miR-186 regulates epithelial-mesenchymal transformation to promote nasopharyngeal carcinoma metastasis by targeting ZEB1

Abstract Objectives Nasopharyngeal carcinoma (NPC) is an aggressive epithelial cancer. The expression of miR-186 is decreased in a variety of malignancies and can promote the invasion and metastasis of cancer cells. This study aimed to explore the role and possible mechanism of miR-186 in the metastasis and epithelial-mesenchymal transformation (EMT) of NPC. Methods The expression of miR-186 in NPC tissues and cells was detected by RT-PCR. Then, miR-186 mimic was used to transfect NPC cell lines C666-1 and CNE-2, and cell activity, invasion and migration were detected by CCK8, transwell and scratch assay, respectively. The expression of EMT-related proteins was analyzed by western blotting analysis. The binding relationship between miR-186 and target gene Zinc Finger E-Box Binding Homeobox 1 (ZEB1) was confirmed by double luciferase assay. Results The expression of miR-186 in NPC was significantly decreased, and transfection of miR-186 mimic could significantly inhibit the cell activity, invasion, and migration, and regulate the protein expressions of E-cadherin, N-cadherin and vimentin in C666-1 and CNE-2 cells. Further experiments confirmed that miR-186 could directly target ZEB1 and negatively regulate its expression. In addition, ZEB1 has been confirmed to be highly expressed in NPC, and inhibition of ZEB1 could inhibit the activity, invasion, metastasis and EMT of NPC cells. And co-transfection of miR-186 mimic and si-ZEB1 could further inhibit the proliferation and metastasis of NPC. Conclusion miR-186 may inhibit the proliferation, metastasis and EMT of NPC by targeting ZEB1, and the miR-186/ZEB1 axis plays an important role in NPC.

Mechanism of miR-186-5p Regulating PRKAA2 to Promote Ferroptosis in Lung Adenocarcinoma Cells / 中国肺癌杂志

BACKGROUND@#Lung adenocarcinoma (LUAD) is the most common type of non-small cell lung cancer, and any change of miRNAs expression will affect the degree of target regulation, thus affecting intracellular homeostasis. This study verified that miR-186-5p could inhibit the proliferation, migration and invasion of LUAD cells by regulating PRKAA2.@*METHODS@#Previous investigations found that the expression of miR-186-5p was markedly suppressed in LUAD. Bioinformatics method is used to predict the target protein related to ferroptosis downstream and inquire about its expression level in LUAD and its influence on the survival of patients. Double luciferase verified the binding site of PRKAA2 and miR-186-5p. Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expression of PRKAA2. The effects of miR-186-5p of LUAD cells as well as the mechanism by which miR-186-5p inhibits Fer-1's sensitivity to ferroptosis were confirmed by EdU, Transwell, and scratch assays. The effect of miR-186-5p on the amount of reactive oxygen species (ROS) in LUAD cells was discovered using ROS experiment. Malondialdehyde (MDA) and glutathione (GSH) experiments were used to detect the effects of miR-186-5p and PRKAA2 on ferroptosis index of LUAD cells. The concentration of lipid ROS (L-ROS) in LUAD cells were measured using the L-ROS tests to determine the effects of miR-186-5p and PRKAA2.@*RESULTS@#The expression of PRKAA2 is up-regulated, and a high level of PRKAA2 expression was associated with a poor prognosis for patients with LUAD. Overexpression of miR-186-5p decreased the gene and protein expression of PRKAA2. By promoting ferroptosis, miR-186-5p overexpression prevented lung cancer cells from proliferating, invading, and migrating. ROS could be produced in higher amounts in LUAD cells due to miR-186-5p. Overexpression of miR-186-5p and knockdown PRKAA2 up-regulated MDA content and reduced GSH content in LUAD cells, respectively. miR-186-5p could increase the content of L-ROS and promote the ferroptosis sensitivity of LUAD cells by targeting PRKAA2.@*CONCLUSIONS@#miR-186-5p promotes ferroptosis of LUAD cells through targeted regulation of PRKAA2, thus inhibiting the proliferation, invasion and migration of LUAD.
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Resveratrol inhibits the migration, invasion and epithelial-mesenchymal transition in liver cancer cells through up- miR-186-5p expression / 浙江大学学报·医学版

To investigate the molecular mechanism of resveratrol inhibiting the metastasis of liver cancer . HepG2 and Huh7 cells were treated with different concentrations of resveratrol, and the cell viability was determined by CCK-8 assay to determine the optimal concentration of resveratrol for subsequent experiments. The expressions of miR-186-5p in liver cancer tissues and liver cancer cells were determined by quantitative real-time RT-PCR. The migration and invasion of HepG2 and Huh7 cells were detected by wound healing assay and Transwell assay, and the expression levels of epithelial-mesenchymal transition (EMT) related proteins were determined by Western blotting. Resveratrol with concentration of had no effect on the viability of HepG2 and Huh7 cells, so the concentration of resveratrol in subsequent experiments was 6.25 μmol/L. Resveratrol inhibited the wound healing and invasion of liver cancer cells; increased the expression of E-cadherin, and decreased the expression of vimentin and Twist1. The expression of miR-186-5p was significantly down-regulated in liver cancer tissues and cells compared with the adjacent tissues and normal liver cells (both <0.05). Furthermore, resveratrol induced the expression of miR-186-5p in liver cancer cells (both <0.01). Overexpression of miR-186-5p suppressed the migration, invasion and EMT of liver cancer cells. Knockdown of miR-186-5p blocked the inhibition effects of resveratrol on the migration, invasion and EMT of liver cancer cells. Resveratrol could inhibit the metastasis of liver cancer , which might be associated with up-regulating miR-186-5p.

Research on the expression of miR-186-5p in serum of patients with colon cancer / 中国临床药理学与治疗学

AIM: To investigate the expression of miR-186-5p in colon cancer and the effect on colon cancer, and provide a new therapeutic target for the treatment and prognosis of colon cancer. METHODS: Serum from eligible patients were collected and divided into five groups (n=20) including the healthy group and clinical stages I, II, III and IV. The miRNAs in patients' serum were extracted by miRVana microRNA isolation kit. The differences of miR-186-5p expression in patients' serum of colon cancer at different stages were detected with real-time quantitative PCR. The full-length gene of miR-186-5p was cloned into pENTR/D-Topo vector to construct cell lines with high expression of miRNA. 5×103 cells were seeded in 96-well plates. The expression of mir-186-5p was decreased by using miR-186-5p inhibitor. The proliferation of cells was detected with cell proliferation ELISA kit after 24 hours of incubation with BrdU and absorbance was measured at 370 and 492 nm. The effect of miR-186-5p on the sensitivity of colon cancer cell lines to chemotherapeutic drugs was detected by MTT assay. RESULTS: The expression of miR-186-5p was down-regulated in the serum of patients with colon cancer compared to normal group. Expression of miR-186-5p in serum of patients with stage I, II, III and IV was significantly decreased (P<0.01) compared with the normal group. Colon cells were transfected with a plasmid integrating the miR-186-5p gene, and miR-186-5p was highly expressed in the HCT116 and SW480 cell lines (P<0.01). The expression of mir-186-5p was decreased by mir-186-5p inhibitor (hsa-miR-186-5p inhibitor) in HCT116 and SW480 cells. The proliferation rate of HCT116 and SW480 cells with high expression of miR-186-5p was lower than control group (P<0.01). After treating with mir-186-5p inhibitor, the cell proliferation rate was higher than that of the control group (P<0.01). After treatment with different concentrations cisplatin for 24 hours, the mortality of HCT116 and SW480 cells with high expression of mir-186-5p was higher than that of the control group (P<0.01). The mortality of the cells with low expression of mir-186-5p was lower than that of the control group (P<0.01). The protein level of PLK1 was decreased after transfecting with miR-186-5p plasmid and increased after stimulating with miR-186-5p inhibitor in HCT116 and SW480 cells. CONCLUSION: The expression of miR-186-5p is decreased in colon cancer patients. Overexpression of miR-186-5p can inhibit the proliferation of colon cancer cells and reduce the drug resistance of colon cancer cells; inhibition of miR-186-5p expression can increase the proliferation and drug resistance of colon cancer cells.

Long Intergenic Non-Protein Coding RNA 665 Regulates Viability, Apoptosis, and Autophagy via the MiR-186-5p/MAP4K3 Axis in Hepatocellular Carcinoma

PURPOSE: Long intergenic non-protein coding RNA 665 (LINC00665) plays a vital role in the development of cancer. Its function in hepatocellular carcinoma (HCC), however, remains largely unknown. MATERIALS AND METHODS: The expressions of LINC00665, miR-186-5p, and MAP4K3 were determined by qRT-PCR. Cell viability and apoptosis were evaluated by MTT and flow cytometry, respectively. Autophagic puncta formation was observed by fluorescence microscopy. Bioinformatics analysis, luciferase reporter assay, RNA immunoprecipitation, and RNA pulldown were performed to identify associations among LINC00665, miR-186-5p, and MAP4K3. Western blot was utilized to examine the expressions of MAP4K3, Beclin-1, and LC3. Tumor growth was evaluated in a xenograft model. RESULTS: Elevations in LINC00665 were observed in HCC tissues and cells. The overall survival of HCC patients with high levels of LINC00665 was shorter than those with low levels. In vitro, LINC00665 depletion inhibited viability and induced apoptosis and autophagy. miR-186-5p interacted with LINC00665 and was downregulated in HCC tissues and cells. Upregulation of miR-186-5p inhibited viability and induced apoptosis and autophagy, which were attenuated by upregulation of LINC00665. MAP4K3 was found to possess binding sites with miR-186-5p and was upregulated in HCC tissues and cells. MAP4K3 depletion inhibited viability and induced apoptosis and autophagy, which were attenuated by miR-186-5p inhibitor. In vivo, miR-186-5p expression was negatively correlated with LINC00665 or MAP4K3 in HCC tissues, while LINC00665 was positively correlated with MAP4K3. LINC00665 knockdown suppressed tumor growth. CONCLUSION: LINC00665 was involved in cell viability, apoptosis, and autophagy in HCC via miR-186-5p/MAP4K3 axis, which may provide a new approach for HCC treatment.

Construction of miR-186 sponge vector and its expression in EA. Hy926 cells / 吉林大学学报(医学版)

Objective: To construct the sponge vector which can target microRNA-186 (miR-186), and to create a stable EA. hy926 cell line that can knockdown miR-186. Methods: The miR-186 sponge sequence was chemically synthesized and cloned into lentiviral vector FV040. Then the FV040-miR-186-sponge recombinant plasmid together with the helper plasmids were cotransfected into the HEK293T cells by using lipofectamine 2000 to package lentivirus and the viral titer was determined. The FV040-control lentivirus (designated as the control group), and the FV040-miR-186-sponge (designated as the experiment group) were used to infect EA. hy926 cells for establishing stable cell lines. The fluorescent quantitative PCR (qPCR) method was used to detect the relative expression levels of miR-186 in the EA. hy926 cells in blank group, FV040-control group and FV040-miR-186 sponge group, respectively. Results: The cloned target sequence was identical with the designed miR-186 sponge sequence. The green fluorescence protein (GFP) was observed in the infected EA. hy926 cells. The lentivirus titre of viruses in the FV040-control group was 2×108 TU middot; mL-1, and which was 6 × 108 TU middot; mL-1 in FV040-miR- 186 sponge group. The EA. hy926 cell line stably expressed miR-186-sponge was established successfully and the infection rate was as high as 95%. The qPCR results indicated that the relative expression level of miR-186 in the EA. hy926 cells in FV040-miR-186-sponge group was lower than those in blank control group and FV040-control group (P<0. 01). Conclusion: The miR-186-sponge vector is successfully constructed, and the EA. hy926-miR- 186-sponge cell line with the stably decreased expression of miR-186 is established successfully.

Construction and identification of miR-186 overexpression lentiviral vector / 吉林大学学报(医学版)

Objective: To construct the miR-186 overexpression lentiviral vector and package the lentivirus, and to explore the infection efficiency and the expression level of miR-186 in the HEK293T cells. Methods: The Hsa-miR-186 precursor sequence was used as a template to design and synthesize the primer, and the the pre-miR-186 gene was amplified by PCR. The pre-miR-186 gene sequence was cloned into the lentiviral vector FV040 carrying EGFP/Puromycin cassette. The recombinant lentiviral vector was digested by EcoR I and Age I restriction endonuclease and confirmed by sequencing. The recombinant FV040 Vector and FV040 miR-186 were co-transfected into the HEK293T cells with the helper plasmids using Lipofectamine 2000, respectively; the FV040 Vector lentivirus (control group) and the FV040 miR-186 lentivirus (experiment group) were collected and used to infect the HEK293T cells 48 h after transfection, respectively. The green fluorescence distribution in the HEK 293T cells was observed 48 h after transfection, and the expression level of miR-186 was determined by real-time fluorescence quantitative PCR. Results: The sequencing analysis results indicated that the sequence of miR-186 overexpressing lentivirus was identical with the sequence of miR-186 published on GenBank. The titers in control group and experiment group were 6×108 TU · mL-1 and 5 × 108 TU · mL-1, respectively. The relative expression level of miR-186 in the HEK293T cells in experiment group (12. 640 0 ± 0. 788 4) was significantly increased by 15. 07 times (t=14. 72, P<0. 01) compared with control group (0. 838 7 ± 0. 145 6). Conclusion: The lentiviral vector which overexpresses miR-186 is constructed successfully and the miR-186 lentivirus is prepared. The HEK 293T cells are infected with miR-186 lentivirus successfully and the expression level of miR-186 in the HEK 293T cells is increased significantly.

Inhibiting miR-186 expression alleviates mitochondrial damage in hypoxic human umbilical vein endothelial cells / 南方医科大学学报

OBJECTIVE@#To investigate the effect of miR-186 inhibition on the expression of hypoxia-inducible factor-1α (HIF-α) and mitochondrial function in hypoxic vascular endothelial cells.@*METHODS@#Human umbilical vein endothelial cells (HUVECs) cultured in routine or hypoxic conditions for 6 h were examined for the expression of miR-186. A miR-186 inhibitor was transfected in the HUVECs, and the cells were subsequently cultured in hypoxic condition for 6 h to observe the changes in the mitochondrial structure under an electron microscope. The changes in the mRNA and protein expressions of HIF-1α in response to miR-186 interference were tested using real-time fluorescent quantitative PCR and Western blotting.@*RESULTS@#The expression of miR-18 was mildly increased in HUVECs after hypoxic exposure for 6 h (=0.0188). Interference of miR-186 expression obviously promoted the mRNA and protein expressions of HIF-1α in HUVECs. In hypoxic conditions, miR-186 interference significantly reduced mitochondrial damage in HUVECs as observed under electron microscope (=0.0297).@*CONCLUSIONS@#Inhibition of miR-186 protects vascular endothelial cells against hypoxic injuries by promoting HIF-α expression to lessen mitochondrial damage, suggesting the possibility of targeted miR-186 interference for treatment of hemorrhagic shock.

NLRP3 inflammasome signaling as an early molecular response is negatively controlled by miR-186 in CFA-induced prosopalgia mice

The NOD-like receptor family pyrin domain containing 3 (NLRP3) inflammasome is the most frequently studied in the central nervous system and has been linked to neuropathic pain. In this study, a post-translational mechanism of microRNA (miR)-186 via regulating the expression of NLRP3 in the complete Freund's adjuvant (CFA)-treated mice was investigated. The injection of CFA was used to induce trigeminal neuropathic pain in mice. miRs microarray chip assay was performed in trigeminal ganglions (TGs). CFA treatment significantly increased the mRNA expression of NLRP3, interleukin (IL)-1β, and IL-18 in TGs compared to the control group. Moreover, 26 miRs were differentially expressed in TGs from trigeminal neuropathic pain mice, and the expression of miR-186 showed the lowest level of all the miRs. Further examination revealed that NLRP3 was a candidate target gene of miR-186. We delivered miR-186 mimics to CFA-treated mice. The head withdrawal thresholds of the CFA-treated mice were significantly increased by miR-186 mimics injection compared with CFA single treatment. The mRNA and protein expression of NLRP3, IL-1β, and IL-18 in TGs from trigeminal neuropathic pain mice were significantly inhibited by miR-186 mimics treatment compared to the CFA group. miR-186 was able to suppress the neuropathic pain via regulating the NLRP3 inflammasome signaling.