Enrichment of miR-17-5p enhances the protective effects of EPC-EXs on vascular and skeletal muscle injury in a diabetic hind limb ischemia model

BACKGROUND/AIMS: Diabetes mellitus (DM) is highly susceptible to diabetic hind limb ischemia (DHI). MicroRNA (MiR)-17-5p is downregulated in DM and plays a key role in vascular protection. Endothelial progenitor cell (EPC)-released exosomes (EPC-EXs) contribute to vascular protection and ischemic tissue repair by transferring their contained miRs to target cells. Here, we investigated whether miR-17-5p-enriched EPC-EXs (EPC-EXsmiR-17-5p) had conspicuous effects on protecting vascular and skeletal muscle in DHI in vitro and in vivo. METHODS: EPCs transfected with scrambled control or miR-17-5p mimics were used to generate EPC-EXs and EPC-EXsmiR-17-5p. Db/db mice were subjected to hind limb ischemia. After the surgery, EPC-EXs and EPC-EXsmiR-17-5p were injected into the gastrocnemius muscle of the hind limb once every 7 days for 3 weeks. Blood flow, microvessel density, capillary angiogenesis, gastrocnemius muscle weight, structure integrity, and apoptosis in the hind limb were assessed. Vascular endothelial cells (ECs) and myoblast cells (C2C12 cells) were subjected to hypoxia plus high glucose (HG) and cocultured with EPC-EXs and EPC-EXsmiR-17-5p. A bioinformatics assay was used to analyze the potential target gene of miR-17-5p, the levels of SPRED1, PI3K, phosphorylated Akt, cleaved caspase-9 and cleaved caspase-3 were measured, and a PI3K inhibitor (LY294002) was used for pathway analysis. RESULTS: In the DHI mouse model, miR-17-5p was markedly decreased in hind limb vessels and muscle tissues, and infusion of EPC-EXsmiR-17-5p was more effective than EPC-EXs in increasing miR-17-5p levels, blood flow, microvessel density, and capillary angiogenesis, as well as in promoting muscle weight, force production and structural integrity while reducing apoptosis in gastrocnemius muscle. In Hypoxia plus HG-injured ECs and C2C12 cells, we found that EPC-EXsmiR-17-5p could deliver their carried miR-17-5p into target ECs and C2C12 cells and subsequently downregulate the target protein SPRED1 while increasing the levels of PI3K and phosphorylated Akt. EPC-EXsmiR-17-5p were more effective than EPC-EXs in decreasing apoptosis and necrosis while increasing viability, migration, and tube formation in Hypoxia plus HG-injured ECs and in decreasing apoptosis while increasing viability and myotube formation in C2C12 cells. These effects of EPC-EXsmiR-17-5p could be abolished by a PI3K inhibitor (LY294002). CONCLUSION: Our results suggest that miR-17-5p promotes the beneficial effects of EPC-EXs on DHI by protecting vascular ECs and muscle cell functions.

miR-17-5p regulates the proliferation and apoptosis of myelodysplastic syndrome SKM-1 cells by targeting SETD2 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨miR-17-5p和含SET结构域蛋白2（SETD2）对骨髓增生异常综合征（MDS）SKM-1细胞增殖与凋亡的影响及其作用机制。方法：收集2019年3月至2021年5月衡水市人民医院就诊的35例MDS患者的骨髓标本（MDS组）、35例健康体检者的骨髓标本（对照组），以及MDS细胞系SKM-1。用qPCR法检测MDS骨髓和SKM-1细胞中miR-17-5p、SETD2 mRNA的表达水平。双荧光素酶报告基因实验验证miR-17-5p与SETD2的靶向关系。利用脂质体转染技术，分别将si-miR-NC、si-miR-17-5p、miR-NC、miR-17-5p mimic、pcDNA、pcDNA-SETD2、si-miR-17-5p+si-NC、si-miR-17-5p+si-SETD2等转染至SKM-1细胞，CCK-8法、流式细胞术检测细胞的增殖和凋亡水平，WB法检测细胞中SETD2、C-caspase-3、C-caspase-9的表达。结果：与对照组相比，MDS组骨髓中miR-17-5p表达水平显著升高、SETD2的mRNA和蛋白表达水平均显著降低（均P<0.01）。与si-miR-NC组相比，si-miR-17-5p组SKM-1细胞增殖能力显著降低、凋亡率显著升高，细胞中C-caspase-3和C-caspase-9表达显著升高（均P<0.01）。miR-17-5p明显抑制野生型SETD2细胞的荧光素酶活性（P<0.01），并负向调控SETD2的表达。过表达SETD2可显著抑制SKM-1细胞的增殖并促进细胞凋亡，同时干扰SETD2表达则可部分逆转干扰miR-17-5p对SKM-1细胞的增殖抑制和凋亡促进作用。结论：MDS骨髓中miR-17-5p呈高表达，干扰miR-17-5p可抑制SKM-1细胞增殖并促进细胞凋亡，其机制与miR-17-5p靶向负调控SETD2的表达有关。

circ_0005379 inhibits the progression of oral squamous cell carcinoma by regulating the miR-17-5p/acyl-CoA oxidase 1 axis / 华西口腔医学杂志

OBJECTIVES@#To investigate the effects of circ_0005379 on the proliferation, apoptosis, migration, and invasion of oral squamous cell carcinoma (OSCC) cells and its mechanism.@*METHODS@#Real-time quantitative polymerase chain reaction (RT-qPCR) was used to detect the expression levels of circ_0005379 and miR-17-5p in OSCC tissues and SCC15 cell lines. Western blot was used to detect the expression levels of acyl-CoA oxidase 1 (ACOX1). The circ_0005379 overexpression vector was transfected into SCC15 cells. Methyl thiazolyl tetrazolium blue staining, flow cytometry, Transwell, and Western blot were used to detect the effects of circ_0005379 overexpression on the proliferation, apoptosis, migration, and invasion of SCC15 cells and the expression of E-cadherin, β-catenin, and Snail proteins. Dual luciferase reporter assay and RNA immunoprecipitation were used to examine the regulation of circ_0005379, miR-17-5p, miR-17-5p, and ACOX1 in SCC15 cells. A nude mouse xenograft model of SCC15 cells stably overexpressing circ_0005379 was established, and the effect of circ_0005379 overexpression on the growth of xenografts in nude mice was observed.@*RESULTS@#Compared with adjacent cancer tissues, the expression levels of circ_0005379 and ACOX1 proteins in OSCC tissues were decreased (@*CONCLUSIONS@#circ_0005379 may inhibit the proliferation, migration, and invasion of OSCC cells by downregulating the expression of miR-17-5p and upregulating ACOX1, which promote apoptosis and inhibit tumor growth

Expression of miR-17-5p in gastrointestinal stromal tumor tissues and its effect on proliferation and apoptosis of GIST882 cells / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探讨miR-17-5p在胃肠道间质瘤（gastrointestinal stromal tumor，GIST）组织中的表达及其对GIST882细胞增殖与凋亡的影响。方法：选取2019年5月至2020年5月广西医科大学第四附属医院胃肠外科手术切除的20例GIST患者的瘤组织及相应的瘤旁组织标本，以及GIST882细胞和人正常肠道上皮细胞HIEC为研究对象。荧光PCR-毛细管电泳测序法检测GIST标本中KIT基因突变情况。分别将miR-17-5p mimic和pc-KIT转染至GIST882细胞中。双荧光素酶报告基因实验验证miR-17-5p与KIT的靶向关系。qPCR和WB法检测GIST组织和细胞中miR-17-5p、KIT mRNA及蛋白的表达，CCK-8法、流式细胞术检测GIST882细胞的增殖、凋亡及细胞周期进程。结果：20例GIST组织中有15例患者发生KIT基因突变。与瘤旁组织比较，GIST组织中miR-17-5p表达水平显著降低、KIT mRNA表达水平显著升高（均P<0.01）；与HIEC细胞比较，GIST882细胞中miR-17-5p表达显著降低、KIT mRNA和蛋白表达显著升高（均P<0.01）。过表达miR-17-5p可显著降低GIST882细胞的增殖能力（P<0.01）、提高细胞凋亡率（P<0.05）、sub-G1期和S期细胞比例显著增加（均P<0.05）、而G0/G1期的细胞比例显著减少（P<0.05），同时KIT蛋白表达水平明显降低（P<0.01）。双荧光素酶报告基因实验证实KIT是miR-17-5p的下游靶基因。同时过表达miR-17-5p和KIT对GIST882细胞的增殖、细胞周期进程和凋亡水平未产生明显影响。结论：过表达miR-17-5p可显著抑制GIST882细胞的增殖并诱导细胞凋亡，同时下调KIT蛋白的表达，miR-17-5p可能是治疗GIST的潜在靶标。

miR-17-5p mitigates endometriosis by directly regulating VEGFA

Endometriosis is a common disease in women, which impairs the quality of life in patients. Recently, accumulating evidences reported that miRNAs play an essential role in diagnosis and treatment of endometriosis.However, the regulatory mechanism of miRNAs has not been fully explored. The expression of miR-17-5p andVEGFA was detected using qRT-PCR. The protein level of VEGFA was measured via Western blot. Cellproliferation was determined by CCK-8 assay. Cell migration and invasion were measured via transwell assay.The relationship of miR-17-5p and VEGFA were verified via luciferase reporter assay. Then miR-17-5p wasremarkably down-regulated in endometriosis tissues, serums and cells, and overexpression of miR-17-5pinhibited cell proliferation, migration and invasion in endometriosis. Results showed that VEGFA was significantly up-regulated in endometriosis tissues and cells and acted as a target of miR-17-5p. Moreover, miR17-5p negatively regulated VEGFA expression in endometriosis. Otherwise, up-regulation of VEGFAimproved cell proliferative, migrated and invasive ability in ECSCs with transfection of miR-17-5p mimicsgroup. Our data showed miR-17-5p inhibits cell proliferation, migration and invasion in endometriosis bydirectly repressing VEGFA expression.

Molecular mechanism of mir-17-5p regulation of hypoxia inducible factor-1α mediated adipocyte differentiation and angiogenesis / 中国组织工程研究

BACKGROUND: miR-17-5p can regulate the differentiation of adipocytes, but its action mechanism is not clear. Hypoxia inducible factor-1α can promote vascular endothelial growth factor gene transcription and promote angiogenesis, and has a regulatory effect on adipocyte formation. However, the specific mechanism of hypoxia inducible factor-1α in regulating adipocyte differentiation and angiogenesis is not clear. OBJECTIVE: To investigate the molecular mechanism of miR-17-5p regulation of hypoxia inducible factor-1α mediated adipocyte differentiation and angiogenesis. METHODS: miR-17-5p expression level and adipocyte differentiation and the expression of angiogenesis markers in mature adipocytes were verified by RT-qPCR. The adipocyte differentiation and angiogenesis markers expression of adipocyte transfecting with miR-17-5p inhibitor, pri-miR-17-5p mimic and hypoxia inducible factor-1α knockdown vector were determined by western blot assay. The proliferation of adipocytes was detected by Oil Red O staining assay and MTT assay after transfection with miR-17-5p inhibitor, pri-miR-17-5p mimic and hypoxia inducible factor-1α knockdown vector. The predicted relationship between miR-17-5p and hypoxia inducible factor-1α was further verified by EGFP report gene assay. RESULTS AND CONCLUSION: (1) Compared to immature adipocyte, miR-17-5p was highly expressed in mature adipocyte (P < 0.05); overexpression of miR-17-5p increased the adipocyte proliferation (P < 0.05), and upregulated adipocyte differentiation and angiogenesis markers level (P < 0.05). (2) The miR-17-5p directly targeted with hypoxia inducible factor-1α 3′UTR; knockdown hypoxia inducible factor-1α inhibited the adipocyte proliferation (P < 0.05), and decreased adipocyte differentiation and angiogenesis markers level (P < 0.001). (3) It is concluded that miR-17-5p regulation of hypoxia inducible factor-1α mediated adipocyte differentiation and angiogenesis.

miR-17-5p regulates proliferation, invasion, migration and apoptosis of nasopharyngeal carcinoma CNE2 cells by down-regulating BRMS1Lexpression / 中国肿瘤生物治疗杂志

@# Objective: To explore the molecular mechanism of miR-17-5p regulating the proliferation and invasion of nasopharyngeal carcinoma cells by regulating the expression of breast cancer metastasis suppressor 1 like (BRMS1-like or BRMS1L) gene. Methods:A total of 40 cases of nasopharyngeal carcinoma tissues and corresponding paracancerous tissues resected from nasopharyngeal carcinoma patients, who were admitted to the General Hospital of Pingdingshan Shenma Medical Group during January 2014 to December 2017, were included in this study; in addition, nasopharyngeal carcinoma cell lines CNE 2, HONE 1, C666-1 and nasopharyngeal immortalized epithelial cell line NP69 were also collected for this study. The expression of miR-17-5p in nasopharyngeal carcinoma tissues and cell lines was detected by qPCR. The targeted relationship between BRMS1L and miR-17-5p was predicted by the StarBase and verified by the Dual luciferase reporter gene assay. Effects of transfection of miR-17-5p mimics and inhibitors on the expression of BRMS1Lin CNE2 cells were detected by WB assay. CCK-8, Transwell and Flow cytometry were used to detect the effects of miR-17-5p/BRMS1L axis on the proliferation, migration, invasion and apoptosis of CNE 2 cells. Results: miR-17-5p was highly expressed in nasopharyngeal carcinoma tissues and cell lines (P<0.05 or P<0.01). Knockdown of miR-17-5p significantly inhibited proliferation, invasion and migration of CNE2 cells but promoted apoptosis (P<0.05 or P<0.01); miR-17-5p targeted BRMS1Land down-regulated its expression. Over-expression of BRMS1Lsignificantly inhibited the proliferation, invasion and migration of CNE2 cells but promoted apoptosis (all P<0.01); while simultaneous over-expression of miR-17-5p and BRMS1L reversed the above effects (all P<0.01). Conclusion: miR-17-5p promoted proliferation, invasion, migration and inhibited apoptosis of CNE 2 cells by down-regulating the expression of BRMS1L.

lncRNA HCG18 regulates the proliferation and metastasis of non-small cell lung cancer cells through targeting miR-17-5p/HMGA2 axis / 中国肿瘤生物治疗杂志

@# Objective: To investigate the mechanism of lncRNA HCG18/miR-17-5p/HMGA2 axis regulating the proliferation and metastasis of non-small cell lung cancer (NSCLC) cells. Methods: Sixty-two pairs of NSCLC tissues and corresponding para-cancerous tissues collected at Central Hospital of Chengde City from June 2017 to June 2018 were used for this study; in addition, NSCLC cell lines (A549, NCI-H1299, H1650, NCI-H460) and human lung epithelial BEAS-B cells were also collected. mRNA expression levels of HCG18, miR-17-5p and high-mobility group AT-hook 2 (HMGA2) in NSCLC tissues and cell lines were measured by quantitative Real-time polymerase chain reaction (qPCR). Si-HCG18, miR-17-5p, miR-17-5p+HCG18 or pcDNA3.1-HMGA2 were transfected into A549 cells and NCI-H460 cells; CCK-8 assay was used to detect the proliferation of transfected cells, Transwell assay was used to detect the migration and invasion ability of cells, and Wb was used to analyze the expressions of HMGA2 and EMT associated proteins (E-cadherin, N-cadherin and vimentin). The target relationships between HCG18 and miR-17-5p, or between miR-17-5p and HMGA2 were confirmed by dual luciferase reporter gene assay. Mice A549 cell xenograft model with HCG18 knockdown was constructed, and the growth of transplanted tumor was observed. Results: lncRNA HCG18 was highly expressed in NSCLC tissues and cells (all P<0.01); HCG18 level was significantly increased in patients at late stage or with lymphnode metastasis; and high HCG18 level was correlated with poor prognosis and low survival rates of NSCLC patients (all P<0.01). Knockdown of HCG18 significantly inhibited NSCLC cell proliferation, migration and invasion (all P<0.01), up-regulated E-cadherin expression but suppressed N-cadherin and vimentin expression (all P<0.01), and the volume of xenograft was obviously decreased (P<0.05). Dual luciferase reporter gene assay confirmed the relationship between HCG18 and miR-17-5p as well as miR-17-5p and HMGA2. miR-17-5p transfection significantly inhibited NSCLC cell proliferation, migration and invasion (all P<0.01), and up-regulated E-cadherin expression, reversely suppressed N-cadherin and vimentin expression (all P<0.01); however, miR-17-5p + HCG18 transfection reversed the effect of miR-17-5p on NSCLCcells.Conclusion:HCG18promotes the proliferationandmigrationofNSCLCcellsthrough regulating miR-17-5p/HMGA2 axis.

Study on the mechanism of miR-17-5p in atherosclerotic mice / 安徽医科大学学报

Objective To investigate the effect of miR-17-5p on the inflammatory response and oxidative stress in the development of atherosclerosis. Methods ApoE-/ - mice were fed with high fat diet for 12 weeks to con-struct atherosclerosis (AS) model, and then miR-17-5p antagonist was injected to the caudal vein of mice subse-quently. HE staining was used to observe the pathological changes of the aorta. The apoptosis of aorta cells was de-tected by TUNEL staining. Western blot was used to detect the expression of apoptosis-related proteins and the ex-pression of inflammatory cytokines weas detected by ELISA. Oxidative stress were assessed by malondialdehyde ( MDA) levels and the activity of superoxide dismutase ( SOD) and myeloperoxidase ( MPO) . Results Inhibition of miR-17-5p alleviated the aortic lesion and apoptosis in AS mice, reduced the expression of Bax, cleaved-caspase-3 and cleaved-PARP and up-regulated the expression of Bcl-2. After inhibition of miR-17-5p, the levels of TNF-α and IL-6 in AS mice decreased, and the level of IL-10 increased. Inhibition of miR-17-5p also resulted in decrease in MDA and MPO activity, but increase in SOD activity. Conclusion Inhibition of miR-17-5p can atten-uate atherosclerotic lesions by reducing the inflammatory response and oxidative stress in the aorta of atherosclerotic mice. MiR-17-5p may be a potential target for the treatment of atherosclerosis.

Detection and analysis of different genotypes of cervical cancer-associated miR-17-5p target loci / 新乡医学院学报

Objective To detect the different genotypes of cervical cancer-associated miR-17-5p target loci,and to analyze the influence of target site polymorphism on the risk of cervical cancer.Methods A total of 250 cervical cancer patients and 250 healthy females were selected in Hanzhong Central Hospital from June 2014 to June 2016.The blood samples were collected from the subjects.The genotypes of the three target loci rs3741216,rs217727 and rs2839702 in miR-17-5p were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP).The associativity between genetic polymorphisms and the risk of cervical cancer was calculated by SPSS 21.0 online software.Results The three candidate SNP loci fitted the Hardy-Weinberg equilibrium law.In the allele model,the rs217727 locus on the H19 gene significantly increased the risk of cervical cancer [OR =1.55,95％ CI(1.21,2.32),P =0.001].Genetic model analysis showed that rs217727 locus in the best model (dominant model),the risk of cervical cancer in the individuals carrying A/G and A/A genotypes increased significantly,and the risk of cervical cancer in the individuals carrying A/G and A/A genotypes was 1.65 times higher than that in the individuals carrying G/G genotype [OR =1.65,95％ CI (1.14,2.28),P =0.006].Conclusion The polymorphism of miR-17-5p target site rs217727 is associated with the risk of cervical cancer,the risk of cervical cancer in the individuals carrying A/G and A/A genotypes is significantly increased.

Research progress on miR-17-5p in tumors / 中国肿瘤临床

As an important gene regulatory molecule, microRNA is closely related with various human diseases. Numerous studies have confirmed that microRNAs function as tumor suppressors or oncogenes in various tumor types. Therefore, microRNA investiga-tion has become a new direction in oncology research. As a member of the miR-17 to-92 cluster, miR-17-5p has been the focus of re-search recently. MicroRNA is involved in many aspects of diseases, such as diabetes mellitus, endometriosis, and a variety of tumors. In this review, the basic characteristics and roles of miR-17-5p in tumors are elaborated.