CircDiaph3 aggravates H/R-induced cardiomyocyte apoptosis and inflammation through miR-338-3p/SRSF1 axis.

Acute myocardial infarction (AMI) is one of the most prevalent cardiovascular diseases, accounting for a high incidence rate and high mortality worldwide. Hypoxia/reoxygenation (H/R)-induced myocardial cell injury is the main cause of AMI. Several studies have shown that circular RNA contributes significantly to the pathogenesis of AMI. Here, we established an AMI mouse model to investigate the effect of circDiaph3 in cardiac function and explore the functional role of circDiaph3 in H/R-induced cardiomyocyte injury and its molecular mechanism. Bioinformatics tool and RT-qPCR techniques were applied to detect circDiaph3 expression in human patient samples, heart tissues of AMI mice, and H/R-induced H9C2 cells. CCK-8 was used to examine cell viability, while annexin-V/PI staining was used to assess cell apoptosis. Myocardial reactive oxygen species (ROS) levels were detected by immunofluorescence. Western blot was used to detect the protein expression of anti-apoptotic Bcl-2 while pro-apoptotic Bax and cleaved-Caspase-3. Furthermore, ELISA was used to detect inflammatory cytokines production. While bioinformatics tool and RNA pull-down assay were used to verify the interaction between circDiaph3 and miR-338-3p. We found that circDiaph3 expression was high in AMI patients and mice, as well as in H/R-treated H9C2 cells. CircDiaph3 silencing ameliorated apoptosis and inflammatory response of cardiomyocytes in vivo. Moreover, the knockdown of cirDiaph3 mitigated H/R-induced apoptosis and the release of inflammatory mediators like IL-1ß, IL-6, and TNF-α in H9C2 cells. Mechanistically, circDiaph3 induced cell apoptosis and inflammatory responses in H/R-treated H9C2 cells by sponging miR-338-3p. Overexpressing miR-338-3p in H/R-treated cells prominently reversed circDiaph3-induced effects. Notably, miR-338-3p inhibited SRSF1 expression in H/R-treated H9C2 cells. While overexpressing SRSF1 abrogated miR-338-3p-mediated alleviation of apoptosis and inflammation after H/R treatment. To summarize, circDiaph3 aggravates H/R-induced cardiomyocyte apoptosis and inflammation through the miR-338-3p/SRSF1 axis. These findings suggest that the circDiaph3/miR-338-3pp/SRSF1 axis could be a potential therapeutic target for treating H/R-induced myocardial injury.

Mettl3 induced miR-338-3p expression in dendritic cells promotes antigen-specific Th17 cell response via regulation of Dusp16.

Pathogenic Th17 cells are critical drivers of multiple autoimmune diseases, including uveitis and its animal model, experimental autoimmune uveitis (EAU). However, how innate immune signals modulate pathogenic Th17 responses remains largely unknown. Here, we showed that miR-338-3p endowed dendritic cells (DCs) with an increased ability to activate interphotoreceptor retinoid-binding protein (IRBP)1-20 -specific Th17 cells by promoting the production of IL-6, IL-1ß, and IL-23. In vivo administration of LV-miR-338-infected DCs promoted pathogenic Th17 responses and exacerbated EAU development. Mechanistically, dual-specificity phosphatase 16 (Dusp16) was a molecular target of miR-338-3p. miR-338-3p repressed Dusp16 and therefore strengthened the mitogen-activated protein kinase (MAPK) p38 signaling, resulting in increased production of Th17-polarizing cytokines and subsequent pathogenic Th17 responses. In addition, methyltransferase like 3 (Mettl3), a key m6A methyltransferase, mediated the upregulation of miR-338-3p in activated DCs. Together, our findings identify a vital role for Mettl3/miR-338-3p/Dusp16/p38 signaling in DCs-driven pathogenic Th17 responses and suggest a potential therapeutic avenue for uveitis and other Th17 cell-related autoimmune disorders.

Cancer-associated fibroblast-derived circFARP1 modulates non-small cell lung cancer invasion and metastasis through the circFARP1/miR-338-3p/SOX4 axis.

The pleiotropic effect of cancer-associated fibroblasts (CAFs) on tumour progression depends on the environment. circFARP1 is critical for CAFs-induced gemcitabine (GEM) resistance in pancreatic cancer. Its specific role and mechanism in non-small cell lung cancer (NSCLC) have not been reported yet. We prepared a cancer-associated fibroblasts-conditioned medium (CAF-CM) to incubate the A549 cells. Quantitative real-time polymerase chain reaction was used to detect RNA levels. We detected protein expression by immunohistochemistry, immunocytochemistry, western blot and immunofluorescence. We also detected the targeting impact between circFARP1, miR-338-3p and SRY-box transcription factor 4 (SOX4) by using dual-luciferase reporter and RNA pull-down assays. We determined cell proliferation, migration and invasion capabilities through Cell Counting Kit-8 and transwell assays. In addition, we measured tumour volume and weight in vivo by establishing a xenograft tumour model. CircFARP1 levels were remarkably high in the CAFs. The transfection experiments found that circFARP1 downregulation in CAFs caused migration, proliferation and invasion inhibition of CAFs and A549 cells, whereas inhibiting miR-38-3p or overexpressing SOX4 in CAFs could significantly reverse the inhibition. In vivo study in nude mice confirmed that CAFs could promote NSCLC tumour growth and knockdown of circFARP1 could inhibit tumour growth of NSCLC, whereas miR-38-3p downregulation or SOX4 overexpression could significantly reverse the inhibition. circFARP1 promotes NSCLC development by stimulating miR-338-3p/SOX4 signalling axis to regulate CAFs.

Bone marrow mesenchymal stem cell-derived exosomes promote osteoblast proliferation, migration and inhibit apoptosis by regulating KLF3-AS1/miR-338-3p.

AIM: This study aimed to investigate the effect and mechanism of bone marrow mesenchymal stem cell-derived exosomes on osteoblast function. METHODS: The expression of KLF3-AS1 and miR-338-3p in serum of fracture patients was detected by qRT-PCR. Exosomes from BMSCs were isolated by ultrafast centrifugation. MC3T3-E1 cells were cultured in vitro as experimental cells. Intracellular gene expression was regulated by transfection of si-KLF3-AS1 or miR-338-3p inhibitors. MTT assay, Transwell assay and flow cytometry were used to evaluate cell viability, migration, and apoptosis. The luciferase reporter gene was used to verify the targeting relationship between KLF3-AS1 and miR-338-3p. Bioinformatics analysis was used to identify the basic functions and possible enrichment pathways of miR-338-3p target genes. RESULTS: The expressions of KLF3-AS1 and miR-338-3p in the serum of fracture patients were down-regulated and up-regulated, respectively. The expression of KLF3-AS1 was increased in MC3T3-E1 cells cultured with BMSCs-Exo, while the viability and migration ability of MC3T3-E1 cells were enhanced, and the apoptosis ability was weakened. Further analysis revealed miR-338-3p was the target gene of KLF3-AS1. The expression of miR-338-3p was downregulated in MC3T3-E1 cells cultured with BMSCs-Exo. Inhibition of miR-338-3p in MC3T3-E1 cells enhanced the viability and migration ability of MC3T3-E1 cells when cultured with BMSCs-Exo, while suppressing apoptosis. Bioinformatics analysis demonstrated that the target genes of miR-338-3p were predominantly localized at the axon's initiation site, involved in biological processes such as development and growth regulation, and mainly enriched in MAPK and ErbB signaling pathways. CONCLUSION: In vitro, BMSCs-Exo exhibits the capacity to enhance proliferation and migration while inhibiting apoptosis of MC3T3-E1 cells, potentially achieved through modulation of KLF3-AS1 and miR-338-3p expression in MC3T3-E1 cells.

CircRNA HLCS regulates lens epithelial cell apoptosis via miR-338-3p/BPNT1 axis.

PURPOSE: The purpose of this study was to investigate the effect of circ_HLCS on age-related cataract (ARC). METHODS: Circ_HLCS, microRNA (miR)-338-3p, and bisphosphate 3'-nucleotidase 1 (BPNT1) were quantified by quantitative real-time polymerase chain reaction or western blot. Cell proliferation and cell viability were assessed by the 5-Ethynyl-2'-deoxyuridinr and cell counting kit-8 assays. Cell apoptosis was detected by flow cytometry. Targeted correlations among circ_HLCS, miR-338-3p, and BPNT1 were verified by the dual-luciferase reporter and RNA pull-down assays. RESULTS: circ_HLCS was diminished in ARC tissues and UV-treated SRA01/04 cells. Elevated content of circ_HLCS undermined UV-induced cell proliferation inhibition and apoptosis. Mechanistically, circ_HLCS directly targeted miR-338-3p, and circ_HLCS regulated BPNT1 expression through miR-338-3p. Furthermore, reduction of miR-338-3p ameliorated UV-induced SRA01/04 cell damage by increasing BPNT1 expression. CONCLUSION: Taken together, these data suggested that circ_HLCS inhibited apoptosis of UV-treated SRA01/04 cells by miR-338-3p/BPNT1 axis. Therefore, circ_HLCS might be a potential therapeutic target for ARC.

Pseudogene TDGF1P3 regulates the proliferation and metastasis of colorectal cancer cells via the miR-338-3p-PKM2 axis.

Research in the past decade has revealed significant roles of pseudogenes in colorectal cancer (CRC). Here, the role of teratocarcinoma-derived growth factor 1 pseudogene 3 (TDGF1P3) in regulating the proliferation and invasion of CRC cells was investigated; in addition, its downstream targets were analyzed, and the underlying mechanisms were elucidated. TDGF1P3 was determined to be upregulated in CRC cells and tissues. Silencing TDGF1P3 substantially repressed cell proliferation, migration, and invasion in vitro. Similarly, in vivo assays showed that TDGF1P3 knockdown attenuated tumor growth in nude mice. Mechanistic investigations revealed that TDGF1P3 directly bound to miR-338-3p, thereby preventing miR-338-3p from binding to its target mRNA pyruvate kinase M2 (PKM2). Functional rescue tests indicated that TDGF1P3 regulates CRC cell proliferation and invasion by restraining the miR-338-3p-PKM2 axis. Thus, these data illustrated that TDGF1P3 exerts its oncogenic activity by upregulating PKM2 via competitively binding miR-338-3p, which may be a therapeutic target for CRC.

CircRBM23 regulates the switch between osteogenesis and adipogenesis of mesenchymal stem cells via sponging miR-338-3p.

BACKGROUND: The disruption of the balance between osteogenic and adipogenic differentiation of mesenchymal stem cells (MSCs) in bone marrow contributes to the adipocytes accumulation and bone loss, which leads to the development of osteoporosis (OP). The circular RNA (circRNA), circRBM23, was generated from the RNA binding motif protein 23 (RBM23) gene. It was reported that circRBM23 was down-regulated in OP patients, but it remains unknown whether its down-regulation is involved in the lineage switch of MSCs. OBJECTIVE: We aimed to explore the role and mechanism of circRBM23 in regulating the switch between osteogenic and adipogenic differentiation of MSCs. METHODS: The expression and function of circRBM23 in vitro were detected by qRT-PCR, alizarin red staining, and oil Red O staining. The interactions between circRBM23 and microRNA-338-3p (miR-338-3p) were analyzed by RNA pull-down assay, FISH, and dual-luciferase reporter assay. MSCs treated with lentivirus overexpression of circRBM23 was applied for both in vitro and in vivo experiments. RESULTS: CircRBM23 was expressed at lower levels in OP patients. Besides, circRBM23 was up-regulated during osteogenesis and down-regulated during adipogenesis of MSCs. CircRBM23 could promote the osteogenic differentiation but inhibit the adipogenic differentiation of MSCs. Mechanistically, circRBM23 acted as a sponge for microRNA-338-3p (miR-338-3p) to enhance the expression of RUNX family transcription factor 2 (RUNX2). CONCLUSIONS: Our research indicates that circRBM23 could promote the switch from adipogenic to osteogenic differentiation of MSCs via sponging miR-338-3p. It might improve the understanding of the lineage switch of MSCs and provide a potential target for diagnosing and treating OP.

MiRNA-338-3p Inhibits Neuroinflammation in the Corpus Callosum of LCV-LPS Rats Via STAT1 Signal Pathway.

Neuroinflammation is a common characteristic of intracranial infection (ICI), which is associated with the activation of astrocytes and microglia. MiRNAs are involved in the process of neuroinflammation. This study aimed to investigate the potential mechanism by which miR-338-3p negatively modulate the occurrence of neuroinflammation. We here reported that the decreased levels of miR-338-3p were detected using qRT-PCR and the upregulated expression of TNF-α and IL-1ß was measured by ELISA in the cerebrospinal fluid (CSF) in patients with ICI. A negative association between miR-338-3p and TNF-α or IL-1ß was revealed by Pearson correlation analysis. Sprague-Dawley (SD) rats were injected with LPS (50 µg) into left cerebral ventricule (LCV), following which the increased expression of TNF-α and IL-1ß and the reduction of miR-338-3p expression were observed in the corpus callosum (CC). Moreover, the expression of TNF-α and IL-1ß in the astrocytes and microglia in the CC of LCV-LPS rats were saliently inhibited by the overexpression of miR-338-3p. In vitro, cultured astrocytes and BV2 cells transfected with mimic-miR-338-3p produced less TNF-α and IL-1ß after LPS administration. Direct interaction between miR-338-3p and STAT1 mRNA was validated by biological information analysis and dual luciferase assay. Furthermore, STAT1 pathway was found to be implicated in inhibition of neuroinflammation induced by mimic miR-338-3p in the astrocytes and BV2 cells. Taken together, our results suggest that miR-338-3p suppress the generation of proinflammatory mediators in astrocyte and BV2 cells induced by LPS exposure through the STAT1 signal pathway. MiR-338-3p could act as a potential therapeutic strategy to reduce the neuroinflammatory response. Diagram describing the cellular and molecular mechanisms associated with LPS-induced neuroinflammation via the miR-338-3p/STAT1 pathway. LPS binds to TLRs on astrocytes or microglia to activate the STAT1 pathway and upregulate the production of pro-inflammatory cytokines. However, miR-338-3p inhibits the expression of STAT1 and reduces the production of inflammatory mediators.

Circ-BICC1 Knockdown Alleviates Lipopolysaccharide (LPS)-Induced WI-38 Cell Injury Through miR-338-3p/MYD88 Axis.

Circular RNAs (circRNAs) play important roles in human diseases, including infantile pneumonia. In this article, we aimed to investigate the functions of circ-BICC1 in lipopolysaccharide (LPS)-induced injury of WI-38 cells. Quantitative real-time polymerase chain reaction (qRT-PCR) assay was performed for the levels of circ-BICC1, BICC1, microRNA-338-3p (miR-338-3p), and myeloid differentiation primary response 88 (MYD88). Cell Counting Kit-8 (CCK-8) assay, 5-ethynyl-2'-deoxyuridine (EdU) assay, and flow cytometry analysis were conducted to evaluate cell viability, proliferation, and apoptosis, respectively. Enzyme-linked immunosorbent assay (ELISA) kits were used for the concentrations of interleukin-1ß (IL-1ß) and tumor necrosis factor-α (TNF-α). The levels of oxidative stress markers were detected with commercial kits. Dual-luciferase reporter assay was adopted to analyze the interaction between circ-BICC1 and miR-338-3p, as well as MYD88 and miR-338-3p. Western blot assay was employed for the protein level of MYD88. Circ-BICC1 level was increased in pneumonia patients' blood samples and LPS-treated WI-38 cells. LPS treatment suppressed WI-38 cell viability and promoted cell apoptosis, inflammation, and oxidative stress. Circ-BICC1 knockdown reversed the effect of LPS-induced WI-38 cell injury. For mechanism analysis, circ-BICC1 could function as the sponge for miR-338-3p and miR-338-3p inhibition reversed the effect of circ-BICC1 knockdown on LPS-induced WI-38 cell injury. MYD88 was identified as the target of miR-338-3p. MiR-338-3p overexpression relieved LPS-induced injury of WI-38 cells, while the impact was abolished by elevating MYD88. Circ-BICC1 silencing remitted LPS-triggered WI-38 cell damage by adsorbing miR-338-3p and regulating MYD88.

Exosomal circKDM4A Induces CUL4B to Promote Prostate Cancer Cell Malignancy in a miR-338-3p-Dependent Manner.

Circular RNA lysine demethylase 4A (circKDM4A) is also named circ_0012098 and its abnormal expression has been confirmed in serum exosomes of prostate cancer (PC) patients. However, whether PC progression involves the exosomal circ_0012098 remains unknown. RNA expression of circKDM4A, microRNA-338-3p (miR-338-3p) and cullin 4B (CUL4B) was detected by quantitative real-time polymerase chain reaction. Protein expression was checked by Western blot. The positive expression rate of nuclear proliferation marker (ki-67) was analyzed by immunohistochemistry assay. Dual-luciferase reporter assay and RNA immunoprecipitation assay were used to identify the interaction between miR-338-3p and circKDM4A or CUL4B. Mouse model assay was performed to determine the effect of exosomal circKDM4A on tumorigenesis in vivo. CircKDM4A expression was significantly upregulated in the serum exosomes from PC patients compared with the exosomes from healthy volunteers. Exosomes treatment promoted the proliferation, migration and invasion of PC cells but inhibited apoptosis; however, these effects were attenuated after circKDM4A knockdown. Meanwhile, circKDM4A depletion restored exosome-increased circKDM4A expression. Additionally, circKDM4A acted as a miR-338-3p sponge, and miR-338-3p bound to CUL4B in PC cells. CircKDM4A regulated the effect of exosome-induced PC cell malignancy by interacting with miR-338-3p and CUL4B. Moreover, circKDM4A silencing relieved exosome-induced tumor growth in vivo. Exosomal circKDM4A promoted PC malignant progression by the miR-338-3p/CUL4B axis, providing a therapeutic target for PC.

The estrogen/miR-338-3p/ADAM17 axis enhances the viability of breast cancer cells via suppressing NK cell's function.

Natural killer (NK) cells are the critical elements of the innate immune response and implicated in rapidly recognizing and eliminating cancer cells. However, the tumor-suppressive ability of NK cells is often impaired in several cancer types. The critical roles of microRNAs have been elucidated by increasing evidences, while the regulation of miR-338-3p in anti-tumor activation of NK cells and its relationship with estrogen in breast cancer (BC) are still confusing. Here, miR-338-3p level was found to be significantly downregulated in BC tissues and estrogen receptor positive (ER+ ) cells, this difference was more obvious in ER+ patients or BC patients at advanced stage (TNM III and IV). MiR-338-3p level was shown to be downregulated by 17ß-estradiol in BC cells (MDA-MB-231 cells and MCF-7) in vitro. MiR-338-3p overexpression decreased disintegrin and metalloprotease-17 (ADAM17) secretion in MDA-MB-231 (ER- ) and MCF-7 (ER+ ) cells. In addition, miR-338-3p overexpression or treatment with anti-ADAM17 antibody could down-regulate granzyme B, CD16, and NKG2D in NK cells, which was reversed by human recombinant ADAM17. Furthermore, these educated NK cells could promote the viability of MDA-MB-231 or MCF-7 cells. Taken together, our results demonstrate that miR-338-3p was negatively regulated by estrogen in BC cells, impairing NK cell's activity by the up-regulation of ADAM17, and conversely promoted the viability of BC cells. Therefore, the estrogen/miR-338-3p/ADAM17 axis is critically implicated in BC pathogenesis and may provide potential targets for BC diagnosis and treatment.

Integrated analysis of miRNA-mRNA regulatory network and functional verification of miR-338-3p in coronary heart disease.

Coronary heart disease is a cardiovascular disease with high morbidity and mortality. Although great progress has been made in treatment, the prognosis is still very poor. Therefore, this project aims to screen potential diagnostic markers and therapeutic targets related to the progression of coronary heart disease. A total of 94 overlapping differentially expressed mRNAs and 70 differentially expressed miRNAs were identified from GSE20681, GSE12288, GSE49823, and GSE105449. Through a series of bioinformatics methods and experiment, we obtained 5 core miRNA-mRNA regulatory pairs, and selected miR-338-3p/RPS23 for functional analysis. Moreover, we found that RPS23 directly targets miR-338-3p by dual luciferase assay, western, and qPCR. And the expression of miR-338-3p and RPS23 is negatively correlated. The AUC value of miR-338-3p is 0.847. Downregulation of miR-338-3p can significantly inhibit the proliferation and migration of HUVEC. On the contrary, overexpression of miR-338-3p promoted the proliferation and migration of HUVEC. In addition, the interference of RPS23 expression can reverse the regulation of miR-338-3p on HUVEC proliferation. In conclusion, miR-338-3p/RPS23 may be involved in the progression of coronary heart disease, and miR-338-3p may be a diagnostic biomarker and therapeutic target for coronary heart disease.

The Colonic Mucosal MicroRNAs, MicroRNA-219a-5p, and MicroRNA-338-3p Are Downregulated in Irritable Bowel Syndrome and Are Associated With Barrier Function and MAPK Signaling.

BACKGROUND & AIMS: Alterations in microRNA (miRNA) and in the intestinal barrier are putative risk factors for irritable bowel syndrome (IBS). We aimed to identify differentially expressed colonic mucosal miRNAs, their targets in IBS compared to healthy controls (HCs), and putative downstream pathways. METHODS: Twenty-nine IBS patients (15 IBS with constipation [IBS-C], 14 IBS with diarrhea [IBS-D]), and 15 age-matched HCs underwent sigmoidoscopy with biopsies. A nCounter array was used to assess biopsy specimen-associated miRNA levels. A false discovery rate (FDR) < 10% was considered significant. Real-time polymerase chain reaction (PCR) was used to validate differentially expressed genes. To assess barrier function, trans-epithelial electrical resistance (TEER) and dextran flux assays were performed on Caco-2 intestinal epithelial cells that were transfected with miRNA-inhibitors or control inhibitors. Protein expression of barrier function associated genes was confirmed using western blots. RESULTS: Four out of 247 miRNAs tested were differentially expressed in IBS compared to HCs (FDR < 10%). Real-time PCR validation suggested decreased levels of miR-219a-5p and miR-338-3p in IBS (P = .026 and P = .004), and IBS-C (P = .02 and P = .06) vs. HCs as the strongest associations. Inhibition of miR-219a-5p resulted in altered expression of proteasome/barrier function genes. Functionally, miR-219a-5p inhibition enhanced the permeability of intestinal epithelial cells as TEER was reduced (25-50%, P < .05) and dextran flux was increased (P < .01). Additionally, inhibition of miR-338-3p in cells caused alterations in the mitogen-activated protein kinase (MAPK) signaling pathway genes. CONCLUSION: Two microRNAs that potentially affect permeability and visceral nociception were identified to be altered in IBS patients. MiR-219a-5p and miR-338-3p potentially alter barrier function and visceral hypersensitivity via neuronal and MAPK signaling and could be therapeutic targets in IBS.

The hsa_circ_0039857/miR-338-3p/RAB32 axis promotes the malignant progression of colorectal cancer.

BACKGROUND: Colorectal cancer (CRC) is a prevalent malignancy of the gastrointestinal. Circular RNAs (circRNAs) act as important roles in CRC malignant progression. However, the role of circ_0039857 in CRC is still unclear. Therefore, this study aimed to explore the function and mechanism of hsa_circ_0039857 in the CRC. METHODS: The mRNA and protein expression were measured via RT-qPCR. RNase R assay and Actinomycin D were employed to evaluate the stability of circ_0039857. Functional experiments, such as proliferation and apoptosis, were applied to study the function of circ_0039857 in CRC cells. The underlying mechanisms of circ_0039857 were then analyzed by bioinformatics, dual-luciferase reporter gene assay, RNA pull-down and rescue experiments. RESULTS: We revealed that circ_0039857 was significantly enhanced in CRC. Circ_0039857 was stabler than linear RNA in cells and valuable for the disease diagnosis. In addition, circ_0039857 knockdown inhibited proliferation and promoted apoptosis. Mechanistically, circ_0039857 positively regulated the expression of RAB32 via sponging miR-338-3p. CONCLUSION: This study demonstrated that circ_0039857 knockdown suppressed CRC malignant progression through miR-338-3p/RAB32 axis. Most importantly, this will help us to better understand the circRNA network in CRC, and may find potential biomarkers and targets for CRC clinical treatment.

MicroRNA-338-3p as a therapeutic target in cardiac fibrosis through FGFR2 suppression.

BACKGROUND: The development of cardiac fibrosis involves the activation of cardiac fibroblasts (CFs) and their differentiation into myofibroblasts, which leads to the disruption of the extracellular matrix network. In the past few years, microRNAs (miRNA) have been described as potential targets for treating cardiac diseases. Although miR-338-3p has been shown to participate in the development of carcinoma, whether it affects cardiac fibrosis is unclear. METHODS: We examined the expression profiles of microRNAs in left ventricular samples of heart failure mice established by thoracic aortic constriction (TAC). Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) was used to detect the expression of miR-338-3p. CCK-8 assay/Transwell migration assay was used to measure the proliferation rate/migration of CFs. Luciferase reporter gene assay was used to test the binding between miR-338-3p and FGFR2. RESULTS: This study demonstrated that miR-338-3p was significantly decreased in thoracic aortic constriction mice. Cardiac miR-338-3p amounts were also reduced in patients with dilated cardiomyopathy (DCM). Interestingly, miR-338-3p overexpression inhibited α-SMA, COL1A1, and COL3A1 expression, as well as cell proliferation and migration in CFs. Bioinformatics analysis and dual-luciferase reporter assays revealed FGFR2 was targeted by miR-338-3p, whose antifibrotic effect could be alleviated by overexpression of FGFR2. Moreover, in DCM cases, serum miR-338-3p levels were markedly elevated in individuals with worse outcomes. CONCLUSIONS: The present study provides evidence that miR-338-3p suppresses cardiac fibroblast activation, proliferation, and migration by directly targeting FGFR2 in mice. Besides, serum miR-338-3p might constitute a potential prognostic biomarker of dilated cardiomyopathy.

Silencing of lncRNA KLF3-AS1 represses cell growth in osteosarcoma via miR-338-3p/MEF2C axis.

BACKGROUND: Osteosarcoma (OS) is a highly recurrent malignancy occurring among adolescents. The goal of this research was to scrutinize the role and action mechanism of KLF3-AS1 in OS. METHODS: Western blotting and quantitative reverse transcription real-time PCR were conducted to ascertain the mRNA expressions of miR-338-3p, KLF3-AS1, and MEF2C in OS cell lines and tissue samples. Colony formation and CCK-8 experiments were done to evaluate the proliferative capacity of the cells. Western blotting was also executed to measure the relative expressions of the proteins Bcl-2 and Bax. RNA immunoprecipitation and dual luciferase reporter experiments were carried out to validate the target relationships among MEF2C, KLF3-AS1, and miR-338-3p. Mouse xenograft models were created to assess the influences of KLF3-AS1 on the growth of tumors in vivo. RESULTS: Elevated levels of KLF3-AS1 and MEF2C and reduced amounts of miR-338-3p were identified in OS. KLF3-AS1 targeted miR-338-3p, and miR-338-3p further targeted MEF2C. Silencing KLF3-AS1 induced apoptosis and attenuated proliferation in vitro and repressed the tumor growth in vivo. Inhibiting miR-338-3p inverted the cancer-suppressing effects of KLF3-AS1 silencing. Meanwhile, loss of MEF2C partially eliminated the effects brought about by miR-338-3p downregulation, namely the stimulation of cell growth and suppression of apoptosis. CONCLUSIONS: Silencing of KLF3-AS1 could repress the growth of cells and induce apoptosis by regulating miR-338-3p/MEF2C in OS. This suggests that the regulatory axis KLF3-AS1/miR-338-3p/MEF2C is a prospective target for OS treatment.

Circ-TFF1 Facilitates Breast Cancer Development via Regulation of miR-338-3p/FGFR1 Axis.

Some circular RNAs (circRNAs) have been verified to act as essential regulators in the progression of breast cancer (BC). We aimed to investigate the role of circRNA trefoil factor 1 (circ-TFF1) in BC progression. The expression of circ-TFF1, microRNA-338-3p (miR-338-3p) and fibroblast growth factor receptor 1 (FGFR1) mRNA was measured by quantitative real-time polymerase chain reaction (qRT-PCR). Cell proliferation was evaluated by methylthiazolyldiphenyl-tetrazolium bromide (MTT), colony formation, and 5-Ethynyl-2'-deoxyuridine (EDU) assays. Cell apoptosis and invasion were assessed by flow cytometry and transwell assay, respectively. Cellular glycolysis, including glucose consumption, lactate production, and ATP/ADP ratio, was detected by commercial kits. All protein levels were measured by western blot assay. The relationship between miR-338-3p and circ-TFF1 or FGFR1 was predicted by online bioinformatics tool and verified by dual-luciferase reporter assay. Xenograft tumor model was established to verify the function of circ-TFF1 in vivo. Circ-TFF1 was overexpressed in BC tissues and cells. Circ-TFF1 knockdown inhibited cell proliferation, invasion and glycolysis and induced apoptosis in BC cells. Circ-TFF1 acted as a sponge of miR-338-3p, and the effects of circ-TFF1 knockdown on BC cell proliferation, apoptosis, invasion, and glycolysis were abolished by miR-338-3p inhibition. FGFR1 was confirmed to be a target gene of miR-338-3p, and miR-338-3p played a tumor-suppressive role in BC by targeting FGFR1. Moreover, circ-TFF1 regulated FGFR1 expression by targeting miR-338-3p. Additionally, circ-TFF1 knockdown hampered tumorigenesis in vivo. Circ-TFF1 knockdown suppressed BC progression by regulating miR-338-3p/FGFR1 axis, providing a promising therapeutic target for BC.

MiR-338-3p Is a Biomarker in Neonatal Acute Respiratory Distress Syndrome (ARDS) and Has Roles in the Inflammatory Response of ARDS Cell Models.

To investigate the association between serum miR-338-3p levels and neonatal acute respiratory distress syndrome (ARDS) and its mechanism. The relative miR-338-3p expression in serum was detected by quantitative real-time RT-PCR. Interleukin-1beta (IL-1ß), IL-6, and tumor necrosis factor-alpha (TNF-α) levels were detected by ELISAs. A receiver operating characteristic (ROC) curve analysis of serum miR-338-3p evaluated the diagnosis of miR-338-3p in neonatal ARDS. Pearson's correlation analysis evaluated the correlation between serum miR-338-3p and neonatal ARDS clinical factors. Flow cytometry evaluated apoptosis, and a CCK-8 assay assessed cell viability. A luciferase assay evaluated the miR-338-3p/AKT3 relationship. The miR- 338-3p expression was decreased in neonatal ARDS patients and in lipopolysaccharide (LPS)-treated cells. The ROC curve showed the accuracy of miR-338-3p for evaluating neonatal ARDS patients. The correlation analysis demonstrated that miR-338-3p was related to PRISM-III, PaO2/FiO2, oxygenation index, IL-1ß, IL-6, and TNF-α in neonatal ARDS patients. MiR-338-3p overexpression inhibited the secretion of inflammatory components, stifled cell apoptosis, and LPS-induced advanced cell viability. The double-luciferase reporter gene experiment confirmed that miR-338-3p negatively regulates AKT3 mRNA expression. Serum miR-338-3p levels were related to the diagnosis and severity of neonatal ARDS, which may be attributed to its regulatory effect on inflammatory response in ARDS.

[CircRNA circTNPO1 promotes the proliferation and metastasis of osteosarcoma by sponging miR-338-3p].

Objective: To explore the effects of circTNPO1 on the proliferation and metastasis of osteosarcoma (OS) by sponging miR-338-3p. Methods: The expression of circTNPO1 on osteoblasts and multiple OS cell lines were detected by qRT-PCR. CircTNPO1 stable knockdown 143B cell line was constructed by sh-circTNPO1. Cell count kit 8 (CCK-8) assay and wound healing assay were applied to evaluate the proliferation and metastasis of this cell. Luciferase reporter assay was used to explore the binding between circTNPO1 and miR-338-3p. In xenograft tumor model, miR-338-3p inhibitor or its control was injected into the circTNPO1 knockdown tumors. The weight and size of the tumors were evaluated and Ki-67 expression was detected by immunohistochemistry. Results: The RNA expression of circTNPO1 in OS cell lines U2OS, HOS, MG63, 143B, ZOS and ZOSM were 2.73±0.27, 3.18±0.54, 4.33±0.52, 5.75±0.65, 4.50±0.49 and 3.96±0.35, respectively, higher than 1.00±0.09 in hFOB1.19 (P<0.001). CCK-8 assay revealed that after 48 h and 72 h, the absorbance of sh-circTNPO1 #1 was 0.81±0.05 and 1.09±0.06, while sh-circTNPO1 #2 143B cells was 0.84±0.04 and 1.2±0.04, which were sharply reduced compared with the control (1.00±0.06 and 1.49±0.06, P<0.001); after 48 h and 72 h, the absorbance of 143B cells transfected with circTNPO1 #1 and miR-338-3p (0.92±0.06 and 1.32±0.07) were higher than those of cells transfected with sh-circTNPO1 cells and miR NC (0.92±0.06 and 1.32±0.07, P<0.050). Wound healing assay demonstrated that the 24 hour-migration rates of sh-circTNPO1 #1 and sh-circTNPO1 #2 cells were (24.43±2.15)% and (39.70±4.20)% respectively, which were significantly lower than that of the control [(56.51±3.27)%, P<0.010]; the migration rates of sh-circTNPO1 #1+ miR NC and sh-circTNPO1 #1+ miR-338-3p inhibitor were (26.70±2.21)% and (46.10±5.71)%, with a significant difference (P<0.005). In xenograft tumor model, the weight and size of tumors in control, sh-circTNPO1 #1+ miR NC and sh-circTNPO1 #1+ miR-338-3p inhibitor mice were (458.80±158.10) mg, (262.50±82.09) mg, (395.40±137.60) mg and (593.00±228.40) mm(2,) (203.30±144.20) mm(2,) (488.60±208.60) mm(2,) respectively. Compared with control, sh-circTNPO1 tumors were significantly smaller (P<0.01). Injection with miR-338-3p inhibitor significantly reversed both the weight and size of tumors (P<0.05). Conclusion: CircTNPO1 promotes the proliferation and metastasis of OS by sponging miR-338-3p, which could be a new target for OS treatments.

[circ-WHSC1 affects the growth, metastasis and radiotherapy sensitivity of nasopharyngeal carcinoma cells by targeting miR-338-3p/ELAVL1 axis].

Objective: To study the effect of circ-WHSC1 on the growth, metastasis and radiosensitivity of nasopharyngeal carcinoma cells and its molecular mechanism. Methods: Cancerous tissues and adjacent tissues were collected from 23 patients with nasopharyngeal carcinoma, and real-time fluorescent quantitative PCR (RT-qPCR) was used to detect the expression levels of circ-WHSC1, miR-338-3p, and ELAVL1 mRNA. Western blot was used to detect the expression of ELAVL1 protein. Nasopharyngeal carcinoma cells 5-8F and SUNE1 were divided into si-NC group, si-circ-WHSC1 group, pCD5-ciR group, circ-WHSC1 group, anti-miR-NC group, anti-miR-338-3p group, miR-NC group, miR-338-3p group, si-circ-WHSC1+ anti-miR-NC group, si-circ-WHSC1+ anti-miR-338-3p group, miR-338-3p+ pcDNA group, miR-338-3p+ ELAVL1 group. Tetramethylazolium salt colorimetric method (MTT) was used to detect cell viability. Clone formation test was used to detect cell clone formation and cell radiosensitivity. Flow cytometry was used to detect cell apoptosis. Transwell was used to detect cell migration and invasion. Dual luciferase assay was used to detect the targeting relationship between circ-WHSC1 and miR-338-3p, miR-338-3p and ELAVL1. The SUNE1 cells stably transfected with sh-circ-WHSC1 were injected into nude mice and irradiated with radiation, and then the tumor volume and weight of mice were detected. Results: The expressions of circ-WHSC1 (1.57±0.94 vs 3.78±1.18, 1.00±0.10 vs 1.64±0.14/2.00±0.21/2.81±0.26/3.36±0.34) and ELAVL1 (1.28±0.74 vs 3.36±0.77, 1.00±0.08 vs 2.51±0.19/3.27±0.27) in nasopharyngeal carcinoma tissues and cells were increased, and the expression of miR-338-3p (3.13±0.96 vs 1.37±0.98, 1.00±0.08 vs 0.48±0.08/0.38±0.07) was decreased (P<0.05). After knockdown of circ-WHSC1, the activity of nasopharyngeal carcinoma cells was decreased [(100.00±8.00)% vs (51.33±8.62)%, (100.00±10.10)% vs (41.02±7.31)%], the number of clone-forming cells was decreased (101.00±8.54 vs 50.33±8.02, 114.00±14.10 vs 42.33±10.01), the rate of apoptosis was increased [(5.37±1.20)% vs (18.3±1.01)%, (6.5±1.18)% vs (22.43±1.40)%], and the numbers of migration (136.00±13.00 vs 72.33±9.50, 154.00±14.10 vs 62.67±11.50) and invasion (113.67±11.59 vs 60.67±9.07, 124.33±15.57 vs 50.33±9.01) were decreased; after different doses of radiation, the cell survival score was decreased (0.23±0.04 vs 0.06±0.01, 0.32±0.07 vs 0.05±0.02) (P<0.05). Circ-WHSC1 targeted and negatively regulated miR-338-3p. Inhibition of miR-338-3p affected the effect of knockdown of circ-WHSC1 on the proliferation, apoptosis, migration, invasion and radiosensitivity of nasopharyngeal carcinoma cells. MiR-338-3p targeted and negatively regulated ELAVL1; ELAVL1 overexpression affected the effects of miR-338-3p on the proliferation, apoptosis, migration, invasion and radiosensitivity of nasopharyngeal carcinoma cells. After the cells stably transfected with sh-circ-WHSC1 were injected into nude mice, the tumor volume [(884.67±95.63)mm(3) vs (487.33±76.51)mm(3)] and weight [(899.01±88.54)mg vs (558.67±75.04) mg] of the nude mice were reduced; after further irradiation, the tumor volume [(395.00±73.50)mm(3) vs 243.13±42.51)mm(3)] and weight[ (452.33±67.30)mg vs (211.09±57.51)mg] of the nude mice were reduced (P<0.05). Circ-WHSC1 regulated the expression of ELAVL1 by targeting miR-382. Conclusion: Knockdown of circ-WHSC1 can inhibit the growth and metastasis of nasopharyngeal carcinoma cells by targeting miR-338-3p/ELAVL1 axis, and enhances the radiosensitivity of nasopharyngeal carcinoma cells.