Urinary miR-185-5p is a biomarker of renal tubulointerstitial fibrosis in IgA nephropathy.

Background: For IgA nephropathy (IgAN), tubular atrophy/interstitial fibrosis is the most important prognostic pathological indicator in the mesangial and endocapillary hypercellularity, segmental sclerosis, interstitial fibrosis/tubular atrophy, and presence of crescents (MEST-C) score. The identification of non-invasive biomarkers for tubular atrophy/interstitial fibrosis would aid clinical monitoring of IgAN progression and improve patient prognosis. Methods: The study included 188 patients with primary IgAN in separate confirmation and validation cohorts. The associations of miR-92a-3p, miR-425-5p, and miR-185-5p with renal histopathological lesions and prognosis were explored using Spearman correlation analysis and Kaplan-Meier survival curves. Bioinformatics analysis and dual luciferase experiments were used to identify hub genes for miR-185-5p. The fibrotic phenotypes of tubular epithelial cells were evaluated in vivo and in HK-2 cells. Results: miRNA sequencing and cohort validation revealed that the expression levels of miR-92a-3p, miR-425-5p, and miR-185-5p in urine were significantly increased among patients with IgAN; these levels could predict the extent of tubular atrophy/interstitial fibrosis in such patients. The combination of the three biomarkers resulted in an area under the receiver operating characteristic curve of 0.742. The renal prognosis was significantly worse in the miR-185-5p high expression group than in the low expression group (P=0.003). Renal tissue in situ hybridization, bioinformatics analysis, and dual luciferase experiments confirmed that miR-185-5p affects prognosis in patients with IgAN mainly by influencing expression of the target gene tight junction protein 1 (TJP1) in renal tubular epithelial cells. In vitro experiment revealed that an miR-185-5p mimic could reduce TJP1 expression in HK-2 cells, while increasing the levels of α-smooth muscle actin, fibronectin, collagen I, and collagen III; these changes promoted the transformation of renal tubular epithelial cells to a fibrotic phenotype. An miR-185-5p inhibitor can reverse the fibrotic phenotype in renal tubular epithelial cells. In a unilateral ureteral obstruction model, the inhibition of miR-185-5p expression alleviated tubular atrophy/interstitial fibrosis. Conclusion: Urinary miR-185-5p, a non-invasive biomarker of tubular atrophy/interstitial fibrosis in IgAN, may promote the transformation of renal tubular epithelial cells to a fibrotic phenotype via TJP1.

Cytotoxic and epigenetic effects of berberine-loaded chitosan/pectin nanoparticles on AGS gastric cancer cells: Role of the miR-185-5p/KLF7 axis, DNMTs, and global DNA methylation.

Poor bioavailability, solubility, and absorption of berberine (Ber) limit its widespread application. Here, we formulated novel chitosan/pectin nanoparticles (NPs) loaded with Ber to address delivery problems and promote the anticancer properties of Ber in AGS gastric cancer cells. The ionic gelification method was used to synthesize NPs-Ber. Physicochemical characterization of NPs-Ber was performed using FE-SEM, DLS, PDI, ζ potential, and FTIR. The cytotoxic effects of NPs-Ber on AGS cells were evaluated using the MTT assay. Apoptosis and cell cycle arrest were examined by flow cytometry. The gene expression levels of miR-185-5p, KLF7, caspase-3, and DNMTs were determined using RT-qPCR. In addition, the 5-methylcytosine level in the genomic DNA was quantified using ELISA. FE-SEM images revealed a denser and more packed matrix for NPs-Ber, and FTIR analysis confirmed the formation of NPs-Ber. The size (550.39 nm), PDI (0.134), and ζ potential (-16.52 mV) confirmed the stability of the prepared NPs-Ber. NPs-Ber showed a continuous release pattern following the Korsmeyer-Peppas model such that 81.36 % of Ber was released from the formulation after 240 min. Compared to NPs and free Ber, NPs-Ber was found to possess higher anticancer activity in AGS cells. This result was indicated by the viability test and further clarified by augmented apoptosis and cell cycle arrest at the G0/G1 phase. The IC50 value of NP-Ber against AGS cells was significantly lower than those of free Ber and NPs. Interestingly, our results showed that NPs-Ber considerably changed the expression levels of miR-185-5p, KLF7, caspase-3, and DNMTs (DNMT1, 3A, and 3B) compared with unloaded NPs and free Ber. Additionally, 5-methylated cytosine (5-mC) levels in cells treated with NPs-Ber were significantly higher than those in cells treated with unloaded NPs or free Ber. In summary, the present study demonstrated that Ber encapsulation in NPs enhances its cytotoxic and epigenetic effects on AGS cells, suggesting the promising potential of NPs-Ber in GC therapy.

THAP9-AS1 promotes nasopharyngeal carcinoma progression through targeted regulation of the miR-185-5p/SOX13 axis.

Background: It has been reported that long non-coding RNA THAP9-AS1 exerts carcinogenic role by mediating miRNAs and target genes in various human cancers. However, whether THAP9-AS1 influences the progression of nasopharyngeal carcinoma (NPC) remains unknown. Methods: The transcriptional levels of THAP9-AS1 and miR-185-5p were estimated via quantitative real time polymerase chain reaction (qRT-PCR) assay. The protein level of SOX13 was detected with western blotting assay. Additionally, methyl thiazolyl tetrazolium (MTT) assay as well as colony formation assay were utilized to measure cell growth. The apoptotic cells were observed by employing Terminal-deoxynucleoitidyl Transferase Mediated Nick End Labeling (TUNEL) staining analysis, and transwell assay was introduced to test cell migration in addition to invasion. Moreover, the relationship between miR-185-5p and THAP9-AS1 or SOX13 was estimated through dual-luciferase reporter gene assay. Results: THAP9-AS1 was overexpressed in head and neck squamous cell carcinoma (HNSCC) tissues and NPC cells. Besides, silencing of THAP9-AS1 depressed the life processes of NPC cells including cell growth, migration as well as invasion but facilitated cell apoptosis. Further investigation proved that miR-185-5p was the direct target of THAP9-AS1. Besides, the knockdown of THAP9-AS1 notably reduced the transcriptional level of miR-185-5p. Furthermore, THAP9-AS1 served as a sponge of miR-185-5p to modulate the expression of SOX13, which regulated the development of NPC cells. Conclusion: This work verified that THAP9-AS1 promoted NPC cell progression at least partly by mediating the miR-185-5p/SOX13 axis.

Vof16-miR-185-5p-GAP43 network improves the outcomes following spinal cord injury via enhancing self-repair and promoting axonal growth.

INTRODUCTION: Self-repair of spinal cord injury (SCI) has been found in humans and experimental animals with partial recovery of neurological functions. However, the regulatory mechanisms underlying the spontaneous locomotion recovery after SCI are elusive. AIMS: This study was aimed at evaluating the pathological changes in injured spinal cord and exploring the possible mechanism related to the spontaneous recovery. RESULTS: Immunofluorescence staining was performed to detect GAP43 expression in lesion site after spinal cord transection (SCT) in rats. Then RNA sequencing and gene ontology (GO) analysis were employed to predict lncRNA that correlates with GAP43. LncRNA smart-silencing was applied to verify the function of lncRNA vof16 in vitro, and knockout rats were used to evaluate its role in neurobehavioral functions after SCT. MicroRNA sequencing, target scan, and RNA22 prediction were performed to further explore the underlying regulatory mechanisms, and miR-185-5p stands out. A miR-185-5p site-regulated relationship with GAP43 and vof16 was determined by luciferase activity analysis. GAP43-silencing, miR-185-5p-mimic/inhibitor, and miR-185-5p knockout rats were also applied to elucidate their effects on spinal cord neurite growth and neurobehavioral function after SCT. We found that a time-dependent increase of GAP43 corresponded with the limited neurological recovery in rats with SCT. CRNA chip and GO analysis revealed lncRNA vof16 was the most functional in targeting GAP43 in SCT rats. Additionally, silencing vof16 suppressed neurite growth and attenuated the motor dysfunction in SCT rats. Luciferase reporter assay showed that miR-185-5p competitively bound the same regulatory region of vof16 and GAP43. CONCLUSIONS: Our data indicated miR-185-5p could be a detrimental factor in SCT, and vof16 may function as a ceRNA by competitively binding miR-185-5p to modulate GAP43 in the process of self-recovery after SCT. Our study revealed a novel vof16-miR-185-5p-GAP43 regulatory network in neurological self-repair after SCT and may underlie the potential treatment target for SCI.

Identification of AP002498.1 and LINC01871 as prognostic biomarkers and therapeutic targets for distant metastasis of colorectal adenocarcinoma.

BACKGROUND: Increasing evidence suggests that lncRNA (Long non-coding RNA, lncRNA)-mediated ceRNA (competing endogenous RNA, ceRNA) networks are involved in the occurrence and progression of colorectal cancer (CRC). However, the roles of the lncRNA-miRNA-mRNA ceRNA network in distant metastasis of CRC are still unclear. METHODS: In this study, we constructed a specific ceRNA network to identify potential biomarkers and therapeutic targets for distant metastasis of CRC. Specifically, RNA-Seq data from The Cancer Genome Atlas (TCGA) were used to screen for differentially expressed lncRNAs (DElncRNAs) and mRNAs (DEmRNAs) related to metastasis. After validation and selection by qRT-PCR and univariate and multivariate analysis of the metastasis- and prognosis-related lncRNAs, the regulated microRNAs (miRNAs) and coexpressed mRNAs were used to construct a ceRNA network for distant metastasis of CRC. RESULTS: Two key distant metastasis-related DElncRNAs, AP002498.1 and LINC01871, were identified by univariate and multivariate analysis in combination with analyses of clinical data and expression levels. Furthermore, lncRNA-associated ceRNA subnetworks were constructed from the predicted miRNAs and 13 coexpressed DEmRNAs (SERPINA1, ITLN1, REG4, L1TD1, IGFALS, MUC5B, CIITA, CXCL9, CXCL10, GBP4, GNLY, IDO1, and NOS2). The AP002498.1- and LINC01871-associated ceRNA subnetworks regulated the expression of the target genes SERPINA1 and MUC5B and GNLY, respectively, through the associated miRNAs. CONCLUSION: The DElncRNA AP002498.1 and the LINC01871/miR-4644 and miR-185-5p/GNLY axes were identified as being closely associated with distant metastasis and could represent independent prognostic biomarkers or therapeutic targets in colorectal adenocarcinoma.

Study on serum miR-185-5p in assessing the injury severity and prognosis of patients with traumatic brain injury.

Background: This study aims to explore whether serum miR-185-5p levels are related to the injury severity and prognosis of traumatic brain injury patients. Methods: Serum miR-185-5p level was quantified in 120 TBI patients. The Glasgow Coma Scale (GCS) was used to grade the damage, and the Glasgow Outcome Scale (GOS) was used to evaluate the prognosis 3 months after TBI. Pearson correlation analysis was performed to determine the relationship between serum miR-185-5p level and injury severity and prognosis, and the value of serum miR-185-5p level to assess injury severity and prognosis was evaluated by receiver operating characteristic (ROC) curve. Results: Serum miR-185-5p level in moderate and severe TBI patients was higher than in mild TBI patients, and serum miR-185-5p was closely related to GCS score and GOS score. Serum miR-185-5p level higher than 0.36 could distinguish patients with mild to moderate TBI injury, with 72.97% sensitivity and 97.62% specificity, while that higher than 0.43 had 46.34% sensitivity and 91.89% specificity to distinguish moderate to severe TBI patients. Moreover, serum miR-185-5p levels higher than 0.36, with a sensitivity of 96.30% and a specificity of 60.24%, distinguished the poor prognosis of TBI patients. Serum miR185-5p level was an independent predictor of poor prognosis in TBI patients after 3 months and was effective in discriminating adverse outcomes at 3 months. Conclusions: Serum miR-185-5p level was significantly correlated with 3-month injury and adverse prognosis in TBI patients, suggesting that serum miR-185-5p level may be a biomarker that provides supplementary prognostic information and can be used to identify the risk of adverse prognosis in TBI patients.

miR-185-5p / ATG101 axis alleviated intestinal barrier damage in intestinal ischemia reperfusion through autophagy.

Objective: Intestinal ischemia-reperfusion (II/R) is a common pathological injury in clinic, and the systemic inflammatory response it causes will lead to multiple organ damage and functional failure. miR-185-5p has been reported to be a regulator of inflammatory response and autophagy, but whether it participates in the regulation of autophagy in II/R is still unclear. Therefore, we aimed to explore the mechanism of miR-185-5p regulating intestinal barrier injury in (II/R). Methods: Caco-2 cells was induced by oxygen-glucose deprivation/reoxygenation (OGD/R) to establish II/R model. The superior mesenteric artery of C57BL/6 mice was clamped for 45 min and then subjected to reperfusion for 4 h for the establishment of II/R mice model. miR-185-5p mimic, miR-185-5p inhibitor, pcDNA-autophagy-related 101 (ATG101) were respectively transfected into Caco-2 cells. Real-time quantitative polymerase chain reaction (RT-qPCR) was performed to assess miR-185-5p expression. Western blot detected the level of ATG101 and tight junction-associated proteins ZO1, Occludin, E-cadherin, ß-catenin, as well as autophagy markers ATG5, ATG12, LC3Ⅰ/Ⅱ, Beclin1 and SQSTM1. Transepithelial electrical resistance (TEER) values was detected by a resistance meter. FITC-Dextran was performed to measure cell permeability. 5-ethynyl-2'-deoxyuridine (EDU) staining measured cell proliferation. Transmission electron microscope was conducted to observe autophagosomes. Hematoxylin & eosin (H&E) staining observed the damage of mice intestinal. Immunohistochemistry (IHC) measured the percentage of ki67 positive cells. TdT-mediated dUTP nick-end labeling (TUNEL) assay assessed cell apoptosis in intestinal tissues of II/R. Dual-luciferase assay verified the targeting relationship between miR-185-5p and ATG101.Results miR-185-5p was overexpressed in OGD/R-induced Caco-2 cells and intestinal tissues of II/R mice. Knocking down miR-185-5p markedly promoted autophagy and TEER values, reduced cell permeability, and alleviated intestinal barrier damage. ATG101 was a target of miR-185-5p, and overexpression of ATG101 promoted autophagy and dampened OGD/R-induced intestinal barrier damage. Overexpression of miR-185-5p reversed the effect of overexpressed ATG101 on OGD/R-induced Caco-2 cells. Conclusion: Knockdown of miR-185-5p enhanced autophagy and alleviated II/R intestinal barrier damage by targeting ATG101.

MALAT1/miR-185-5p mediated high glucose-induced oxidative stress, mitochondrial injury and cardiomyocyte apoptosis via the RhoA/ROCK pathway.

To explore the underlying mechanism of lncRNA MALAT1 in the pathogenesis of diabetic cardiomyopathy (DCM). DCM models were confirmed in db/db mice. MiRNAs in myocardium were detected by miRNA sequencing. The interactions of miR-185-5p with MALAT1 and RhoA were validated by dual-luciferase reporter assays. Primary neonatal cardiomyocytes were cultured with 5.5 or 30 mmol/L D-glucose (HG) in the presence or absence of MALAT1-shRNA and fasudil, a ROCK inhibitor. MALAT1 and miR-185-5p expression were determined by real-time quantitative PCR. The apoptotic cardiomyocytes were evaluated using flow cytometry and TUNEL staining. SOD activity and MDA contents were measured. The ROCK activity, phosphorylation of Drp1S616 , mitofusin 2 and apoptosis-related proteins were analysed by Western blotting. Mitochondrial membrane potential was examined by JC-1. MALAT1 was significantly up-regulated while miR-185-5p was down-regulated in myocardium of db/db mice and HG-induced cardiomyocytes. MALAT1 regulated RhoA/ROCK pathway via sponging miR-185-5p in cardiomyocytes in HG. Knockdown of MALAT1 and fasudil all inhibited HG-induced oxidative stress, and alleviated imbalance of mitochondrial dynamics and mitochondrial dysfunction, accompanied by reduced cardiomyocyte apoptosis. MALAT1 activated the RhoA/ROCK pathway via sponging miR-185-5p and mediated HG-induced oxidative stress, mitochondrial damage and apoptosis of cardiomyocytes in mice.

MicroRNA-185-5p targets tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta to regulate non-small cell lung cancer progression.

BACKGROUND: Lung cancer (LC) is one of the most frequent cancers worldwide, as well as the leading cause of cancer-related death. Non-small cell lung cancer (NSCLC, which accounts for 85% of occurrences) is the main type of LC. MiRNAs appear to play a role in the occurrence and progression of many malignancies, according to mounting data. The underlying mechanism of miRNAs in regulating NSCLC cell biological activity and progression, on the other hand, is still being investigated. METHODS: QRT-PCR were used to detect miR-185-5p expression and YWHAZ mRNA in NSCLC. The CCK-8 assay was used to determine the tumor cells' ability to proliferate. Transwall assay was used to test the migratory and invasive properties of cells. Cell apoptosis was detected using flow cytometry. Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activation protein zeta (YWHAZ), E-Cadherin, N-Cadherin and cleaved-caspase3 protein expression were assessed using Western Blot. The bioinformatics analysis software StarBase2.0 predicted miR-185-5p downstream targets. To confirm the target association between miR-185-5p and YWHAZ, a luciferase experiment was used. In addition, an NCl-H1299 xenograft model was created to assess the anti-tumor impact of miR-185-5p in vivo. The expression level of YWHAZ in tumor tissues of small xenograft tumor model was detected by immunohistochemistry assay. RESULTS: Decreased miR-185-5p expression levels were observed in NSCLC. In vitro, over-expressed miR-185-5p decreased cell viability, proliferation, invasion/migration, and induced cell apoptosis, while inhibiting tumor growth in vivo. Dual-luciferase gene experiments confirmed that YWHAZ binds to miR-185-5p. Overexpression of YWHAZ partially restored the inhibitory effects of miR-185-5p on cell behaviors. CONCLUSION: MiR-185-5p was down-regulated in NSCLC, and that overexpressed miR-185-5p inhibited malignant behaviors of cells and tumor growth by negatively regulating YWHAZ.

CircSLC39A8 attenuates paclitaxel resistance in ovarian cancer by regulating the miR­185­5p/BMF axis.

Chemoresistance to paclitaxel (PTX) is one of the main reasons for treatment failure and poor prognosis in patients with advanced ovarian cancer. Therefore, it is imperative to explore the mechanisms related to chemotherapy resistance in ovarian cancer to find potential therapeutic targets. Circular RNAs (circRNAs) play important roles in cancer development and progression. However, their biological functions and clinical significance in ovarian cancer have not been fully elucidated. Therefore, in this study, we aimed to investigate the function and underlying mechanism of hsa_circ_0002782 (circSLC39A8), identified by circRNA sequencing, in regulating PTX resistance. The effects of circSLC39A8 on PTX resistance was assessed by cell viability, colony formation, flow cytometry assays and an in vivo subcutaneous xenografted tumor mouse model. RNA immunoprecipitation and dual-luciferase reporter assays were performed to verify the interaction between circSLC39A8 and the miR-185-5p/BMF signal axis. We found that circSLC39A8 was downregulated in PTX-resistant ovarian cancer cells and tissues, and its low expression was associated with poor prognosis. Biologically, circSLC39A8 knockdown promoted PTX resistance in vitro and in vivo, while circSLC39A8 overexpression showed the opposite effect. Mechanistically, circSLC39A8, acting as an endogenous sponge for miR-185-5p, could relieve the inhibition of miR-185-5p on the expression of its downstream target, BMF; thus enhancing the sensitivity of ovarian cancer to PTX. Our findings demonstrate that circSLC39A8 can promote PTX sensitivity by regulating the miR-185-5p/BMF axis. This may be a valuable prognostic biomarker and a promising therapeutic target for patients with ovarian cancer.

MiR-185-5p measurement assists in reflecting Th1/Th2 cell imbalance, inflammatory cytokine production, and exacerbation risk for childhood asthma.

BACKGROUND: MicroRNA (miR)-185-5p participates in the pathology of asthma by regulating immune imbalance, inflammation, periostin synthesis, and smooth muscle contraction. This study intended to explore the dysregulation of miR-185p and its correlation with T-helper (Th)1, Th2 cells, and inflammatory cytokines in childhood asthma. METHODS: In 150 childhood asthma patients and 30 healthy controls (HCs), miR-185-5p from peripheral blood mononuclear cells was detected using reverse transcription-quantitative polymerase chain reaction, Th cells from peripheral blood samples were detected using flow cytometry, inflammatory cytokines from serum samples were detected using enzyme-linked immunosorbent assay. RESULTS: MiR-185-5p was increased in childhood asthma patients versus HCs [median (interquartile range (IQR)): 2.315 (1.770-3.855) versus 1.005 (0.655-1.520)] (P < 0.001). Meanwhile, miR-185-5p was negatively associated with Th1 cells (P = 0.035) but positively correlated with Th2 cells (P = 0.006) and IL-4 (P = 0.003) in childhood asthma patients; however, miR-185-5p was not linked to Th1 cells, Th2 cells, IFN-γ, or IL-4 in HCs (all P > 0.05). In addition, miR-185-5p was positively related to TNF-α (P < 0.001), IL-1ß (P = 0.015), and IL-6 (P = 0.008) in childhood asthma patients, miR-185-5p was only linked to TNF-α (P = 0.040) but not IL-1ß or IL-6 (both P > 0.05) in HCs. Moreover, miR-185-5p was increased in exacerbated childhood asthma patients versus remissive patients [median (IQR): 3.170 (2.070-4.905) versus 1.900 (1.525-2.615)] (P < 0.001). Besides, miR-185-5p was highest in patients with severe exacerbation followed by patients with moderate exacerbation, and lowest in patients with mild exacerbation (P = 0.010). CONCLUSION: MiR-185-5p is associated with imbalanced Th1/Th2 cells, increased inflammatory cytokines along with elevated exacerbation risk, and severity in childhood asthma patients.

MiR-185-5p measurement assists in reflecting Th1/Th2 cell imbalance, inflammatory cytokine production, and exacerbation risk for childhood asthma

Background: MicroRNA (miR)-185-5p participates in the pathology of asthma by regulating immune imbalance, inflammation, periostin synthesis, and smooth muscle contraction. This study intended to explore the dysregulation of miR-185p and its correlation with T-helper (Th)1, Th2 cells, and inflammatory cytokines in childhood asthma. Methods: In 150 childhood asthma patients and 30 healthy controls (HCs), miR-185-5p from peripheral blood mononuclear cells was detected using reverse transcription-quantitative polymerase chain reaction, Th cells from peripheral blood samples were detected using flow cytometry, inflammatory cytokines from serum samples were detected using enzyme-linked immunosorbent assay. Results: MiR-185-5p was increased in childhood asthma patients versus HCs [median (interquartile range (IQR)): 2.315 (1.770–3.855) versus 1.005 (0.655–1.520)] (P < 0.001). Meanwhile, miR-185-5p was negatively associated with Th1 cells (P = 0.035) but positively correlated with Th2 cells (P = 0.006) and IL-4 (P = 0.003) in childhood asthma patients; however, miR-185-5p was not linked to Th1 cells, Th2 cells, IFN-γ, or IL-4 in HCs (all P > 0.05). In addition, miR-185-5p was positively related to TNF-α (P < 0.001), IL-1β (P = 0.015), and IL-6 (P = 0.008) in childhood asthma patients, miR-185-5p was only linked to TNF-α (P = 0.040) but not IL-1β or IL-6 (both P > 0.05) in HCs. Moreover, miR-185-5p was increased in exacerbated childhood asthma patients versus remissive patients [median (IQR): 3.170 (2.070–4.905) versus 1.900 (1.525–2.615)] (P < 0.001). Besides, miR-185-5p was highest in patients with severe exacerbation followed by patients with moderate exacerbation, and lowest in patients with mild exacerbation (P = 0.010). Conclusion: MiR-185-5p is associated with imbalanced Th1/Th2 cells, increased inflammatory cytokines along with elevated exacerbation risk, and severity in childhood asthma patients (AU)

CircRNA ZKSCAN1 promotes lung adenocarcinoma progression by miR-185-5p/TAGLN2 axis.

BACKGROUND: Circ-ZKSCAN1 has been found to accelerate non-small cell lung cancer (NSCLC) progression; however, the role and mechanism of circ-ZKSCAN1 in lung adenocarcinoma (LUAD) cisplatin (DDP) resistance remain unclear. METHODS: Levels of genes and proteins were examined using qRT-PCR. Functional experiments were performed using CCK-8 assay, flow cytometry, transwell assay and xenograft model assay, respectively. Glucose metabolism was calculated by detecting glucose consumption, lactate production, ATP and HK-2 levels. The interaction between miR-185-5p and circ-ZKSCAN1 or transgelin 2 (TAGLN2) was validated by dual-luciferase reporter assay. RESULTS: Circ-ZKSCAN1 was highly expressed in DDP-resistant LUAD tissues and cell lines, and circ-ZKSCAN1 knockdown weakened DDP resistance and suppressed cell viability, migration, invasion, and glycolysis in LUAD. Circ-ZKSCAN1 acted as a sponge of miR-185-5p, and the regulatory effects of circ-ZKSCAN1 knockdown on LUAD were reversed by miR-185-5p downregulation. Meanwhile, miR-185-5p directly targeted TAGLN2, and performed anticancer effects by regulating TAGLN2. Importantly, silencing of circ-ZKSCAN1 hindered tumor growth and promoted DDP sensitivity in vivo via regulating miR-185-5p and TAGLN2. CONCLUSION: Circ-ZKSCAN1 promoted LUAD tumorigenesis and DDP resistance by regulating miR-185-5p/TAGLN2 axis.

Highly-expressed lncRNA FOXD2-AS1 in adipose mesenchymal stem cell derived exosomes affects HaCaT cells via regulating miR-185-5p/ROCK2 axis.

The healing of skin wounds is a highly coordinated multi-step process that occurs after trauma including surgical incisions, thermal burns, and chronic ulcers. In this study, the authors investigated lncRNA FOXD2-AS1 function in adipose mesenchymal exosomes from ADMSCs that were successfully extracted. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes accelerated HaCaT cell migration and proliferation. LncRNA FOXD2-AS1 negatively targeted miR-185-5p, and miR-185-5p negatively targeted ROCK2. Highly expressed lncRNA FOXD2-AS1 in ADMSCs-exosomes promoted HaCaT cell migration and proliferation via down-regulating miR-185-5p and further up-regulating ROCK2. In conclusion, LncRNA FOXD2-AS1 overexpression in ADMSCs derived exosomes might accelerate HaCaT cell migration and proliferation via modulating the miR-185-5p/ROCK2 axis.

Circ_0008529 Contributes to Renal Tubular Cell Dysfunction in High Glucose Stress via miR-185-5p/SMAD2 Pathway in Diabetic Nephropathy.

Circular RNA (circRNA) is key regulator of diabetic nephropathy (DN) progression. However, the role of circ_0008529 in DN progression remains to be better deciphered. Cell viability, cell cycle, apoptosis and inflammation were measured by MTS assay, flow cytometry and corresponding assay kits. RT-qPCR was used to assess the expression of circ_0008529, miR-185-5p and SMAD family member 2 (SMAD2). Also, western blotting was performed to measure protein expression. Target relationship was validated by RNA pull-down assay, dual-luciferase reporter assay and RNA immunoprecipitation assay. Urinary exosome was isolated using ultracentrifugation method and identified by transmission electron microscopy. Receiver operating characteristic curve was used to analyze the diagnostic value of circ_0008529 in DN patients. Circ_0008529 and SMAD2 were upregulated, while miR-185-5p was downregulated in high glucose (HG)-induced renal tubular HK-2 cells. Under HG treatment, cell viability and cell cycle process were suppressed, while apoptosis, inflammation and extracellular matrix accumulation were enhanced. However, interfering circ_0008529 could attenuate HG-induced effects, and this protection was abated by miR-185 inhibition or SMAD2 re-expression. Mechanically, circ_0008529 and SMAD2 were competing endogenous RNAs for miR-185-5p via target binding, and circ_0008529 regulated SMAD2 expression via miR-185-5p. Notably, circ_0008529 expression was upregulated in urinary exosomes of DN patients, and showed diagnostic value (Sensitivity: 70.21%; Specificity: 86.67%). Circ_0008529 might be a potential target for DN, which regulated DN progression via miR-185-5p/SMAD2 pathway.

Upregulation of circFOXP1 attenuates inflammation and apoptosis induced by ox-LDL in human umbilical vein endothelial cells by regulating the miR-185-5p/BCL-2 axis.

The pathogenesis of coronary artery disease (CAD) is closely related to an abnormal function of the coronary arteries due to myocardial ischemia, hypoxia, or necrosis, which poses a threat to human health. Therefore, this study was conducted to evaluate the role of circFOXP1 in controlling endothelial cell function during atherosclerosis (AS), and further investigate its potential molecular mechanism of regulation. Through Starbase database analysis, we predicted that circFOXP1 can sponge miR-185-5p that targets BCL-2. We found that interleukin (IL)-6, tumor necrois factor (TNF)-α, and IL-1ß were significantly upregulated in high-fat diet (HFD)-induced apolipoprotein E-deficient (ApoE-/-) mice compared with those in the control mice. CircFOXP1 was also significantly upregulated in the AS-mice model and AS-cell model. Moreover, miR-185-5p overexpression was found to inhibit BCL-2 protein expression, which consequently reduced the proliferation, and increased the oxidized low-density lipoprotein (ox-LDL)-induced human umbilical vein endothelial cells (HUVECs) apoptotic rate. Taken together, our data show that circFOXP1 can further aggravate endothelial cell injury by regulating the miR-185-5p/BCL-2 signal axis.

Circ_0114428 promotes proliferation, fibrosis and EMT process of high glucose-induced glomerular mesangial cells through regulating the miR-185-5p/SMAD3 axis.

Circular RNA (circRNA) has been confirmed to be the key regulators of diabetic nephropathy (DN) progression. However, the role of circ_0114428 in the DN progression remains unclear. Glomerular mesangial cells (GMCs) were treated with high glucose (HG) to mimic DN cell models in vitro. The expression levels of circ_0114428, microRNA (miR)-185-5p, and SMAD3 mRNA were examined by quantitative real-time PCR. Cell proliferation ability was detected by MTT assay, EdU staining and flow cytometry. The protein levels of proliferation marker, fibrosis markers, epithelial-mesenchymal transition (EMT) markers and SMAD3 were measured by western blot assay. The interaction between miR-185-5p and circ_0114428 or SMAD3 was confirmed via dual-luciferase reporter assay, RIP assay and RNA pull-down assay. Our data showed that circ_0114428 was upregulated in HG-induced GMCs. Circ_0114428 overexpression could aggravate the promotion effect of HG on the proliferation, fibrosis and EMT process of GMCs, while its knockdown had an opposite effect. In the terms of mechanisms, circ_0114428 could sponge miR-185-5p to regulate SMAD3. MiR-185-5p inhibitor could reverse the suppressive effect of circ_0114428 knockdown on the proliferation, fibrosis and EMT process in HG-induced GMCs. Also, SMAD3 overexpression abolished the inhibition of miR-185-5p on the proliferation, fibrosis and EMT process in HG-induced GMCs. Taken together, our data suggested that circ_0114428 might promote DN progression by regulating the miR-185-5p/SMAD3 axis.

Circular RNA hsa_circ_0004381 Promotes Neuronal Injury in Parkinson's Disease Cell Model by miR-185-5p/RAC1 Axis.

The study aims to explore the molecular mechanism involved in Parkinson's disease (PD). Hsa_circ_0004381, microRNA-185-5p (miR-185-5p), and Rac family small GTPase 1 (RAC1) level were measured by real-time quantitative polymerase chain reaction (RT-qPCR). Furthermore, cell viability and apoptosis rate were assessed by Cell Counting Kit-8 (CCK-8) and flow cytometry assays, respectively. Protein levels of B cell lymphoma-2 (Bcl-2), Bcl-2-related X protein (Bax), cleaved-caspase 3 (c-caspase 3), and RAC1 were determined by western blot assay. The levels of tumor necrosis factor-α (TNF-α), interleukin-1ß (IL-1ß), and IL-6 were detected by enzyme-linked immunosorbent assay (ELISA). The ROS generation and LDH and SOD activity were detected by the corresponding kits. The binding relationship between miR-185-5p and hsa_circ_0004381 or RAC1 was predicted by Starbase and then verified by a dual-luciferase reporter and RNA Immunoprecipitation (RIP) assays. Hsa_circ_0004381 and RAC1 were increased, and miR-185-5p was decreased in MPP+-triggered SK-N-SH cells. Moreover, hsa_circ_0004381 silencing promoted cell viability, and repressed apoptosis, inflammatory response, and oxidative stress in MPP+-treated SK-N-SH cells. The mechanical analysis suggested that hsa_circ_0004381 served as a sponge of miR-185-5p to affect RAC1 expression. Hsa_circ_0004381 could contribute to MPP+-triggered neuron injury by targeting the miR-185-5p/RAC1 axis, which provided a novel insight into the pathogenesis and treatment of PD.

LncRNA FAF attenuates hypoxia/ischaemia-induced pyroptosis via the miR-185-5p/PAK2 axis in cardiomyocytes.

Pyroptosis is associated with various cardiovascular diseases. Increasing evidence suggests that long noncoding RNAs (lncRNAs) have been implicated in gene regulation, but how lncRNAs participate in the regulation of pyroptosis in the heart remains largely unknown. In this study, we aimed to explore the antipyroptotic effects of lncRNA FGF9-associated factor (FAF) in acute myocardial infarction (AMI). The expression patterns of lncRNA FAF, miR-185-5p and P21 activated kinase 2 (PAK2) were detected in hypoxia/ischaemia-induced cardiomyocytes. Hoechst 33342/PI staining, lactate dehydrogenase (LDH) release assay, immunofluorescence and Western blotting were conducted to assay cell pyroptosis. The interaction between lncRNA FAF, miR-185-5p and PAK2 was verified by bioinformatics analysis, small RNA sequencing luciferase reporter assay and qRT-PCR. The expression of LncRNA FAF was downregulated in hypoxic cardiomyocytes and myocardial tissues. Overexpression of lncRNA FAF could attenuate cardiomyocyte pyroptosis, improve cell viability and reduce infarct size during the procession of AMI. Moreover, lncRNA FAF was confirmed as a sponge of miR-185-5p and promoted PAK2 expression in cardiomyocytes. Collectively, our findings reveal a novel lncRNA FAF/miR-185-5p/PAK2 axis as a crucial regulator in cardiomyocyte pyroptosis, which might be a potential therapeutic target of AMI.

The elevation of miR-185-5p alleviates high-fat diet-induced atherosclerosis and lipid accumulation in vivo and in vitro via SREBP2 activation.

OBJECTIVE: SREBP2, a member of the SREBP family, is a primary regulator of lipid metabolism. In recent years, an increasing number of studies have suggested that miRNAs regulate lipid metabolism-related genes. It was speculated in this study that miRNAs may be implicated in the regulation of lipid accumulation in macrophages by SREBP2 protein. METHODS AND RESULTS: GSE34812, GSE132651 and GSE28829 datasets comprised of atherosclerosis samples were downloaded to explore the gene expression profiles related to the miRNAs and SREBP2, and miR-185-5p was predicted to be a target of SREBP2. The GO annotations and KEGG pathway analysis were adopted for functional classification of differentially expressed genes, and lipid metabolic process was an enriched pathway in atherosclerosis. Besides, the effects of SREBP2 on increasing lipid accumulation were investigated in vivo using miR-185-5p mimic/apoE-/- mice and miR-185-5p NC/apoE-/- mice. All mice fed with a HFD suffered from atherosclerosis. Moreover, the effects of miR-185-5p on atherosclerotic plaque formation in mice were analyzed. An in vitro assay was also performed to determine the effect of miR-185-5p on ox-LDL-stimulated RAW 264.7 macrophages. Finally, miR-185-5p mimic was transfected into cultured macrophages. The results showed that the miR-185-5p elevation might regulate lipid accumulation in mice by targeting SREBP2. Furthermore, miR-185-5p mimic repressed the activation of SREBP1, SREBP2, LDLR, SCD-1, HMGCR as well as NLRP3, IL-1ß, TNF-α in HFD fed mice or ox-LDL-stimulated macrophages. CONCLUSIONS: our study demonstrated that miR-185-5p effectively alleviates atherosclerosis and lipid accumulation by regulating the miR-185-5p/SREBP2 axis.