Exploring the oncogenic potential of circSOD2 in clear cell renal cell carcinoma: a novel positive feedback loop.

BACKGROUND: Existing studies have found that circular RNAs (circRNAs) act as sponges for micro RNAs (miRNAs) to control downstream genes. However, the specific functionalities and mechanisms of circRNAs in human clear cell renal cell carcinoma (ccRCC) have yet to be thoroughly investigated. METHODS: Patient cohorts from online databases were used to screen candidate circRNAs, while another cohort from our hospital was obtained for validation. CircSOD2 was identified as a potential oncogenic target, and its relevant characteristics were investigated during ccRCC progression through various assays. A positive feedback loop containing downstream miRNA and its target gene were identified using bioinformatics and validated by luciferase reporter assays, RNA pull-down, and high-throughput sequencing. RESULTS: CircSOD2 expression was elevated in tumor samples and significantly correlated with overall survival (OS) and the tumor stage of ccRCC patients, which appeared in the enhanced proliferation, invasion, and migration of tumor cells. Through competitive binding to circSOD2, miR-532-3p can promote the expression of PAX5 and the progression of ccRCC, and such regulation can be salvaged by miR-532-3p inhibitor. CONCLUSION: A novel positive feedback loop, PAX5/circSOD2/miR-532-3p/PAX5 was identified in the study, indicating that the loop may play an important role in the diagnosis and prognostic prediction in ccRCC patients.

Melatonin enhances the sensitivity of colorectal cancer cells to 5-fluorouracil through the regulation of the miR-532-3p/ß-catenin pathway.

This research aimed to investigate whether melatonin affected sensitivity to 5-fluorouracil (5-FU) in colorectal cancer (CRC) as well as to show the underlying molecular mechanism. Melatonin and 5-FU were added to CRC cells at varying doses. The effect of melatonin on sensitivity to 5-FU was investigated by measuring cell activity and apoptosis, and the potential underlying mechanism was further explored by detecting miR-532-3p expression and the associated pathway proteins. Melatonin could suppress cell malignancy in SW480 and HCT116 cells. Melatonin also significantly promoted sensitivity to 5-FU in CRC cells. miR-532-3p expression was downregulated in CRC and was also markedly enhanced when treated with 1 mmol/L melatonin. The inhibitory ability of the co-cultured melatonin, 5-FU, and miR-532-3p inhibitor on SW480 and HCT116 cells was markedly diminished, and the IC50 value was significantly enhanced. Relative to the melatonin group, melatonin+miR-532-3p inhibitor markedly declined apoptosis rate. Bioinformatics analysis predicted the target of miR-532-3p. ß-catenin level presented obvious downregulation in the melatonin group, while it was notably upregulated in the co-culture group in relative to with that in the melatonin group. Overall, melatonin promotes sensitivity to 5-FU in CRC cells by regulating the miR-532-3p/ß-catenin pathway.

Long non-coding RNA LINC00565 regulates ADAM19 expression through sponging microRNA-532-3p, thereby facilitating clear cell renal cell carcinoma progression.

Proven by publications, long non-coding RNAs (lncRNAs) play critical roles in the development of clear cell renal cell carcinoma (ccRCC). Although lncRNA LINC00565 has been implicated in the progression of various cancers, its biological effects on ccRCC remain unknown. This study aimed to investigate the biological functions of LINC00565, as well as its potential mechanism in ccRCC. Here, the expression data of mature microRNAs (miRNAs) (normal: 71, tumor: 545), messenger RNAs (mRNAs), and lncRNAs (normal: 72, tumor: 539) of ccRCC were acquired from The Cancer Genome Atlas (TCGA) database and subjected to differential expression analysis. Quantitative reverse transcriptase polymerase chain reaction analyzed the expression levels of LINC00565, miR-532-3p, and ADAM19 mRNA. TCGA database, dual-luciferase report detection, and Argonaute 2 RNA immunoprecipitation were utilized to confirm the relationships between LINC00565 and miR-532-3p and between miR-532-3p and ADAM19, respectively. The progression of ccRCC cells was determined via CCK-8, colony formation, scratch healing, and transwell assays. Western blot was applied to detect the protein levels of epithelial-mesenchymal transition markers and ADAM19. We herein suggested that LINC00565 was prominently upregulated in ccRCC tissues and cells. Knockdown of LINC00565 repressed cell progression. We further predicted and validated miR-532-3p as a target of LINC00565, and miR-532-3p could target ADAM19. Knockdown of LINC00565 resulted in ADAM19 level downregulation in ccRCC cells and suppressed miR-532-3p could restore ADAM19 level. Thus, the three RNAs constructed a ceRNA network. Overexpressed ADAM19 could eliminate the anticancer effects caused by knocking down LINC00565 on ccRCC cells. In conclusion, LINC00565 upregulated ADAM19 via absorbing miR-532-3p, thereby facilitating the progression of ccRCC cells.

LncRNA NORAD regulates the mechanism of the miR-532-3p/Nectin-4 axis in pancreatic cancer cell proliferation and angiogenesis.

Backgound: Pancreatic cancer (PC) is one of the deadliest cancers worldwide, and cell proliferation and angiogenesis play an important role in its occurrence and development. High levels of lncRNANORAD have been detected in many tumors, including PC, yet the effect and mechanism of lncRNA NORAD on PC cell angiogenesis are unexplored. Methods: qRT.PCR was applied to quantify lncRNA NORAD and miR-532-3p expression in PC cells, and a dual luciferase reporter gene was used to verify the targeting effects of NORAD, miR-532-3p and Nectin-4. Then, we regulated NORAD and miR-532-3p expression in PC cells and detected their effects on PC cell proliferation and angiogenesis using cloning experiments and HUVEC tube formation experiments. Results: LncRNA NORAD was upregulated and miR-532-3p was downregulated in PC cells compared with normal cells. Knockdown of NORAD inhibited PC cell proliferation and angiogenesis. LncRNA NORAD and miR-532-3p competitively bound to promote the expression of the miR-532-3p target gene Nectin-4, thereby promoting proliferation and angiogenesis of PC cells in vitro. Conclusion: LncRNA NORAD promotes the proliferation and angiogenesis of PC cells by regulating the miR-532-3p/Nectin-4 axis, which may be a potential biological target in the diagnosis and treatment of clinical PC.

LncRNA TSIX aggravates spinal cord injury by regulating the PI3K/AKT pathway via the miR-532-3p/DDOST axis.

Long noncoding RNA (lncRNA)-X-inactive-specific transcript (TSIX) expression is upregulated in spinal cord tissues following spinal cord injury (SCI). However, the role of lncRNA-TSIX in SCI remains elusive. SCI animal model was established using C57BL/6 mice. LncRNA TSIX and miR-532-3p expression were determined using quantitative reverse transcription polymerase chain reaction (qRT-PCR). Apoptosis, cell proliferation, and migration were evaluated by transferase dUTP nick end labeling staining, CCK-8, and Transwell assays, respectively. The interaction of miR-532-3p with lncRNA TSIX and DDOST was explored via a dual-luciferase reporter system. Hematoxylin-eosin staining and the Basso, Beattie, and Bresnahan locomotor rating (BBB) scale were performed to investigate SCI progression. The expression of the lncRNA TSIX was found to be significantly upregulated in the serum of SCI patients and spinal cord tissues of SCI mice. The overexpression of lncRNA TSIX enhanced spinal cord neural stem cell (SC-NSC) proliferation and migration in vitro while inhibiting apoptosis and inflammatory cell infiltration in vivo. Moreover, lncRNA TSIX acted as a molecular sponge for miR-532-3p, and the knockdown of miR-532-3p promoted proliferation and migration and inhibited apoptosis of SC-NSCs. Moreover, DDOST was found to be the downstream target of miR-532-3p, and DDOST overexpression showed a similar effect as miR-532-3p silencing on the proliferation, migration, and apoptosis of SC-NSCs. Furthermore, we found that lncRNA TSIX overexpression promoted the activation of the PI3K/AKT signaling pathway. LncRNA TSIX aggravates SCI by regulating the PI3K/AKT pathway via the miR-532-3p/DDOST axis, indicating potential applications for targeted therapy of SCI regeneration.

Abnormal expression pattern of lncRNA H19 participates in multiple myeloma bone disease by unbalancing osteogenesis and osteolysis.

BACKGROUND: Accumulating genetic and epigenetic alterations in multiple myeloma (MM) have been demonstrated to be closely associated with osteolytic bone disease, generally characterized as increased osteoclast formation and decreased osteoblast activity. Previously, serum long non-coding RNA (lncRNA) H19 has been proved to be a biomarker for the diagnosis of MM. Whereas, its role in MM-associated bone homeostasis remains largely elusive. METHODS: A cohort of 42 MM patients and 40 healthy volunteers were enrolled for evaluating differential expressions of H19 and its downstream effectors. The proliferative capacity of MM cells was monitored by CCK-8 assay. Alkaline phosphatase (ALP) staining and activity detection, either with Alizarin red staining (ARS) were employed to assess osteoblast formation. Osteoblast- or osteoclast-associated gene were detected using qRT-PCR and western blot analysis. Bioinformatics analysis, RNA pull-down, RNA immunoprecipitation (RIP), and chromatin immunoprecipitation (ChIP) were subjected to verify H19/miR-532-3p/E2F7/EZH2 axis, which was accounted for epigenetic suppression of PTEN. The functional role of H19 on MM development through unbalancing osteolysis and osteogenesis was also confirmed in the murine MM model. RESULTS: Upregulation of serum H19 was observed in MM patients, suggesting its positive correlation with the poor prognosis of MM patients. Loss of H19 dramatically weakened cell proliferation of MM cells, promoted osteoblastic differentiation, and impaired osteoclast activity. While reinforced H19 exhibited the opposite effects. Akt/mTOR signaling plays an indispensable role in H19-mediated osteoblast formation and osteoclastgenesis. Mechanistically, H19 served as a sponge for miR-532-3p to upregulate E2F7, a transcriptional activator of EZH2, thereby accounting for modulating epigenetic suppression of PTEN. The in vivo experiments further validated that H19 exerted important impacts on tumor growth through breaking the balance between osteogenesis and osteolysis via Akt/mTOR signaling. CONCLUSION: Collectively, increased enrichment of H19 in MM cells exhibits an essential role in MM development by disturbing bone homeostasis.

Xanthohumol improves cognitive impairment by regulating miRNA-532-3p/Mpped1 in ovariectomized mice.

RATIONALE: Studies have shown the potential neuroprotective effect of xanthohumol, while whether xanthohumol has the ability of repairing cognitive impairment and its underlying mechanism still remains obscure. OBJECTIVES: To unravel the mechanism of xanthohumol repairing cognitive impairment caused by estrogen deprivation. METHODS: C57BL/6 J female mice that underwent bilateral ovariectomy to establish cognitive decline model were randomly divided into three xanthohumol-treated groups and a saline-treated model group. For identifying the neuroprotective function of xanthohumol, Morris water maze (MWM) test and open field test (OFT) were conducted. After extracting total RNA of mouse hippocampus of different groups, mRNA-seq and microRNA (miRNA)-seq analysis were performed, and the differentially expressed miRNAs (DEMIs) and their target genes were further validated by qPCR. MiR-532-3p and its downstream gene Mpped1 were screened as targets of xanthohumol. Influence of miR-532-3p/Mpped1 to cognitive ability was examined via MWM test and OFT after stereotactic brain injection of Mpped1 overexpressed adeno-associated virus. The regulation of miR-532-3p on Mpped1 was confirmed in hippocampal neuronal cell line HT22 by luciferase reporter gene assay. RESULTS: Xanthohumol treatment reversed the cognitive decline of OVX mice according to behavioral tests. By comparing miRNA levels of xanthohumol-treated groups with saline-treated group, we found that the main changed miRNAs were miR-122-5p, miR-532-3p, and miR-539-3p. Increased miR-532-3p in OVX mice was suppressed by xanthohumol treatment. Furthermore, the downstream gene of miR-532-3p, Mpped1, was also increased by xanthohumol and showed the capability of relieving cognitive impairment of OVX mice after overexpressed in hippocampus. The 3' untranslated region of Mpped1 was identified as the target region of miR-532-3p, and agomiR-532-3p remarkably reduced the expression of Mpped1 mRNA. CONCLUSIONS: Xanthohumol has the ability of repairing cognitive impairment through removing the inhibition of miR-532-3p on Mpped1 in mouse hippocampus. This finding not only advances the understanding of neuroprotective mechanism of xanthohumol, but also provides novel treatment targets for dementia of postmenopausal women.

Targeted delivery of 5-fluorouracil, miR-532-3p, and si-KRAS to the colorectal tumor using layer-by-layer liposomes.

Co-delivery of siRNA or miRNA with chemotherapeutic drugs into tumor sites is an attractive synergetic strategy for treating colorectal cancer (CRC) due to their complementary mechanisms. In the current work, a liposome nanoparticle (Huang et al., Cancer Metastasis Rev., 2018, 37, 173-187) coated by cationic chitosan (CS) using a controlled layer-by-layer (LbL) process was designed to deliver simultaneous si-KRAS, miRNA-532-3p, and 5-Fluorouracil (5-FU) into CRC cells. The LbL NPs exhibited a spherical structure with an average size of 165.9 nm and effectively protected si-KRAS and miRNA-532-3p against degradation by serum and nucleases. Interestingly, the LbL NPs were successfully entered into cells and efficiently promoted cytotoxicity and suppressed cancer cell migration and invasion. In vivo, the LbL NPs reduced tumor growth in SW480-tumor-bearing mice models. In conclusion, these results suggested that the LbL NPs co-loaded with 5-FU and miR-532-3p/si-KRAS might provide a promising potential strategy for inhibiting the malignant phenotypes of CRC cells.

LncRNA MHENCR Predicts Poor Outcomes in Patients with Colorectal Carcinoma and Modulates Tumorigenesis by Impairing MiR-532-3p.

The long noncoding RNAs (lncRNAs) are widely involved in the progression of various malignant tumors. The current study investigated the role and mechanism of lncRNA melanoma highly expressed noncoding RNA (MHENCR) in colorectal carcinoma. The expression of MHENCR was measured by real-time quantitative reverse transcription PCR (RT-qPCR). The chi-square analysis was used to analyze the correlation between MHECNR and miR-532-3p. Kaplan-Meier curve and multivariate Cox regression analysis were conducted to assess the significance of MHENCR in clinic. The interaction of MHENCR and miR-532-3p was probed using Pearson analysis and dual-luciferase reporter assay. Cellular experiments were implemented to explore the effects of MHENCR/miR-532-3p on colorectal carcinoma cells. Compared with para-cancerous tissues, MHENCR expression was increased and miR-532-3p expression was decreased in tumor tissues. High expression of MHENCR exhibited shorter overall survival. Interfering of MHENCR suppressed cellular activities while the silence of miR-532-3p diminished the decreased cellular behaviors in colorectal carcinoma cells. Interfering with MHENCR expression represses colorectal carcinoma cell proliferation, migration, and invasion by regulating miR-532-3p. MHENCR may act as a novel prognostic marker in colorectal carcinoma and MHENCR/miR-532-3p may serve as a potential target for treating colorectal carcinoma.

[Mechanism Underlying the Inhibitory Effect of MiR-532-3p on the Cells Proliferation of Diffuse Large B-Cell Lymphoma].

OBJECTIVE: To investigate the effects and underlying mechanism of miR-532-3p and resibufogenin (RES) by regulating Wnt/ß-catenin signaling on diffuse Large B-cell lymphoma (DLBCL) cells proliferation. METHODS: DLBCL tissues and adjacent normal tissues were collected from patients had been diagnosed with DLBCL at the First Hospital of Lanzhou University from October 2019 to October 2021. Four groups including mimics-NC, miR-532-3p mimics, RES control and RES treatment in SU-DHL-4 cells were designed. The expression level of miR-532-3p was detected by RT-qPCR. The protein content of ß-catenin was detected by Western blot. MTT assay was used to detect the proliferation activity of SU-DHL-4 cells. RESULTS: miR-532-3p expression was significantly decreased in DLBCL tissues compared with adjacent normal tissues (P<0.001). The miR-532-3p content in lymphoma cells was significantly lower than that in normal lymphocytes (P<0.001). After overexpression of miR-532-3p, the viability of SU-DHL 4 cells was significantly decreased (P<0.001), with a reduced expression of ß-catenin (P<0.05). RES treatment inhibited the proliferation of SU-DHL-4 cells and decreased ß-catenin expression in SU-DHL-4 cells compared with the control group. CONCLUSION: Overexpression of miR-532-3p reduced Wnt/ß-catenin signaling and inhibited the proliferation of lymphoma cells. Moreover, RES treatment inhibited lymphoma cells growth partially through Wnt/ß-catenin signaling suppression.

MiR-532-3p suppresses cell proliferation, migration and invasion of colon adenocarcinoma via targeting FJX1.

OBJECTIVE: We attempted to probe into mechanism of FJX1 regulating cell behaviors of colon adenocarcinoma, and to provide ideas for targeted therapy of colon adenocarcinoma. METHODS: Differential mRNAs were screened out from TCGA-COAD dataset. K-M analysis was done to compare correlation between FJX1 expression and patient's survival status. Upstream miRNAs of FJX1 were identified by bioinformatics methods. Targeted relationship of miR-532-3p and FJX1 was verified by dual-luciferase reporter gene assay. FJX1 level in colon adenocarcinoma cell lines was assayed via qRT-PCR and western blot. The impact of regulation of miR-532-3p to FJX1 on biological functions of colon adenocarcinoma cells was evaluated by MTT, wound healing and Transwell invasion assays. RESULTS: TCGA-COAD data and qRT-PCR manifested that FJX1 was increased in colon adenocarcinoma tissue, while miR-532-3p was conspicuously less expressed. Survival analysis denoted that FJX1 is potential to be an independent prognosticator in colon adenocarcinoma. Dual-luciferase assay manifested that miR-532-3p targeted FJX1 to repress expression of FJX1. Overexpression of FJX1 could stimulate cell malignant behaviors of colon adenocarcinoma, whereas forced expression of miR-532-3p reversed this promotive effect. CONCLUSION: This study revealed that FJX1 was an oncogene in colon adenocarcinoma cells, which was regulated by miR-532-3p. MiR-532-3p targeted and regulated FJX1 expression to suppress cell malignant behaviors of colon adenocarcinoma.

Circ_0000064 promotes high glucose-induced renal tubular epithelial cells injury to facilitate diabetic nephropathy progression through miR-532-3p/ROCK1 axis.

BACKGROUND: Circular RNA (circRNA) has been shown to mediate diabetic nephropathy (DN) development by regulating renal tubular epithelial cells (RTECs) injury. However, the role and mechanism of circ_0000064 in high glucose (HG)-induced RTECs injury have not been fully elucidated. METHODS: Human RTECs (HK-2) were exposed to HG to induce cell injury. Cell oxidative stress was assessed by detecting the levels of oxidative stress-markers. Moreover, cell proliferation and apoptosis were determined by CCK8 assay, EDU assay and flow cytometry. The protein levels of proliferation markers, apoptosis markers and Rho-associated coiled-coil-containing kinase 1 (ROCK1) were measured using western blot analysis. Furthermore, quantitative real-time PCR was performed to assess the expression of circ_0000064, microRNA (miR)-532-3p and ROCK1. The interaction between miR-532-3p and circ_0000064 or ROCK1 was confirmed by dual-luciferase reporter assay and RNA pull-down assay. RESULTS: Our results revealed that HG treatment could promote HK-2 cells oxidative stress, apoptosis, fibrosis, and inhibit proliferation. Circ_0000064 expression was increased in the serum of DN patients and HG-induced HK-2 cells, and silenced circ_0000064 could relieve HG-induced HK-2 cells injury. MiR-532-3p could be sponged by circ_0000064, and its overexpression also alleviated HG-induced HK-2 cells injury. Besides, the regulation of circ_0000064 knockdown on HG-induced HK-2 cells injury could be reversed by miR-532-3p inhibitor. Additionally, ROCK1 was a target of miR-532-3p, and its expression was inhibited by circ_0000064 knockdown. The inhibition effect of circ_0000064 knockdown on HG-induced HK-2 cells injury also could be reversed by overexpressing ROCK1. CONCLUSION: In summary, circ_0000064 knockdown might alleviate HG-induced HK-2 cells injury via regulating the miR-532-3p/ROCK1 axis, which provided a new perspective for DN treatment.

CircWHSC1 expedites cervical cancer progression via miR-532-3p/LTBP2 axis.

Dysregulated circRNAs have potential roles in the progression of various cancer types, including cervical cancer (CaCx). The carcinogenic roles of circRNA Wolf-Hirschhorn syndrome candidate gene-1 (circWHSC1) are described in the development of diverse cancers. The objective of this study was to investigate the expression and the underlying role of circWHSC1 in CaCx. The expression of circWHSC1 was detected by real-time PCR. After the suppression of circWHSC1 expression, the changes in the proliferation, migration, invasion, and apoptosis capacities were detected by CCK-8 assay, colony formation assay, Transwell assays, flow cytometry, and the determination of apoptosis-related proteins. The interplay among circWHSC1, miR-532-3p, and latent transforming growth factor-ß binding protein 2 (LTBP2) was confirmed by luciferase reporter and biotinylated RNA pull-down assays. A nude mice xenograft tumor model was established to evaluate the anti-tumorigenic role of circWHSC1 silencing in vivo. CircWHSC1 was overexpressed in CaCx tissues and cell lines and its high expression was inversely associated with the survival rate of patients with CaCx. CircWHSC1 silencing was capable of suppressing the proliferation, metastasis, and invasion of tumor cells and inducing apoptosis. Investigation to its molecular mechanism revealed that circWHSC1 functioned as a competitive endogenous RNA (ceRNA), mediating LTBP2 expression by targeting miR-532-3p. The in vivo experiments further confirmed the inhibition of tumor growth and metastasis by circWHSC1 knockdown. The circWHSC1-mediated miR-532-3p/LTBP2 signaling axis might be a novel therapeutic target for CaCx.

Circ_0025033 deficiency suppresses paclitaxel resistance and malignant development of paclitaxel-resistant ovarian cancer cells by modulating the miR-532-3p/FOXM1 network.

BACKGROUND: Ovarian cancer (OC) is the main cause of cancer-related death in women, and drug resistance is a leading cause of treatment failure. Recently, the involvement of circular RNAs (circRNAs) in cancer progression has become an area of increased investigation. The objective of this study is to uncover the function and regulatory mechanism of circ_0025033 in paclitaxel (PTX)-resistant OC cells. METHODS: The expression of circ_0025033, FOXM1 and miR-532-3p was investigated using quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expression of FOXM1 was quantified by western blot. Cell biological functions, including cell viability, migration/invasion and apoptosis, were explored using 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays, transwell assays and flow cytometry assays. The interaction between miR-532-3p and circ_0025033 or FOXM1 predicted by bioinformatics analysis was validated by pull-down assay and dual-luciferase reporter assay. Exosomes were isolated to determine the further function of circ_0025033. RESULT: Circ_0025033 and FOXM1 were highly expressed, while miR-532-3p was poorly expressed in OC tissues and cells, and the expression pattern was greater in PTX-resistant OC cells. Circ_0025033 knockdown lessened PTX resistance, suppressed migration/invasion and promoted apoptosis of PTX-resistant cells. With respect to mechanism, circ_0025033 upregulated the expression of FOXM1 by targeting miR-532-3p, and circ_0025033 knockdown blocked the malignant activities of PTX-resistant OC cells by enriching miR-532-3p and suppressing FOXM1. Exosomes derived from PTX-resistant cells with circ_0025033 knockdown also could repress the malignant actions of PTX-resistant OC cells. CONCLUSION: Circ_0025033 downregulation impaired PTX resistance and malignant activities of PTX-resistant OC cells by regulating the miR-532-3p/FOXM1 network.

lncRNA SNHG10 promotes the invasion and migration of colorectal cancer SW620 cells by targeting miR-532-3p / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the expression of long non-coding RNA (lncRNA) small nucleolar RNA host gene 10 (SNHG10) in colorectal cancer tissues and cells and its effect on the invasion and migration of colorectal cancer cells and the underlying mechanism. Methods: From January 2018 to December 2019, 78 pairs of colorectal cancer tissue and para-cancerous tissues from the patients who had radical colorectal cancer resection in Henan Provincial People's Hospital were collected. Quantitative PCR (qPCR) was performed to quantify the levels of lncRNA SNHG10 and miR-532-3p in colorectal cancer tissues, colorectal cancer cell lines (SW480, SW620, HT-29 and LoVo) and human normal colorectal mucosal FHC cells; and their correlations with the clinicopathological features of colorectal cancer patients were further analyzed. Dual-luciferase reporter gene assay was used to validate the targeted relationship between lncRNA SNHG10 and miR-532-3p. After transfection with si-SNHG10 or miR-532-3p mimic or co-transfection of si-SNHG10 and miR-532-3p inhibitor, the invasion and migration of SW620 cells were detected by Transwell assay, and the protein expression of E-cadherin, N-cadherin and vimentin were detected by WB. Results: SNHG10 was highly expressed in colorectal cancer tissues and cells (all P<0.05), and its expression was related to TNM stage and distant metastasis (all P<0.05). miR-532-3p was lowly expressed in colorectal cancer tissues and cells, and its expression was correlated with TNM stage, lymphonode metastasis and distant metastasis (all P<0.05). The expression of SNHG10 and miR-532-3p in colorectal cancer tissues was negatively correlated (r=-0.225, P=0.048). Dual-luciferase reporter gene assay confirmed that SNHG10 targetedly regulated miR-532-3p. Both down-regulation of SNHG10 and up-regulation of miR-532-3p significantly inhibited the invasion and migration of SW620 cells (all P<0.05), up-regulated the expression of E-cadherin (P<0.05), while down-regulated the expression of N-cadherin and vimentin (all P<0.05). After transfection with miR-532-3p inhibitor, the inhibitory effect of knocking down the expression of lncRNA SNHG10 on the invasion and migration of colorectal cancer cells was reversed (all P<0.05). Conclusions: LncRNA SNHG10 is highly expressed in colorectal cancer and is associated with TNM stage and distant metastasis. LncRNA SNHG10 affects the invasion and metastasis of colorectal cancer cells by targeting miR-532-3p and regulating EMT.

Lipid metabolism disorders effects of 6:2 chlorinated polyfluorinated ether sulfonate through Hsa-miRNA-532-3p/Acyl-CoA oxidase 1(ACOX1) pathway.

6:2 Chlorinated polyfluorinated ether sulfonate (6:2 Cl-PFESA), an alternative product of perfluorooctane sulfonate (PFOS), has been frequently detected in various environmental, wildlife, and human samples. A few studies revealed the hepatotoxicity of 6:2 Cl-PFESA in animals, but the underlying toxicity mechanisms remain largely unknown. In this study, we investigated the lipid metabolism disorders of 6:2 Cl-PFESA through miRNA-gene interaction mode in Huh-7 cells. Our results showed that 6:2 Cl-PFESA significantly promoted cellular lipid accumulation and increased the expression of Acyl-CoA oxidase 1 (ACOX1), with the lowest effective concentrations (LOECs) of 3 µM. In silico analysis showed that hsa-miR-532-3p is a potential miRNA molecule targeting ACOX1. Fluorescent-based RNA electrophoretic mobility shift assay (FREMSA) and ACOX1-mediated luciferase reporter gene assays showed that hsa-miR-532-3p could directly bind to ACOX1 and inhibit its transcription activity. Besides, 6:2 Cl-PFESA decreased the expression of hsa-miR-532-3p in the PPARα-independent manner. Overexpression of hsa-miR-532-3p promoted 6:2 Cl-PFESA-induced cellular lipid accumulation and decreased the ACOX1 production in Huh-7 cells. Taken together, at human exposure relevant concentrations, 6:2 Cl-PFESA might upregulate the expression levels of ACOX1 through downregulating hsa-miR-532-3p, and disturbed lipid homeostasis in Huh-7 cells, which revealed a novel epigenetic mechanism of 6:2 Cl-PFESA-induced hepatic lipid toxic effects.

Circ-PGC increases the expression of FOXR2 by targeting miR-532-3p to promote the development of non-small cell lung cancer.

This study was to explore the function of circular progastricsin (circ-PGC) in NSCLC. The histological morphology of tumor tissues was observed by hematoxylin and eosin (HE) staining. The expression of circ-PGC, miR-532-3p and forkhead box R2 (FOXR2) mRNA was measured by real-time quantitative polymerase chain reaction (RT-qPCR). The protein level of FOXR2 was checked by western blot. In functional analyses, cell viability, colony formation, cell apoptosis, cell migration and cell invasion were investigated using cell counting kit-8 (CCK-8) assay, colony formation assay, flow cytometry assay, wound healing assay and transwell assay. Besides, glycolysis metabolism was assessed by the levels of glucose consumption, lactate production and adenosine triphosphate (ATP) production. The predicted relationship between miR-532-3p and circ-PGC and FOXR2 was verified by dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. The results showed that circ-PGC and FOXR2 were upregulated in NSCLC tissues and cells. Circ-PGC knockdown or FOXR2 knockdown inhibited NSCLC cell viability, colony formation, cell migration, invasion and glycolysis metabolism, and FOXR2 overexpression rescued these inhibitory effects caused by circ-PGC knockdown. MiR-532-3p harbored the same binding site with circ-PGC and FOXR2, and circ-PGC positively regulated FOXR2 expression by targeting miR-532-3p. The expression of ß-catenin and c-Myc was decreased in cells after circ-PGC knockdown but recovered with miR-532-3p inhibition or FOXR2 overexpression. Circ-PGC downregulation also inhibited tumor growth in vivo. In conclusion, circ-PGC positively regulated FOXR2 expression by competitively binding to miR-532-3p, thereby promoting the development of NSCLC, and the Wnt/ß-catenin signaling pathway might be activated by the circ-PGC/miR-532-3p/FOXR2 network.

Knockdown of circNRIP1 sensitizes colorectal cancer to 5­FU via sponging miR­532­3p.

The present study aimed to investigate the influence of circular RNA nuclear receptor­interacting protein 1 (circNRIP1) on the chemotherapeutic effect of 5­fluorouracil (5­FU) in colorectal cancer (CRC) and reveal its potential molecular mechanisms. The effects of circNRIP1 on cell proliferation, migration and invasion, and apoptosis were evaluated using Cell Counting Kit­8, Transwell and flow cytometric assays, respectively. A dual­luciferase reporter assay was performed to verify the potential interaction between circNRIP1 and microRNA (miR)­532­3p. The results of the present study indicated that circNRIP1 was upregulated in CRC and its increased expression was associated with CRC progression. Furthermore, overexpression of circNRIP1 promoted CRC cell proliferation, invasion and migration, while it inhibited apoptosis. Knockdown of circNRIP1 significantly enhanced the 5­FU­induced inhibition of the viability of HCT116 and SW480 cells. Bioinformatics analysis predicted that miR­532­3p was a direct target of circNRIP1, which was further confirmed by a dual­luciferase reporter assay. miR­532­3p silencing reversed the effects of circNRIP1 knockdown on the sensitivity of 5­FU in the chemotherapy of CRC. The results suggested that circNRIP1 and miR­532­3p may be utilized to improve the diagnosis of CRC and serve as diagnostic markers. In conclusion, overexpression of circNRIP1 promoted the progression of CRC, while circNRIP1 silencing sensitized CRC cells to 5­FU via sponging miR­532­3p.

MiR-532-3p suppresses cell viability, migration and invasion of clear cell renal cell carcinoma through targeting TROAP.

Clear cell renal cell carcinoma (ccRCC) is a subtype of renal cell cancer with the highest mortality, infiltration, and metastasis rate, threatening human health. Despite oncogenic role of TROAP in various cancers, its function in ccRCC remains to be unraveled. The differentially expressed mRNAs (DEmRNAs) and miRNAs (DEmiRNAs) were obtained by analyzing the related data sets of ccRCC in TCGA. The expression levels of mRNAs and miRNAs in the cell were detected by qRT-PCR, while the protein levels were characterized by western blot. The viability, migratory and invasive abilities of ccRCC cells were determined by MTT, wound healing and cell invasion assays. The combination of miRNA target site prediction and dual-luciferase reporter gene assay verified the binding relationship between miR-532-3p and TROAP. Research on ccRCC displayed that TROAP expression was upregulated, while miR-532-3p was down-regulated. Besides, upregulation of TROAP could accelerate viability, migratory and invasive potentials of ccRCC cells. On the contrary, miR-532-3p could downregulate TROAP level, but TROAP upregulation reversed the viability, migration, and invasion of ccRCC cells. MiR-532-3p could attenuate the viability, migration and invasion of ccRCC cells by targeting TROAP. This may generate novel insights into molecular therapeutic targets for ccRCC.

Circular RNA hsa_circ_0013958 Functions as an Oncogenic Gene Through Modulating miR-532-3p/WEE1 Axis in Hepatocellular Carcinoma.

BACKGROUND: circ0013958 was identified as a biomarker, which can be used for the diagnosis and screening of lung cancer. However, the role of circ0013958 in hepatocellular carcinoma (HCC) remains unclear. METHODS: In our study, quantitative real-time polymerase chain reaction was performed to determine the levels of circ0013958 in HCC tissues and cell lines. EdU, CCK-8, transwell, flow cytometry and tumorigenesis assays were applied to assess the functions of circ0013958 in HCC in vitro and in vivo. Western blot assay was to detect the expression of WEE1. Luciferase reporter assay, bioinformatics analysis and rescue experiments were used to examine the interaction among circ0013958, miR-532-3p and WEE1. RESULTS: It revealed that circ0013958 was significantly up-regulated in HCC, which was positively correlated with poor prognosis of HCC patients. Circ0013958 promoted HCC cell proliferation and invasion, inhibited cell apoptosis in vitro, and promoted tumorigenesis in vivo. Circ0013958 acted as a miR-532-3p sponge to regulate WEE1 expression, thus promoting the progression of HCC. CONCLUSIONS: Circ0013958 promotes HCC progression through miR-532-3p/WEE1 axis. Circ0013958 may serve as a potential diagnostic biomarker and therapeutic target of HCC.