Overexpression of lncRNA-MEG3 inhibits endometrial cell proliferation and invasion via miR-21-5p/DNMT3B/Twist

Abstract Recent studies have found that lncRNA-MEG3(MEG3) plays an important role in the development of EMs (Endometriosis), but the specific mechanism needs to be further explored. This study aimed to investigate the effect of MEG3 on the proliferation, invasion of EMs cells. The authors used RT-qPCR to detect the expression of MEG3 and miR-21-5p in EMs tissues and hESCs cells, MTT and Transwell to detect cell proliferation and invasion, western blotting assay to detect the expression of DNMT3B and Twist, MSP to detect the methylation of Twist. The present study's detection results showed that MEG3 was lowly expressed in EMs tissues and hESCs cells, and overexpression of MEG3 could down-regulate miR-21-5p and inhibit endometrial cell proliferation and invasion. In addition, overexpression of MEG3 upregulated the expression of DNMT3B and promoted the methylation of TWIST. In conclusion, the present findings suggest that MEG3 is downregulated in EMs tissues, and overexpression of MEG3 can promote the activity of DNA methyltransferase DNMT3B by downregulating miR-21-5p, thereby promoting the methylation of Twist, downregulating Twist level to inhibits hESCs cells proliferation and invasion.

Overexpression of serum extracellular vesicle microRNA-215-5p is associated with early tumor recurrence and poor prognosis of gastric cancer

OBJECTIVES: Extracellular vesicle microRNAs (EV-miRNAs) have been demonstrated to be reliable candidate biomarkers for clinical applications. However, the clinical application potential of serum EV-miR-215-5p for gastric cancer (GC) remains poorly understood. The goal of our study was to determine the efficacy of serum EV-miR-215-5p in predicting the prognosis of GC. METHODS: Blood samples were collected from 118 patients with GC, 60 patients with benign gastric disease and BGD and 70 healthy controls. The relative levels of serum EV-miR-215-5p were measured using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Compared to patients with BGD and normal controls, GC patients exhibited remarkably higher serum EV-miR-215-5p level, especially those with early tumor recurrence (ETR). Receiver operating characteristic (ROC) curve analysis showed that serum EV-miR-215-5p was able to distinguish GC patients from BGD patients or healthy controls and GC patients with ETR from those without ETR. In addition, increased serum EV-miR-215-5p levels were notably correlated with invasive depth, TNM stage, and lymph node metastasis. Moreover, serum EV-miR-215-5p levels were greatly decreased after surgical treatment, but increased at the time of ETR. Survival analysis showed that patients with higher serum EV-miR-215-5p had shorter survival. Furthermore, serum EV-miR-215-5p was an independent risk factor for GC. CONCLUSIONS: Serum EV-miR-215-5p might be a novel biomarker for predicting ETR and prognosis of GC.

Overexpression of serum extracellular vesicle microRNA-215-5p is associated with early tumor recurrence and poor prognosis of gastric cancer

OBJECTIVES: Extracellular vesicle microRNAs (EV-miRNAs) have been demonstrated to be reliable candidate biomarkers for clinical applications. However, the clinical application potential of serum EV-miR-215-5p for gastric cancer (GC) remains poorly understood. The goal of our study was to determine the efficacy of serum EV-miR-215-5p in predicting the prognosis of GC. METHODS: Blood samples were collected from 118 patients with GC, 60 patients with benign gastric disease and BGD and 70 healthy controls. The relative levels of serum EV-miR-215-5p were measured using quantitative real-time polymerase chain reaction (qRT-PCR). RESULTS: Compared to patients with BGD and normal controls, GC patients exhibited remarkably higher serum EV-miR-215-5p level, especially those with early tumor recurrence (ETR). Receiver operating characteristic (ROC) curve analysis showed that serum EV-miR-215-5p was able to distinguish GC patients from BGD patients or healthy controls and GC patients with ETR from those without ETR. In addition, increased serum EV-miR-215-5p levels were notably correlated with invasive depth, TNM stage, and lymph node metastasis. Moreover, serum EV-miR-215-5p levels were greatly decreased after surgical treatment, but increased at the time of ETR. Survival analysis showed that patients with higher serum EV-miR-215-5p had shorter survival. Furthermore, serum EV-miR-215-5p was an independent risk factor for GC. CONCLUSIONS: Serum EV-miR-215-5p might be a novel biomarker for predicting ETR and prognosis of GC.

Bone marrow mesenchymal stem cell derived exosomes miR-21-5p promotes proliferation, migration and invasion of prostate cancer PC-3 cell by downregulating PHLPP2 / 中国肿瘤生物治疗杂志

@#[Abstract] Objective: To investigate the effects of exosome originated from bone marrow mesenchymal stem cell (BMSCs) on proliferation, migration and invasion of prostate cancer PC-3 cell and its mechanism. Methods: qPCR was used to detect the expression level of miR-21-5p in prostate cancer cell lines. The morphology of exosomes isolated from BMSCs was observed with an electron microscope. Western blotting was used to detect the expressions of exosome surface markers and the epithelial-mesenchymal transition (EMT)-related proteins (E-cadherin, N-cadherin and Vimentin). Dual luciferase reporter gene experiment was used to detect the targeted regulation relationship between miR-21-5p and PH domain leucine-rich repeat protein phosphatase 2 (PHLPP2). PC-3 cells were co-cultured with 10 μl BMSCs exosomes suspension (Exo group), transfected with sh-PHLPP2 or antagomiR, then CCK-8 and Transwell experiments were used to detect changesinproliferation,migrationandinvasionofPC-3cell.Results: miR-21-5p was highly expressed in prostate cancer PC-3 cell line. The exosomes in the supernatant of BMSCs culture fluid were successfully isolated, and the typical vesicle-like structures of exosomes were observed under transmission electron microscope. Exosomes expressed specific proteins such as CD9, CD63 and CD81. In the Exo group, the proliferation, invasion, migration, as well as the expressions of N-cadherin, Vimentin and miR-21-5p in PC-3 cells were significantly higher than those in the control group (all P<0.05). PHLPP2 is a target gene of miR-21-5p. Compared with the control group, the expression of PHLPP2 in PC-3 cells of Exo group and sh-PHLPP2 group was significantly reduced (0.66±0.09, 0.42±0.05 vs 1.09±0.08, all P<0.01); cell viability, invasion and migration were significantly improved (all P<0.01); and E-cadherin expression level was significantly reduced while N-cadherin and Vimentin expressions were significantly increased (both P<0.05). Conclusion: miR-21-5p is highly expressed in prostate cancer PC-3 cell line. BMSC exosome miR-21-5p can increase the proliferation, migration and invasion ability of PC-3 cells through targeted down-regulation of PHLPP2.

Application of bioinformatics in screening of miRNA biomarkers in triple-negative breast cancer / 吉林大学学报(医学版)

Objective: To analyze the differentially expressed miRNAs in triple negative breast cancer (TNBC) and predict their target genes through The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) databases, to explore their biological functions and molecular mechanisms, and to find the prognosis-related targets of TNBC. Methods: A total of 343 miRNAs expression data related to breast cancer tissue and adjacent tissue were downloaded from the TCGA database to screen the differentially expressed miRNAs in breast cancer and adjacent tissue. The GEO database was used to validate the expressions of miRNAs in 26 kinds of cell lines of TNBC and the changes in serum miRNAs in the TNBC patients before and after chemotherapy. The target gene function of candidate miRNAs was analyzed by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) signal pathway enrichment and protein interaction network. Results: The TCGA database showed that the expression level of miR-21-5p in breast cancer tissue was significantly higher than that in adjacent tissue (logFC = 5. 557, P<0. 01). The results of GEO database showed that the expression level of miR-21-5p increased in TNBC cell line was significantly higher; the relative expression levels in more than 20 kinds of cell lines from 26 TNBC cell lines were over 70 000, and the expression level of miR-21-5p in the TNBC patients after combined chemotherapy was significantly decreased (logFC= -5.07, P<0. 01). The GO analysis showed that miR-21-5p played a regulatory role in DNA replication, transcription and vascular remodeling. The KEGG enrichment analysis showed that miR-21-5p mainly affected the occurrence and development of TNBC through mitogen activated protein kinase (MAPK) and transforming growth factor-β (TGF-J3) pathways. Conclusion: miR-21-5p is up-regulated in TNBC tissue and plays a positive regulatory role in the progression of TNBC, which may be a key biomarker for identifying the prognostic extent of TNBC. DUSP8 may be involved in the regulation of the occurrence and development of TNBC as a target gene of miR-21-5p.

Effect of metformin on the proliferation of and expressions of miR-21-5p and PDCD4 in HaCaT human keratinocytes / 中华皮肤科杂志

Objective To evaluate the effect of metformin on the proliferation of keratinocytes,and to investigate its possible mechanism.Methods HaCaT human keratinocytes were divided into several groups to remain untreated (control group) or be treated with different concentrations (25,50,75,100 mmol/L) of mefformin for 24 hours (intervention groups).Subsequently,CCK8 assay was conducted to evaluate the proliferation of HaCaT cells,real-time quantitative PCR to measure the mRNA expressions of miR-21-5p and its downstream target gene PDCD4,and Western blot to detect the expression of PDCD4 protein in HaCaT cells.Statistical analysis was done by using one-way analysis of variance for multiple group comparisons and SNK-q test for paired comparisons.Results After 24-hour treatment,the proliferation of HaCaT cells was inhibited by (5.43 ± 3.67)％,(19.61 ± 6.95)％,(45.93 ± 9.56)％ and (61.91 ± 6.93)％ by metformin of 25,50,75 and 100 mmol/L,respectively,with significant differences observed in cell proliferation inhibition rates among these intervention groups (F =246.90,P ＜ 0.05).Cellular proliferative activity was similar between the control cells (0.00 ± 3.00％) and those treated with 25 mmol/L metformin,but significantly higher in the control cells than in the other 3 metformin-treated groups (all P ＜ 0.05),and significantly different between the 4 metformin-treated groups (all P ＜ 0.05).The relative mRNA expression level (2-△△Q) of miR-21-5p was 0.90 ± 0.11,0.33 ± 0.05,0.21 ± 0.07 and 0.14 ± 0.04 (F =36.99,P ＜ 0.01),while that of PDCD4 was 2.11 ± 0.64,7.22 ± 1.13,11.16 ± 1.23 and 19.12 ± 3.16 (F =96.26,P ＜ 0.05),and the expression level of PDCD4 protein was 1.22 ± 0.08,2.09 ± 0.20,2.26 ± 0.1 1 and 2.37 ± 0.07 (F=75.37,P＜ 0.05),respectively,in HaCaT cells treated with metformin of 25,50,75 and 100 mmol/L.Similarly,no significant difference was observed between the control cells and those treated with 25 mmol/L metformin in the expression level of miR-21-5p mRNA,PDCD4 mRNA or protein,but decreased expression of miR-21-5p mRNA and increased expression of PDCD4 mRNA and protein were noted in cells treated with the other 3 concentrations of metformin compared with the control cells (all P＜ 0.05),and significant differences were also found in the expression levels of miR-21-5p mRNA as well as PDCD4 mRNA and protein among the 4 intervention groups (all P ＜ 0.05).Conclusion Metformin can markedly inhibit the proliferation of HaCaT cells in vitro,likely by downregulating miR-21-5p expression and upregulating PDCD4 expression.

The molecular mechanism of antiapoptosis of typeⅡ alveolar epithelial cell by microRNA-21-5p / 中华危重病急救医学

ObjectiveTo study the effect of hydrogen peroxide (H2O2) in inducing apoptosis of typeⅡalveolar epithelial cell (AECⅡ) after overexpression by adenoviral transfection of micro RNA-21-5p (miR-21-5p), and to explore the mechanism of its anti-apoptosis.Methods Subculture AECⅡ were randomly divided into four groups: normal control group (normal saline), H2O2 challenge group ( 0.5 mmol/L H2O2), miR-21-5p overexpression group (miR-21-5p adenovirus+ 0.5 mmol/L H2O2), miR-21-5p negative transfection group (adenovirus void+0.5 mmol/L H2O2). Transmission electron microscopy and flow cytometry were used to detect apoptotic morphology and early apoptotic rate. Real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of miR-21-5p in AECⅡ, and Western Blot was used to detect the protein expressions of Bcl-2, Bax, and caspase-3 at the highest transfection efficiency at different time points (6, 12, 24, 48 hours).Results ① AECⅡ identification: fluorescence microscopy showed the presence of characteristic structure of AECⅡ, i.e. microvilli and osmiophilic lamellar bodies.② Apoptotic morphology: transmission electron microscopy showed cytoplasmic retraction, chromatin condensation, margination, lack of cell surface microvilli, and emptying of osmiophilic lamellar bodies in AECⅡ.③ The expression of miR-21-5p in AECⅡ: the highest transfection efficiency was found at 48 hours. The expression of miR-21-5p in miR-21-5p overexpression group was significantly higher than that of the normal control group, H2O2 challenge group and miR-21-5p negative transfection group (A value: 1.54±0.02 vs. 1.02±0.02, 0.56±0.03, 0.58±0.02, allP< 0.05).④ The rate of early apoptosis: compared with normal control group, the early apoptotic rates in H2O2 challenge group, miR-21-5p negative transfection group and miR-21-5p overexpression group were gradually elevated with the prolongation of injury time. The early apoptotic rate in miR-21-5p overexpression group was significantly lower than that of the H2O2 challenge group and miR-21-5p negative transfection group at all time points except 6 hours [12 hours: (10.73±2.80)% vs. (16.26±0.59)%, (16.04±0.70)%; 24 hours:(16.00±3.44)% vs. (23.29±2.78)%, (23.58±2.31)%; 48 hours: (31.30±3.55)% vs. (50.53±2.17)%, (49.41±1.97)%, allP< 0.05]. There was no significant difference in early apoptotic rate between miR-21-5p negative transfection group and H2O2 challenge group at each time point.⑤ Protein expression: the expressions of Bax and caspase-3 in miR-21-5p overexpression group were significantly lower than those of the H2O2 challenge group and miR-21-5p negative transfection group [Bax (A value): 0.07±0.01 vs. 0.18±0.01, 0.13±0.01; caspase-3 (A value): 0.07±0.01 vs. 0.23±0.01, 0.12±0.01, allP< 0.05], and Bcl-2 protein expression was significantly higher than that of the H2O2 challenge group and miR-21-5p negative transfection group (A value: 0.26±0.01 vs. 0.06±0.01, 0.10±0.01, both P< 0.05).Conclusions① miR-21-5p has the function of anti-apoptosis of AECⅡ.② Adenoviral vector is a successful gene transfer vector when transfected with AECⅡ.③ The anti-apoptosis of AECⅡ by miR-21-5p may be associated with reduced Bax and caspase-3 protein levels and raised expression levels of Bcl-2 protein.