Effect and mechanism of miR-34a-5p targeting Notchl onH/R-induced apoptosis of human cardiomyocytes / 安徽医科大学学报

Objective@#To investigate the improvement of endoplasmic reticulum stress mediated by microRNANotchl)signaling axis on hypoxia/reoxygenation(H/R)human(miR)-34a-5p/transmembrane fusion protein 1 (es were randomly divided into Control group, H/R group, mimiccardiomyocytes. @*Methods@#Human cardiomyocytNC group, mimic group, inhibitor NC group andinhibitor group. Except the Control group, H/R injury model wasof miR-34a-5p and Notchl were detected by quantitative real.established in other groups. The expression levesurvival rate was detected by thiazolyl blue ( MTT), cell apopto-time polymerase chain reaction(qRT-PCR), celtargeting relationship between miR-34a-5p and Notchl was detec-sis rate was detected by flow cytometry, and theexpressions of transcriptional activator 6(ATF6), inositol demandted by dual luciferase gene reporting method. Thesmic reticulum kinase (PERK) and glucose regulatory protein 78 (GRP78)were detected by Western blot. @*Results@#miR-34a-5p targeted Notchl(P<0.05);compared with Con-apoptosis rate and protein expressions of ATF6, IREl, PERK andtrol group, the expression level of miR-34a-5pGRP78 in H/R group increased, while the cellsurvival rate and Notchl mRNA and protein expressions decreased(P<0.05). Compared with H/R group and minic NC group, miR-34a-5p expression, apoptosis rate , and proteinexpressions of ATF6, IRE1, PERK and GRP78in mimic group increased, while cell survival rate and Notchl mRNA and protein expressions decreased (P <0. 05).Compared with H/R group and inhibitor NC group, the exprespressions of ATF6 , IREl , PERK and GRP78 decreased in inhibi-sion of miR-34a-5p, apoptosis rate and protein etor group,while cell survival rate and Notch1 mINA and protein expressions increased ( P <0.05). @*Conclusion@#miR-34a-5p can inhibit the apoptosis of H/R human cardiomyocytes, possibly through the targeted inhibition ofNotchl-mediated endoplasmic reticulum stress.

Mechanism by which miR-34a-5p/PLCD3 axis regulates osteoarthritis progression / 中国组织工程研究

BACKGROUND:Molecular mechanisms targeting the miRNA/mRNA axis to regulate osteoarthritis disease process have been studied.We identified the mRNA:phospholipase C delta 3(PLCD3)and its target miRNA(miR-34a-5p)with clinical predictive value through previous bioinformatics studies,while experiments to verify their specific roles and mechanisms in regulating osteoarthritis are still lacking. OBJECTIVE:To investigate the regulatory role and mechanism of miR-34a-5p/PLCD3 axis on osteoarthritis progression. METHODS:The synovium of 15 patients with knee osteoarthritis was selected as the osteoarthritis group,and the synovium of 15 young patients with internal fixation of patellar fracture caused by trauma during the same period was selected as the control group.The expression of PLCD3 and miR-34a-5p in the synovium was detected by real-time PCR.Human fibroblast like synovial cells-osteoarthritis(HFLS-OA)cells were treated by cell transfection and divided into miR-34a-5p mimic group,pCDH-PLCD3 group,miR-34a-5p mimic+pCDH-PLCD3 group,miR-34a-5p inhibitor group,si-PLCD3 group,and miR-34a-5p inhibitor+si-PLCD3 group.The relationship between PLCD3 and miR-34a-5p expression was detected by real-time PCR.The effects of HFLS-OA cell viability and cell migration in each group were detected by CCK-8 assay and cell scratch test.Western blot assay was used to detect the expression level of apoptosis marker protein.The expression of inflammatory factors was detected by ELISA. RESULTS AND CONCLUSION:(1)PLCD3 was a direct target of miR-34a-5p,and the expression levels of PLCD3 and miR-34a-5p were negatively correlated.(2)Upregulation of PLCD3 promoted proliferation of HFLS-OA cells and inhibited cell migration.The up-regulation of miR-34a-5p significantly inhibited the activity of HFLS-OA cells and enhanced cell migration.Overexpression of miR-34a-5p significantly increased the levels of Casp3 and Casp9 proteins in HFLS-OA cells,while overexpression of PLCD3 showed the opposite trend.(3)PLCD3 overexpression significantly increased the expression of interleukin 6 and tumor necrosis factor alpha in HFLS-OA cells,while miR-34a-5p mimics showed protective activity.(4)The miR-34a-5p/PLCD3 axis may affect the progression of osteoarthritis by regulating the inflammatory process or apoptosis of synovial cells.

Effects of miR-34a on polarization and inflammatory factor content of mouse macrophage cell line RAW264.7 under high glucose conditions / 基础医学与临床

Objective To detect the role of miR-34a on macrophage inflammation and polarization under high glucose conditions.Methods Mouse macrophages were collected and cultured during high glucose for 3－28 days.RT-qPCR was used to detect the expression of miR-34a,iNOS,MCP-1 and Arg-1 mRNA.Then miR-34a was over-expressed or silenced,ELISA was used to detect the expression of IL-6,IL-1β,TNF-α and qRT-PCR was used to detect the expression of iNOS and MCP-1 mRNA.Western blot was used to detect the expres-sion of Notch1.Results Expression of miR-34a increased under high glucose conditions in RAW264.7 cells continuously.Over-expression of miR-34a promoted the expression of MCP-1 and iNOS observed by RT-qPCR and increased the expression of IL-6,IL-1β and TNF-α detected by ELISA.Further studies showed that siRNA-Notch1 down-regulated the expression of miR-34a,MCP-1,iNOS,IL-6,IL-1β and TNF-α.Conclusions Chronic high glucose condition stimulates the expression of miR-34a which promotes M1 macrophage polarization and releasing of pro-inflammatory factors.

Regulation of Notch1 and Foxp1 by MiR-34a in psoriasis vulgaris / Regulación de Notch1 y Foxp1 por MiR-34ª en psoriasis vulgaris

SUMMARY: This study is to investigate the regulation of Notch1 and Foxp1 by miR-34a in the development of psoriasis vulgaris. RT-PCR was used to compare the levels of miR-34a in the skin lesions of 20 patients with psoriasis vulgaris and 20 normal skin tissues. Immunohistochemistry was used to detect the expression of Notch1 and Foxp1 in 51 patients with psoriasis vulgaris, which were further compared with that in 29 normal control tissues. In addition, in HaCaT cells, we used miR-34a mimics and inhibitors to overexpress and inhibit miR-34a, respectively, and detected the mRNA and protein levels of miR-34a, Notch1, and Foxp1. The level of miR-34a in the skin lesions of patients with psoriasis vulgaris was significantly higher than that in normal skin tissues (t=2.192, P<0.05). The positive rate of Notch1 in the skin lesions of patients with psoriasis vulgaris was 76.47 %, which was significantly higher than that in normal skin tissues (13.79 %) (t=29.215, P<0.01). The positive rate of FOXP1 in the psoriasis vulgaris group was 92.16 %, which was also significantly higher than that in the normal skin group (65.52 %) (t=9.087, P<0.01). In addition, overexpression of miR-34a significantly promoted the expression of Notch1 and Foxp1. However, inhibition of miR-34a significantly reduced Notch1 and Foxp1 levels. miR- 34a is highly expressed in the skin tissues of patients with psoriasis vulgaris, and may participate in the development of psoriasis vulgaris by regulating Notch1 and Foxp1.

RESUMEN: El objetivo de este estudio fue investigar la regulación de Notch1 y Foxp1 por miR-34a en el desarrollo de la psoriasis vulgar. Se utilizó RT-PCR con el fin de comparar los niveles de miR-34a en las lesiones cutáneas de 20 pacientes con psoriasis vulgar y 20 tejidos de piel normales. Se utilizó inmunohistoquímica para detectar la expresión de Notch1 y Foxp1 en 51 pacientes con psoriasis vulgar, que se compararon además con la de 29 tejidos normales control. Además, en las células HaCaT, usamos miméticos e inhibidores de miR-34a para sobreexpresar e inhibir miR-34a, respectivamente, y detectamos los niveles de ARNm y proteína de miR-34a, Notch1 y Foxp1. El nivel de miR- 34a en las lesiones cutáneas de pacientes con psoriasis vulgar fue significativamente mayor que en los tejidos normales de la piel (t=2,192, P<0,05). La tasa de positividad de Notch1 en las lesiones cutáneas de pacientes con psoriasis vulgar fue del 76,47 %, que fue significativamente mayor que la de los tejidos normales de la piel (13,79 %) (t=29,215, P<0,01). La tasa positiva de FOXP1 en el grupo de psoriasis vulgar fue del 92,16 %, que también fue significativamente mayor que la del grupo de piel normal (65,52 %) (t=9,087, P<0,01). Además, la sobreexpresión de miR-34a promovió significativamente la expresión de Notch1 y Foxp1. Sin embargo, la inhibición de miR-34a redujo de manera importante los niveles de Notch1 y Foxp1. miR-34a se expresa en gran medida en los tejidos de la piel en pacientes con psoriasis vulgar y puede participar en el desarrollo de la psoriasis vulgar mediante la regulación de Notch1 y Foxp1.

Correlation between Serum miR-34a Level and Thrombocytopenia after Chemotherapy in Patients with Diffuse Large B-Cell Lymphoma / 中国实验血液学杂志

OBJECTIVE@#To analyze the relationship between serum miR-34a level and thrombocytopenia after chemotherapy in patients with diffuse large B-cell lymphoma (DLBCL).@*METHODS@#A total of 69 eligible DLBCL patients who received chemotherapy in our hospital from January 2018 to January 2020 were prospectively included as the research subjects, all patients received R-CHOP 21 regimen (rituximab + cyclophosphamide + adriamycin + vincristine + prednisone) for chemotherapy, 3 weeks was 1 cycle, and 2 cycles of chemotherapy were used. The patients were divided into a reduction group and a non reduction group according to whether there was thrombocytopenia after chemotherapy, the general data and laboratory indexes of the two groups were investigated and compared, the relationship between serum miR-34a before chemotherapy and thrombocytopenia after chemotherapy in patients was analyzed.@*RESULTS@#Among the 69 DLBCL patients, 36 patients developed thrombocytopenia after 2 cycles of R-CHOP 21 regimen for chemotherapy, the incidence was 52.17%; the level of serum IL-11 and the relative expression of miR-34a mRNA in the reduction group were significantly lower than the non reduction group (P<0.05), compared other data between groups, there was no statistical significant difference (P>0.05); after Logistic regression analysis, the results showed that the level of serum IL-11 and the relative expression of miR-34a mRNA were related to thrombocytopenia after chemotherapy in DLBCL patients, low expression of each index may be a risk factor of thrombocytopenia after chemotherapy in DLBCL patients (OR>1, P<0.05); ROC curve was drawn, and the results showed that the AUC of serum IL-11 level and miR-34a mRNA relative expression before chemotherapy alone and in combination predicted the risk of thrombocytopenia after chemotherapy in DLBCL patients were all >0.80, and the predictive value was ideal, when the cut-off value of serum IL-11 level before chemotherapy was 42.094 pg/ml, and the cut-off value of miR-34a mRNA relative expression was 3.894, the combined prediction value was the best.@*CONCLUSION@#The relative expression of miR-34a mRNA is associated with thrombocytopenia after chemotherapy in DLBCL patients, which may be a risk factor for thrombocytopenia in patients after chemotherapy, has certain value in predicting the risk of thrombocytopenia of patients after chemotherapy.

Polysaccharide of Atractylodis Macrocephalae Rhizoma inhibits expression of immune checkpoint PD-L1 by targeting miR-34a in esophageal carcinoma cells / 中国中药杂志

The immune checkpoint programmed cell death-ligand 1(PD-L1)-mediated immunosuppression is among the important features of tumor. PD-L1, an immunosuppressant, can induce T cell failure by binding to programmed cell death-1(PD-1). Thus, the key to restoring the function of T cells is inhibiting the expression of PD-L1. The Chinese medicinal Atractylodis Macrocephalae Rhizoma(AMR) has the anti-tumor, anti-inflammatory, antioxidant, and hypoglycemic activities, and the polysaccharide in AMR(PAMR) plays a crucial role in immunoregulation, but the influence on the immune checkpoints which are closely related to immunosuppression has not been reported. MicroRNA-34 a(miR-34 a) expression in esophageal carcinoma tissue is significantly lower than that in normal tissue. This study aims to investigate the inhibitory effect of PAMR on esophageal carcinoma cells, and the relationship between its inhibitory effect on PD-L1 expression and miR-34 a, which is expected to clarify the anti-tumor mechanism of PAMR. Firstly, different human esophageal carcinoma cell lines(EC9706, EC-1, TE-1, EC109 cells) were screend out, and expression of PD-L1 was determined. Then, EC109 cells, with high expression of PD-L1, were selected for further experiment. The result showed that PAMR suppressed EC109 cell growth. According to the real-time quantitative PCR(qPCR) and Western blot, it significantly suppressed the mRNA and protein expression of PD-L1, while promoting the expression of tumor suppressor miR-34 a. The confocal microscopy and luci-ferase assay proved that PAMR alleviated the inhibitory effect of PD-L1 while blocked miR-34 a. Additionally, the expression of PD-L1 was controlled by miR-34 a, and the combination of miR-34 a inhibitor with high-dose PAMR reversed the inhibitory effect of PAMR on PD-L1 protein expression. Thus, the PAMR may inhibit PD-L1 by increasing the expression of miR-34 a and regulating its downstream target genes. In conclusion, PAMR inhibits the expression of PD-L1 mainly by inducing miR-34 a.

Effects of irinotecan combined with XELOX regimen on prognosis and serum CD cells, miR-34a and let-7i in patients with advanced gastric cancer / 中国医师杂志

Objective:To investigate the effect of irinotecan combined with XELOX regimen on serum CD cells, miR-34a and let-7i in patients with advanced gastric cancer.Methods:A total of 72 patients with advanced gastric cancer treated in the Affiliated Hospital of Hubei University of Arts And Science from January 2015 to January 2020 were prospectively selected, and they were divided into control group and observation group with 36 cases in each group by random number table method. The control group was treated with XELOX regimen, while the observation group was treated with irinotecan on the basis of the control group. The efficacy, serum immune level, serum miR-34a and let-7i contents, incidence of toxic and side effects and life cycle of the two groups were compared.Results:The objective response rate (ORR) of the observation group was 33.33% , which were significantly higher than that of the control group (13.89%, χ 2=3.770, P>0.05). There was no significant difference in serum immune level, miR-34a and let-7i between the two groups before treatment (all P>0.05). After treatment, the levels of CD3 + , CD4 + and CD8 + in the two groups were significantly increased (all P<0.05), and the improvement of CD3 + , CD4 + and CD8 + in observation group was significantly better than that in control group (all P<0.05). After treatment, the serum miR-34a level increased significantly and serum let-7i level decreased significantly in the two groups (all P<0.05), and the serum miR-34a level in the observation group was higher than that in the control group, and the serum let-7i level was significantly lower than that in the control group (all P<0.05). Among the grade Ⅰ-Ⅱ adverse reactions, nausea and vomiting and leukocyte decline were the most common; The incidence of grade Ⅲ-Ⅳ leukopenia in the control group and the observation group were 5.56%(2/36) and 5.56%(2/36), respectively; The incidence of grade Ⅲ-Ⅳ thrombocytopenia was 5.56%(2/36) and 2.78%(1/36), respectively; One patient in the control group had grade Ⅲ-Ⅳ abnormal liver function, and one patient in the observation group had grade Ⅲ-Ⅳ nausea and vomiting. All patients with adverse reactions were effectively relieved after symptomatic treatment, and there were no treatment-related deaths. The progression free survival (PFS) of the observation group was 24.90 months (95% CI: 0.469-1.955), and that of the control group was 21.85 months (95% CI: 0.512-2.131). The PFS of the observation group was significantly longer than that of the control group (log rank=1.357, P=0.007). Conclusions:Irinotecan combined with XELOX regimen in the treatment of advanced gastric cancer can effectively improve the level of serum miR-34a and immune function, reduce the content of let-7i, and has good safety.

Combination therapy with miR34a and doxorubicin synergistically inhibits Dox-resistant breast cancer progression

Resistance to breast cancer (BCa) chemotherapy severely hampers the patient's prognosis. MicroRNAs provide a potential therapeutic prospect for BCa. In this study, the reversal function of microRNA34a (miR34a) on doxorubicin (Dox) resistance of BCa and the possible mechanism was investigated. We found that the relative level of miR34a was significantly decreased in Dox-resistant breast cancer cell MCF-7 (MCF-7/A) compared with Dox-sensitive MCF-7 cells. Transfection with miR34a significantly suppressed the invasion, migration, adhesion of MCF-7/A cells without inhibiting their growth obviously. The combination of miR34a and Dox could significantly inhibit the proliferation, migration, invasion and induce the apoptosis of MCF-7/A cells. The synergistic effect of this combination on resistant MCF-7/A cells has no obvious relation with the expressions of classical drug-resistant proteins P-GP, MRP and GST-

Berberine exerts antidepressant-like effects via regulating miR-34a-synaptotagmin1/Bcl-2 axis / 中草药·英文版

Objective: Berberine, a cationic alkaloid first isolated in 1917, has been approved by the China Drug Administration for decades. Accumulating evidence demonstrated its antidepressant-like activities in vivo. Our previous study has shown that chronic stress leads to the upregulation of miR-34a in the hippocampus of mice. This study aims to evaluate the underlying miR-34a mediated mechanism of berberine in chronic stress-induced depression in mice. Methods: In the present study, mice were administered with berberine during chronic stress. Levels of miR-34a, dendritic density, mitochondrial morphology, and neurogenesis were assessed in the hippocampus. Subsequently, miR-34a agomir was used as a pharmacological intervention for the investigation of berberine. Results: The results showed that berberine reversed the decrease in sucrose preference and the increase in latency to feed without altering total food consumption. Furthermore, chronic stress-induced overexpression of miR-34a decreased synaptotagmin-1 and Bcl-2 levels, thereby impairing spinal morphology, mitochondria and neurogenesis. Berberine inhibited miR-34a expression, in turn restored synaptotagmin-1 and Bcl-2 levels, and thus improved spinal morphology, mitochondria and neurogenesis in the hippocampus. However, the improvements induced by berberine were totally blocked by the pretreatment of miR-34a agomir, which caused the elevation of miR-34a levels in the hippocampus. Conclusion: This finding demonstrated that miR-34a downregulation was involved in the antidepressant-like effects of berberine in mice exposed to chronic stress.

Expression of miR-34a-5p and AKT1 gene in endometrial tissue of endometriosis and its effect on migration and invasion of endometrial stromal cells / 解放军医学杂志

Objective To investigate the expression of miR-34a-5p and AKT1 genes in endometrium tissues of patients with endometriosis (EM) and their effects on migration and invasion of endometrial stromal cells (ESCs). Methods A total of 91 patients, undergone hysterectomy in the Department of Obstetrics and Gynecology of Zhujiang Hospital of Southern Medical University from Jan. 2018 to Jun. 2019 due to benign gynecological diseases, were collected and divided into EM group (68 cases) and non-EM group (23 cases). The expressions of miR-34a-5p and AKT1 genes in endometrium tissues of patients were detected by in situ hybridization and immunohistochemistry. ESCs were transfected with miR-34a-5p mimic and negative control RNA (miR-NC) using liposome-3000 to construct the cell models of miR-34a-5p mimic group and miR-NC group. The cell proliferation rate was detected by CCK-8 method, and cell migration, invasion, apoptosis and autophagy ability experiments were performed to determine the effect of miR-34a-5p on ESCs' proliferation, migration, invasion, apoptosis and autophagy. Results The positive expression rate of miR-34a-5p was lower, and of AKT1 was higher in EM group than those in non-EM group (16.2% vs. 82.6%, X2=34.323; 72.1% vs. 30.4%, X2=12.581, P<0.001). After culturing for 12, 24 and 48 h, the cell proliferation rate was higher in miR-NC group than that in miR-34a-5p mimic group (P<0.05). The cell migration ability and invasion ability were higher in miR-NC group than those in miR-34a-5p mimic group with statistically significant difference [(65.00%±5.00%) vs. (30.67%±4.04%); (88.0±8.5) vs. (32.3±6.1), t=9.179, P<0.05]. The cell apoptosis rate and the expression level of LC3 gene were obviously lower in miR-NC group than those in miR-34a-5p mimic group [(9.33%±3.51%) vs. (18.00%±2.00%); (0.19±0.04) vs. (0.39±0.03), t=8.02, P<0.05]. Conclusion miR-34a-5p may be involved in the pathogenesis of EM by targeting AKT1 genes to affect the proliferation, migration, invasion, apoptosis and autophagy function of ESC.

LncRNA SNHG7/miR-34a-5p/SYVN1 axis plays a vital role in proliferation, apoptosis and autophagy in osteoarthritis

BACKGROUND: Osteoarthritis (OA) is one of the most common rheumatic diseases of which clinical symptoms includes swelling, synovitis and inflammatory pain, affect patients' daily life. It was reported that non-coding RNAs play vital roles in OA. However, the regulation mechanism of ncRNA in OA pathogenesis has not been fully elucidated. METHODS: The expression of SNHG7, miR-34a-5p and SYVN1 was detected using qRT-PCR in tissues, serum and cells. The protein expression of SYVN1, PCNA, cleavage-caspase 3, beclinl and LC3 were measured using western blot. The RNA immunoprecipitation (RIP), RNA pulldown, and luciferase reporter assays were used to verify the relationship between SNHG7, miR-34a-5p and SYVN1. The MTT and flow cytometry assay was performed to detected cell proliferation and cell apoptosis respectively. RESULTS: In this study, SNHG7 and SYVN1 expression were down-regulated, but miR-34a-5p was up-regulated in OA tissues and IL-1P treated cells compared with normal tissues and chondrocyte. Functional investigation revealed that up-regulated SNHG7 or down-regulated miR-34a-5p could promote cell proliferation and inhibit cell apoptosis and autophagy in OA cells. More than that, RIP, pulldown and luciferase reporter assay was applied to determine that miR-34a-5p was a target miRNA of SNHG7 and SYVN1 was a target mRNA of miR-34-5p. Rescue experiments showed that overexpression of miR-34a reversed high expression of SNHG7-mediated suppression of apoptosis and autophagy as well as promotion of proliferation, while its knockdown inhibited cell apoptosis and autophagy and promoted cell proliferation which could be impaired by silencing SYVN1. In addition, SNHG7 regulated SYVN1 through sponging miR-34a-5p. CONCLUSION: SNHG7 sponged miR-34a-5p to affect cell proliferation, apoptosis and autophagy through targeting SYVN1 which provides a novel sight into the pathogenesis of OA.

MiR-34a inhibits cell invasion and proliferation of hepatocellular carcinoma by repressing c-Met signaling / 医学研究生学报

ObjectiveTo explore the role of miR-34a in the proliferation, migration and invasion of hepatocellular carcinoma (HCC) cells.MethodsHepG2 and Huh-7 cells were transfected with miR-34a constructs by using lipofection. qPCR was applied to detect the expression of miR-34a. Cell proliferation assay, colony formation assay, cell migration and invasion assay were used to elucidate the invasive growth of transfected and un-transfected cells. The subcutaneous xenograft in nude mice was used to verified the anti-tumor effect of miR-34a in vivo. TargetScan, miR-WALK and luciferase assay were performed to explore the target of miR-34a in HCC.ResultsCompared with the overexpression control group and blank group, the expression of miR-34a in the miR-34a overexpression group cells were (2.727±0.173) and (4.042±0.104), respectively (P<0.05). The levels of miR-34a in overexpression control and blank groups were (0.003±0.030) and (0.005±0.027) (P＞0.05). Compared with the overexpression control groups, cell proliferation, colony formation, migration and invasion of HCC cells overexpressing miR34a were all impeded with statistically significant differences (P<0.05). In vivo, tumor weight and tumor volume were significantly reduced in the miR-34a treatment group. The results of luciferase reporter gene indicated that c-Met was the direct target of miR-34a in HCC.ConclusionsUp-regulation of miR-34a in hepatocellular carcinoma can inhibit the proliferation, migration and invasion of HCC cells, and its repression may be benefited by declining c-Met signaling in HCC.

LncRNA OIP5-AS1 upregulates snail expression by sponging miR-34a to promote ovarian carcinoma cell invasion and migration

BACKGROUND: Although OIP5-AS1 has been characterized as an oncogenic lncRNA in many types of cancer, its role and underlying mechanism in ovarian carcinoma (OC) remains unknown. This study aimed to investigate the role of OIP5-AS1 in OC. METHODS: OC tissues and non-tumor tissues (ovary tissues within 3 cm around tumors) were collected from 58 OC patients (age range 36 to 67 years old, mean age 51.4 ± 5.9 years old). The expression of OIP5-AS1 and snail in paired tissues were determined by RT-qPCR. The interaction between OIP5-AS1 and miR-34a was predicted by IntaRNA2.0 and confirmed by dual luciferase reporter assay. The effects of overexpression of OIP5-AS1 and miR-34a on the expression of snail were analyzed by RT-qPCR and Western blotting. Cell invasion and migration were analyzed by Transwell assay. RESULTS: We observed that the expression of OIP5-AS1 and snail was upregulated and positively correlated with each other in OC. RNA-RNA interaction analysis showed that OIP5-AS1 might sponge miR-34a. In OC cells, overexpression of OIP5-AS1 resulted in the upregulated expression of snail, while overexpression of miR-34a downregulated the expression of snail. In addition, overexpression of miR-34a reduced the effects of overexpression of OIP5-AS1 on the expression of snail. In cell invasion and migration assay, overexpression of OIP5-AS1 and snail resulted in increased OC cell invasion and migration, while overexpression of miR-34a decreased OC cell invasion and migration. Moreover, overexpression of miR-34a attenuated the effects of OIP5-AS1 overexpression on OC cell invasion and migration. CONCLUSIONS: Therefore, OIP5-AS1 may upregulate snail expression in OC by sponging miR-34a to promote OC cell invasion and migration.

Synergistic effect of microRNA and albumin-bound nanoparticles for inhibition of glioblastoma cancer cell proliferation

The functional significance of upregulation miR-34a in combination with albumin-bound paclitaxel nanoparticles in U251 glioblastoma cell line has been evaluated. The MTT assay determined that miR-34a and albumin-bound paclitaxel nanoparticles can reduce cell viability, but the combination of both factors has a stronger effect on cell viability. The application of qRT-PCR has demonstrated that the transduction of miR-34a could lead to exogenous upregulation of miR-34a level and downregulation of SURVIVIN. Moreover, treatment of U251 cells with miR-34a and nanoparticles together considerably inhibit SURVIVIN expression compared to miR-34a and nanoparticles alone. Flow cytometry showed that upon miR-34a overexpression cell cycle arrested in G1 phase, while treatment with nanoparticles increased the cell population in G2 phase. Upregulation of miR-34a along with treatment with nanoparticles elevated the number of cells arrested in G1/ G2 phases of the cell cycle. Expression of miR-34a with albumin-bound paclitaxel nanoparticles reduced cell viability, downregulated SURVIVIN and enhanced cell cycle arrest in G1/G2 phases. Thus, the upregulation of miR-34a with these nanoparticles are potential candidates therapeutic for glioblastoma cancer.

Expression and clinical significance of serum miR-23a and miR-34a in patients with age-related macular degeneration / 国际眼科杂志(Guoji Yanke Zazhi)

@#AIM: To investigate the expression levels of serum miR-23a and miR-34a in patients with age-related macular degeneration(ARMD)and its relationship with the development of ARMD. <p>METHODS: Totally 102 patients with ARMD who were treated in our hospital from May 2015 to February 2018 were enrolled in the case group, and 70 healthy subjects in the same period were used as control group. The relative expression levels of miR-23a and miR-34a in serum were detected by RT-PCR, and the levels of serum tumor necrosis factor alpha(TNF-α)and nuclear factor kB(NF-kB)were detected by enzyme linked immunosorbent assay(ELISA). Analysis on the relationship of miR-23a, miR-34a expression levels with TNF-a, NF-kB in patients with ARMD and its diagnostic value for ARMD were taken.<p>RESULTS: The relative expression levels of serum miR-23a and miR-34a in the case group were significantly higher than those in the control group(<i>P</i><0.01). The relative expression levels of serum miR-23a and miR-34a in the advanced group were significantly higher than those in the middle and early stage(<i>P</i><0.01). The relative expression levels of serum miR-23a and miR-34a in the middle term patients were significantly higher than those in the early stage(<i>P</i><0.01). The serum levels of TNF- α and NF-κB in the case group were significantly higher than those in the control group(<i>P</i><0.01). There was a significant positive correlation between serum miR-23a and TNF-α and NF-kB in the case group(<i>r</i>=0.798, 0.720, both <i>P</i><0.01), and serum miR-34a was significantly positively correlated with TNF-α and NF-kB(<i>r</i>=0.814, 0.740, both <i>P</i><0.01). The area under the ROC curve(AUC)of serum miR-23a and miR-34a for diagnosis of ARMD was 0.831 and 0.867, respectively.<p>CONCLUSION: The expression of miR-23a and miR-34a in serum of ARMD patients is up-regulated, which may be involved in the development and progression of ARMD by promoting inflammation and oxidative stress. Detection of serum miR-23a and miR-34a may be helpful for early diagnosis and prevention of ARMD.

Involvement of miR-34a and p53 protein of cerebral cortex in electroacpuncture analgesia in mice with neuropathic pain / 针刺研究

OBJECTIVE: To explore the involvement of miR-34a in cerebral cortex mediated anti-hyperalgesic effect of electroacupuncture (EA) in mice with neuropathic pain induced by chronic constriction injury (CCI) of sciatic nerve, so as to reveal its mechanisms underlying improvement of neuropathic pain. METHODS: A total of 75 male C57BL/6 mice were equally randomized into 3 groups: sham, CCI model and CCI+EA (n=25 in each group). Mice of the sham group received simple separation of the right sciatic nerve without ligation. The CCI model was established by liagation of the right sciatic nerve. EA (2 Hz /15 Hz, 1 mA) was applied to bilateral "Zusanli" (ST36) and "Sanyinjiao" (SP9) for 30 min, once every other day. The mechanical and thermal pain threshold of the bilateral hind-paws was detected at the 3rd, 5th and 7th day after modeling, and the expression of miR-34a of bilateral cerebral cortex tissues and that of p53 protein of the left cerebral cortex were determined by using quantitive real time PCR and Western blot, respectively. RESULTS: The mechnical paw withdrawal frequency were significantly higher and the thermal paw withdrawal latencies (PWLs) were significantly shorter at the affected hind-limb (rather than at the healthy hind limb) on day 3, 5 and 7 in the CCI model group than those in the sham group (P<0.05), and considerably reversed at the affected hind-limb (rather than at the healthy hind limb) in the EA group than in the CCI model group (P<0.05), suggesting an analgesic effect of EA intervention. After modeling, the expression levels of miR-34a and p53 on day 3, 5 and 7 were significantly up-regulated in the left cerebral cortex tissue (rather than in the right cerebral cortex) of the CCI model group in comparison with the sham group (P<0.05). After EA intervention, the up-regulated expression levels of miR-34a and p53 in the left cerebral cortex tissue (rather than in the right cerebral cortex) were obviously suppressed in the EA group relevant to the CCI model group (P<0.05). CONCLUSION: EA stimulation of ST36 and SP9 can down-regulate the expression of miR-34a and p53 in the contra-lateral cerebral cortex tissue of the CCI mice, which may contribute to its anti-hyperalgesic effect.

Inhibitory effect of miR-34a on biological behavior of uveal melanoma cells and its mechanism / 中华实验眼科杂志

Objective To study the effect of microRNA-34a(miR-34a) on the biological behavior of uveal melanoma cells and its mechanism.Methods Uveal melanoma M23 cells were used as research objects,miR-34a mimics and mimics negative control were transfected into the cells respectively as miR-34a transfection group and the negative control group,and the non-transfected cells served as the normal control group.The overexpression effect was validated by real-time PCR.MTT assay was used to detect cell proliferation.Cell invasion and migration were detected by Transwell test.Target gene prediction library predicted target genes of miR-34a,and the target gene was identified by luciferase activity report.Real-time PCR and Western blot were used to detect the mRNA and protein expression of target genes.MiR-34a mimics and microphthalmia-associtated transcription factor (MITF) overexpression vectors were cotransfected into M23 cells.Cell proliferation,invasion and migration abilities were detected by MTT assay and Transwell test,respectively.The mRNA and protein expressions of MITF were detected by real-time PCR and Western blot.Results The expression of miR-34a in M23 cells transfected with miR-34a mimics increased.The cell proliferation (A570),number of invasive cells and migrating cells were significantly different among the miR-34a transfection group,negative control group and normal control group (F =18.000,P =0.003;F =20.345,P =0.002;F=15.717,P=0.004).The proliferation,invasion and migration ability of M23 cells in the miR-34a transfection group were significantly decreased compared with the negative control group and normal control group (all at P＜0.05).Target gene prediction library and luciferase activity report showed that MITF was the target gene of miR-34a.The relative expression levels of MITF mRNA and protein were 0.45 ±0.06 and 0.36± 0.04 in the miR-34a transfection group,0.99± 0.11 and 0.62 ± 0.05 in the negative control group,1.00 ± 0.07 and 0.63 ± 0.08 in the normal control group,respectively,and compared with the negative control group and normal control group,the expression of MITF in miR-34a transfection group were significantly decreased (all at P＜0.05).Cell proliferation (A570),the number of invasived cells and the number of migrated cells were 0.35±0.02,29.48±3.20 and 41.87±5.82 in the miR-34a + MITF group,0.26 ± 0.03,18.53 ± 1.47 and 27.64 ± 2.45 in the miR-34a + Vector group,respectively,the proliferation,invasion and migration ability of the cell.s in the miR-34a+MITF group was significantly higher than that in the miR-34a+Vector group (all at P＜0.05).Conclusions miR-34a can inhibit the malignant phenotype of uveal melanoma cells by inhibiting the expression of the target gene MITF.

miR-34a mediates the multidrug resistance of breast cancer by regulating the expression of downstream gene / 临床检验杂志

Objective@#To identify the therapeutic target of multidrug resistance of breast cancer by investigating the regulatory mechanism of the abnormal expression of miR-34a and FUT8 on the multidrug resistance of breast cancer. @*Methods@#The expression levels of miR-34a and FUT8 in MCF-7 and MCF-7/ADR cells were detected by the real-time PCR and western blot. The transfection efficiency of miR-34a in breast cancer cells was determined by the real-time PCR. The targeting relationship between miR-34a and FUT8 was predicted by the bioinformatics method, and further verified by the dual-luciferase reporter assay. After the expression of miR-34a was specifically regulated, the changes of FUT8 mRNA and protein levels in transfected cells were detected by the real-time PCR, Western blot and immunofluorescence staining, respectively. After the expression of miR-34a was specifically regulated, the proliferation and multidrug resistance of MCF-7 and MCF-7/ADR cells were determined by the CCK8 and immunofluorescence assays. @*Results@#The expression levels of miR-34a in MCF-7 cells were significantly higher than that in MCF-7/ADR cells ( P =0.002 6). The expression levels of FUT8 gene in MCF-7/ADR cells were significantly higher than that in MCF-7 cells ( P =0.001 6). FUT8 was the target of miR-34a regulating the drug resistance of breast cancer ( P =0.001 9). The up-regulation of miR-34a in MCF-7/ADR cells could significantly inhibit the expression of FUT8 , and the multidrug resistance and proliferation of MCF-7/ADR cells. While, the down-regulation of miR-34a increased the expression of FUT8 , and enhanced the multidrug resistance and proliferation of MCF-7 cells. @*Conclusion@#MiR-34a mediates the multidrug resistance of breast cancer by regulating the expression of downstream gene FUT8 .

Dynamic expressions of miR-34a and related target genes sirtuin 1 and nuclear factor-E2-related factor 2 and their significances in subependymal zone of rats with cerebral ischemia / 中华神经医学杂志

Objective To observe the expression changes ofmiR-34a and its related target genes sirtuin 1 (Sirtl) and nuclear factor-E2-related factor 2 (Nrf2) in subependymal zone (SVZ) of rats after cerebral ischemia,and to explore the effect of miR-34a on proliferation of endogenous neural stem cells (NSCs) after cerebral ischemia.Methods Forty SD rats were randomly divided into control group,cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group (n=10).Middle cerebral artery occlusion models were established by thread embolization in the latter three groups,and the SVZs were taken one,three and 7 d after model making,respectively.Immunofluorescence staining was used to detect the expression of nestin,real-time PCR was used to detect the mRNA expressions ofmiR-34a,Sirtl and Nrf2,and Western blotting was used to detect the protein expressions of Sirt1 and Nrf2.Results (1) In SVZs of cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group,the number of nestin positive cells increased gradually ([2.013±0.526],[4.821 ±1.154],and [6.394±1.027]) cells/filed,with statistical differences (P＜0.05).(2) In SVZs of cerebral ischemia one d group,cerebral ischemia three d group and cerebral ischemia 7 d group,the expression levels of miR-34a mRNA increased gradually (0.295±0.145,0.701 ±0.075,and 1.136±0.018),the Sirt1 mRNA expression levels showed a downward trend (0.835±0.024,0.620±0.133,and 0.278±0.110),the Nrf2 mRNA expression levels obviously decreased (1.032±0.256,0.833±0.187,and 0.630±0.123),the Sirt1 protein expression levels showed a downward trend (0.832±0.018,0.731±0.156,and 0.454±0.132),and the Nrf2 protein expression levels obviously decreased (1.003±0.133,0.813±0.111,and 0.671 ±0.071),with statistically significant differences (P＜0.05).(3)Statistical correlation analysis showed that the number of nestin positive cells was positively correlated with miR-34a mRNA expression level and negatively correlated with Sirt1 and Nrf2 mRNA and protein expression levels after cerebral ischemia (r=0.887,P=0.003;r=-0.746,P=0.005;r=-0.521,P=0.013;r=-0.344,P=0.025;r=-0.863,P=0.000).Conclusions MiR-34a may regulate the proliferation of NSCs through Sirt1/Nrf2 pathway after cerebral ischemia.

The Long Noncoding RNA NEAT1 Targets miR-34a-5p and Drives Nasopharyngeal Carcinoma Progression via Wnt/β-Catenin Signaling

PURPOSE: Long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEAT1) has been deemed an oncogene in many human cancers. However, the underlying mechanism of NEAT1 in nasopharyngeal carcinoma (NPC) progression remains largely unclear. MATERIALS AND METHODS: Quantitative real-time PCR assay was performed to assess the expression of NEAT1 and miR-34a-5p in NPC tissues and cells. Western blot analysis was used to observe cell epithelial to mesenchymal transition (EMT) and the activation of Wnt/β-catenin signaling in 5-8F cells. MiRNA directly interacting with NEAT1 were verified by dual-luciferase reporter assay and RNA immunoprecipitation. Cell proliferation ability was determined by CCK-8 assay, and cell migration and invasion capacities were assessed by transwell assays. An animal model was used to investigate the regulatory effect of NEAT1 on tumor growth in vivo. RESULTS: Our data revealed that NEAT1 is upregulated, while miR-34a-5p is downregulated in NPC tissues and cell lines. NEAT1 knockdown repressed tumor growth in vitro and in vivo. Additionally, we discovered that NEAT1 directly binds to miR-34a-5p and suppresses miR-34a-5p expression. Moreover, NEAT1 knockdown exerted suppression effects on cell proliferation, migration, invasion, and EMT by miR-34a-5p. NEAT1 knockdown blocked Wnt/β-catenin signaling via miR-34a-5p. CONCLUSION: Our study demonstrated that NEAT1 targets miR-34a-5p at least partly to drive NPC progression by regulating Wnt/β-catenin signaling, suggesting a potential therapeutic target for NPC.