Expression and diagnostic value of miRNA-29c in type 1 diabetic patients with early nephropathy / 中华内分泌外科杂志

Objective:To study the expression of miRNA-29c in type 1 diabetic patients with early nephropathy and its diagnostic value for early nephropathy.Methods:168 patients with type 1 diabetes who were treated in our hospital from Jan. 2019 to Mar. 2022 were retrospectively selected as the research subjects. According to the occurrence of nephropathy, they were divided into simple diabetes group (122 cases) and diabetic nephropathy group (46 cases). Serum miRNA-29c levels were detected by RT-PCR. The gender, age, course of disease, and serum miRNA-29c levels were compared between the two groups. Logistic regression was used to analyze the influencing factors of early nephropathy in patients with type 1 diabetes. The ROC curve was used to analyze the diagnostic value of miRNA-29c for early nephropathy in type 1 diabetes.Results:The course of disease, blood pressure (systolic blood pressure, diastolic blood pressure), HbA1c, TC and blood uric acid in early nephropathy group were higher than those in simple diabetes group, while albumin, total bilirubin and miRNA-29c were lower than those in simple diabetes group, and the difference was statistically significant ( P<0.05). Multivariate Logistic analysis showed: long disease duration ( OR=2.061, 95% CI=1.090-3.896), systolic blood pressure ( OR=1.143, 95% CI=1.023-1.279), diastolic blood pressure ( OR=1.151, 95% CI=1.022) -1.298), high HbA1c ( OR=1.317, 95% CI=1.049~1.653), high blood uric acid ( OR=1.306, 95% CI=1.028-1.659), low miRNA-29c ( OR=0.845,95% CI= 0.730-0.979) were the risk factors for early nephropathy in patients with type 1 diabetes ( P<0.05). ROC curve analysis showed that the cut-off value of miRNA-29c for the diagnosis of early renal disease was 0.952, the area under the curve (AUC) was 0.863 (95% CI: 0.801-0.925), and the sensitivity and specificity were 84.78% and 80.33%, respectively. Conclusion:Serum miRNA-29c in patients with early stage nephropathy of type 1 diabetes is in a low expression state, which is an influencing factor for early stage nephropathy in patients with type 1 diabetes, and has a good diagnostic value for early stage nephropathy.

Diagnostic value of miR-29 family in cancers: a Meta-analysis / 中国肿瘤生物治疗杂志

@#Objective: To determine the potential diagnostic value of miRNA-29 (miR-29) for malignant tumor. Methods: A systematic search of literature regarding miR-29 was performed in three English databases (PubMed, Web of Science, and Embase) and two Chinese databases (Chinese National Knowledge Infrastructure [CNKI] and WanFang). The retrieval was ended until September 15, 2018. Search terms included miRNA-29 (miR-29), tumor, cancer, serum, plasma, diagnosis, etc. Quality Assessment of Diagnostic Accuracy Studies-2 (QUADAS-2) was carried out to evaluate the quality of the selected articles. STATA12.0 was used to calculate the combined sensitivity, specificity, positive likelihood ratio (PLR), negative likelihood ratio (NLR) and diagnostic odds ratio (DOR). Subgroup analysis and Meta-regression analysis were carried out to explore the origin of heterogeneity. Results: Twenty eligible articles were selected from 1 172 literatures related to tumors and miR-29. The combined sensitivity was 0.76 (95%CI: 0.68-0.83), combined specificity was 0.83 (95%CI: 0.74-0.89), combined PLR was 4.5 (95%CI: 2.7-7.4), combined NLR was 0.28 (95%CI: 0.20-0.41), DOR was 16 (95%CI: 7-35), and theAUC was 0.86 (95%CI: 0.83-0.89). The combined specificity of plasma samples was higher than that of serum samples, and the difference was statistically significant (P<0.01). There was a higher diagnostic value of miR-29 for breast cancer and pancreatic cancer (DOR=101.52, 11.22), but lower diagnostic value for colorectal cancer and non-small cell lung cancer (DOR=5.05, 6.57); miR-29b showed a high diagnostic value for cancer (DOR=60.91). The publication bias was not obvious in this study (P>0.05). Conclusion: This systematic review and Meta-analysis suggests that miR-29 family is a potential biomarker in the diagnosis of cancers with great sensitivity and specificity.

Exosomes secreted from miRNA-29b-modified mesenchymal stem cells repaired spinal cord injury in rats

Exosomes, a kind of extracellular vesicle, are promising therapeutic agents for spinal cord injury (SCI). This article aimed to investigate effects of exosomes secreted from miRNA-29b-modified bone marrow mesenchymal stem cells (BMSCs) on SCI. Exosomes were extracted from BMSCs transfected with miRNA-29b or negative control (miR NC). SCI rats were injected intravenously with exosomes (control exosomes, miRNA-29b exosomes) and BMSCs (miR NC, miRNA-29b) through the tail vein. The expression of miRNA-29b in spinal cord tissues of SCI rats was detected by qRT-PCR. The hind limb motor function was evaluated by Basso Beattie Bresnahan (BBB) score. The histopathological damage and neuronal regeneration in spinal cord tissues was observed by HE staining and immunohistochemistry, respectively. The injection of miRNA-29b exosomes and miRNA-29b BMSCs both significantly increased the expression of miRNA-29b in spinal cord tissues of SCI rats (P<0.05). Compared with SCI rats, rats in the miRNA-29b exosomes and the miRNA-29b groups exhibited improved SCI, including increased BBB score, NF200 and GAP-43 positive neurons, as well as decreased contractile nerve cell numbers and GFAP positive neurons (all P<0.05). The relieving degree of SCI was significantly higher in the miRNA-29b exosomes group than in the miRNA-29b BMSCs group (P<0.05). Exosomes secreted from miRNA-29b-modified BMSCs were effective in the repair of SCI in rats.

Expression of miRNA-29a in U937 macrophages infected with Mycobacterium tuberculosis and its regulation of target genes / 中华临床感染病杂志

Objective To analyze the expression of miRNA-29a in U937 macrophages infected with Mycobacterium tuberculosis and its regulation of target genes.Methods The target genes of miRNA-29a were predicted with softwares miRnada,RNAhybrid and targetscan.The differentiation of U937 macrophages was induced by phorbol ester(PMA), the induced U937 cells were infected with bacilli calmette guerin (BCG).The expression levels of miRNA-29a and its target genes in U937 cells were detected with real-time fluorescence quantitative PCR(RT-fqPCR).The miRNA-29a over-and low-expression U937 macrophage cell lines were constructed and the levels of miRNA-29a and its garget genes were detected.The SPSS 18.0 software was used to analyze the data.Results As predicted by relevant softwares,the miRNA-29a regulate the expression of VEGFA,NFATC3 and TSC22D3 genes.After BCG infection,the expression of miRNA-29a increased to 1.33 fold(P <0.05), and the expression levels of VEGFA, NFATC3 and TSC22D3 were increased to 5.34,99.25 and 2.12 fold,respectively(P<0.01).In the miRNA-29a over-expressing U937 macrophages,the expression levels of VEGFA,NFATC3 and TSC22D3 were up-regulated to 1.35,1.29 and 3.38 fold,respectively(P<0.05 or <0.01).While in the U937 macrophages with miRNA-29a knock-down,the expression levels of VEGFA, NFATC3 and TSC22D3 were down-regulated to 0.07, 0.08 and 0.55 fold, respectively(P <0.01).Conclusion The results suggest that Mycobacterium tuberculosis infection can increase the expression of miRNA-29a in U937 macrophages,further targeting the regulation of VEGFA, NFATC3 and TSC22D3 gene expression, which may participate in the pathogenesis and development of tuberculosis.

Construction of miRNA-29b1 knockout mice based on CRISPR/Cas9 technology / 中国比较医学杂志

Objective To construct miRNA-29b1 gene knockout mice based on CRISPR/Cas9 technology. Methods To design and synthesize sgRNA according to the miRNA-29b1 sequence in Genbank .sgRNA and Cas9 were transcribed to RNA in vitro, these RNA were then microinjected into zygotes of C 57BL/6 mice.After mouse birth, the genome DNA was extracted and sequenced to identify its genotype; meanwhile , real-time PCR was used to assay the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney of mutated mice .Result A 20 bp sgRNA targeted on miRNA-29b1 was synthesized and transcribed to RNA with Cas 9.After microinjection, miRNA-29b1 gene-mutated mice were obtained.The sequencing results showed that there were two types of genotype for the mutated mice , one was 10 bp deletion, and another was 23 bp deletion accompanied with a 3 bp insertion.Compared with the wild-type mice, the expression of miRNA-29b1 in the heart, liver, spleen, lung and kidney was reduced significantly .Conclusions miRNA-29b1 gene knockout mice are constructed successfully by using CRISPR /Cas9 technology.

Effects of microRNA-29b on invasion and proliferation of human high peritoneal metastatic gastric cancer cells / 中华消化杂志

Objective To investigate the expression of microRNA-29b (miR-29b) in gastric cancer cell line GC9811 and high peritoneal metastatic gastric cancer cell line GC9811-P and its effect on invasion,proliferation and apoptosis.Methods The relative quantitative expression of miR-29b was detected by quantitative realtime polymerase chain reaction(qRT PCR).GC9811 cells were divided into three groups,miRNA down-regulated and transfected with lentiviruses LV-miR-29b inhibitor group,negative control group with negative transfection,and untransfected blank control group.GC9811-P cells were divided into three groups,miR-29b up regulated and transfected with lentiviruses LV miR 29b group,negative control with negative transfection group,and untransfected blank control group.The cell invasion ability was detected with Transwell assay,the cell proliferation ability was measured by methyl-thiazolyl tetrazolium (MTT) test,the colony forming ability was determined by plate colony formation assay,and the apoptosis was tested by flow cytometry.The expressions of miR 29b and secreted protein,acidic and rich in cystenie (SPARC) in gastric cell line GC9811-P,GC9811,MKN28M,MKN28NM and normal gastric cell line GES were determined by qRT-PCR,and the correlation was analyzed.Two independent samples t test or SNK-q test was performed for mean comparison between two groups,and one way analysis of variance was used for mean comparison among three groups.Results The relative quantitative expression of miR-29b inGC9811-P (0.21±-0.04) was significantly lower than that of GC9811 (1.00±0.03,t 28.140,P＜ 0.01).After GC9811 cells transfected with lentiviruses LV-miR-29b inhibitor,the expression of miR-29b (0.21±0.04) was significantly lower than that of control group (0.89±0.07) and blank control group (1.00±0.04,q 12.76,14.73,both P＜0.01).Compared with negative group,the transmembrane cell number and the clonality of miRNA up-regulated group raised(274.33± 9.03 vs 110.67 ± 13.69,t=9.981,P＜0.01;131.33±4.91 vs69.67±2.33,t 11.340,P＜0.01),and the results of MTT test also shows the proliferation was increased.After GC9811-P cells transfected with LV-miR-29b,the expression of miR 29b (4.08±0.20) was significantly higher than that of negative control group (1.15±0.05) and blank control group (1.00±0.10,q=21.73,22.81,both P＜0.01).Compared with negative group,the transmembrane cell number and the clonality of miRNA up regulated group reduced (51.33±5.55 vs 104.00±6.24,t 6.305,P＜0.01; 48.00±5.51 vs 113.33±5.17,t 11.340,P＜0.01),and the results of MTT test also shows the proliferation was weakened.Apoptosis assays demonstrated miR-29b promoted apoptosis; however,the difference was not statistically significant.The expression of miR-29b was negatively correlated with SPARC mRNA in gastric cancer cells (r=-0.97,P=0.03).Conclusions The low expression of miR-29b in high peritoneal metastatic gastric cancer cell inhibited the ability of invasion and proliferation.MiR-29b might be a new target of inhibiting peritoneal metastasis in gastric cancer.

The effect of miRNA-29a on the proliferation and apoptosis of HVSMCs cells / 重庆医学

Objective To detect the expression of miRNA‐29a in abdominal aortic aneurysm(AAA) patients and construct a recombinant lentiviral vector carrying miRNA‐29a ,transfection of human abdominal aortic vascular smooth muscle cells (HVSMCs) ,and to investigate the alteration on the proliferation in HVSMCs .Methods Collected 25 AAA patients′serum and 25 normal elderly serum specimens from March 2011 to March 2014 ,the mean age were 62 .9 ± 13 .6 and 61 .5 ± 11 .8 separately .Em‐ploy RT‐PCR analysis the miRNA‐29 expression in different group .The MCS‐CMV‐miRNA‐29a was transfected into HVSMCs cells ,the transfection efficiency was observed with fluorescence microscope ,the mRNA and COL1A1 protein expresstion was detec‐ted by RT‐PCR and Western blot respectively .MTS method was used to determine effects of cell inhibition rates of different groups ,cell apoptosis was analysised by Annexin V/7‐AAD double staining after 72 h .Results The expression of miRNA‐29a with AAA was lower than normal group .The transfection efficiency was (74 .25 ± 10 .10)% .miRNA‐29a mRNA and its target protein COL1A1 expression increased significantly compared with the control group after transfection ,MTS and Annexin V/7‐AAD results showed that cell apoptosis was decreased after miRNA‐29a overexpression ,cell proliferation was increased compared with the con‐trol group .Conclusion The miRNA‐29 expression decreased associated with the occurrence of abdominal aortic aneurysm ,but overexpress miRNA‐29a in HVSMC cells by lentivirus mediated ,which could promote the proliferation of cells ,inhibit its apoptosis .