Expression of plasma miR-320c in patients with osteoarthritis and its effect on proliferation and apoptosis of chondrocyte / 中华老年医学杂志

Objective:To investigate the expression level of plasma miR-320c in patients with osteoarthritis(OA), and explore the clinical significance and the role in pathogenesis of OA.Methods:The clinical data and peripheral blood of 30 patients with OA, 30 patients with connective tissue diseases and 30 healthy control individuals were collected.The levels of plasma miR-320c were detected byfluorescentquantitative reverse transcription PCR(qRT-PCR). Correlation analysis was used to explore the correlation of plasma miR-320c level with knee X-ray data and VAS pain score in OA patients.Finally the miR-320c mimic, the miR-320c inhibitor, and the control material were transfected to the chondrocyte HC-a.The proliferative capacity of HC-a chondrocytes was examined at different time points as determined by the CCK-8 assay.Results:The expression level of plasma miR-320c was significantly higher in OA group(3.26±0.55)than that in the connective tissue diseases group(1.62±0.50)and in healthy control group(1.21±0.66)( F=107.66, P<0.001). Plasma miR-320c expression was positively correlated with radiographic grade( r=0.830, P<0.001), and had no correlation with VAS pain score in OA group( P>0.05). Through repeated measurement variance analysis, the time effect, the group effect and the interaction effect between group and time showed statistically significant differences in chondrocyte proliferation between NC mimic group and miR-320c mimic group( Ftime=5256.767, Fgroup=1947.436, Ftime×group=114.314, all P<0.001). The level of proliferation was significantly reduced.Apoptosis rate of chondrocytes was significantly increased in the group transfected with miR-320c( t=7.85, P<0.01). Conclusions:The expression level of plasma miR-320c is significantly higher in osteoarthritis patients and associated with knee radiographic severity grade.Furthermore, over-expression of miR-320c could suppress the proliferation of chondrocytes.Plasma miR-320c might be potential bio-marker for osteoarthritis knee severity assessment, and involves in regulating chondrocyte growth in the pathogenesis of osteoarthritis.

Effects of Linc00460 on Aerobic Glycolysis in Breast Cancer Cells via Sponge Adsorption of miR-320a / 肿瘤防治研究

Objective To explore the effect of Linc00460 on the aerobic glycolysis of breast cancer (BC) cells through sponge adsorption of miR-320a. Methods The qRT-PCR method was used to detect Linc00460 and miR-320a expression levels in normal breast epithelial cell line MCF-10A and five BC cell lines. The effect of interfering Linc00460 on miR-320a expression was detected by qRT-PCR. The double luciferase reporter gene experiment was used to analyze the targeting relationship between miR-320a and Linc00460. In addition, the si-Linc00460 and miR-320a inhibitor were co-transfected into MDA-MB-231 cells, and the expression level of miR-320a in the cells was detected by qRT-PCR; cell proliferation ability was measured by the MTT method; glucose uptake rate was detected by 2-NBDG method; the content of lactic acid in the cell supernatant was detected by colorimetric method; the key enzymes of glycolysis was detected by the enzyme activity kit; and the expression levels of the key proteins in the glycolysis pathway were detected by Western blot. Results Linc00460 was highly expressed in five BC cell lines, while miR-320a was lowly expressed as compared with MCF-10A cells. The expression of miR-320a in MDA-MB-231 cells significantly increased after interfering with Linc00460. The double luciferase reporter gene experiment confirmed that miR-320a and Linc00460 could target binding. Interfering with the expression of Linc00460 could inhibit MDA-MB-231 cells proliferation (all P=0.000), reduce the rate of cellular glucose uptake and the content of lactic acid in the cell supernatant (all P=0.000), inhibit the activities of PFK, PK, and LDH enzymes (all P=0.000), and downregulate the protein expression levels of PFKM, GLUT1, and LDHA (all P=0.000). Meanwhile, inhibition of miR-320a could reverse the inhibitory effect of si-Linc00460 on the proliferation and glycolysis of MDA-MB-231 cells (all P=0.000 or 0.001). Conclusion Linc00460 might adsorb miR-320a, consequently leading to upregulation of PFKM expression, thereby promoting aerobic glycolysis in BC cells.

The effect of aquaporin 4 regulated by microRNA-320a on a cell model of Alzheimer′s disease / 中华物理医学与康复杂志

Objective:To explore the effect of aquaporin 4 (AQP4) regulated by miR-320a on a cell model of Alzheimer′s disease.Methods:A rat adrenal pheochromocytoma cell line (PC12) was induced into neurons using nerve growth factor (NGF). The morphology of the PC12 cells and the neurons was observed, and ubiquitin carboxy terminal hydrolase L1 (Uch-L1) and neurofilament protein (NFP) were detected. Levels of microtubule-associated protein (MAP2) and AQP4 target genes were related to the mRNA expression of NFP to determine the neuron-inducing effect. The neurons were then randomly divided into a control group (given no treatment), an miR-320a mimic transfection group (cultured by adding 50nmol/L miR-320a as a mimic agent), an miR-320a inhibitor group (cultured by adding 50nmol/L miR-320a as an inhibitor), an Aβ treatment group (cultured by adding Aβ), an Aβ+ miR-320a mimic group (cultured by adding both 50nmol/L miR-320a and Aβ), and an Aβ+ miR-320a inhibitor group (also cultured by adding Aβ, but with 50nmol/L miR-320a as an inhibitor). Cell activity was measured by the CCK8 method. Reverse-transcription polymerase chain reactions were used to detect the relative expression of the target gene miR-320a, AQP4, B-cell bcl2-associated X (Bax), and B-cell bcl-2 (Bcl-2) mRNA. Western blotting was employed to detect the relative expression of AQP4, Bax and Bcl-2 proteins.Results:After PC12 was induced by 50μg/L NGF, the expression of Uch-L1 genes in the induced neuron was significantly down-regulated compared with the PC12. The expressions of NFP, MAP2 and AQP4 genes were significantly up-regulated, and the relative expressions of MAP2 and AQP4 proteins increased significantly. Compared with the control group, the apoptosis and cell activity of neurons in the treatment group increased, the mRNA and protein expressions of miR-320a, AQP4, bcl-2, AQP4 and Bcl-2 decreased significantly, and the mRNA and protein expressions of Bax increased significantly. Compared with the Aβ-treated group, the cell activity of the Aβ+ Mir-320a mimic group increased significantly, the mRNA and protein expressions of miR-320a, AQP4 and Bcl-2 increased significantly, and the mRNA and protein expressions of Bax decreased significantly. Compared with the Aβ+ miR-320a mimic group, the cell activity of the Aβ+ miR-320a inhibitor group decreased significantly, the mRNA and protein expressions of miR-320a, AQP4 and Bcl-2 decreased significantly, and the mRNA and protein expressions of Bax increased significantly.Conclusion:miR-320a can up-regulate AQP4 expression in a cell model of Alzheimer′s disease, reduce apoptosis and increase the cell survival rate.

The value of serum miR-320 expression in predicting the prognosis of sepsis children complicated with acute kidney injury / 中华实用儿科临床杂志

Objective:To investigate the prognostic value of serum levels of miR-320 in sepsis children complicated with acute kidney injury (AKI).Methods:A total of 158 sepsis children with complicated with AKI who were admitted to Hainan Women and Children′s Medical Center from January 2017 to June 2019 were divided into survival group (105 cases) and death group (53 cases) according to their 28-day survival.Serum levels of miR-320, neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and serum creatinine (Scr) were detected in the two groups.Multivariate Logistic regression was used to analyze the risk factors of death in children with sepsis complicated with AKI.The receiver operating characteristic curve (ROC) was drawn to analyze the value of serum levels of miR-320, NGAL, KIM-1 and Scr in predicting the death of children with sepsis complicated with AKI.The correlation between serum levels of miR-320 and NGAL, KIM-1 and Scr was analyzed using Pearson correlation analysis. Results:The serum levels of miR-320 (1.28±0.47 vs. 0.54±0.12), NGAL [(537.40±48.26) mg/L vs. (285.60±29.40) mg/L], KIM-1 [(26.80±5.72) μg/ L vs. (16.35±3.17) μg/L] and Scr[(573.70±105.46) μmol/L vs. (390.64±74.38) μmol/L] in the death group were significantly higher than those in the survival group (all P<0.001). Multivariate Logistic regression analysis showed that serum levels of miR-320 ( OR=2.280, 95% CI: 1.483-4.380), NGAL ( OR=2.753, 95% CI: 1.826-5.227), KIM-1 ( OR=1.985, 95% CI: 1.274-3.518) and Scr ( OR=1.714, 95% CI: 1.105-2.986) were independent risk factors for death in sepsis children with AKI (all P<0.001). ROC curve analysis showed that the area under the curve (0.952, 95% CI: 0.894-0.990) of serum miR-320, NGAL, KIM-1 and Scr levels combined to predict the death of children with sepsis and AKI was the largest, with a high sensitivity and specificity of 95.8% and 90.6%.Correlation analysis showed that the expression level of serum miR-320 in the death group was positively correlated with NGAL, KIM-1 and Scr ( r=0.874, 0.830, 0.702, all P<0.01). Conclusions:Serum levels of miR-320 are significantly increased in sepsis children with AKI, which is an independent risk factor for death in sepsis children with AKI.The combination of NGAL, KIM-1 and Scr levels has important value in predicting the prognosis of children with AKI.

Expression of miR-320a and CYLD in gastric cancer and its relationship with clinicopathological characteristics and prognosis / 实用肿瘤学杂志

Objective The aim of this study was to investigate the expression of miR-320a and cephalospermoma syndrome protein(CYLD)in patients with gastric cancer and its relationship with clinicopathological characteristics and prognosis. Methods A total of 460 patients with gastric cancer underwent tumor resection in our hospital from March 2013 to November 2014 were enrolled. Tumor tissues,non-tumor gastric mucosa tissues and normal tissues were collected. The expression of miR-320a and CYLD at levels of mRNA and protein were detected by Real-Time PCR,immunohistochemistry and Western blotting. The relationship between the expression of miR-320a and CYLD,and the clinicopathological features&prognosis of gastric cancer patients was analyzed. Results The relative expression of miR-320a and CYLD at the mRNA level in tumor tissues was(0. 37 ± 0. 09),(0. 91 ± 0. 23),and the relative expression in non-tumor tissues was(0. 86 ± 0. 15),(1. 56 ± 0. 42),respectively. The relative expression of miR-320a and CYLD mRNA in tumor tissues was significantly different from non-tumor tissues(t=60. 078,29. 113,P=0. 000),the positive ex-pression rate of CYLD protein in tumor tissues was 43. 48% when compared to 73. 91% in non-tumor tissues. The difference was statistically significant(χ2=86. 624,P=0. 003). The expression level of miR-320a was significantly associated with the diameter of the tumor and lymph node metastasis(χ2=25. 859 and 13. 742,P<0. 05). The expression of CYLD was also significantly associated with the TNM stage and degree of tumor differentiation(χ2=37. 725 and 59. 323,P<0. 05). The median survival of patients with low miR-320a expression(20. 36 months,95% CI:19. 252 ~21. 462 months) and those with high miR -320a expression(28. 29 months,95% CI:27.158~29.412 months)were statistically significant(χ2=87.967,P<0.001).The median survival of patients with CYLD negative expression(17. 70 months,95% CI:16. 599~18. 796 months)and those with CYLD positive expression(26. 74 months,95% CI:25. 474~27. 997 months)were statistically significant(χ2=109. 887,P<0. 001);The median survival of patients with the co-expressed miR-320a and CYLD was(29. 01 months,95% CI:26. 831~28. 946 months)and those with the non-co-expressed miR-320a and CYLD(17. 13 months,95% CI:17. 214~19. 568 months)were statistically significant(χ2=117. 680,P<0. 001). There showed a positive correlation between the expression of miR-320a and CYLD at mRNA level(r=0. 607,P<0. 001);miR-320a at the low expression,CYLD at the negative expression,TNM staging,lymph node metastasis and degree of tumor differen-tiation were independent risk factors for the prognosis of gastric cancer(HR=1. 939,2. 180,1. 561,1. 719,1. 608,95% CI:1. 141~3.295,1.252~3.796,1.014~2.403,1.115~2.650,1.097~2.357,respectively)(P<0.05).Conclusion The expressions of miR-320a and CYLD in tumor tissues of patients with gastric cancer is significantly decreased,which is related to the occurrence and development of diseases,and poor prognosis. It is a potential target for diagnosis and treatment of gastric cancer.

Role and molecular mechanism of miR-320 in the genesis and progression of tumors / 基础医学与临床

miR-320 is a newly discovered microRNA .In recent years, the abnormal expression of miR-320 has been found in various kinds of tumors .Therefore, its function and molecular mechanism in the tumorigenesis and tumor progression has getting more and more attention .

MiR-320 inhibits the growth of glioma cells through downregulating PBX3

BACKGROUND: MiR-320 is downregulated in multiple cancers, including glioma and acts as tumor suppressor through inhibiting tumor cells proliferation and inducing apoptosis. PBX3 (Pre-B cell leukemia homeobox 3), a putative target gene of miR-320, has been reported to be upregulated in various tumors and promote tumor cell growth through regulating MAKP/ERK pathway. This study aimed to verify whether miR-320 influences glioma cells growth through regulating PBX3. METHODS: Twenty-four human glioma and paired adjacent nontumorous tissues were collected for determination of miR-320 and PBX3 expression using RT-qPCR and western blot assays. Luciferase reporter assay was performed to verify the interaction between miR-320 and its targeting sequence in the 3' UTR of PBX3 in glioma cells U87 and U251. Increased miR-320 level in U87 and U251 cells was achieved through miR-320 mimic transfection and the effect of which on glioma cells growth, proliferation, cell cycle, apoptosis and activation of Raf-1/MAPK pathway was determined using MTT, colony formation, flow cytometry and western blot assays. PBX3 knockdown was performed using shPBX3 and the influence on MAPK pathway activation was evaluated. RESULTS: MiR-320 downregulation and PBX3 upregulation was found in glioma tissues. Luciferase reporter assays identified miR-320 directly blinds to the 3' UTR of PBX3 in glioma cells. MiR-320 mimic transfection suppressed glioma cells proliferation, and induced cell cycle arrest and apoptosis. Both miR-320 overexpression and PBX3 knockdown inhibited Raf-1/MAPK activation. CONCLUSION: MiR-320 may suppress glioma cells growth and induced apoptosis through the PBX3/Raf-1/MAPK axis, and miR-320 oligonucleotides may be a potential cancer therapeutic for glioma.

The impact of miR-320-3p on adipocyte differentiation / 天津医药

Objective To study the role of miR-320-3p in adipocyte differentiation. Methods Marrow mesenchymal stem cells were isolated from mice and cultured then induced with adipogenic agents for 3 days. The transcription level of miR-320-3p was examined by qRT-PCR. Stromal ST2 cells were transfected with miR-320-3p, followed by adipogenic treatment. Oil-red O staining and qRT-PCR were employed to assess the differentiation of adipocytes induced by miR-320-3p. Results The expression level of miR-320-3p increased in MSCs after adipogenic treatment (P < 0.01). Addition of miR-320-3p in ST2 cells promoted the formation of oil-red O positive adipocytes and up-regulated the expression levels of adipogenic transcription factors such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer binding protein-α(C/EBPα) and the marker gene adipocyte fatty acid binding protein 4 (FABP4),compared to cells that transfected with miR-320-3p mimics (P<0.05). Conclusion miR-320-3p promotes ST2 cells differentiation into adipocytes.