LncRNA NR2F2-AS1 inhibits the progression of oral squamous cell carcinoma by mediating the miR-32-5p/SEMA3A axis.

Previous studies have supported a tumor-suppressive role of semaphorin 3A (SEMA3A) in several tumors including oral squamous cell carcinoma (OSCC). However, in-depth characterization of the role of SEMA3A in OSCC and the underlying molecular mechanisms is lacking. Gene and protein expressions were detected using quantitative real-time PCR, western blot assay, and immunohistochemistry. OSCC cell metastasis was evaluated using Transwell and angiogenesis of human umbilical vein endothelial cells (HUVECs) was determined using tube formation assay. The interactions among molecules were predicted using bioinformatics analysis and validated using luciferase activity experiment and RNA immunoprecipitation assay. Pulmonary metastasis was evaluated using hematoxylin and eosin staining after constructing a lung metastasis tumor model in mice. SEMA3A expression was decreased in OSCC cells and its overexpression led to suppression of epithelial-mesenchymal transition (EMT), migration, and invasion of OSCC cells and angiogenesis of HUVECs. miR-32-5p was identified as an upstream molecule of SEMA3A and long non-coding RNA NR2F2 antisense RNA 1 (NR2F2-AS1) was validated as an upstream gene of miR-32-5p. Further experiments revealed that the inhibitory effects of NR2F2-AS1 overexpression on EMT, migration, invasion of OSCC cells, and angiogenesis of HUVECs as well as tumor growth and metastasis in mice were mediated via the miR-32-5p/SEMA3A axis. To conclude, NR2F2-AS1 may attenuate OSCC cell metastasis and angiogenesis of HUVECs and suppress tumor growth and metastasis in mice via the miR-32-5p/SEMA3A axis.

Sodium Houttuyfonate Ameliorates DSS-induced Colitis Aggravated by Candida albicans through Dectin-1/NF-κB/miR-32-5p/NFKBIZ Axis Based on Intestinal microRNA Profiling.

Our previous research indicated that Sodium houttuyfonate (SH) can effectively ameliorate dextran sulfate sodium (DSS)-induced colitis exacerbated by Candida albicans. However, the underlying protective mechanism of SH remains unclear. Therefore, in this study, a mice colitis model was infected with C. albicans, and the total colonic miRNAs were assessed. Furthermore, the differentially expressed miRNAs were enriched, clustered, and analyzed. Moreover, based on the dual luciferase analysis of NFKBIZ modulation by miR-32-5p, the in vitro and in vivo therapeutic effects of SH on inflammatory response, fungal burden, oxidative stress, and apoptosis were assessed at transcriptional and translational levels in the presence of agonist and antagonist. A total of 1157 miRNAs were identified, 84 of which were differentially expressed. Furthermore, qRT-PCR validated that SH treatment improved 17 differentially expressed miRNAs with > fourfold upregulation or > sixfold downregulation. Similar to most differentially altered miRNA, C. albicans significantly increased Dectin-1, NF-κB, TNF-α, IL-1ß, IL-17A, and decreased miR-32-5p which negatively targeted NFKBIZ. In addition, SH treatment reduced inflammatory response and fungal burden in a colitis model with C. albicans infection. Further analyses indicated that in C. albicans infected Caco2 cells, SH inhibited fungal growth, oxidative stress, and apoptosis by increasing Dectin-1, NF-κB, NFKBIZ, TNF-α, IL-1ß, IL-17A, and decreasing miR-32-5p. Therefore, SH can ameliorate the severity of colitis aggravated by C. albicans via the Dectin-1/NF-κB/miR-32-5p/NFKBIZ axis.

A CEBPB/miR-32-5p/GATA6 axis promotes vascular calcification in type 2 diabetes.

Vascular calcification in diabetes patients is a major independent risk factor for developing diabetic cardiovascular complications. However, the mechanisms by which diabetes leads to vascular calcification are complex and not yet fully understood. Our previous study revealed that miR-32-5p is a potential new diagnostic marker for coronary artery calcification. In this study, we found that miR-32-5p levels were significantly greater in the plasma of type 2 diabetes patients with coronary artery calcification and were positively correlated with the coronary artery calcification score. In type 2 diabetic mice, miR-32-5p levels were also elevated in the aorta, and knockout of miR-32-5p inhibited the osteogenic differentiation of vascular smooth muscle cells in vivo. Furthermore, overexpression of miR-32-5p promoted vascular smooth muscle cell calcification, while antagonism of miR-32-5p inhibited vascular smooth muscle cell calcification under high-glucose conditions. GATA binding protein 6 (GATA6) was identified as the key target gene through which miR-32-5p promotes vascular smooth muscle cell calcification. Overexpression of GATA6 antagonized the effects of miR-32-5p on vascular calcification. Additionally, high glucose levels were shown to induce the upregulation of miR-32-5p by activating CCAAT/enhancer binding protein beta (CEBPB). These results suggest that miR-32-5p is an important procalcification factor in vascular calcification associated with type 2 diabetes and identify the CEBPB/miR-32-5p/GATA6 axis as a potential biomarker and therapeutic target for preventing and treating vascular calcification in type 2 diabetes.

miR-32-5p induces hepatic steatosis and hyperlipidemia by triggering de novo lipogenesis.

BACKGROUND AND OBJECTIVES: MicroRNA-dependent regulation of hepatic lipid metabolism has been recognized recently as a key pathological mechanism contributing to the development of NAFLD. However, whether miR-32-5p (miR-32) plays a role in lipid metabolism or contributes to NAFLD remains unclear. METHODS AND RESULTS: A marked increase in miR-32 expression was observed in liver samples from patients and mice with NAFLD, as well as in palmitate-induced hepatocytes. Hepatocyte-specific miR-32 knockout (miR-32-HKO) dramatically ameliorated hepatic steatosis and metabolic disorders in high-fat diet-fed mice. Conversely, hepatic miR-32 overexpression markedly exacerbated the progression of these abnormalities. Further, combinational analysis of transcriptomics and lipidomics suggested that miR-32 was a key trigger for de novo lipogenesis in the liver. Mechanistically, RNA sequencing, luciferase assay and adenovirus-mediated downstream gene rescue assay demonstrated that miR-32 directly bound to insulin-induced gene 1 (INSIG1) and subsequently activated sterol regulatory element binding protein-mediated lipogenic gene programs, thereby promoting hepatic lipid accumulation and metabolic disorders. Notably, pharmacological administration of miR-32 antagonist significantly inhibited palmitate-induced triglyceride deposition in hepatocytes and markedly mitigated hepatic steatosis and metabolic abnormalities in obesity-associated NAFLD mice. CONCLUSION: miR-32 is an important checkpoint for lipogenesis in the liver, and targeting miR-32 could be a promising therapeutic approach for NAFLD treatment.

Long non-coding RNA SNHG17 contributes to the progression of pancreatic adenocarcinoma by modulating miR-32-5p/EZH2/STAT3 signaling.

BACKGROUND: Adenocarcinoma of the pancreas (PAAD) is one of the most malignant tumors in the gastrointestinal tract. Long-chain noncoding RNAs (lncRNAs) are non-coding RNAs that are expressed in a variety of cancers. The purpose of this study is to study the expression, biology functions, and molecular mechanism of lncRNA SNHG17 in PAAD. METHODS: In this study, qRT-PCR was used to measure the relative expression of SNHG17 and miR-32-5p in PAAD. In order to investigate the effect of SNHG17 and miR-32-5p on the proliferation, migration and invasion of PAAD cells, we performed a variety of tests including CCK-8, colony formation, scratch and transwell assays. Furthermore, SNHG17 and miR-32-5p interactions were confirmed by a luciferase reporter gene test. RESULTS: Our results indicate that the expression of SNHG17 in PAAD is elevated, and in vitro studies have shown that SNHG17 enhances the proliferation of PAAD cells, Mechanistically, it has been shown that SNHG17 can direct target miR-32-5p in PAAD cells, thus promoting the proliferation of PAAD cells, migration, and invasion. Furthermore, SNHG17 has been found to activate EZH2/STAT3 signaling pathway through miR-32-5p in PAAD cells. CONCLUSION: Our results show that SNHG17 plays a key role in the progression of PAAD by activating STAT3 signaling via regulation of miR-32-5p and EZH2.Identifying these new regulatory pathways may shed light on the underlying mechanism of PAAD and offer a potential therapeutic target for this fatal disease.

Circ_0078607 inhibits the progression of ovarian cancer via regulating the miR-32-5p/SIK1 network.

BACKGROUND: Circular RNA (circRNA) has been shown to be involved in the regulation of human disease progression, including ovarian cancer (OC). Circ_0078607 was found to participate in OC progression. But its function and mechanism in OC deserve further exploration. METHODS: The expression levels of circ_0078607, salt-inducible kinase 1 (SIK1) and microRNA (miR)-32-5p were examined by qRT-PCR. And the protein expression levels of SIK1, metastasis marker and apoptosis marker were determined using western blot analysis. EDU staining, colony formation assay, transwell assay and flow cytometry were used to detect the proliferation, migration, invasion and apoptosis of cells. Moreover, dual-luciferase reporter assay was employed to verify the interaction between miR-32-5p and circ_0078607 or SIK1. Xenograft models were constructed to perform in vivo experiments. RESULTS: Circ_0078607 and SIK1 were downregulated in OC tissues and cells. Overexpressed circ_0078607 and SIK1 could inhibit OC cell proliferation, migration, invasion, and promote apoptosis. MiR-32-5p could be sponged by circ_0078607, and its overexpression could reverse the suppressive effect of circ_0078607 on OC progression. Furthermore, SIK1 was a target of miR-32-5p, and circ_0078607 could regulate SIK1 by sponging miR-32-5p. The inhibitory effect of circ_0078607 on OC progression also could be reversed by SIK1 silencing. In vivo experiments showed that circ_0078607 reduced OC tumorigenesis by regulating the miR-32-5p/SIK1 axis. CONCLUSION: Circ_0078607 could serve as a sponge of miR-32-5p to regulate SIK1 expression, thereby inhibiting OC progression.

MiR-32-5p promoted epithelial-to-mesenchymal transition of oral squamous cell carcinoma cells via regulating the KLF2/CXCR4 pathway.

Oral squamous cell carcinoma (OSCC) is one of the most common carcinomas of the oral cavity. However, the regulatory mechanisms on miR-32-5p remain poorly understood in OSCC. The expression of miR-32-5p, Krüppel-like factor 2 (KLF2), C-X-C motif chemokine receptor 4 (CXCR4), and epithelial-to-mesenchymal transition (EMT)-related proteins (E-cadherin, Vimentin, N-cadherin, and Snail) were evaluated were assessed using RT-qPCR and Western blot. 3-(4, 5-Dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide assay, wound healing assay, and transwell assay were employed to detect cell proliferation, migration, and invasion of OSCC cells. Finally, dual-luciferase reporter assay was performed to verify the binding relationship between KLF2 and miR-32-5p. MiR-32-5p was highly expressed while KLF2 was lowly expressed in OSCC cells, and miR-32-5p knockdown or KLF2 overexpression could markedly reduce cell proliferation, migration, invasion, and EMT of OSCC cells. What is more, KLF2 was the target of miR-32-5p, and knockdown of KLF2 abolished the inhibitory effect of miR-32-5p inhibitor on progression of OSCC. Finally, CXCR4 expression was negatively regulated by KLF2, and inhibition of CXCR4 obviously alleviated the biological effects of si-KLF2 on the progression of OSCC. MiR-32-5p could enhance cell proliferation, migration, invasion, and EMT of OSCC cells, and the discovery of miR-32-5p/KLF2/CXCR4 axis might provide potential therapeutic targets for OSCC.

miR-32-5p inhibits the malignant biological behaviors and the growth of nude mice xenograft of pancreatic cancer PANC-1 cells by targeted down-regulation of EZH2 expression / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：探究miR-32-5p通过靶向zeste基因增强子人类同源物2（EZH2）对胰腺癌PANC-1细胞恶性生物学行为的影响及其分子机制。方法：利用GEPIA数据库分析胰腺癌组织中EZH2的表达水平及其与患者的预后生存期的关系，并分析miR-32-5p表达与患者临床病理因素的关系。qPCR法检测正常胰腺HPDE6-C7细胞和胰腺癌PANC-1、AsPC-1、SW1990细胞中miR-32-5p和EZH2 mRNA的表达，通过LipofectamineTM 2000将miR-NC、miR-32-5p mimic、miR-32-5p inhibitor、pcDNA-NC、pcDNA EZH2质粒分别转染PANC-1细胞，其分为对照组（不转染）、 miR-NC组（转染miR-NC）、 miR-32-5p mimic组（转染miR-32-5p mimic）、Anti-miR-32-5p组（转染miR-32-5p inhibitor）、miR-NC+pcDNA-NC组（转染miR-NC+pcDNA-NC）、miR-NC+pcDNA EZH2组（转染miR-NC+pcDNA EZH2）、miR-32-5p mimic+pcDNA-NC组（转染miR-32-5p mimic+pcDNA-NC）、miR-32-5p mimic+pcDNA EZH2组（转染miR-32-5p mimic+pcDNA EZH2）。双荧光素酶报告基因实验验证miR-32-5p与EZH2的靶向关系；MTT法及克隆形成实验检测各组细胞的增殖能力，划痕愈合实验检测各组细胞的迁移能力；Transwell小室实验检测各组细胞的侵袭能力，WB法检测各组细胞EZH2、E-cadherin、N-cadherin的表达；裸鼠成瘤实验检测转染后各组PANC-1细胞移植瘤的生长情况，免疫组化染色法观察移植瘤组织中Ki67和MMP-2的表达。结果：GEPIA数据库显示胰腺癌组织中EZH2的表达水平高于癌旁组织，患者预后生存期与EZH2的表达水平呈负相关（均P<0.05）；miR-32-5p表达水平与胰腺癌神经浸润、肿瘤分化程度、TNM分期、淋巴结转移有明显的关联（均P<0.05）；与HPDE6-C7细胞相比，PANC-1、AsPC-1、SW1990细胞中miR-32-5p呈高表达、EZH2 mRNA呈低表达、miR-32-5p表达水平与EZH2表达水平呈负相关（均P<0.05）。miR-32-5p靶向EZH2且抑制其表达（均P<0.05）；过表达miR-32-5p能够下调Ki67、MMP-2、N-cadherin表达水平、上调E-cadherin表达水平，抑制PANC-1细胞的增殖、迁移和侵袭能力，抑制移植瘤质量增加（均P<0.05）。结论：miR-32-5p能够靶向调控EZH2从而影响胰腺癌PANC-1细胞的增殖、迁移、侵袭、EMT进程及裸鼠体内移植瘤的生长。

GATA1-Activated HNF1A-AS1 Facilitates the Progression of Triple-Negative Breast Cancer via Sponging miR-32-5p to Upregulate RNF38.

BACKGROUND: Triple-negative breast cancer (TNBC) is a highly invasive subtype of breast cancer with a high mortality rate. Recently, long non-coding RNAs (lncRNAs) are confirmed to modulate the progression of assorted cancers, including TNBC. However, the functions of lncRNA HNF1 homeobox A antisense RNA 1 (HNF1A-AS1) in TNBC are still unclear. AIM: We aimed to investigate the function and mechanism of HNF1A-AS1 in TNBC. METHODS: The expression of genes in TNBC cells was tested by quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. In vitro loss-of-function assays and in vivo xenograft experiments were conducted for evaluating the impact of HNF1A-AS1 on TNBC progression. RNA pull-down, luciferase reporter and RNA immunoprecipitation (RIP) assays were utilized for assessing the correlations between molecules. RESULTS: We discovered that HNF1A-AS1 was highly expressed in TNBC tissues and cells. Knockdown of HNF1A-AS1 restrained cell proliferation but accelerated cell apoptosis. Besides, GATA-binding protein 1 (GATA1) activated HNF1A-AS1 transcription in TNBC. MicroRNA-32-5p (miR-32-5p) was slowly expressed in TNBC cells and sponged by HNF1A-AS1, and its overexpression hinders TNBC cell growth. Ring finger protein 38 (RNF38) was verified as the target of miR-32-5p, and HNF1A-AS1 was a competing endogenous RNA (ceRNA) of RNF38 through sponging miR-32-5p. Rescue experiments indicated that upregulation of RNF38 reversed the inhibited impacts of silencing HNF1A-AS1 on TNBC cell growth. CONCLUSION: GATA1-activated HNF1A-AS1 facilitated TNBC progression via miR-32-5p/RNF38 axis. The findings may provide new roads for developing targeted therapies of TNBC.

CUL4B renders breast cancer cells tamoxifen-resistant via miR-32-5p/ER-α36 axis.

Tamoxifen (TAM) resistance is a significant clinical challenge in endocrine therapies for estrogen receptor (ER)-positive breast cancer patients. Cullin 4B (CUL4B), which acts as a scaffold protein in CUL4B-RING ubiquitin ligase complexes (CRL4B), is frequently overexpressed in cancer and represses tumor suppressors through diverse epigenetic mechanisms. However, the role and the underlying mechanisms of CUL4B in regulating drug resistance remain unknown. Here, we showed that CUL4B promotes TAM resistance in breast cancer cells through a miR-32-5p/ER-α36 axis. We found that upregulation of CUL4B correlated with decreased TAM sensitivity of breast cancer cells, and knockdown of CUL4B or expression of a dominant-negative CUL4B mutant restored the response to TAM in TAM-resistant MCF7-TAMR and T47D-TAMR cells. Mechanistically, we demonstrated that CUL4B renders breast cancer cells TAM-resistant by upregulating ER-α36 expression, which was mediated by downregulation of miR-32-5p. We further showed that CRL4B epigenetically represses the transcription of miR-32-5p by catalyzing monoubiquitination at H2AK119 and coordinating with PRC2 and HDAC complexes to promote trimethylation at H3K27 at the promoter of miR-32-5p. Pharmacologic or genetic inhibition of CRL4B/PRC2/HDAC complexes significantly increased TAM sensitivity in breast cancer cells in vitro and in vivo. Taken together, our findings thus establish a critical role for the CUL4B-miR-32-5p-ER-α36 axis in the regulation of TAM resistance and have important therapeutic implications for combined application of TAM and the inhibitors of CRL4B/PRC2/HDAC complex in breast cancer treatment. © 2021 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

miR-32-5p regulates the biological behaviors of breast cancer MDA-MB-231 cells by targeting the expression of Dickkopf-related protein 3 / 中国肿瘤生物治疗杂志

@#[摘 要] 目的：通过生物信息学手段筛选乳腺癌中差异表达的关键miRNA及其靶基因，干预其在乳腺癌细胞中的表达并观察对乳腺癌细胞功能的影响。方法：利用GEO数据库筛选在乳腺癌中差异表达的miRNA，ENCORI数据库验证差异miRNA的表达，以选定最显著的差异表达miRNA为研究对象；利用Starbase、miRDB和miRWalk数据库预测miR-32-5p的靶基因，利用DAVID数据库对靶基因进行GO分析和KEGG分析，利用String数据库联合Cytoscape3.6.2软件进行PPI网络分析及核心基因的筛选，从核心基因中选择相互联系紧密“度值”最显著的Dickkopf相关蛋白3（DDK3）基因进行后续实验。qPCR检测miR-32-5p在人正常乳腺细胞 MCF10A和人乳腺癌细胞MCF7、MDA-MB-231、MDA-MB-453细胞中的表达。向MDA-MB-231细胞中转染miR-32-5p mimic、miR-32-5p inhibitor及各自的对照（NC）序列，分别用CCK-8法、流式细胞术和Transwell实验检测过表达或抑制miR-32-5p对细胞增殖、凋亡和侵袭的影响。结果：从GEO数据库中获取的两个数据集共识别出两个差异miRNA，ENCORI数据库验证差异miRNA的表达发现miR-32-5p的表达水平与GEO数据库的结果一致，故选择其进行研究；预测得到198个miR-32-5p潜在的靶基因并鉴定出10个核心基因（DKK3、WNT2B、SFRP5、SFRP2、SFRP1、LRP6、WNT6、KREMEN1、NEDD4L、TRIP12），其中DKK3的度值最大可能在乳腺癌中较为重要，于是选择miR-32-5p/DKK3轴进行后续研究。miR-32-5p在3种乳腺癌细胞中的表达水平显著高于正常乳腺细胞（均P<0.01），其中以MDA-MB-231细胞中表达最高。双荧光素酶基因报告实验验证了miR-32-5p与DKK3基因的靶向结合及其对后者表达的负向调控。转染miR-32-5p mimic、miR-32-5p inhibitor后成功提高或抑制了MDA-MB-231细胞中miR-32-5p的表达。与对照组相比，过表达miR-32-5p可抑制MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭（P<0.05或P<0.01），敲低miR-32-5p则起相反的作用（均P<0.01）。结论：miR-32-5p/DKK3轴可能是影响乳腺癌发生发展的关键通路，过表达miR-32-5p能够抑制乳腺癌MDA-MB-231细胞的凋亡而促进细胞增殖和侵袭。

miR-32-5p Regulates Lipid Accumulation in Intramuscular Fat of Erhualian Pigs by Suppressing KLF3.

Intramuscular fat (IMF) and subcutaneous fat (SCF) are important traits affecting the economics of the pork industry, in which less SCF and more IMF content is desirable. However, the mechanisms that regulate IMF and SCF content are not clear yet. In this study, we demonstrate that KLF3 (Krüppel-like factor 3) was negatively correlated with IMF content in the longissimus dorsi muscle of Erhualian pigs. In addition, the expression level of KLF3 was significantly higher in IMF than SCF. Overexpression and knockdown experiments revealed that KLF3 could suppress adipocyte differentiation in vitro by downregulating adipogenic markers, including PPARG, C/EBPA, and FABP4. Luciferase activity analysis proved that miR-32-5p was able to suppress KLF3. Notably, miR-32-5p level was negatively correlated to KLF3 mRNA level in both IMF and SCF tissues. The same relationship was proved in samples with different IMF content. Further studies showed that miR-32-5p could promote adipocyte differentiation via inhibiting KLF3. Our results suggest that the miR-32-5p-KLF3 pathway is involved in the regulation of differential fat deposition of IMF and SCF tissues.

MiR-32-5p knockdown inhibits epithelial to mesenchymal transition and renal fibrosis by targeting SMAD7 in diabetic nephropathy.

Diabetic nephropathy (DN) is primary cause of end-stage renal disease. A previous study has shown that miR-32-5p (miR-32) is highly expressed in kidney tissue during chronic allograft dysfunction with interstitial fibrosis and tubular atrophy. However, the role of miR-32-5p (miR-32) in DN is still unclear. In this study, streptozotocin-induced DN rat models and high glucose (HG)-incubated human kidney proximal tubular epithelial (HK-2) cells were established to investigate the role and underlying mechanisms of miR-32 in DN. Results of real-time PCR revealed that miR-32 levels were greatly increased in DN rats and HG-incubated HK-2 cells. Downregulation of miR-32 effectively relieved HG-induced autophagy suppression, fibrosis, epithelial-mesenchymal transition (EMT) and inflammation in HK-2 cells. Besides, miR-32 overexpression significantly down-regulated the expression of mothers against decapentaplegic homolog 7 (SMAD7), whereas knockdown of miR-32 markedly up-regulated the level of SMAD7. Dual-luciferase reporter gene assay confirmed that SMAD7 was a target of miR-32. Reintroduction of SMAD7 expression rescued miR-32-induced HK-2 cells autophagy suppression, EMT and renal fibrosis. Our findings indicate that miR-32 may play roles in the progression of EMT and fibrosis in DN.

Long Noncoding RNA HNF1A-AS1 Regulates Osteosarcoma Advancement Through Modulating the miR-32-5p/HMGB1 Axis.

Background: Osteosarcoma (OS) is a primary malignant tumor in children and adolescents. Long noncoding RNA HNF1A antisense RNA 1 (HNF1A-AS1) is connected with OS development. However, there are few reports on the role and mechanism of HNF1A-AS1 in OS. Materials and Methods: Quantitative real-time polymerase chain reaction (qRT-PCR) was employed to assess the expression of HNF1A-AS1, miR-32-5p, and high-mobility group protein B1 (HMGB1). Western blot analysis was performed to detect the protein level of HMGB1. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), colony formation, transwell, or flow cytometer assays were applied to determine the proliferation, migration, invasion, and apoptosis of OS cells. The interaction between HNF1A-AS1 and miR-32-5p or HMGB1 was predicted by the starBase database and confirmed by dual-luciferase reporter assay. Enzyme-linked immunosorbent assay was employed to analyze levels of HMGB1 in the OS cell supernatant. Results: HNF1A-AS1 and HMGB1 were upregulated, while miR-32-5p was downregulated, in OS tissues and cells. Functionally, HNF1A-AS1 depletion induced apoptosis and impeded proliferation, migration, and invasion of OS cells. Interestingly, HNF1A-AS1 bound to miR-32-5p to regulate the expression of HMGB1. Furthermore, miR-32-5p knockdown overturned the effects of HNF1A-AS1 knockdown on apoptosis, proliferation, migration, and invasion of OS cells. In addition, the effects of HNF1A-AS1 silencing on the malignant behaviors of OS cells were reserved by HMGB1 overexpression. In addition, HNF1A-AS1 regulated the HMGB1 level in the OS cell supernatant through the miR-32-5p/HMGB1 axis. Conclusion: Downregulation of HNF1A-AS1 blocked OS progression through the miR-32-5p/HMGB1 axis, which provides a possible target and prognostic biomarker for treatment of OS.

LncRNA Xist Contributes to Endogenous Neurological Repair After Chronic Compressive Spinal Cord Injury by Promoting Angiogenesis Through the miR-32-5p/Notch-1 Axis.

Endogenous repair after chronic compressive spinal cord injury (CCSCI) is of great clinical interest. Ischemia-hypoxia-induced angiogenesis has been proposed to play an important role during this repair process. Emerging evidence indicates that long non-coding RNAs (lncRNAs) are involved in the pathophysiological processes of various diseases. Here, we identified a lncRNA (Xist; X-inactive specific transcript) with upregulated expression in cervical spine lesions during endogenous neurological repair in CCSCI rats. Therapeutically, the introduction of Xist to rats increased neurological function in vivo as assayed using the Basso, Beattie, and Bresnahan (BBB) score and inclined plane test (IPT). We found that the introduction of Xist enhanced endogenous neurological repair by promoting angiogenesis and microvessel density after CCSCI, while depletion of Xist inhibited angiogenesis and cell sprouting and migration. Mechanistically, Xist promoted angiogenesis by sponging miR-32-5p and modulating Notch-1 expression both in vitro and in vivo. These findings suggest a role of the Xist/miR-32-5p/Notch-1 axis in endogenous repair and provide a potential molecular target for the treatment of ischemia-related central nervous system (CNS) diseases.

LncRNA WEE2-AS1 promotes proliferation and inhibits apoptosis in triple negative breast cancer cells via regulating miR-32-5p/TOB1 axis.

Triple negative breast cancer (TNBC) is a malignant breast cancer subtype with poor prognosis. Recent studies have revealed the critical roles of dysregulated long non-coding RNAs (lncRNAs) in many cancer types, including TNBC. LncRNA WEE2 antisense RNA 1 (WEE2-AS1) has been reported to be able to promote the progression of hepatocellular carcinoma, but the function of WEE2-AS1 in TNBC is still unknown. Therefore, in this study, we specifically researched the role of WEE2-AS1 and probed its molecular mechanism in TNBC cells. Our results showed that WEE2-AS1 was up-regulated in TNBC cell lines, and WEE2-AS1 knockdown could inhibit TNBC cell proliferation, promote apoptosis, and suppress migration and invasion. Further, we found that miR-32-5p was down-regulated in TNBC cells and could be sponged by WEE2-AS1. Moreover, miR-32-5p could target its downstream gene transducer of ERBB2, 1 (TOB1), which was highly expressed and could play the oncogenic role in TNBC cells. Through rescue assays, we proved that WEE2-AS1/miR-32-5p/TOB1 axis could modulate cancer progression in TNBC cells. In conclusion, our results demonstrated the oncogenic function of lncRNA WEE2-AS1 in TNBC cells, providing a novel insight into TNBC therapy.

miR32-5p promoted vascular smooth muscle cell calcification by upregulating TNFα in the microenvironment.

BACKGROUND: Vascular calcification is often associated with chronic inflammation and is a risk factor for brain arterial stiffness. Our previous results showed that miR32-5p was positively correlated with vascular smooth muscle cells (VSMC) calcification, but it is unclear whether miR32-5p promoted VSMC calcification by regulating inflammatory factor production. RESULTS: In this study, bioinformatics analysis was used to select tumour necrosis factor α (TNFα) as a candidate inflammatory factor associated with calcification. Moreover, alizarin red staining and qRT-PCR analysis revealed that TNFα produced by BV2 cells was the key promoting factor of VSMC calcification. Interestingly, the expression of TNFα was significantly increased at the mRNA and protein levels after miR32-5p mimic treatment but significantly decreased after miR32-5p antagomir treatment. To explore the mechanism of the regulation of TNFα expression by miR32-5p, bioinformatics analysis indicated that PIKfyve was a candidate target gene of miR32-5p, and luciferase assays verified that the expression of PIKfyve was significantly repressed by miR32-5p mimics. Importantly, rescue experiments showed that the expression of TNFα in BV2 cells treated with miR32-5p antagomir and the PIKfyve inhibitor YM201636 was significantly increased. CONCLUSIONS: The production of TNFα in microglia could be affected by miR32-5p targeting PIKfyve, and these results will be beneficial to reveal the mechanism of brain arterial calcification.

Mechanism of miR-32-5p targeting TOB1 gene in regulating radiosensitization, migration and invasion of colorectal cancer cells / 中华放射肿瘤学杂志

Objection@#To investigate the effect of miR-32-5p on the radiosensitivity, migration and invasion of colorectal cancer cells and the underlying mechanism.@*Methods@#Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured. The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC, anti-miR-32-5p, pcDNA, pcDNA-TOB1, anti-miR-32-5p+ si-NC and anti-miR-32-5p+ si-TOB1, respectively). The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot. The radiosensitivity of the transfected cells was determined by colony formation assay. The migration and invasion ability of the transfected cells were detected by Transwell assay. Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.@*Results@#Compared with human colonic epithelial cells, the expression of miR-32-5p was significantly up-regulated, whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P<0.05). Compared with the anti-miR-NC, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group. Compared with the pcDNA group, the quantity of cell migration and invasion was significantly decreased (both P<0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group. Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein. Compared with the anti-miR-32-5p+ si-NC group, the quantity of cell migration and invasion was significantly increased (both P<0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+ si-TOB1 group.@*Conclusions@#Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion. The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.

Mechanism of miR-32-5p targeting TOB1 gene in regulating radiosensitization, migration and invasion of colorectal cancer cells / 中华放射肿瘤学杂志

Objection To investigate the effect of miR-32-5p on the radiosensitivity,migration and invasion of colorectal cancer cells and the underlying mechanism.Methods Human colorectal cancer SW480 cells and normal colonic epithelial NCM460 cells were cultured.The colorectal cancer cells were divided into the non-transfected and transfected groups (transfected with anti-miR-NC,anti-miR-32-5p,pcDNA,pcDNA-TOB1,anti-miR-32-5p+si-NC and anti-miR-32-5p+si-TOB1,respectively).The expression of miR-32-5p and TOB1 at the mRNA and protein levels was detected by RT-qPCR and Western blot.The radiosensitivity of the transfected cells was determined by colony formation assay.The migration and invasion ability of the transfected cells were detected by Transwell assay.Whether miR-32-5p targeted TOB1 was validated by dual luciferase reporter gene assay and Western blot.Results Compared with human colonic epithelial cells,the expression of miR-32-5p was significantly up-regulated,whereas the expression of TOB1 mRNA and protein was remarkably down-regulated in the colon cancer cells (all P＜0.05).Compared with the anti-miR-NC,the quantity of cell migration and invasion was significantly decreased (both P＜0.05) and the radiosensitivity ratio was 1.801 in the anti-miR-32-5p group.Compared with the pcDNA group,the quantity of cell migration and invasion was significantly decreased (both P＜0.05) and the radiosensitivity ratio was 1.764 in the pcDNA-TOB1 group.Dual luciferase reporter gene assay and Western blot confirmed that miR-32-5p negatively regulated the expression of TOB1 protein.Compared with the anti-miR-32-5p+si-NC group,the quantity of cell migration and invasion was significantly increased (both P＜0.05) and the radiosensitivity ratio was 0.591 in the anti-miR-32-5p+si-TOB1 group.Conclusions Inhibition of miR-32-5p expression can significantly enhance the radiosensitivity of colorectal cancer cells and suppress cell migration and invasion.The underlying mechanism might be related to the targeted up-regulation of TOB1 expression.

MYC-activated lncRNA HNF1A-AS1 overexpression facilitates glioma progression via cooperating with miR-32-5p/SOX4 axis.

Mounting literatures have revealed the crucial effects of long noncoding RNA (lncRNA) in various cancers, including glioma. HNF1A-AS1, a novel lncRNA, is reported to modulate tumorigenesis and development of multiple cancers. However, the tumorigenic function of lncRNA HNF1A-AS1 in glioma remains largely unknown. quantitative reverse transcription and polymerase chain reaction and western blot assays were applied to evaluate the expression of relevant mRNAs and proteins. 5-Ethynyl-2'- deoxyuridine, terminal deoxynucleotidyl transferase dUTP nick-end labeling, flow cytometry, and transwell assays were conducted for examining the influence of HNF1A-AS1 on glioma cell functions. The relationship among RNAs was investigated by mechanical experiments. The results demonstrated that HNF1A-AS1 was predominantly highly expressed in glioma cell lines compared with nontumor glial epithelial cell, which was associated with the stimulation of transcription factor myelocytomatosis oncogene. Knockdown of HNF1A-AS1 remarkably inhibited glioma cells proliferation, migration, and invasion, while accelerating cell apoptosis in vitro. Mechanically, HNF1A-AS1 served as a miR-32-5p sponge. Moreover, SOX4 was discovered as a target of miR-32-5p. Inhibited miR-32-5p or upregulated SOX4 could markedly counteract the inhibitory effects of silencing HNF1A-AS1 on glioma malignant biological behaviors. HNF1A-AS1 exerted oncogenic property in glioma progression via upregulating miR-32-5p-mediated SOX4 expression, suggesting potential novel therapeutic target for future glioma treatment.