Inhibitory effect of siRNA-KCNQ1OT1 on human lens epithelial cell apoptosis by targeting miR-199a-5p / 中华实验眼科杂志

Objective:To explore the inhibitory effect of long non-coding RNA (lncRNA) KCNQ1 overlapping transcript 1 (KCNQ1OT1) by targeting microRNA-199a-5p (miR-199a-5p) on the apoptosis of human lens epithelial cells (LECs).Methods:The anterior lens capsule tissue of 23 age-related cataract patients who underwent cataract surgery in Xinxiang First People's Hospital from December 2018 to August 2019 was collected.At the same time, anterior lens capsules from 20 healthy donor were collected.The expressions of KCNQ1OT1 and miR-199a-5p in the tissues were detected by real-time fluorescence PCR.Human LECs SRA01/04 cultured in vitro were divided into blank control group, model control group, small interfering RNA-negative control (siR-NC) group, siR-KCNQ1OT1 group, miR-NC group, miR-199a-5p group, siR-KCNQ1OT1+ anti-miR-NC group and siR-KCNQ1OT1+ anti-miR-199a-5p group.No intervention was administered to blank control group.Cells in model control group were cultured with 100 μmol/L H 2O 2 for 24 hours to establish oxidative stress injured model, and cells in the other six groups were transfected with corresponding transfection reagents for 6 hours by liposome method according to grouping, and then treated with 100 μmol/L H 2O 2 for 24 hours.The expressions of KCNQ1OT1 and miR-199a-5p in lens anterior capsule tissue and LECs cells were determined by real-time fluorescent quantitative PCR.Cell viability was detected with thiazolyl blue (MTT). Cell apoptosis was analyzed by flow cytometry.The expressions of B-cell lymphoma/leukemia-2 (bcl-2) and bcl-2 related X protein (Bax) proteins were assayed by Western blot.The superoxide dismutase (SOD) activity and malondialdehyde (MDA) content were measured by enzyme-linked immunosorbent assay (ELISA). The targeting relationship between KCNQ1OT1 and miR-199a-5p was verified by dual luciferase reporter experiment.The study protocol was approved by an Ethics Committee of Xinxiang First People's Hospital (No.2019-001). Written informed consent was obtained from relatives of patient. Results:The relative expression of KCNQ1OT1 in the anterior capsule of patients with age-related cataract was 2.41±0.42, which was significantly higher than 0.97±0.19 of normal people, and the relative expression of miR-199a-5p in the capsule of patients with age-related cataract was 0.36±0.12, which was lower than 1.04±0.15 of normal people, and the differences were statistically significant ( t=14.112, 16.507; both at P<0.001). Compared with blank control group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content were significantly increased, and the relative expressions of miR-199a-5p and bcl-2 protein, cell viability and SOD activity were significantly reduced in model control group, showing statistically significant differences (all at P<0.001). Compared with siR-NC group, the relative expressions of KCNQ1OT1 and bax protein, cell apoptosis rate and MDA content in cells of siR-KCNQ1OT1 group were decreased, while the relative expression of bcl-2 protein, cell survival rate and SOD activity were increased, and the differences were statistically significant (all at P<0.05). Compared with miR-NC group, the KCNQ1OT1-wild type (WT) luciferase activity in miR-199a-5p group was significantly decreased, with a statistically significant difference ( t=21.131, P<0.001). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly increased, and the relative expression of bax protein, cell apoptosis rate and MDA content were significantly decreased in miR-199a-5p group than those in miR-NC group, and the differences were statistically significant (all at P<0.05). The relative expression levels of miR-199a-5p and bcl-2 proteins, cell survival rate and SOD activity were significantly lower, and the cell apoptosis rate, relative expression of bax protein and MDA content were significantly higher in siR-KCNQ1OT1+ anti-miR-199a-5p group than those in siR-KCNQ1OT1+ anti-miR-NC group, and the differences were statistically significant (all at P<0.05). Conclusions:The inhibition of KCNQ1OT1 can promote the cell viability of human LECs, inhibit H 2O 2-induced cell apoptosis and oxidative stress, and the mechanism may be related to the up-regulation of miR-199a-5p.

Expression and clinical significance of visfatin, hsa-miR-199a and extracellular signal regulating protein kinase 1/2 in colorectal cancer / 解剖学报

Objective To investigate the expression and correlation of visfatin, hsa-miR-199a and extracellular regulated protein kinase 1/2 (ERK1/2) in colorectal cancer (CRC) tissues. Methods The expression levels of visfatin mRNA and hsa-miR-199a were detected by Real-time PCR in 64 primary cases of radical resection of colorectal cancer who underwent pathological clinical examination in the clinicopathological examination in our hospital center from January 2017 to December 2017, the protein expression levels of visfatin and ERK1/2 were detected by immunohistochemistry and Western blotting. The relative expression levels of hsa-miR-199a in each group were compared according to the expression levels of visfatin and ERK1/2. Results The levels of visfatin gene and protein in colorectal cancer tissue (observation group, 64 cases) were higher than those in normal colorectal cancer (control group, 64 cases), and the expression was consistent. The colorectal cancer tissue(observation group) expression level of hsa-miR-199a was lower than that of normal colorectal tissue(control group), and visfatin mRNA was significantly negatively correlated. The colorectal cancer tissue (observation group) expression of ERK1/2 was higher than normal colorectal tissue (control group) and visfatin was significantly positively correlated. The relative expression of hsa-miR-199a in the visfatin and ERK1/2 positive group was significantly different from that in the test variables. Conclusion The overexpression of visfatin in colorectal cancer affects the down-regulation of hsa-miR-199a and is related to the ERK signaling pathway.

miR-199a-5p enhances expression of myocardial fibrosis-related genes by targeting SIRT1 / 中国病理生理杂志

AIM:To investigate the role of microRNA (miR)-199a-5p in myocardial fibrosis and the potential target of miR-199a-5p.METHODS:C57BL/6 mouse cardiac fibroblasts were isolated and cultured for cellular experimen-tal study.Dual-luciferase reporter assay was performed to confirm the interaction between miR-199a-5p and the 3'-untrans-lated region (3'-UTR) of silent information regulator 1 (SIRT1).The expression of SIRT1 and fibrosis markers collagen (Col) 1a1, Col3a1 and α-smooth muscle actin (α-SMA) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively.RESULTS:The expression levels of miR-199a-5p, Col1a1, Col3a1 andα-SMA were marked-ly increased in cardiac fibroblasts after treatment with angiotensin Ⅱ(AngⅡ).The over-expression of miR-199a-5p signif-icantly increased the expression of Col1a1, Col3a1 andα-SMA in cardiac fibroblasts.Moreover, the results of dual-lucifer-ase reporter assay revealed that miR-199a-5p interacted with the 3'-UTR of SIRT1.miR-199a-5p inhibited SIRT1 expres-sion at post-transcriptional level.Meanwhile, miR-199a-5p mimic, in parallel to SIRT1 siRNA, inhibited SIRT1 expres-sion, increased the expression of Col1a1, Col3a1 and α-SMA in cardiac fibroblasts.Inactivation of NF-κB signaling con-tributed to the decrease in miR-199a-5p in Ang II-treated cardiac fibroblasts .CONCLUSION:SIRT1 is a target gene of miR-199a-5p, which mediates the pro-fibrotic effect of miR-199a-5p on cardiac fibroblasts .

miR-199a-5p enhances expression of myocardial fibrosis-related genes by targeting SIRT1 / 中国病理生理杂志

AIM:To investigate the role of microRNA (miR)-199a-5p in myocardial fibrosis and the potential target of miR-199a-5p.METHODS:C57BL/6 mouse cardiac fibroblasts were isolated and cultured for cellular experimen-tal study.Dual-luciferase reporter assay was performed to confirm the interaction between miR-199a-5p and the 3'-untrans-lated region (3'-UTR) of silent information regulator 1 (SIRT1).The expression of SIRT1 and fibrosis markers collagen (Col) 1a1, Col3a1 and α-smooth muscle actin (α-SMA) at mRNA and protein levels was determined by RT-qPCR and Western blot, respectively.RESULTS:The expression levels of miR-199a-5p, Col1a1, Col3a1 andα-SMA were marked-ly increased in cardiac fibroblasts after treatment with angiotensin Ⅱ(AngⅡ).The over-expression of miR-199a-5p signif-icantly increased the expression of Col1a1, Col3a1 andα-SMA in cardiac fibroblasts.Moreover, the results of dual-lucifer-ase reporter assay revealed that miR-199a-5p interacted with the 3'-UTR of SIRT1.miR-199a-5p inhibited SIRT1 expres-sion at post-transcriptional level.Meanwhile, miR-199a-5p mimic, in parallel to SIRT1 siRNA, inhibited SIRT1 expres-sion, increased the expression of Col1a1, Col3a1 and α-SMA in cardiac fibroblasts.Inactivation of NF-κB signaling con-tributed to the decrease in miR-199a-5p in Ang II-treated cardiac fibroblasts .CONCLUSION:SIRT1 is a target gene of miR-199a-5p, which mediates the pro-fibrotic effect of miR-199a-5p on cardiac fibroblasts .

Identification of targeting relationship between mouse lncRNA-H19 and miR-199 a-5 p by dual-luciferase reporter assay / 中国病理生理杂志

AIM:To validate the association between long noncoding (lncRNA)-H19 and microRNA-199a-5p (miR-199a-5p) through the dual-luciferase reporter gene system by construction of a luciferase reporter vector containing the gene of lncRNA-H19.METHODS:The potential complementary binding sites of lncRNA-H19 and miR-199a-5p were predicted by RegRNA 2.0.The H19 gene or its mutant ( Mut) fragment was cloned into luciferase reporter vector psi-CHECK-2.Restriction enzyme analysis and sequence analysis were used to identify whether the recombinant plasmids of the H19 and H19-Mut were successfully constructed .miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimics neg-ative control or miR-199a-5p inhibitor negative control was co-transfected into the 293T cells with the luciferase reporters containing H19 or H19-Mut.Dual-luciferase reporter assay was performed to detect the luciferase activity in different groups in order to verify the relationship between lncRNA-H19 and miR-199a-5p.RESULTS:The results of double enzyme diges-tion and DNA sequencing showed that the sequence of luciferase reporter vector was correct .The results of dual-luciferase reporter assay indicated that the H 19 reporter gene luciferase activity significantly decreased in miR-199a-5p mimics group by 49%(P<0.01), and the H19 reporter gene luciferase activity was obviously upregulated in miR-199a-5p inhibitor group compared with miR-199a-5p mimics group ( P<0.01).However, miR-199a-5p mimics, miR-199a-5p inhibitor, miR-199a-5p mimics negative control and miR-199a-5p inhibitor negative control showed no effect at H 19-Mut reporter gene.CONCLUSION:lncRNA-H19 binds to miR-199a-5p to exert an inhibitory effect at transcriptional level .

Effect of miR-199a/b-3p on cell proliferation of TNBC cells / 中国免疫学杂志

Objective:To analyze the inhibiting mechanism of microRNA-199a/b-3p(miR-199)on cell proliferation of triple negative breast cancer(TNBC)cells.Methods:Expression of miR199 in BT549 and MDA-MB-231 cells transfected with miR-199a/b-3p was detected by qRT-PCR.The proliferation of BT549 and MDA-MB-231 cells transfected with miR-199 were analysed by MTT as-say.Cell cycle of TNBC cells transfected with miR-199 was detected by Flow Cytometry assay.Results:MiR-199a/b-3p could suppress the proliferation of BT549 and MDA-MB-231 cells.Comparing with normal control,the proliferation rate were up to(41.02±2.34)%and(28.42 ±6.70 )%,and the cell cycle were arrest at G 1 phase.Conclusion: MiR-199a/b-3p could suppress the proliferation of TNBC,and may be a promising anti-cancer drug for TNBC.

Value of serum microRNA-199a/b-3p on early diagnosis of hepatocellular carci-noma / 中国免疫学杂志

Objective:To analyze the expression levels of serum microRNA-199a/b-3p ( miR-199-3p) in hepatocellular carcinoma (HCC) patients and to explore whether miR-199a/b-3p could be a novel biomarker of early HCC.Methods: Patients with normal liver,liver cirrhosis and early HCC in clinic were included in this study.Blood samples of the patients were collected for analy-sis.Real time quantitative PCR technique was used to measure the expression level of serum miRNA-199a/b-3p.The AFP expression was got from clinical data.Results: Comparing with normal liver and liver cirrhosis patients, miRNA-199a/b-3p presented lower expressions in early HCC patients (P<0.05);the accuracy and positive rate of serum miR-199a/b-3p detection in cirrhosis and HCC were better than AFP detection.Conclusion:Data indicated that miRNA-199a/b-3p had diagnostic value for indicating liver cirrhosis and early HCC.

Effect of miR-199a/b-3p on cell motility of breast cancer cells / 中国免疫学杂志

Objective:To analyze the inhibiting mechanism of microRNA-199a/b-3p ( miR-199a/b-3p) on cell motility of breast cancer cells.Methods:The expression of PAK4 in MDA-MB-231 cells transfected with miR-199a/b-3p was detected by Western blot.The biology behaviors of MDA-MB-231 cells transfected with miR-199a/b-3p or PAK4 SiRNA were analysed by cell migration assay,invasion assay and protrusion dynamics.Results: MiR-199a/b-3p could suppress the expression of PAK 4 in MDA-MB-231 cells.Comparing with normal control ,miR-199a/b-3p or PAK4 SiRNA could suppress the migration ,invasion and membrane protrusion of MDA-MB-231 cells.Conclusion:miR-199a/b-3p could suppress the expression of PAK4,which are considered key breast cancer suppressors and inhibit the cell motility of breast cancer cells.

Inhibition effect of interruption PAK4 expression on migration and invasion of hepatocellular carcinoma / 中国免疫学杂志

Objective:To analyze the effect of PAK4 interruption by microRNA-199a/b-3p (miR-199a/b-3p) on migration and invasion of hepatocellular carcinoma (HCC).Methods: To test targeting of PAK4 by miR-199a/b-3p,we used luciferase assay in HEK293T cells cotransfected miR-199a/b-3p mimcs and pmirGLO-PAK4 3′UTR.The expression of PAK4 in SMMC-7721 transfected with miR-199a/b-3p was detected by Western blot.The biology behaviors of SMMC-7721 cells transfected with miR-199a/b-3p or PAK4 Si were analysed by cell migration assay and invasion assay.Results:MiR-199a/b-3p could suppress the mRNA and protein ex-pression of PAK4 by targeting PAK4 3′UTR,and the downregulating PAK 4 expression suppress the migration and invasion of SMMC-7721 cells.Conclusion: MiR-199a/b-3p could suppress the expression of PAK 4, which are considered key HCC suppressors and inhibit the migration and invasion of HCC cells.

Relationship of clinical outcomes and expression of microRNA-199 a/b-3 p in HCC / 中国免疫学杂志

Objective:To investigate the expression of microRNA-199a/b-3p (miR-199a/b-3p) in hepatocellular carcinoma ( HCC) tissues,and to explore the relationship with clinical outcomes. Methods: Real time quantitative PCR technique was used to measure the expression of miRNA-199a/b-3p in HCC tissues. The correlation between miR-199a/b-3p expression and the clinic pathological features of patients were analyzed. Results: Comparing with adjacent control, miRNA-199a/b-3p presented lower expressions in HCC tissues (P<0. 05);lower miR-199a/b-3p was found correlated with metastasis and poor survival. Conclusion:MiR-199a/b-3p take a crucial role in HCC metastasis and recurrence.