miR-335 targets ENPP4 and FADS1 genes to mediate obesity in high-fat mice / 中华内分泌代谢杂志

Objective:To detect the differential expression of microRNA(miRNA) in abdominal fat of mice fed with high fat and to explore the role of highly expressed miR-335 in lipid metabolism.Methods:MicroRNA microarray was used to detect the differential expression of miRNAs in abdominal fat of mice fed with high fat. The target genes of miR-335 were predicted by Targetscan, the target genes of ectonucleotide pyrophosphatase/phosphordiester-ase 4(ENPP4) and fatty acid desaturase1(FADS1) were verified with double luciferase reporter system and point mutation test. miR-335 mimics was transfected into 3T3L1 cells to detect the expression of target gene mRNA and protein; Realtime PCR was used to detect the expression levels of miR-335 and target genes in different tissues of mice.Results:After high-fat feeding, the size of mice in the model group was significantly larger than the control group, and the serum total cholesterol and triglyceride levels of mice were significantly increased( P=0.016, P=0.014). miRNAs were differentially expressed in adipose tissues of mice fed with high fat, and the expression of miR-335 increased significantly( P=0.024). Double luciferase reporter system showed that miR-335 combined with the predicted target sites of ENPP4 and FADS1 through the " seed region" to inhibit the expression of ENPP4 and FADS1 genes, and the point mutation test confirmed the binding target sites between miR-335 and ENPP4/FADS1. MiR-335 mimics were transfected into 3T3L1 cells, the expression level of FADS1 mRNA decreased significantly( P=0.038), and the protein levels of ENPP4 and FADS1 decreased significantly( P=0.033, P=0.007). Realtime PCR showed that, miR-335 was significantly higher expressed in liver, brown adipose tissue, and brain after high-fat feeding, especially in white adipose tissue( P=0.002). The expression of ENPP4 and FADS1 in brain( P=0.006, P=0.034) and brown adipose tissue( P=0.014, P=0.037) decreased significantly, and the expression of ENPP4 in liver also decreased significantly after high-fat diet( P<0.001). Conclusion:miR-335 is a miRNA related to lipid metabolism and fat deposition. ENPP4 and FADS1 are the target genes of miR-335.

Serum exosomal miR-335-5p serves as a diagnostic biomarker of triple-negative breast cancer / 中华内分泌外科杂志

Objective:To investigate the expression and diagnostic value of microRNA-335-5p (miR-335-5p) and Human Vascular endothelial growth factor receptor 1 (VEGFR-1, FLT1) in serum exosomal bodies of patients with triple negative breast cancer (TNBC) .Methods:Gene Expression Omnibus database (GEO) analysis was performed and differentially expressed miRNA in breast cancer tissues was screened. From Jan. 2016 to Nov. 2017, the peripheral blood samples of 56 TNBC patients in People’s Hospital Affiliated to Ningbo University were collected, and the exosomes were isolated and identified. FLT1 was selected as the target gene of miR-335-5p by using bioinformatics analysis. Expression levels of miR-335-5p and FLT1 in serum exosome were detected by qRT-PCR. The relationship between miR-335-5p and clinicopathological parameters of TNBC patients was analyzed. The diagnostic value of miR-335-5p was evaluated by receiver operating characteristic (ROC) , and the survival prognosis of miR-335-5p and FLT1 was analyzed by Kaplan Meier survival curve.Results:The expression of miR-335-5p in the serum exosome of TNBC patients was lower than that of the control group, while the expression of FLT1 in the serum exosome was higher than that of the control group ( P<0.05) . The expression of miR-335-5p was related to tissue grade ( χ2=22.02, P<0.000 1) , degree of differentiation ( χ2=20.67, P<0.000 1) and lymph node metastasis ( χ2=4.667, P=0.030 8) in patients with TNBC ( P<0.05) . The area under ROC diagnosed by miR-335-5p was 0.809 8 (95% CI: 0.726 3-0.893 2, P<0.000 1) . Kaplan-Meier survival analysis showed that the overall survival time of patients with low expression of miR-335-5p was significantly shorter than that of patients with high expression of miR-335-5p ( P=0.004 5) . The high expression of its target gene FLT1 was associated with low survival rate ( P=0.048 0) . Conclusion:Serum exosomal miR-335-5p can be used as an important index to predict the prognosis of TNBF and is expected to play a role in diagnosis and treatment of TNBC.

circ_0005075 promotes proliferation and invasion of liver cancer HCCC9810 cells by sponging miR-335 / 中国肿瘤生物治疗杂志

@#Objective: To explore the effect of circ_0005075 on the proliferation and invasion of liver cancer cells and its underlying mechanism. Methods:Atotal of 35 cases of cancer tissues and corresponding para-cancerous tissues from liver cancer patients, who underwent surgical resection in Tangshan Workers’Hospital from March 2015 to March 2018, were collected for this study. qPCR was used to detect the expression levels of circ_0005075 and miR-335 in liver cancer tissues, para-cancerous tissues, liver cancer cell lines (HCCC9810, HepG2, HLE and hepatic epithelial THLE-3 cells). Dual luciferase reporter gene assay was used to verify the targeting relationship among circ 0005075, mir-335 and CCND1. By using liposome-mediated method, Sh-circ_0005075, miR-335 mimics, miR335 mimics+pcDNA-CCND1, sh-circ_0005075+pcDNA-CCND1, pcDNA-circ_0005075+miR-335 mimics, sh-CCND1+pcDNA-circ_ 0005075 were transfected into HCCC9810 cells, respectively. The effects of circ_0005075/miR-335/CCND1 molecular axis on the proliferation and invasion of HCCC9810 cells were detected by MTT and Transwell methods. Results: circ_0005075 was highly expressed in liver cancer tissues and cell lines (P<0.01) ,and the highest expression in HCCC9810 cells (P<0.05). Dual luciferase reporter gene results showed that circ_0005075 negatively regulated miR-335 (P<0.05), and CCND1 was a target gene of miR-335 (P<0.05). Further experiments proved that knockdown of circ_0005075 or overexpression of miR-335 could inhibit the proliferation and invasion of HCCC9810 cells by regulating CCND1（P<0.05 or P<0.01） . Conclusion: Circ_0005075 upregulates the expression level of CCND1 by sponging miR-335, thereby promoting the proliferation and invasion of HCCC9810 cells.

miR-335 regulate cell proliferation by targeting survivin in breast cancer cells MCF-7 / 重庆医学

Objective To evaluate the effect of miR‐335 regulate cell proliferation by targeting survivin in breast cancer cells MCF‐7 .Methods (1)Chose breast cancer tissue and para‐carcinoma tissue ,and used RT‐PCR or Western blot to detect the expres‐sion of miR‐335 and survivin protein .(2)Chose breast cancer cells MCF‐7 ,respectively transfected miR‐335 mimic and mimic con‐trol .The expression of miR‐335 and survivin protein were detected by RT‐PCR or Western blot .(3)Chose breast cancer cells MCF‐7 ,respectively co‐transfection wild type survivin 3′‐UTR(survivin‐wt)or mutant type urvivin 3′‐UTR(survivin‐Mut)and miR‐335 mimic or negative control(NC)to breast cancer cells MCF‐7 .The cell luciferase activity were detected by dual‐luciferase report gene experiment .(4)Chose breast cancer cells MCF‐7 ,respectively transfected mimic control ,miR‐335 mimic and miR‐335 mimic+ sur‐vivin .The cell proliferation activity of each group were detected by MTT method .Results (1)Compared with para‐carcinoma tis‐sue ,the miR‐335 expression of breast cancer tissue significantly decreased(P0 .05) .(4)Compared with transfection mimic control ,the MCF‐7 proliferative activity significantly decreased after transfection miR‐335mimic(P<0 .05) .Compared with transfection miR‐335 mimic ,the MCF‐7 proliferative activity signifi‐cantly increased after transfection miR‐335 mimic+survivin(P<0 .05) ,but still lower than transfection mimic control(P<0 .05) . Conclusion In breast cancer tissue ,the miR‐335 show low expression ,and survivin show high expression .miR‐335 can target sur‐vivin to inhibit the proliferation activity of breast cancer cells MCF‐7 .

The identification of human microRNA335′s predicated target gene CCL1 1,CCL26 and SOX4 / 重庆医学

Objective To identify the targeted-regulating relationship between human MicroRNA335 (hsa-miR-335 )and CCL1 1,CCL26 and SOX4.Methods The potential fragments of hsa-miR-335 target genes CCL1 1,CCL26 and SOX4 were predicted by the bioinformatics analyzing tools online.The 3′untranslated regions(3′UTR)of the CCL1 1,CCL26 and SOX4 were connected to the eukaryotic expression vectors pMIR REPORT.The constructs of pMIR-REPORT-CCL1 13′UTR,pMIR-REPORT-CCL26 3′UTR,pMIR-REPORT-SOX4 3′UTR and positive control were co-transfected with Pre-miRTM miRNA335 Precursor or negative control into 293 T7/1 7 cell line by lipofectamine 2000,respectively.Both Firefly and Renilla luciferase activity were detected by dual luciferase reporter assay system.Results Compared with the negative control group,luciferase assay revealed that has-miR-335 could significantly diminish luciferase activity from SOX4 reporter vector (P 0.05).Conclusion The results suggested that hsa-miR-335 targeted regu-lated SOX4,but not targeted CCL1 1 and CCL26.

Effects of miR-335-5p on the osteoblast function in high glucose condition / 中华内分泌代谢杂志

Objective To investigate the effects of miR-335-5p on the proliferation and apoptosis of osteoblasts which were exposed to high glucose condition, and explore its possible molecular mechanisms. Methods MC3T3-E1 osteoblasts were divided into four groups:control group(5. 5 mmol/L glucose), high glucose group(HG group, 22. 0 mmol/L glucose), agomir-335-5p group(transfected with agomir-335-5p and exposed to 22. 0 mmol/L glucose) , and agomir negative control group( agomir NC group, transfected with agomir negative control and exposed to 22. 0 mmol/L glucose), cultured for 7 days. Cell proliferaton, cell apoptosis, expressions of miR-335-5p and dickkopfhomolog1(DKK1)mRNA,proteinlevelsofDKK1andcysteinylaspartate-specificproteinase-3(caspase-3) were detected using MTT, flow cytometry, quantitative realtime PCR and western blot, respectively. Results Compared with control group, the expression of miR-335-5p mRNA and cell proliferation in HG group were significantly decreased(P0. 05). The miR-335-5p mRNA expression and cell proliferation in agomir-335-5p group were higher than those in HG group and agomir NC group(P<0. 05). However, Cell apoptosis and the protein levels of DKK1 and caspase-3 in agomir-335-5p group were lower than those in HG group(P<0. 05). Conclusion High glucose inhibits the proliferation and induce the apoptosis of MC3T3-E1 osteoblast through decreasing the expression of miR-335-5p and subsequently increasing the DKK1 expression.

miR-335 inhibits migration and invasion of human osteosarcoma cell line MG-63 by targeting ROCK1 / 中国癌症杂志

Background and purpose:MicroRNAs are endogenous small RNAs and involved in target gene regulation in post-transcription level. As a tumor suppressor, microRNA-335 (miR-335) was participated in the occurrence and progression in many types of human tumors. This study aimed at investigating whether miR-335 can regulate cell migration and invasion by negative targeting Rho associated coiled-coil forming protein kinase (ROCK1) in human osteosarcoma cell line MG-63.Methods:The speciifc binding ability of miR-335 to ROCK1 3’-untranslated region (UTR) was theoretically predicted and detected by the luciferase reporter gene assay. Western blot and real-time PCR were used to evaluate the effect of miR-335 on the expression of ROCK1 at protein and mRNA levels. Cell migration and invasion assays were performed by transwell chambers in MG-63 cells intervened by over-expression of miR-335 and knockdown of ROCK1.Results:Luciferase reporter gene assay showed that miR-335 could target ROCK1 3’-UTR. Lower miR-335 but higher ROCK1 was expressed in MG-63 cells. The results of Western blot showed that ROCK1 protein expression was decreased by transfection of miR-335 mimic or siROCK1. Transwell chambers results showed decreased cells invading through the substrate membrane after over-expression of miR-335 or knockdown ofROCK1 gene.Conclusion:miR-335 inhibits the migration and invasion ability of MG-63 human osteosarcoma cell line, which may through targeting ROCK1 3’-UTR and subsequent down-regulating ROCK1 expression.

Relative role of free fatty acids on expression of miR-335 in human matured adipocytes / 中华实用儿科临床杂志

Objective To explore the role of free fatty acids(FFA) on expression of miR-335 in human matured adipocytes.Methods In order to induce differentiation,confluent human pre-adipocytes (day 0) were subsequently cultured in serum-free PAM containing 50 nmol/L insulin,100 nmol/L dexamethasone,0.5 mmol/L 3-isobutyl1-methylxanthine,and 100 μmol/L rosiglitazone.Then human matured adipocytes (day 16) were treated with 1 mmol/L FFA cocktail composed of lauric acid,myristic acid,linoleic acid,oleic acid,and arachidonic acids for 4,8 and 24 hours.Meanwhile,untreated cells were collected as control group.Total RNA from these adipocytes were extracted and the levels of miR-335 expression were evaluated by real-time PCR.Results The expression of miR-335 at 4,8 and 24 hours showed no statistical significance when compared to 0 hour in untreated matured adipocytes (all P ＞ 0.05).The relative expression of miR-335 after the intervention of FFA in human matured adipocytes were 9.03 ± 0.31,9.85 ±2.41 and 11.23 ± 0.62,respectively at 4,8 and 24 hours when used with snRU6 for normalization,and there was statistical signi-ficance compared with 0 hour in control group (all P ＜ 0.05).The levels of miR-335 were 4.73 ± 0.60,5.38 ± 1.25 and 4.57 ±0.52 at the same time point when used with miR-103 for normalization,and there was statistical significance compared with 0 hour in control group (all P ＜ 0.05).Conclusions FFA exert a positive effect on the miR-335 expression in human matured adipocytes,which provide the basis for the further study about the role of miR-335 in human adipocytes.