Remyelination Regulated by microRNAs in Neurodegenerative Diseases： A Review / 中国实验方剂学杂志

Demyelination of the central nervous system often occurs in neurodegenerative diseases， such as multiple sclerosis （MS）. The myelin sheath， a layer of myelin membrane wrapping the axon， plays a role in the rapid conduction and metabolic coupling of impulses for neurons. The exposure of the axon will lead to axonal degeneratio， and further neuronal degeneration， which is the main cause of dysfunction and even disability in patients with demyelinating neurodegenerative diseases. In addition to the demyelination of mature myelin sheath， remyelination disorder is also one of the major reasons leading to the development of the diseases. The myelin sheath is composed of oligodendrocytes （OLs） derived from oligodendrocyte progenitor cells （OPCs） which are differentiated from neural stem cells （NSCs）. The process of myelin regeneration， i.e.， remyelination， is the differentiation of NSCs into OLs. Recent studies have shown that this process is regulated by a variety of genes. MicroRNAs， as important regulators of neurodegenerative diseases， form a complex regulatory network in the process of myelin regeneration. This review summarizes the main molecular pathways of myelin regeneration and microRNAs involved in this process and classifies the mechanisms and targets. This review is expected to provide a theoretical reference for the future research on the treatment of demyelinating diseases by targeting the regulation of microRNAs.

MiR-15b-5p Promotes Growth and Metastasis of Renal Clear Cell Carcinoma / MiR-15b-5p Promueve el Crecimiento y la Metástasis del Carcinoma Renal de Células Claras

SUMMARY: We investigated the expression and clinical significance of miR-15b-5p in clear cell renal cell carcinoma (RCC) through bioinformatics analysis and experimental verification. The differentially expressed miRNAs were screened in the GEO database. Venn diagram showed that there were 5 up-regulated miRNAs (has-miR-210, has-miR-142-3p, has-miR-142-5p, has-miR-15b-5p, and has-miR-193a-3p) and only 1 down-regulated miRNA (has-miR-532-3p) that were commonly expressed between GSE189331 and GSE16441 datasets. This was further confirmed in TCGA. Further analysis showed that the has-miR-193a-3p, has-miR-142-3p, has- miR-142-5p, and has-miR-15b-5p were closely related to tumor invasion, distant metastasis and survival probability. The expression of miR-15b-5p in ccRCC tissues was significantly higher than that in adjacent normal kidney tissues (P0.05). Following inhibition of miR-15b-5p expression, RCC cells had attenuated proliferation, increased apoptosis, and attenuated migration and invasion. has-miR-15b-5p-WEE1, has-miR-15b-5p-EIF4E, has-miR-15b-5p-PPP2R1B may be three potential regulatory pathways in ccRCC. miR-15b-5p is highly expressed in cancer tissues of ccRCC patients. It may promote proliferation, inhibit apoptosis and enhance cell migration and invasion of RCC cells. The has-miR-15b-5p-WEE1, has-miR-15b-5p-EIF4E, and has-miR-15b-5p-PPP2R1B may be three potential regulatory pathways in ccRCC.

Investigamos la expresión y la importancia clínica de miR-15b-5p en el carcinoma de células renales (CCR) de células claras mediante análisis bioinformático y verificación experimental. Los miARN expresados diferencialmente se examinaron en la base de datos GEO. El diagrama de Venn mostró que había 5 miARN regulados positivamente (has-miR-210, has-miR-142-3p, has-miR-142-5p, has-miR-15b-5p y has-miR-193a-3p). ) y solo 1 miARN regulado negativamente (has-miR-532-3p) que se expresaron comúnmente entre los conjuntos de datos GSE189331 y GSE16441. Esto fue confirmado aún más en TCGA. Un análisis más detallado mostró que has-miR-193a-3p, has-miR-142-3p, has- miR-142-5p y has-miR-15b-5p estaban estrechamente relacionados con la invasión tumoral, la metástasis a distancia y la probabilidad de supervivencia. La expresión de miR-15b-5p en tejidos ccRCC fue significativamente mayor que la de los tejidos renales normales adyacentes (P 0,05). Tras la inhibición de la expresión de miR-15b-5p, las células RCC tuvieron una proliferación atenuada, un aumento de la apoptosis y una migración e invasión atenuadas. has-miR-15b-5p-WEE1, has- miR-15b-5p-EIF4E, has-miR-15b-5p-PPP2R1B pueden ser tres posibles vías reguladoras en ccRCC. miR-15b-5p se expresa altamente en tejidos cancerosos de pacientes con ccRCC. Puede promover la proliferación, inhibir la apoptosis y mejorar la migración celular y la invasión de células RCC. has-miR-15b-5p-WEE1, has- miR-15b-5p-EIF4E y has-miR-15b-5p-PPP2R1B pueden ser tres posibles vías reguladoras en ccRCC.

miR604A>G gene polymorphism is associated with recurrent pregnancy loss in Turkish women

SUMMARY OBJECTIVE: Recurrent pregnancy loss is considerably a reproductive health problem for couples. Genetic, epigenetic, and environmental factors play an important role in the development of recurrent pregnancy loss. While there are many causes, genetic and epigenetic factors are common. In this study, we aimed to examine the association between miR604 (rs2368393) A>G gene polymorphism and the risk of recurrent miscarriage in the Turkish population. METHODS: The study included 250 participants (i.e., 150 patients and 100 controls). DNA samples were isolated from peripheral blood, and polymerase chain reactions and restriction fragment length polymorphism methodologies were applied. RESULTS: The genotype distribution and allele frequencies of miR604A>G gene showed statistically significant differences between patients and control groups (p=0.002 and p<0.002, respectively). CONCLUSION: As a result of the study, we found that the AA genotype and A allele of the miR604A>G gene were statistically significant for the risk of recurrent pregnancy loss in Turkish women.

Total oxidant capacity, total antioxidant capacity, ischemic modified albumin, microRNA levels, and their relationship with psoriasis area and severity index

Aims: To examine the differences in the levels of microRNA, ischemic modified albumin (IMA), total oxidant capacity (TOC), and total antioxidant capacity (TAC) of persons with and without psoriasis and, in the case group, the relationship between these parameters and psoriasis area and severity index (PASI). Methods: Blood samples were collected from patients and healthy participants to examine levels of these parameters. Results: The mean serum TOC level was higher in the case group. The mean serum TAC and IMA levels were significantly lower in the case group (P <0.001). It was observed that the mean serum miR-203 and miR-146a levels were increased in psoriasis patients. It was determined that there was only a significant positive weak correlation between miR-203 and PASI (r = 0.232, P = 0.027). Limitations: The small sample size, not controlling serum albumin and not evaluating the effects of the treatment agents used by the patients on oxidative and inflammatory processes. Conclusion: In the case group changes in the mean serum TOC and TAC levels provide evidence that oxidative stress may play a critical role in disease pathogenesis. The increase in the mean serum miR-203 and miR-146a levels suggest the possibility of therapies targeting these microRNAs as a new option

MicroRNA-150-5p inhibits the proliferation and invasion of human larynx epidermiod cancer cells though regulating peptidyl-prolyl cis/trans isomerase

Abstract Objective This study aimed to investigate the molecular mechanism of miR-150-5p regulating the malignant biological behavior of Human Epidermoid cancer cell (HEp-2) by targeting peptidyl-prolyl cis/trans isomerase NIMA-Interacting-1 (PIN1). Methods Firstly, qRT-PCR and Western blot were adopted to detect the expression levels of miR-150-5p and PIN1 in cancer tissue and paracancerous tissues of patients with LSCC, and those in human bronchial epithelial cells (16 HBE) and HEp-2. Next, the targeted relationship between miR-150-5p and PIN1 was assessed by bioinformatics website and dual-luciferase reporter assay, followed by their correlation analysis. Besides, after interfering with miR-150-5p or PIN1 expression in HEp-2 cells, CCK-8, cell colony formation assay, and transwell assay were utilized to detect the proliferation, viability, and invasion of cells, respectively. Subsequently, the protein levels of MMP-2, MMP-9, and EMT-related proteins in HEp-2 cells were checked by Western blot. Results Expression of miR-150-5p was down-regulated in LSCC tissues and HEp-2 cells. Moreover, miR-150-5p suppressed proliferation and invasion of HEp-2 cells, affected protein expression related to MMP and EMT, thereby inhibiting development of cancer. The expression of PIN1 was significantly increased in cancer tissues and HEp-2 cells, and there was a targeted relationship and negative correlation between miR-150-5p and PIN1 in cancer tissue. However, overexpression of PIN1 could reverse the effect of miR-150-5p on the proliferation and invasion of HEp-2 cells. Conclusion In a nutshell, there is a targeted relationship between PIN1 and miR-150-5p. Besides, miR-150-5p can inhibit the proliferation and invasion of HEp-2 cells by regulating the expression of PIN1. Level of evidence 3.

The role and clinical significance of microRNA-29a-3p in the development of hypopharyngeal carcinoma

Abstract Objective MicroRNA-29a-3p has been reported in a variety of cancers, but its role in hypopharyngeal cancer remains unclear. This study was to determine the role of microRNA-29a-3p in the occurrence and development of hypopharyngeal cancer. Methods 40 patients with hypopharyngeal cancer who underwent surgery in the Affiliated Hospital of Jining Medical University from April 2013 to November 2017 were selected for this study. The cancer tissue samples of the patients were collected, and the patients were followed up for three years. The expression of microRNA-29a-3p in tissue samples was detected by in situ hybridization with fluorescent probe, and the relationships among microRNA-29a-3p and clinicopathological factors, postoperative recurrent-metastasis, survival time were studied. Immunohistochemical was used to detect the expression of Ki67 and E-cadherin in tissue samples. Results Combined with HE staining results showed that microRNA-29a-3p expression was relatively high in non-cancer tissue cells (red blood cells and fibroblasts in tumor interstitial vessels), but was relatively low in cancer tissue and cells. According to the follow-up data of 40 patients with hypopharyngeal cancer, tumor size, T-stage, tumor differentiation, postoperative recurrent-metastasis of hypopharyngeal cancer patients were significantly negatively correlated with microRNA-29a-3p (p< 0.05). Immunohistochemica results further confirmed that microRNA-29a-3p was negatively correlated with the expression of Ki67 and E-cadherin. The survival time of patients positively related with microRNA-29a-3p expression (p< 0.05). Moreover, ROC curve analysis showed that the area under the curve of the combined detection of miRNA-29a-3p+Ki67+E-cadherin was larger than that of the single detection of the three indexes. Conclusions The expression of microRNA-29a-3p is closely related to the occurrence, development and prognosis of hypopharyngeal cancer, and it affects the proliferation and invasion. This indicates that microRNA-29a-3p serves as a therapeutic target for the occurrence and development of hypopharyngeal cancer. The evidence of study designs of this study is IV using "Oxford Centre for Evidence-Based Medicine 2011 Levels of Evidence".

Targeting microRNA-125b inhibited the metastasis of Alisertib resistance cells through mediating p53 pathway / 中华肿瘤杂志

Objective: To clarify the mechanisms involvement in Alisertib-resistant colorectal cells and explore a potential target to overcome Alisertib-resistance. Methods: Drug-resistant colon cancer cell line (named as HCT-8-7T cells) was established and transplanted into immunodeficient mice. The metastasis in vivo were observed. Proliferation and migration of HCT-8-7T cells and their parental cells were assessed by colony formation and Transwell assay, respectively. Glycolytic capacity and glutamine metabolism of cells were analyzed by metabolism assays. The protein and mRNA levels of critical factors which are involved in mediating glycolysis and epithelial-mesenchymal transition (EMT) were examined by western blot and reverse transcription-quantitative real-time polymerase chain reaction(RT-qPCR), respectively. Results: In comparison with the mice transplanted with HCT-8 cells, which were survival with limited metastatic tumor cells in organs, aggressive metastases were observed in liver, lung, kidney and ovary of HCT-8-7T transplanted mice (P<0.05). The levels of ATP [(0.10±0.01) mmol/L], glycolysis [(81.77±8.21) mpH/min] and the capacity of glycolysis [(55.50±3.48) mpH/min] in HCT-8-7T cells were higher than those of HCT-8 cells [(0.04±0.01) mmol/L, (27.77±2.55) mpH/min and(14.00±1.19) mpH/min, respectively, P<0.05]. Meanwhile, the levels of p53 protein and mRNA in HCT-8-7T cells were potently decreased as compared to that in HCT-8 cells (P<0.05). However, the level of miRNA-125b (2.21±0.12) in HCT-8-7T cells was significantly elevated as compared to that in HCT-8 cells (1.00±0.00, P<0.001). In HCT-8-7T cells, forced-expression of p53 reduced the colon number (162.00±24.00) and the migration [(18.53±5.67)%] as compared with those in cells transfected with control vector [274.70±40.50 and (100.00±29.06)%, P<0.05, respectively]. Similarly, miR-125b mimic decreased the glycolysis [(25.28±9.51) mpH/min] in HCT-8-7T cells as compared with that [(54.38±12.70)mpH/min, P=0.003] in HCT-8-7T cells transfected with control. Meanwhile, in comparison with control transfected HCT-8-7T cells, miR-125b mimic also significantly led to an increase in the levels of p53 and β-catenin, in parallel with a decrease in the levels of PFK1 and HK1 in HCT-8-7T cells (P<0.05). Conclusions: Silencing of p53 by miR-125b could be one of the mechanisms that contributes to Alisertib resistance. Targeting miR-125b could be a strategy to overcome Alisertib resistance.

MicroRNA21-5p alleviates hyperoxia-induced acute lung injury in rats through activating phosphatidylinositol 3 kinase/serine-threonine protein kinase signaling pathway by regulating type Ⅱ alveolar epithelial cell apoptosis / 中华危重病急救医学

Objective:To investigate whether microRNA-21-5p (miR-21-5p) alleviates hyperoxia-induced acute lung injury (HALI) through activating the phosphatidylinositol 3 kinase/serine-threonine protein kinase (PI3K/Akt) signaling pathway by regulating apoptosis of type Ⅱ alveolar epithelial cell (AECⅡ).Methods:Seventy-two male Sprague-Dawley (SD) rats were divided into normozone-controlled group, HALI group, PI3K/Akt signaling pathway inhibitor LY294002+HALI group (LY+HALI group), miR-21-5p overexpression+LY294002+HALI group (miR-21-5p+LY+HALI group), miR-21-5p overexpression+HALI group (miR-21-5p+HALI group), and dimethyl sulfoxide (DMSO)+HALI group by random number table method with 12 rats in each group. Animal models of HALI were prepared using 95% concentrations of oxygen. The animals in the normozone-controlled group were fed normally under normoxia. Transfection of lung tissue by miR-21-5p adeno-associated viral vector AAV6-miR-21-5p was performed by instillation of 200 μL titer (1×10 12 TU/mL) through a tracheal catheter 3 weeks prior to modeling. DMSO and LY294002 were administered via the tail vein at 0.3 mg/kg 1 hour before modeling. After 48 hours of modeling, carotid artery blood was collected to detect oxygenation index (OI) and respiratory index (RI), and real-time fluorescence quantitative reverse transcription-polymerase chain reaction (RT-PCR) was used to detect miR-21-5p expression. Lung tissue was collected, and the levels of inflammatory factors including tumor necrosis factor-α (TNF-α) and interleukins (IL-6, IL-1β) were measured by enzyme-linked immunosorbent assay (ELISA), and the ratio of pulmonary wet/dry weight (W/D) was determined, and the pathological changes of lung histopathology were observed under the light microscopy after hematoxylin-eosin (HE) staining. Each group was purified AECⅡ cells from 6 rats, the apoptosis rate was detected by flow cytometry, and the expression levels of phosphatase and tensin homologous gene (PTEN), and proteins from the PI3K/Akt signaling pathway were detected by Western blotting. Results:Compared with the normozone-controlled group, alveolar septal thickening and massive inflammatory cell infiltration were found after hyperoxia exposure, RI, inflammatory factors, lung W/D ratio, pathological score, AECⅡ cells early apoptosis rate, PTEN protein expression and phosphorylation level of Akt were increased, while OI and miR-21-5p expression were decreased, indicating the successful preparation of the model. After pretreatment, LY294002 could aggravate the pathological injury of lung tissue in HALI rats, RI, inflammatory factors and lung W/D ratio were further increased, and OI was further reduced compared with HALI group. At the same time, it could promote the AECⅡ cell apoptosis, further up-regulate the expression of PTEN, and reduce the phosphorylation of Akt. However, miR-21-5p pretreatment could negatively regulate PTEN, activate PI3K/Akt signal pathway, inhibit AECⅡ cell apoptosis, and reduce HALI, which was shown by the decreased level of inflammatory factors in miR-21-5p+LY+HALI group compared with LY+HALI group [TNF-α (μg/L): 100.33±3.48 vs. 116.55±2.53, IL-6 (ng/L): 141.06±3.70 vs. 161.31±3.59, IL-1β (μg/L): 90.82±3.69 vs. 112.23±2.87, all P < 0.05], RI, lung injury pathology score, lung W/D ratio, and AECⅡ cell early apoptosis rate were significantly decreased [RI: 0.81±0.02 vs. 1.05±0.07, pathology score: 0.304±0.008 vs. 0.359±0.007, lung W/D ratio: 5.29±0.03 vs. 5.52±0.08, apoptosis rate: (27.20±2.34)% vs. (34.17±1.49)%, all P < 0.05], OI and expressions of miR-21-5p were significantly increased [OI (mmHg, 1 mmHg≈0.133 kPa): 266.71±2.75 vs. 230.12±4.04, miR-21-5p (2 -ΔΔCt): 2.21±0.13 vs. 0.33±0.03, both P < 0.05], and PTEN protein expression in AECⅡ cell was significantly reduced (PTEN/GAPDH: 0.50±0.06 vs. 0.93±0.06, P < 0.05), and phosphorylation level of Akt was significantly increased [phosphorylated Akt (p-Akt) protein (p-Akt/GAPDH): 0.86±0.05 vs. 0.56±0.06, P < 0.05]. Conclusion:miR-21-5p attenuates HALI by inhibiting AECⅡ cell apoptosis, possibly through negative regulation of PTEN to activate PI3K/Akt signaling pathway.

Expression and significance of microRNA-21 and microRNA-181b in peripheral blood of patients with schizophrenia / 中国基层医药

Objective:To investigate the expression and significance of microRNA-21 (miRNA-21) and microRNA-181b (miRNA-181b) in the peripheral blood of patients with schizophrenia.Methods:A total of 100 patients with schizophrenia who received treatment in Shaoxing 7 th People's Hospital from March 2020 to March 2022 were included in the study group. An additional 30 healthy controls who concurrently underwent physical examination were included in the control group. The expression of miRNA-21 and miRNA-181b in peripheral blood was compared between the two groups. The 100 patients with schizophrenia received standardized clinical treatment. Their mental symptoms were evaluated with the Positive and Negative Symptom Scale (PANSS). miRNA-21 and miRNA-181b expression and PANSS scores before and 1, 4, 8, and 12 weeks after treatment were collected and compared between the two groups. The receiver operating characteristic curve was plotted to analyze the value of miRNA-21 and miRNA-181b expression in the diagnosis of schizophrenia. Results:Serum miRNA-21 and miRNA-181b expression in the study group were (2.41 ± 1.12) and (15.62 ± 2.26), respectively, which were significantly higher than (0.73 ± 0.37) and (8.11 ± 0.98) in the control group ( t = 8.07,17.67, both P < 0.05). With the prolongation of treatment time, serum miRNA-21 and miRNA-181b expression and PANSS score in the study group gradually decreased (all P < 0.001). The area under the receiver operating characteristic curve plotted for evaluating the value of miRNA-21 and miRNA-181b expression in the diagnosis of schizophrenia was 0.616 and 0.683, respectively. The area under the receiver operating characteristic curve plotted for evaluating the value of miRNA-21 combined with miRNA-181b expression in the diagnosis of schizophrenia was 0.788, which was markedly higher than that for the detection of miRNA-21 or miRNA-181b expression alone. Conclusion:miRNA-21 and miRNA-181b are abnormally highly expressed in the peripheral blood of patients with schizophrenia. Both of them can be used as objective and effective indicators for early diagnosis of schizophrenia. Combined detection of miRNA-21 and miRNA-181b provides higher accuracy in the diagnosis of schizophrenia than the detection of miRNA-21 or miRNA-181b alone.

The value of fibroblast growth factor 2 and microRNA-206 in predicting postoperative delayed union of closed tibial shaft fractures / 中国基层医药

Objective:To investigate the value of fibroblast growth factor 2 (FGF-2) and microRNA-206 (miR-206) in predicting postoperative delayed union of closed tibial shaft fractures.Methods:The clinical data of 136 patients who underwent closed tibial shaft fracture surgery in Hospital of the 80 th Group Army of Chinese People's Liberation Army Ground Forces from May 2018 to May 2021 were retrospectively analyzed. Eighty-six patients who had delayed union of closed tibial shaft fractures were included in the observation group, and fifty patients who had normal union of closed tibial shaft fractures were included in the control group. Serum FGF-2 level was measured using the enzyme-linked immunosorbent assay, and serum miR-206 expression was detected using the real-time fluorescence polymerase chain reaction. The relationship between FGF-2 expression and miR-206 expression and closed tibial shaft fractures was analyzed. Results:At 1 day, 1, and 4 weeks after surgery, serum FGF-2 level was significantly lower in the observation group than the control group [(14.24 ± 2.15) ng/L vs. (20.36 ± 3.42) ng/L, (21.38 ± 3.27) ng/L vs. (30.45 ± 4.29) ng/L, (23.59 ± 4.36) ng/L vs. (36.67 ± 4.51) ng/L, t = 7.42, 8.42, 16.66, all P < 0.001]. Serum FGF-2 level gradually increased with time in each group. At 1 day after surgery, serum miR-206 expression was significantly lower in the observation group than the control group ( t = 7.50, P < 0.001). At 4 weeks after surgery, serum miR-206 expression was significantly higher in the observation group than the control group ( t = 17.24, P < 0.001). At 1 week after surgery, there was no significant difference in serum miR-206 expression between the two groups ( P > 0.05). Univariate analysis results showed that postoperative infection, FGF-2, and miR-206 were closely related to the delayed union of closed tibial shaft fractures after surgery (all P < 0.05). Multivariate logistic regression analysis results showed that postoperative infection ( OR = 1.93, 95% CI: 1.20-3.07), FGF-2 ( OR = 2.10, 95% CI: 1.31-3.36), miR-206 ( OR = 2.30, 95% CI: 1.35-3.89) were independent risk factors for delayed union of closed tibial shaft fractures after surgery (all P < 0.05). The receiver operating characteristic (ROC) curves plotting serum FGF-2 level and serum miR-206 expression after closed tibial shaft fractures showed that at 4 weeks after surgery, the optimal cut-off value of FGF-2 for predicting delayed union of closed tibial shaft fractures was 29.83 ng/L, with the area under the curve, sensitivity, and specificity of 0.76 (95% CI: 1.23-3.25), 79.34%, and 68.82%, respectively; at 4 weeks after surgery, the optimal cut-off value of miR-206 for predicting delayed union of closed tibial shaft fractures was 0.63, with the area under the curve, sensitivity and specificity of 0.72 (95% CI: 1.10-2.45), 75.33%, and 67.25%, respectively. The area under the curve, the sensitivity, and specificity of combined use of FGF-2 and miR-206 in predicting delayed union of closed tibial shaft fractures were 0.81 (95% CI: 1.35-3.26), sensitivity and specificity were 83.45% and 67.36% respectively. Conclusion:The decrease in serum FGF-2 level and the increase in serum miR-206 expression at 4 weeks after surgery are independent risk factors for delayed union of closed tibial shaft fractures. Combined use of FGF-2 and miR-206 can better predict the delayed union of closed tibial shaft fractures.

The expression of miR-122 in the liver of rats infected with Clonorchis sinensis and its correlation with inflammatory cytokines / 中华地方病学杂志

Objective:To observe the expression changes of microRNA(miR)-122 in liver tissue of rats infected with Clonorchis sinensis and its correlation with expression level of inflammatory cytokines. Methods:Totally 24 SPF grade Wistar male rats were selected and randomly divided into a control group (100 μl physiological saline gavage), a 4-week infection group (100 Clonorchis sinensis metacercariae gavage), and an 8-week infection group (100 Clonorchis sinensis metacercariae gavage) based on body weight (100-120 g) using a random number table method, with 8 rats in each group. Starting from the third week of infection, rat feces were collected and directly smeared with physiological saline for identification of Wistar rat animal models infected with Clonorchis sinensis. After 4 and 8 weeks of infection, the rats in the 4- and 8-week infection groups were euthanized, while 4 rats in the control group were euthanized, respectively. The heart blood and left lobe liver tissue and serum samples were collected from each group of rats. Using hematoxylin-eosin (HE) staining to observe liver pathological damage under the light microscope, real-time fluorescence quantitative PCR to detect the expression level of miR-122 in liver tissue, and Luminex 200 liquid suspension chip to detect the expression levels of serum inflammatory cytokines [tumor necrosis factor-α(TNF-α), interleukin-1β(IL-1β), interleukin-6 (IL-6)]. The correlation between miR-122 and inflammatory cytokines was analyzed using Pearson correlation. Results:Under the light microscope, the morphology of hepatocytes in control group was normal, and no inflammatory cell infiltration was observed. There was inflammatory cells such as lymphocyte, eosinophil and other inflammatory cell infiltration around the portal area in the 4-week infection group. The hepatocytes of the 8-week infected rats were arranged in a disordered manner, with varying degrees of swelling, loose and lightly stained cytoplasm, and some hepatocytes showed watery degeneration; additionally, bile duct dilation and thickening of the bile duct wall were observed in the liver tissue. There were statistically significant differences of liver miR-122 (1.00 ± 0.32, 2.57 ± 0.60, 3.63 ± 1.63), serum TNF-α [(0.14 ± 0.06), (0.43 ± 0.09), (0.61 ± 0.10) ng/ml], and IL-6 expression levels [(0.03 ± 0.01), (1.06 ± 0.24), (1.48 ± 0.33) ng/ml] in control group, 4- and 8-week infection groups ( F = 13.36, 69.99, 82.23, P < 0.001). There was no statistically significant difference in expression level of IL-1β between different groups ( F = 2.15, P = 0.141). The Pearson correlation analysis showed that the expression level of miR-122 was positively correlated with the expression levels of inflammatory cytokines TNF-α and IL-6 ( r = 0.67, 0.80, P < 0.001). Conclusion:Clonorchis sinensis infection can increase the expression of miR-122 in the host liver tissue, and the miR-122 is closely related to the expression levels of inflammatory cytokines TNF-α and IL-6.

Regulatory effect of miR-210 in hypoxia-induced epithelial-mesenchymal transition of pancreatic cancer PANC1 cells / 中华胰腺病杂志

Objective:To explore the regulatory role of miR-210 in hypoxia-induced epithelial-mesenchymal transition (EMT) of pancreatic cancer PANC1 cells.Methods:PANC1 cells cultured in normoxia and hypoxia were established in normoxia group and hypoxia group. Recombinant plasmid carrrying miR-210 mimics and miR-210 antagomirs were constructed. The recombinant plasmids were transfected with PANC1 cells cultured in normoxia and hypoxia by liposome method to establish cell lines of miR-210 overexpression (miR-210 mimics normoxia group) and miR-210 expression inhibition (miR-210 antagomirs hypoxia group). The blank plasmids were transfected to establish blank plasmid normoxia group and blank plasmid hypoxia group. Relative expression levels of miR-210 for PANC1 cells were determined by qRT-PCR in each group. Western blot was used to measure the expressions of HIF-1α, NF-κB and EMT related protein such as E-cadherin, β-catenin, vimentin and N-cadherin. Cell relative viability under gemcitabine and in vitro cell invasion ability were detected by CCK8 and Transwell chamber experiments, respectively. Results:The relative expressions of miR-210 in hypoxia group and miR-210 mimics normoxia group were significantly higher than those in normoxia group and blank plasmid normoxia group. However, there were significantly lower in miR-210 antagomirs hypoxia group than those in blank plasmid hypoxia group. The expression levels of HIF-1α, NF-κB and mesenchymal cell markers such as vimentin and N-cadherin in hypoxia group were significantly higher than those in normoxia group (0.74±0.06 vs 0.40±0.05, 1.58±0.16 vs 1.09±0.13, 0.46±0.04 vs 0.17±0.02, 1.27±0.07 vs 0.40±0.03) and the epithelial cell markers such as E-cadherin and β-catenin were significantly lower (0.40±0.07 vs 0.77±0.10, 0.35±0.02 vs 0.94±0.08). The expression levels of HIF-1α, NF-κB, vimentin and N-cadherin in miR-210 mimics normoxia group were significantly higher than those in blank plasmid normoxia group (0.91±0.08 vs 0.40±0.06, 1.52±0.17 vs 1.05±0.14, 0.82±0.06 vs 0.66±0.07, 0.76±0.04 vs 0.46±0.03) and E-cadherin and β-catenin were significantly lower (0.38±0.07 vs 0.65±0.09, 0.50±0.03 vs 0.94±0.08). The expression levels of HIF-1α, NF-κB, vimentin and N-cadherin in miR-210 antagomirs hypoxia group were significantly lower than those in blank plasmid hypoxia group (0.31±0.05 vs 0.55±0.06, 0.68±0.05 vs 1.11±0.13, 0.41±0.03 vs 0.74±0.07, 0.69±0.06 vs 0.78±0.05), while E-cadherin and β-catenin were significantly higher (0.72±0.13 vs 0.50±0.07, 0.71±0.04 vs 0.54±0.05). All the differences among the groups were statistically significant (all P value <0.05). Under gemcitabine, the relative viability of PANC1 cells in hypoxia group and miR-210 mimics normoxia group were significantly higher than those in normoxia group and blank plasmid normoxia group at 48 h (1.10±0.10 vs 0.76±0.05, 1.46±0.11 vs 1.12±0.09) and 72 h (1.12±0.13 vs 0.76±0.05, 1.54±0.13 vs 1.12±0.09) accordingly. However, there were significantly lower in miR-210 antagomirs hypoxia group than those in blank plasmid hypoxia group at 48 and 72 h (0.75±0.09 vs 1.10±0.10, 1.19±0.12 vs 1.46±0.11). All the differences among the groups were statistically significant (all P value <0.05). The number of transmembrane cells in hypoxia group and miR-210 mimics normoxia group was significantly higher than those in normoxia group and blank plasmid normoxia group, respectively (417.50±81.22 vs 228.30±47.71, 371.30±72.81 vs 245.00±33.62 per high field), while those in miR-210 antagomirs hypoxia group was significantly lower than those in blank plasmid hypoxia group (228.30±54.01 vs 433.30±65.63 per high field). All the differences among the groups were statistically significant (all P value <0.05). Conclusions:miR-210 could weaken the sensitivity to gemcitabine and promote the invasion of PANC1 cells by regulating the occurrence of the hypoxia-induced epithelial-mesenchymal transition.

Inhibitory effect of miR-497 on the repair of diabetic mice corneal epithelial damage by targeting wnt3a / 中华实验眼科杂志

Objective:To investigate the inhibitory effect of miR-497 on the corneal epithelial healing in diabetic mice and its possible mechanism.Methods:Forty healthy clean-grade wild-type C57BL/J6 mice were randomly divided into a blank control group and a model control group, with 20 mice in each group.Another 20 CRISPR/Cas9-mediated miR-497 knockout mice and miR-497 overexpression mice were taken as miR-497 knockout and miR-497 overexpression groups, respectively.The diabetes model was constructed by continuous intraperitoneal injection of streptozotocin (STZ) to the mice in model control, miR-497 knockout and miR-497 overexpression groups, and the mice in blank control group were injected with an equal amount of citrate buffer, followed by 8-week normal feeding.After the establishment of diabetes model, the corneal epithelial injury model was further constructed by scraping off part of the corneal epithelium with a central diameter of 2 mm.The corneal epithelial defect area of mice in 0, 12, 24 and 36 hours after corneal epithelial injury was observed by corneal fluorescein sodium staining.The expression of Wnt3a and β-catenin proteins in mice corneal tissues was detected by Western blot.The expression of miR-497 as well as the mRNA expression levels of cell proliferation-associated factor genes CyclinD1, c-Myc, and Ki-67 mRNA was detected by real-time quantitative fluorescence PCR.The targeting relationship between miR-497 and wnt3a was detected by a dual luciferase reporter gene assay.Human corneal epithelial cells (HCEC) were cultured in vitro and transfected with miR-497 mimics, miR-497 mimics negative control, miR-497 inhibitor, and miR-497 inhibitor negative control by Lipo8000 as miR-497 mimics group, mimics negative control group, miR-497 inhibitor group, andmiR-497 inhibitor negative control group, respectively, all of which were cultured in high glucose medium containing 25% glucose.Another two groups of HCEC were taken and cultured in medium containing 5% and 25% glucose as control and high glucose groups, respectively.The cell proliferation viability was determined by CCK8 method.The use and care of animals complied ith the ARVO statement.The study protocol was approved by the Ethics Committee of Renmin Hospital of Wuhan University (2019K-K010). Results:Eight weeks after STZ injection, the blood glucose of mice was significantly higher and the weight was significantly lower in each diabetic model group than those of blank control group (all at P<0.05). At 12, 24 and 36 hours after the corneal epithelial injury, the percentages of corneal epithelial defect area observed by slit-lamp microscopy in model control group were significantly higher than those in blank control group and miR-497 knockout group and lower than those in miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expressions of wnt3a and β-catenin proteins in the corneal tissues of model control group were significantly lower than those of blank control group and miR-497 knockout group, but higher than those of miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expressions of CyclinD1, c-Myc and Ki-67 mRNA in model control group were lower than those in miR-497 knockout group, but higher than those in miR-497 overexpression group, and the differences were statistically significant (all at P<0.05). The relative expression of miR-497 in model control group, miR-497 knockout group and miR-497 overexpression group was 1.00±0.02, 0.63±0.06 and 1.48±0.03, respectively, with a statistically significant difference ( F=19.62, P<0.01). The luciferase activity of miR-497-5p mimics group in wild-type wnt3a transfected cells was lower than that of miR-497-5p negative control group and empty vector group, and the differences were statistically significant (all at P<0.05). In the mutant wnt3a transfected cells, there was no significant difference in the luciferase activity among various groups ( F=0.73, P=0.59). The cell proliferation A value of high glucose group was 0.59±0.03, which was significantly lower than 0.59±0.03 of normal control group and 0.88±0.08 of miR-497 inhibitor group, but significantly higher than 0.48±0.11 of miR-497 mimics group (all at P<0.05). Conclusions:The silencing of miR-497 may promote the repair of diabetic corneal epithelial defects by targeting wnt/β-catenin pathway.

Regulation of tumor suppressor microRNA-218 on malignant progression and cancer stem cell properties of prostate cancer / 现代泌尿外科杂志

【Objective】 To investigate the role of microRNA-218 (miR-218) in regulating prostate cancer (PCa) cell stemness and epithelial-mesenchymal transition (EMT). 【Methods】 PCa cell line stably overexpressing miR-218 was constructed with lentivirus transfection. The expression of miR-218 was detected with real-time fluorescence quantitative polymerase chain reaction (q-PCR). The migration ability was detected with Transwell assay. The expression of EMT related proteins were detected with Western blot. The properties of cells were determined with colony formation and tumor sphere formation assays. 【Results】 The results of q-PCR showed that the mRNA level of miR-218 was significantly lower in PCa cell lines LNCaP and C4-2 than in BPH-1. Transwell assay showed that miR-218 inhibited the migration of PCa cells. Western blot showed that the expression of EMT related proteins were inhibited by miR-218. Colony formation and tumor sphere formation assays showed that overexpression of miR-218 significantly inhibited the properties of cells. 【Conclusion】 The expression of miR-218 is downregulated in PCa cell lines. miR-28 can inhibit cell migration, EMT and cancer stem cell properties.

Correlation of serum exosomal miR-301a with cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy / 西安交通大学学报(医学版)

【Objective】 To investigate the correlation of serum exosomal microRNA-301a (miR-301a) with cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy. 【Methods】 A total of 211 patients with diabetic nephropathy treated with peritoneal dialysis from June 2019 to June 2020 in the First Hospital Affiliated of Hebei North University were selected as study subjects. Serum exosomal miR-301a was detected by real-time fluorescence quantitative polymerase chain reaction. The patients were divided into high miR-301a group and low miR-301a group based on the median of miR-301a; the clinical data of the two groups were compared. The correlation of miR-301a with high-sensitivity C-reactive protein (hs-CRP) was analyzed by Spearman. Linear regression was applied to analyze the factors associated with the effect of miR-301a. The patients were followed up for two years. Kaplan-Meier and Log-Rank were conducted to compare the cumulative incidence of cardiovascular and cerebrovascular events between the two groups, and COX regression and restricted cubic spline were used to analyze the level-effect relationship between miR-301a and cardiovascular and cerebrovascular events after peritoneal dialysis. 【Results】 Thirty-seven cases (17.54%) of cardiovascular and cerebrovascular events occurred during follow-up. The hs-CRP level and dialysis duration were lower in low miR-301a group than in high miR-301a group (P<0.05). There was a positive correlation between miR-301a and hs-CRP (rs=0.237, P=0.001). Linear regression analysis showed that hs-CRP was independently associated with miR-301a (P<0.05). The cumulative cardiovascular and cerebrovascular event rate in low miR-301a group was 3.70% (4/108), which was lower than that in high miR-301a group [32.04% (33/103), P<0.001]. COX regression analysis showed that high serum albumin level was an independent protective factor for cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy,while high hs-CRP level and miR-301a >1.46 were independent risk factors for cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy. Restricted cubic spline fitting COX regression analysis showed a non-linear relationship between miR-301a and cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy (P<0.05). 【Conclusion】 hs-CRP is independently associated with miR-301a in diabetic nephropathy peritoneal dialysis patients. High miR-301a level suggests a high risk of cardiovascular and cerebrovascular events after peritoneal dialysis in diabetic nephropathy.

Identification of diagnostic signatures associated with tumor mutation burden in gastric cancer based on TCGA and machine-learning methods / 西安交通大学学报(医学版)

【Objective】 To select and identify miRNA signatures to predict TMB level in gastric cancer based on The Cancer Genome Atlas (TCGA) database and machine learning methods. 【Methods】 MiRNA expression and somatic mutation profiles of gastric cancer (GC) were downloaded from TCGA database. R "limma" package was performed to select differentially expressed miRNAs between high-TMB and low-TMB groups. Two machine learning algorisms, random forest (RF), and Support Vector Machine-Recursive Feature Elimination were utilized to identify miRNAs with the highest discriminative ability. ROC was used to test the predictive ability of these signatures in multiple datasets. Besides, immune cells of different TMB levels were compared by the CIBERSORT method. 【Results】 A total of 56 differentially expressed miRNAs (DE-miRNAs) were filtered. Functional enrichment analysis showed that these DE miRNAs are mainly enriched in signaling pathways related to tumor occurrence and development as well as immunity-related biological processes. The RF and SVM-RFE algorithms jointly identified 10 diagnostic features of miRNAs, among which only hsa-miR-210-3p is considered the most relevant predictive biomarker for TMB classification. The AUC value of hsa-miR-210-3p in the training, testing, and total sets is 0.822, 0.721, and 0.793, respectively, and has been validated in other cancer types. Besides, CIBERSORT analysis suggests differences in immune cell infiltration between high- and low-TMB groups. Meanwhile, there is a significant positive correlation between the expression of immune checkpoint related genes and mismatch repair related genes and hsa-miR-210-3p. 【Conclusion】 This study successfully identified hsa-miR-210-3p as a predictive biomarker for TMB classification, which can effectively predict TMB values in gastric cancer and other cancer patients and may provide some guidance for immunotherapy.

Effects of LncRNA MEG3 on the invasion and migration of prostate cancer cells by regulating microRNA-181b-5p/TIMP3 / 现代泌尿外科杂志

【Objective】 To explore the effects of long non-coding RNA maternally expressed gene 3 (lncRNA MEG3) on the invasion and migration of prostate cancer cells (PC3 cells) by regulating microRNA-181b-5p (miR-181b-5p)/tissue inhibitor of metalloproteinase 3 (TIMP3). 【Methods】 The prostate cancer tissues and adjacent tissues were collected from 20 prostate cancer patients treated in our hospital during Dec.2020 and Dec.2021. The expressions of MEG3 and miR-181b-5p in tissues were detected with quantitative real-time PCR (qRT-PCR). P3 cells were randomly divided into control group (untreated), pcDNA3.1-NC (transfected with pcDNA3.1-NC), pcDNA3.1-MEG3 group (transfected with pcDNA3.1-MEG), pcDNA3.1-MEG3+miR-NC group (pcDNA3.1-MEG3 co-transfected with miR-NC), pcDNA3.1-MEG3+miR-181b-5p mimic group (pcDNA3.1-MEG3 co-transfected with miR-181b-5p mimic). The expressions of MEG3 and miR-181b-5p in PC3 cells were detected with qRT-PCR. The cell viability, invasion and migration ability were determined with MTT assay, Transwell assay and scratch assay. The protein expressions of TIMP3, matrix metalloproteinase (MMP)9 and MMP2 in PC3 cells were detected with Western blot. The targeting relationship of MEG3, miR-181b-5p and TIMP3 was analyzed with dual luciferase assay. 【Results】 The expressions of MEG3 in prostate cancer tissues ( 0.37±0.05 vs. 1.00±0.04) and cells (0.31±0.06 vs. 1.00±0.01) were significantly decreased (P<0.05). Compared with the control group, the pcDNA3.1-MEG3 group had significantly decreased expression of miR-181b-5p (0.26±0.04 vs.1.00±0.02 ), cell survival rate (53.60±5.22 vs.100.00±0.00), number of invasive cells (62.33±9.85 vs.162.34±21.30), cell migration rate (32.85±3.80 vs.75.22±5.96), expressions of MMP9 (0.61±0.08 vs.1.62±0.23) and MMP2 (0.73±0.10 vs.1.20±0.16), but significantly higher expressions of MEG3 (2.31±0.36 vs. 1.00±0.01) and TIMP3 (1.32±0.24 vs. 0.53±0.08) (P<0.05). Overexpression of miR-181b-5p reversed the above changes (P<0.05). MiR-181b-5p had a targeting relationship with MEG3 and TIMP3. 【Conclusion】 Overexpression of lncRNA MEG3 can inhibit miR-181b-5p to promote the expression of TIMP3, thereby inhibiting invasion and migration of PC3 cells.

Improvement effect and mechanism of total flavonoids from Alpinia zerumbet on gastric mucosa injury induced by absolute ethanol / 中国药房

OBJECTIVE To explore the improvement effect and potential mechanism of total flavonoids from Alpinia zerumbet on gastric mucosa injury induced by absolute ethanol through microRNA-146a-5p （miR-146a-5p）. METHODS Using human gastric mucosa GES-1 cells as objects， the acute gastric ulcer model was established by absolute ethanol； based on the investigation of the effects of different concentrations of total flavonoids from A. zerumbet on cell activity and the selection of action concentration， the relative expression level of miR-146a-5p in GES-1 cells was detected， the protein expressions of tumor necrosis factor （TNF） receptor-associated factor 6（TRAF6）， nuclear factor-κB p65 （NF-κB p65） and TNF-α were detected， and the levels of interleukin- 1β （IL-1β）， IL-6 and prostaglandin E2 （PGE2） in cell supernatant were determined. The targeting relationship between miR-146a- 5p and TRAF6 was verified； the effects of overexpressed miR-146a-5p and TRAF6 knockdown on the levels of IL-1β， IL-6 and PEG2 in supernatant of model cells as well as the effects of miR-146a-5p knockdown on anti-gastric ulcer effect of total flavonoids from A. zerumbet were observed. RESULTS Compared with the blank group， the relative expression of miR-146a-5p in cells and the level of PGE2 in cell supernatant were decreased significantly in the model group （P＜0.01）， while the protein expressions of TRAF6， NF-κB p65 and TNF-α in cells and the levels of IL-1β and IL-6 in cell supernatant were increased significantly （P＜ 0.01）. Compared with the model group， the relative expression of miR-146a-5p in cells and the level of PGE2 in cell supernatant were increased significantly in model+A. zerumbet total flavonoids （60 mg/L） group （P＜0.01）， while the protein expressions of TRAF6， NF-κB p65 and TNF-α in cells and 82260767） the levels of IL-1β and IL-6 in cell supernatant were decreased significantly （P＜0.05 or P＜0.01）. There was a targeted relationship and a negative correlation between miR-146a-5p E-mail：3113836821@qq.com and TRAF6. After overexpression of miR-146a-5p or TRAF6 knockdown， the levels of IL-1β and IL-6 were decreased significantly in cell supernatant， while the level of PGE2 was increased significantly （P＜0.05）. After miR-146a-5p knockdown， the levels of IL-1β and IL-6 in cell supernatant and the protein expression of TRAF6 in cells administered with total flavonoids of A. zerumbet were increased significantly， while the level of PGE2 was decreased significantly （P＜0.05）. CONCLUSIONS Total flavonoids of A. zerumbet can improve the gastric mucosa injury induced by absolute ethanol. The mechanism may be related to up-regulating the expression of miR-146a-5p， inhibiting the expression of TRAF6， and further inhibiting the secretion of related inflammatory factors.

Research advance of mesenchymal stem cell-derived exosomes in graft-versus-host disease / 中华器官移植杂志

Mesenchymal stem cells (MSCs) are pluripotent stem cells with self-renewing differentiation, immunoactivity and anti-inflammatory potentials.Allogeneic hematopoietic stem cell transplantation (allo-HSCT) is currently the most effective treatment for hematologic malignancies.However, the presence of graft-versus-host disease (GVHD) after transplantation has hindered the development of allo-HSCT.MSC-derived exosomes (MSC-exo) derived from mesenchymal stem cells have been confirmed to have broad therapeutic prospects in allo-HSCT and GVHD.This review focused upon immunomodulatory effects of MSC, biological activities of MSC-exo and research advances of MSC-exo on managing GVHD, aiming to provide new therapeutic rationales for GVHD in the future.