aimed to explore the function and functional mechanism of circ_0006768 in oxygen–glucose deprivation/reoxygenation (OGD/R)-induced brain injury models of ischemic stroke. Human brain microvascular endothelial cells (HBMECs) were induced by OGD/R to mimic ischemic stroke models in vitro. The expression of circ_0006768, microRNA-222-3p (miR-222-3p) and vascular endothelial zinc finger 1 (VEZF1) was detected by quantitative real-time PCR (qPCR). Cell viability, angiogenesis ability and cell migration were assessed by cell counting kit-8 (CCK-8) assay, tube formation assay and wound healing assay, respectively. The releases of pro-inflammatory factors were determined by commercial enzyme-linked immunosorbent assay (ELISA) kits. The protein levels of vascular endothelial growth factor A (VEGFA), N-cadherin and VEZF1 were detected by western blot. The putative relationship between miR-222-3p and circ_0006768 or VEZF1 was validated by dual-luciferase reporter assay, RNA Immunoprecipitation (RIP) assay and pull-down assay. Circ_0006768 was poorly expressed in ischemic stroke plasma and OGD/R-induced HBMECs. OGD/R inhibited cell viability, angiogenesis and cell migration and promoted the releases of pro-inflammatory factors, while circ_0006768 overexpression or miR-222-3p inhibition partially abolished the effects of OGD/R. MiR-222-3p was targeted by circ_0006768, and VEZF1 was a target of miR-222-3p. Circ_0006768 enriched the expression of VEZF1 via mediating miR-222-3p inhibition. Rescue experiments presented that the effects of circ_0006768 overexpression were reversed by miR-222-3p restoration or VEZF1 knockdown. Circ_0006768 overexpression attenuates OGD/R-induced HBMEC injuries by upregulating VEZF1 via miR-222-3p inhibition.

     

Circ-LARP1B knockdown restrains the tumorigenicity and enhances radiosensitivity by regulating miR-578/IGF1R axis in hepatocellular carcinoma

Introduction and ObjectivesCircular RNA La Ribonucleoprotein 1B (circ-LARP1B) was reported to serve as an oncogene in many types of cancers. Radiotherapy (RT) is an important element of the multimodal treatment concept in malignancies. Here, this work aimed to investigate the role of circ-LARP1B in the tumorigenesis and radiosensitivity of hepatocellular carcinoma (HCC).Patients or Materials and MethodsQuantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of genes and proteins. In vitro experiments were conducted using cell counting Kit-8 (CCK-8), colony formation, EDU, transwell, and tube formation assays, respectively. Dual-luciferase reporter assay was employed to identify the target relationship between miR-578 and circ-LARP1B or IGF1R (insulin-like growth factor 1 receptor). In vivo assay was performed using murine xenograft model.ResultsCirc-LARP1B was highly expressed in HCC tissues and cells, and high expression of circ-LARP1B was closely associated with poor prognosis. Functional experiments demonstrated that circ-LARP1B silencing impaired cell proliferation, invasion, angiogenesis and reduced radioresistance in vitro. Mechanistically, circ-LARP1B could competitively bind with miR-578 to relieve the repression of miR-578 on the expression of its target gene IGF1R. Further rescue assay confirmed that miR-578 inhibition reversed the inhibitory effects of circ-LARP1B knockdown on HCC cell malignant phenotypes and radioresistance. Moreover, miR-578 overexpression restrained tumorigenicity and enhanced radiosensitivity in HCC cells, which were attenuated by IGF1R up-regulation. Besides that, circ-LARP1B knockdown impeded tumor growth and enhanced irradiation sensitivity in HCC in vivo.ConclusionsCirc-LARP1B knockdown restrained HCC tumorigenicity and enhanced radiosensitivity by regulating miR-578/IGF1R axis, providing a new target for the treatment of HCC.     

Circular RNA circVAPA promotes chemotherapy drug resistance in gastric cancer progression by regulating miR-125b-5p/STAT3 axis

BACKGROUND Gastric cancer(GC)is a prevalent malignancy,leading to a high incidence of cancer-associated death.Cisplatin(DDP)-based chemotherapy is the principal therapy for clinical GC treatment,but DDP resistance is a severe clinical challenge and the mechanism remains poorly understood.Circular RNAs(circRNAs)have been identified to play crucial roles in modulating the chemoresistance of gastric cancer cells.AIM To explore the effect of circVAPA on chemotherapy resistance during GC progression.METHODS The effect of circVAPA on GC progression and chemotherapy resistance was analyzed by MTT assay,colony formation assay,Transwell assay,wound healing assay,and flow cytometry analysis in GC cells and DDP resistant GC cell lines,and tumorigenicity analysis in nude mice in vivo.The mechanism was investigated by luciferase reporter assay,quantitative real-time PCR,and Western blot analysis.RESULTS CircVAPA expression was up-regulated in clinical GC tissues compared with normal samples.CircVAPA depletion inhibited proliferation,migration,and invasion and increased apoptosis of GC cells.The expression of circVAPA,STAT3,and STAT3 downstream genes was elevated in DDP resistant SGC7901/DDP cell lines.CircVAPA knockdown attenuated the DDP resistance of GC cells.Mechanically,circVAPA was able to sponge miR-125b-5p,and miR-125b-5p could target STAT3 in the GC cells.MiR-125b-5p inhibitor reversed circVAPA depletion-enhanced inhibitory effect of DDP on GC cells,and STAT3 knockdown blocked circVAPA overexpression-induced proliferation of DDPtreated SGC7901/DDP cells.The depletion of STAT3 and miR-125b-5p inhibitor reversed circVAPA depletion-induced GC cell apoptosis.Functionally,circVAPA contributed to the tumor growth of SGC7901/DDP cells in vivo.CONCLUSION CircVAPA promotes chemotherapy resistance and malignant progression in GC by miR-125b-5p/STAT3 signaling.Our findings present novel insights into the mechanism by which circVAPA regulates chemotherapy resistance of GC cells.CircVAPA and miR-125b-5p may be considered as the potential targets for GC therapy.     

Circ-PRMT5 enhances the proliferation,migration and glycolysis of hepatoma cells by targeting miR-188-5p/HK2 axis

Introduction and objectivesCircular RNA (circRNA) has been demonstrated as a critical regulator in human cancer, including hepatocellular carcinoma (HCC). Nevertheless, the role of circ-PRMT5 in HCC remains largely unknown.Patients or materials and methodsThe real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess the expression levels of circ-PRMT5, miR-188-5p and anti-Hexokinase II (HK2) in HCC tissues and cells. The cell proliferation, migration and glycolysis were determined by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl-2H-tetrazol-3-ium bromide (MTT), transwell migration assay, and indicated kits, respectively. The interaction relationship between miR-188-5p and circ-PRMT5 or HK2 was analyzed by the bioinformatics database, dual-luciferase reporter assay, and RNA immunoprecipitation (RIP) assay. The western blot assay was used to analyze the expression level of HK2. The functional role of circ-PRMT5 in vivo was assessed by a xenograft experiment.ResultsCirc-PRMT5 was elevated in HCC tissues and cells than matched control groups. Furthermore, loss-of-functional experiments revealed that the silencing of circ-PRMT5 could repress proliferation, migration, glycolysis in vitro and tumor growth in vivo. Moreover, we also confirmed that overexpression of circ-PRMT5 abolished the effects on HCC cells induced by upregulating miR-188-5p. In addition, overexpression of miR-188-5p could repress the development of HCC. More importantly, HK2 was a target gene of miR-188-5p, and miR-188-5p regulated proliferation, migration, glycolysis of HCC cells by specifically binding to HK2. Mechanistically, circ-PRMT5 could act as a sponge of miR-188-5p to regulate the expression of HK2.ConclusionIn summary, circ-PRMT5 might play a key role in proliferation, migration, glycolysis of HCC cells via miR-188-5p/HK2 axis, which indicated that circ-PRMT5 might be a potential therapeutic target for HCC treatment.     

CircRNA_0084927 promotes colorectal cancer progression by regulating miRNA-20b-3p/glutathione S-transferase mu 5 axis

BACKGROUNDColorectal cancer (CRC) is the third most common cancer and the second most common cause of cancer-related death worldwide. The 5-year survival rate of patients with early-stage CRC could reach 90%, but it is very low in patients with advanced-stage CRC. Recent studies have shown that circular RNAs play important roles in regulating the migration and invasion of CRC cells.AIMTo elucidate the role of circRNA_0084927 (circ_0084927) in the migration and invasion of CRC cells and its underlying mechanism.METHODSClinical tissue samples and cells were collected, and the expression of circ_0084927 was detected by quantitative polymerase chain reaction (qPCR). The diagnostic performance of circ_0084927 was assessed by receiver operating characteristic curve analysis. The role of circ_0084927 in CRC cell proliferation, migration, and invasion was determined using cell counting kit-8 assay, wound healing assay, and transwell assay, respectively. The regulatory relationship among circ_0084927, miRNA-20b-3p (miR-20b-3p), and glutathione S-transferase mu 5 (GSTM5) was identified using databases, luciferase reporter assay, qPCR, and Western blot analysis. AKT-mTOR signaling was also verified after circ_0084927 knockdown or miR-20b-3p mimic treatment.RESULTSThe expression of circ_0084927 was significantly increased in CRC tissues and cells, and it was higher in advanced-stage CRC compared with early-stage CRC. The area under the curve (AUC) of circ_0084927 was 0.806 [95% confidence interval (CI): 0.683-0.896]. In addition, the AUC was 0.874 (95%CI: 0.738-0.956) in patients with advanced-stage CRC and 0.713 (95%CI: 0.555-0.840) in those with early-stage CRC. Knockdown of circ_0084927 inhibited the migration and invasion of HCT116 cells. Moreover, circ_0084927 was found to act as a sponge of miR-20b-3p. MiR-20b-3p activation reduced the circ_0084927 level, whereas miR-20b-3p inhibition increased the circ_0084927 level. But the effect was not found after circ_0084927 mutation. In addition, miR-20b-3p expression in CRC patients was also reduced and negatively correlated with circ_0084927 expression. The function of circ_0084927 in HCT116 cells with circ_0084927 knockdown was rescued by miR-20b-3p. Moreover, GSTM5 expression was significantly decreased after overexpressing miR-20b-3p or inhibiting circ_0084927, but its expression was rescued when circ_0084927 and miR-20b-3p were both inhibited. Finally, AKT-mTOR signaling was markedly regulated by circ_0084927 and miR-20b-3p.CONCLUSIONThe expression of circ_0084927 is significantly increased in CRC and higher in advanced-stage CRC than in early-stage CRC. Moreover, circ_0084927 potentially regulates CRC cell migration and invasion via the miR-20b-3p/GSTM5/ AKT/mTOR pathway.     

Long noncoding RNA LINC00488 functions as a ceRNA to regulate hepatocellular carcinoma cell growth and angiogenesis through miR-330-5

BackgroundLong non-coding RNAs (lncRNAs) recently have been identified as influential indicators in a variety of malignancies. Hence, the aim of the present study was to identify a functional lncRNA and its associated effects on hepatocellular carcinoma (HCC) in terms of cellular growth and a ngiogenesis.Methods and resultsMicroarray-based analysis revealed a possible regulatory mechanism involving LINC00488, microRNA-330-5p (miR-330-5p) and talin-1 (TLN1) in HCC. Targetscan and RNA22 online tools predicted the relationship among LINC00488, miR-330-5p and TLN1, which were further validated by dual luciferase reporter gene assay, RNA pull-down and RIP. To evaluate the effects of LINC00488 and miR-330-5p on the cellular process of HCC, we performed a series of in vitro and in vivo experiments, with the expression of LINC00488, miR-330-5p, and TLN1 altered by delivering plasmids into Hep3B cell line. The obtained results demonstrated that cells with siRNA-mediated depletion of LINC00488 or restoration of miR-330-5p displayed suppressed abilities of in vitro proliferation as well as of in vivo tumor growth and angiogenesis, while in vitro apoptosis was notably induced.ConclusionThe fundamental findings of the present study collectively propose that lncRNA LINC00488 can competitively sponge miR-330-5p to regulate TLN1 in relation to the cell growth and angiogenesis in HCC.