Objective To clarify that the vascular endothelial cell injury caused by obstructive sleep apnoea hypopnea syndrome (OSAHS) is partly mediated by miRNA-92a. Methods Serum miRNA-92a level was measured in patients who underwent polysomnography between January 2018 and December 2018. The correlation between miRNA-92a and OSAHS was analyzed. Meanwhile, endothelial cells were cultured in vitro, and morphological changes and JC-1 staining results of endothelial cells were observed after OSAHS serum stimulation, so as to further clarify the injury of endothelial cells. The changes of miRNA-92a target gene were detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blot to further clarify the mechanism of endothelial cell injury. Results Seventy-two patients received polysomnography, including 22 cases in the non-OSAHS group, 18 in the mild OSAHS group, 10 in the moderate OSAHS group, and 22 in the severe OSAHS group. Serum miRNA-92a level was significantly increased in the OSAHS patients, and it also increased with the aggravation of OSAHS severity. OSAHS serum significantly damaged endothelial cells. Endothelial cells were swollen, disordered arrangement, and unclear boundaries. JC-1 staining showed that green fluorescence was significantly enhanced compared with the control group. RT-PCR and Western blot showed that the expressions of Krüppel-like factor-2 (KLF-2), Krüppel-like factor-4 (KLF-4) and endothelial nitric oxide synthase (eNOS) were significantly decreased under OSAHS serum stimulation. Conclusion Serum miRNA-92a of OSAHS patients is significantly increased, and reduces the expression of target genes KLF-2, KLF-4 and eNOS, affects the mitochondrial function of endothelial cells, and injures endothelial cells.
Objective To explore the diagnostic value of miRNAs for pancreatic cancer. Methods PubMed, Scopus, Web of Science, CBM, CNKI, WanFang Data and VIP databases were retrieved from inception to December 31st 2015, to collect diagnostic accuracy studies about miRNAs for pancreatic cancer. Two reviewers independently screened literature, extracted data and assessed the risk of bias of included studies by using the QUADAS-2 tool. Then meta-analysis was performed using MetaDiSc 1.4 and Stata 12.0 softwares. Results A total of 40 articles involving 109 studies were included. The results of meta-analysis showed that the pooled Sen, Spe, +LR, –LR and DOR were 0.81 (95%CI 0.80 to 0.82), 0.77 (95%CI 0.75 to 0.78), 3.15 (95%CI 2.78 to 3.58), 0.27 (95%CI 0.24 to 0.31) and 13.58 (95%CI 10.89 to 16.94), respectively. The AUC of SROC was 0.86 (95%CI 0.84 to 0.88). Subgroups analysis showed that: as to diagnostic accuracy, Caucasian was superior to Asian (AUC=0.89vs. 0.84); multiple-miRNAs profiling-based assays was superior to single miRNA assays (AUC=0.91vs. 0.84). Conclusion Current evidence suggests that miRNA has potential diagnostic value for pancreatic cancer, particularly using multiple miRNAs. Due to limited quality of the included studies, more high quality studies are needed to verify the above conclusion.
ObjectiveTo investigate the expression of miRNA-1 in denervated skeletal muscle at different periods, and to explore effects of passive movement on the expression of miRNA-1 and differentiation of myoblasts in denervation-induced skeletal muscle atrophy in rats. MethodsTwenty-seven Sprague Dawley rats, weighing (200±10) g, were randomly divided into sham-operated group (group A, n=3), denervated group (group B, n=12), and passive movement group (group C, n=12). After the right sciatic nerve was exposed and dissociated, the sciatic nerve of 1 cm in length was removed in groups B and C; resection was not performed in group A. At 1 day after operation, passive flexion and extension movement was performed on the right hind limb in group C. At 6 hours in group A and at 3, 7, 14, and 28 days in groups B and C, 3 rats were sacrificed to measure the wet weight ratio of gastrocnemius muscle, to observe the diameter of the gastrocnemius muscle cell and evaluate the muscle atrophy by HE staining; RT-PCR was used to detect the mRNA expression of miRNA-1 and myocyte differentiation factor (MyoD), and immunohistochemistry to determine the protein expression of MyoD. ResultsAtrophy in various degrees was observed in denervated gastrocnemius muscle of groups B and C. The muscle fiber arranged in disorder and the diameter of the muscle cells decreased gradually with the time, without normal structure and morphology. The wet weight ratio and the cell diameter of the gastrocnemius in groups B and C were significantly less than those in group A (P<0.05); the wet weight ratio at 7, 14, 28 days and the cell diameter at 7, 14 days of group B were significantly greater than those of group A (P<0.05). The expressions of miRNA-1 and MyoD mRNA gradually increased with time in groups B and C, but were significantly less than those of group A at each time point (P<0.05). At 7, 14, and 28 days after operation, the expressions of miRNA-1 and MyoD mRNA in group C were significantly higher than those in group B (P<0.05). Immunohistochemical staining showed positive expression of MyoD in groups A, B, and C at each time point, but higher expression was observed in groups B and C than group A; the expression increased with time in groups B and C, and it was significantly higher in group C than group B. The correlation analysis results showed that the overall change trend of miRNA-1 and MyoD had no relation with the gastrocnemius wet weight ratio at 3 and 7 days (P>0.05), and had positive correlation at 14 and 28 days (P<0.05); positive correlation was found between the relative expression of MyoD and miRNA-1 mRNA (P<0.05). ConclusionPassive movement can prevent amyotrophy by increasing the expression of miRNA-1 and promoting the differentiation of myoblasts.
ObjectiveTo systematically review the diagnostic value of miRNAs for Alzheimer’s disease (AD).MethodsPubMed, Web of Science, EMbase, The Cochrane Library, CNKI, WanFang Data, VIP, and CBM databases were electronically searched to collect diagnostic tests of miRNAs for AD from inception to October 31, 2020. Two researchers independently screened literature, extracted data, and assessed the risk of bias of the included studies. RevMan 5.3 and Stata 14.0 software were used for meta-analysis. ResultsA total of 22 studies involving 4 006 subjects were included. The meta-analysis results showed that the pooled sensitivity, specificity, positive and negative likelihood ratios, diagnostic odds ratio, and the areas under the working characteristic curve of miRNA in AD diagnosis were 0.83 (95%CI 0.79 to 0.87), 0.80 (95%CI 0.76 to 0.83), 4.07 (95%CI 3.37 to 4.92), 0.21 (95%CI 0.17 to 0.27), 19.20 (95%CI 12.96 to 28.48) and 0.88 (95%CI 0.85 to 0.90), respectively. ConclusionThe current evidence shows that miRNAs have a high diagnostic value for AD. However, because of the limited quality and quantity of the included studies, more high-quality studies are required to verify the above conclusion.
ObjectiveTo explore expression, clinical and biological significance of plasma miRNA-196a from patients with advanced gastric cancer.MethodsReal time quantitative RT-PCR (qRT-PCR) method was used to detect the miRNA-196a levels in tissues and plasma from 75 gastric cancer patients and 35 benign gastric lesions controls. Then clinic pathological correlations of plasma miRNA-196a in 75 gastric cancer patients were analyzed. Twenty-five gastric cancer patients were randomized selected from 75 patients, to compare plasma miRNA-196a levels between preoperation and postoperation. Meanwhile, the effect of miRNA-196a on the invasion ability of gastric cancer MGC-803 cell line was observed in vitro.ResultsThe levels of miRNA-196a in both plasma and tissues from 75 gastric cancer patients were significantly increased compared with 35 benign gastric lesions controls (P<0.000 1). Clinic pathological data of 75 gastric cancer patients showed that the expressions of miRNA-196a were significantly up-regulated in gastric cancer patients with serosal invasion (P<0.001), lymph node metastasis (P=0.004), distant metastasis (P<0.001) and late clinical stage (P<0.001). The expression of miRNA-196a in peripheral plasma of patients with gastric cancer was significantly down regulated after operation (P<0.000 1). In vitro, overexpression of miRNA-196a significantly increased the invasion ability of MGC-803 cells (P<0.05), whereas knockdown of endogenous miRNA-196a significantly inhibited the invasion ability of MGC-803 cells (P<0.05).ConclusionsThe expression of miRNA-196a is up-regulated not only in peripheral plasma of patients with gastric cancer, but also with the progression of gastric cancer (serosal invasion, lymph node metastasis and distant metastasis). The up-regulation of miRNA-196a expression in peripheral plasma is mainly due to the release of primary tumor tissue. miRNA-196a is expected to be a prognostic marker and a potential therapeutic target for advanced gastric cancer.
Objective Hepatitis B virus X (HBx) protein is involved in the initiation and progression of hepatocellular carcinoma (HCC) by regulating the host protein-coding genes. Herein, we want to explore whether HBx protein can alter the expression of microRNAs (miRNAs) to promote proliferation and transformation in malignant hepatocytesin vitro. Methods MiRNA microarray and quantitative reverse-transcription polymerase chain reactions (qRT-PCRs) were performed to identify miRNAs that were differentially regulated by HBx protein in HCC cells. Protein and mRNA expression analyses, cell cycle and apoptosis analyses, and luciferase reporter assays were performed to delineate the consequences of miR-16 family repression in HepG2 cells. Results HBx protein induced widespread deregulation of miRNAs in HepG2 cells, and the downregulation of the miR-16 family was reproducible in HepG2, SK-HEP-1, and Huh7 cells. CCND1, a target gene of the miR-16 family, was derepressed by HBx protein in HepG2 cells. C-myc mediated the HBx-induced repression of miR-15a/16 in HepG2 cells. Ectopically expressed miR-15a/16 suppressed the proliferation, clonogenicity, and anchorage-independent growth of HBx-expressing HepG2 cells by arresting them in the G1 phase and inducing apoptosis, whereas reduced expression of miR-16 accelerated the growth and cell-cycle progression of HepG2 cells. Conclusions HBx protein altered thein vitro expression of miRNAs in host malignant hepatocytes, particularly downregulating the miR-16 family. Repression of miR-15a/16 is c-myc mediated and is required for the HBx-induced transformation of HepG2 cellsin vitro. Therefore, miR-16 family may serve as a therapeutic target for hepatitis B virus (HBV)-associated HCC.
Objective To screen pyroptosis-related miRNAs of acute aortic dissection (AAD) from the GEO database, and analyze and verify their functions. MethodsThe microarray data set based on the miRNA chip in the GEO database was downloaded, the differentially expressed miRNAs were screened, and the target genes were predicted by the miRWalk database. Pyroptosis-related genes (PRGs) were searched in the PubMed database with "pyroptosis" as the keyword, and the intersection of PRGs and differential miRNAs predicting target genes were taken as AAD PRGs by Venn diagram. GO and KEGG enrichment analyses were performed. CytoHubba was used to screen the critical AAD PRGs and then the AAD pyroptosis-related miRNAs were identified. Aortic tissues were collected from gender- and age-matched AAD patients and healthy people, and the critical PRGs and miRNAs were verified by Western blotting and RT-qPCR. ResultsA total of 46 AAD differentially expressed miRNAs were screened, and 49 AAD PRGs were obtained by Venn diagram. GO enrichment analysis showed that the genes played a vital role in apoptosis regulated by cysteine endopeptidases. KEGG analysis showed that the genes enriched in Salmonella infection, necroptosis, and Nod-like receptor signaling pathways. CytoHubba screened the critical AAD PRGs such as cysteine aspartase-1 (Caspase-1), tumor necrosis factor (IL)-1β, and tumor necrosis factor (TNF), then obtained 12 AAD pyroptosis-related miRNAs. Aortic tissues were collected from 6 AAD patients and 6 healthy people. There were 5 males and 1 females in the AAD group with an average age of 48.70±6.35 years, and 4 males and 2 females in the healty control group with an average age of 45.30±4.58 years. There was no statistical difference between the two groups in terms of gender, age, smoking history, hypertension, diabetes, or coronary heart disease (P>0.05). Western blotting and RT-qPCR results showed that Caspase-1 was up-regulated in the AAD patients' aortic tissues compared with the healthy aorta, and the corresponding miRNAs were miR-198, miR-3202, and miR-514b-5p, which were all down-regulated. Conclusion Through bioinformatics analysis and verification, the critical AAD PRGs are Caspase-1, IL-1β, and TNF, and Caspase-1 is up-regulated and 3 corresponding pyroptosis-related miRNAs are down-regulated, which provides new ideas for the molecular mechanism and targeted therapy of AAD cell pyroptosis.
ObjectiveTo investigate the effect of Smad4 on the fibrosis of tendon derived fibroblasts (TDFs) induced by transforming growth factor β1(TGF-β1) by targeted regulation of miRNA219-5P (miR219-5P). MethodsThe tendons donated by the volunteers were harvested to isolate and culture TDFs. The 3rd generation cells were used for experiment. Chemically synthesized miR219-5P mimics, miR219-5P inhibitor, and negative control sequences were transfected into TDFs. The gene expression of miR219-5P in TDFs was detected by real-time PCR, and the protein expression of Smad4 in TDFs was detected by Western blot at 48 hours after transfection. The combining sites of miR219-5P and Smad4 in 3'UTR district were predicted by informatics software. Wild type and mutant type reporter gene expression vectors were constructed and then targeted verification was carried out by the luciferase reporter gene test. Transfected TDFs were then induced by TGF-β1. The proliferation activity of the cells were measured by the cell counting kit 8 after culturing for 24, 48, and 72 hours. The expressions of fibrosis related proteins in TDFs were detected by Western blot at 72 hours. ResultsAfter TDFs were transfected by miR219-5P mimics, miR219-5P expression was significantly up-regulated, but the expressions of Smad4 was decreased subsequently (P<0.05). Intracellular expression of miR219-5P was inhibited by miR219-5P mimics inhibitor, however, the protein expression of Smad4 was significantly increased (P<0.05). Luciferase reporter gene test showed that luciferase activities were significantly decreased in pGL3-WT-Smad4+mimics group, but were significantly increased in pGL3-WT-Smad4+inhibitor group when compared with pGL3-WT-Smad4 transfected group (P<0.05), but no significant difference was found between GL3-MT-Smad4+mimics and pGL3-MT-Smad4+inhibitor groups (P>0.05). Cell proliferation and the fibrosis related proteins were increased in TGF-β1 induced TDFs, however, decreased in TGF-β1 induced TDFs after transfected by miR219-5P inhibitor (P<0.01). ConclusionmiR219-5P can significantly inhibit fibrosis of TDFs induced by TGF-β1 by down-regulating Smad4 expression.
ObjectiveTo explore the influence of miRNA-155/PU.1 signaling pathway blockade on bone marrow-derived dendritic cells (DCs) maturation and immune function of rat small intestinal transplantation. MethodsThe DCs were induced by adherent culture.The critical transcription factor gene PU.1 was designed and PU.1 siRNA was synthe-sized.The DCs were transfected by liposome transfection and a pair of PU.1 siRNA was screened according to the high silencing efficiency.The expressions of DCs surface markers CD80, CD86, and MHC-Ⅱamong three groups (PU.1 silent group, negative control group, and control group) were analyzed by flow cytometry.The IL-10 and IL-12p70 secretion levels in the supernatant were tested by ELISA method.The allogeneic T lymphocyte proliferation was tested by mixed lymphocyte reaction.The transfected cells were intravenously injected into the recipient rat on day 7 before intestinal transplantation.The survival conditions as well as pathological changes were observed in each group recipients. Results①The surface molecules CD80, CD86, and MHC-Ⅱin the PU.1 silent group were (27.0±5.6)%, (23.6±4.8)%, and (36.8±6.8)%, respectively; versus (74.0±9.4)%, (76.5±8.7)%, and (87.8±11.3)% in the negative control group, respectively, which were significantly lower in former and showing an in creased trend (P < 0.05).②Compared with the negative control group, IL-10 secretion level was significantly increased (P < 0.05), IL-12p70 secretion level significantly decreased (P < 0.05) in the PU.1 silent group.③The proliferation of T lymphocytes in the PU.1 silent group was significantly lower than that in the negative control group (P < 0.05).④When the transfected DCs were injected into the intestinal transplantation rats on day 7 before operation, the survival time was (14.3±3.3) d, (7.8±1.5) d, and (8.0±2.5) d in the PU.1 silent group, negative control group, and control group, respectively, which in the PU.1 silent group were significantly longer than that in the other two groups (P < 0.05), and the graft pathology showed that there were mild intestinal tissue damage, lymphocyte infiltration or villus edema in the PU.1 silent group. ConclusionmiRNA-155/PU.1 signaling pathway blockade could reduce DCs maturation and induce receptor-specific immune tolerance, which are proved both in vivo and in vitro.
ObjectiveTo explore the dynamic expression changes of neuronal growth and differentiation-associated miR-124a and miR-9 in the process of epileptogenesis. MethodsEstablish the lithium-pilocarpine induced status epilepticus (SE) rat model. Animal behavior change induced by SE as well as in the period of chronic epilepsy was observed by naked-eye or video-recording. Major time points for the study were chosen at 1d, 7d, 14d and 28d post-SE, on which the post-SE rats were decapitated and their hippocampal specimens were obtained. Total RNA from each specimen was extracted and qPCR was exploited to detect miR-124a and miR-9 expression in the specimens. Statistical analysis was used to show the dynamic expressional changes of miR-124a and miR-9 in rat hippocampus at 1d, 7d, 14d and 28d post-SE during the process of epileptogenesis. ResultsCompared with normal rats, the expression level of miR-124a in rat hippocampus did not show a significant difference at 1d post-SE, but it had shown markedly differences at 7d, 14d and 28d post-SE(P < 0.05), with a declining trend. Compared with normal rats, the expression level of miR-9 had demonstrated significant differences at 1d, 7d, 14d and 28d post-SE(P < 0.05)with a generally increasing trend, although there was slight fluctuation of expressional up-regulation at 7d post-SE. ConclusionNeuronal growth and differentiation-associated miR-124a and miR-9 had shown dynamic changes of down-regulation or up-regulation in the process of epileptogenesis. It can be suspected that miR-124a and miR-9 take part in hippocampal neurogenesis post-SE and be involved in epileptogenesis process.