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This study aimed to investigate the effect of Wenyang Zhenshuai Granules(WYZSG) on autophagy and apoptosis of myocardial cells in rats with sepsis via regulating the expression of microRNA-132-3p(miR-132-3p)/uncoupling protein 2(UCP2). Sixty SD rats were randomly divided into modeling group(n=50) and sham operation group(n=10). The sepsis rat model was constructed by cecal ligation and perforation in the modeling group. The successfully modeled rats were randomly divided into WYZSG low-, medium-and high-dose groups, model group and positive control group. Rats in the sham operation group underwent opening and cecum division but without perforation and ligation. Hematoxylin-eosin(HE) staining was used to observe the pathological changes of rat myocardial tissue. Myocardial cell apoptosis was detected by TdT-mediated dUTP nick end labeling(TUNEL) assay. Real-time quantitative polymerase chain reaction(RT-qPCR) was performed to detect the expression of miR-132-3p and the mRNA expressions of UCP2, microtubule-associated protein light chain 3(LC3-Ⅱ/LC3-Ⅰ), Beclin-1 and caspase-3 in rat myocardial tissue. The protein expressions of UCP2, LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3 in myocardial tissue were detected by Western blot. Dual luciferase reporter assay was used to verify the regulatory relationship between miR-132-3p and UCP2. The myocardial fibers of sepsis model rats were disordered, and there were obvious inflammatory cell infiltration as well as myocardial cell edema and necrosis. With the increase of the WYZSG dose, the histopathological changes of myocardium were improved to varying degrees. Compared with the conditions in the sham operation group, the survival rate and left ventricular ejection fraction(LVEF) of rats in the model group, positive control group and WYZSG low-, medium-and high-dose groups were decreased, and the myocardial injury score and apoptosis rate were increased. Compared with the model group, the positive control group and WYZSG low-, medium-and high-dose groups had elevated survival rate and LVEF, and lowered myocardial injury score and apoptosis rate. The expression of miR-132-3p and the mRNA and protein expressions of UCP2 in myocardial tissue in the model group, positive control group and WYZSG low-, medium-and high-dose groups were lower, while the mRNA and protein expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3 were higher than those in the sham operation group. Compared with model group, the positive control group and the WYZSG low-, medium-and high-dose groups had an up-regulation in the expression of miR-132-3p and the mRNA and protein expressions of UCP2, while a down-regulation in the mRNA and protein expressions of LC3-Ⅱ/LC3-Ⅰ, Beclin-1 and caspase-3. WYZSG inhibited excessive autophagy and apoptosis of myocardial cells in septic rats and improved myocardial injury, possibly by regulating the expression of miR-132-3p/UCP2.
Subject(s)
Animals , Rats , Rats, Sprague-Dawley , Caspase 3 , Beclin-1/genetics , Stroke Volume , Ventricular Function, Left , Apoptosis/genetics , Autophagy/genetics , Heart Injuries , MicroRNAs/geneticsABSTRACT
Objective:To validate the effect of TUT4 on the radiosensitivity of esophageal epithelial cells (HEEC) by regulating the uridylation of miR132/212 clusters.Methods:The expression of TUT4 in HEEC at 0, 6, 18, 24 and 48 h after 0, 2, 4, 6 and 8 Gy X-ray irradiation was detected by PCR. The HEEC cells were divided into four groups: NC group, shTUT4 group, 6 Gy group, and 6 Gy+ shTUT4 group. The effects of TUT4 on cell radiosensitivity, cell proliferation, cell cycle, mitochondrial damage, and oxygen free radical production were detected respectively. The effect of down-regulated TUT4 expression on miR132/212 uridylation was detected by RT-PCR, and the radiosensitivity of HEEC with overexpression of miR132/212 or miR132/212+ UUU was detected by clone formation and proliferation assay, respectively. Proliferation assay was performed to detect the proliferation of HEEC when TUT4 expression was down-regulated and miR132/212 or miR132/212+ UUU was overexpressed.Results:TUT4 expression increased after different doses of X-ray irradiation ( t=12.84, 62.06, 27.86, 32.43, P<0.05). Downregulation of TUT4 expression increased the radiosensitivity of HEEC ( t=13.2, 5.85, 7.31, P<0.05) with a SER D0of 1.41 and D0=0.79, Dq=1.61, SF2=0.47. Compared with 6 Gy group, cell proliferation in 6 Gy+ shTUT4 group was decreased ( t=7.12, 13.63, P<0.05), cells in S phase were increased ( t=11.98, P<0.05), mitochondrial damage was increased ( t=11.98, P<0.05), and ROS level was increased ( t=15.65, P<0.05). Down-regulation of TUT4 expression increased miR132/212 expression and decreased miR132/212+ UUU expression ( t=27.90, 60.99, P<0.05). Overexpression of miR132/212 increased the radiosensitivity of HEEC, and overexpression of miR132/212+ UUU decreased the radiosensitivity of HEEC, with SER D0 of 1.20 and 0.71, respectively. Cell proliferation of shTUT4 + miR132/212 group waslower than that of shTUT4 group( t=4.76, 7.65, P<0.05), and cell proliferation of shTUT4 + miR132/212+ UUU group was higher than that of shTUT4 ( t=7.22, P<0.05). Conclusions:X-ray irradiation increased the expression of TUT4 in HEEC, and the down-regulation of TUT4 reduced HEEC radiosensitivity and radiation damage, where the uridylation of miR132/212 was involved in.
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BACKGROUND: Mechanical stress can influence the proliferation and differentiation of MC3T3-E1 cells and trigger differential expression of miR-132-3p. However, further research is warranted concerning whether tensile stress can influence the proliferation and differentiation of osteoblasts by regulating miR-132-3p. OBJECTIVE: To determine the expression of osteogenic differentiation markers and miR-132-3p in MC3T3-E1 cells under 12% cyclic stretch and to explore the effect of miR-132-3p on cell proliferation and differentiation. METHODS: MC3T3-E1 cells were loaded with 0% and 12% tensile stress, and alkaline phosphatase activity, osteocalcin mRNA and miR-132-3p expression levels were detected. MC3T3-E1 cells were transiently transfected with miR-132-3p mimics and a negative control transfection group was set up. The expression of alkaline phosphatase, osteocalcin and Runx2 mRNA in transfected cells were detected by qRT-PCR, and the effect of miR-132-3p on cell proliferation were detected by cell counting kit-8 assay. RESULTS AND CONCLUSION: The alkaline phosphatase activity and osteocalcin mRNA expression were down-regulated in MC3T3-E1 cells under 12% stretch stress (P < 0.01), and the expression of miR-132-3p was significantly increased (P < 0.05). QRT-PCR results showed the expression levels of osteogenic differentiation markers alkaline phosphatase activity, osteocalcin, and Runx2 mRNA in miR-132-3p mimics group were significantly decreased after intracellular transfection of miR-132-3p (P < 0.05). Compared with the negative control transfection group, the cell proliferation in the miR-132-3p mimic group was decreased at 24, 48, and 72 hours after transfection (P < 0.001), and the most obvious reduction was observed after 48-hour transfection. These findings indicate that 12% cyclic tensile stress can negatively regulate the proliferation and differentiation ability of MC3T3-E1 cells by overexpressing miR-132-3p.
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MicroRNA-132 (miR-132), a small RNA that regulates gene expression, is known to promote neurogenesis in the embryonic nervous system and adult brain. Although exposure to psychoactive substances can increase miR-132 expression in cultured neural stem cells (NSCs) and the adult brain of rodents, little is known about its role in opioid addiction. So, we set out to determine the effect of miR-132 on differentiation of the NSCs and whether this effect is involved in opioid addiction using the rat morphine self-administration (MSA) model. We found that miR-132 overexpression enhanced the differentiation of NSCs in vivo and in vitro. Similarly, specific overexpression of miR-132 in NSCs of the adult hippocampal dentate gyrus (DG) during the acquisition stage of MSA potentiated morphine-seeking behavior. These findings indicate that miR-132 is involved in opioid addiction, probably by promoting the differentiation of NSCs in the adult DG.
Subject(s)
Animals , Male , Cell Differentiation , Cell Line, Tumor , Dentate Gyrus , Metabolism , Gene Expression Regulation , MicroRNAs , Metabolism , Neural Stem Cells , Metabolism , Opioid-Related Disorders , Metabolism , Rats, Sprague-DawleyABSTRACT
@#AIM:To analyze the expression and significance of miR-132 in the plasma of patients with diabetic retinopathy.<p>METHODS: From July 2015 to October 2015, a total of 55 patients with diabetes who were treated in our hospital were divided into 5 groups according to diabetic retinopathy clinical staging international standard, including 13 cases of no obvious retinopathy as group A, 10 cases of mild NPDR as group B, 11 cases of moderate NPDR as group C, 12 cases of severe NPDR as group D and 9 cases of PDR as group E, at the same time, the other 12 healthy people were enrolled as control group F. Real-time quantitative PCR(qRT-PCR)technique was used to detect the relative expression of miR-132 in the plasma of 55 patients with different stages of diabetic retinopathy and the expression difference between different groups were compared. <p>RESULTS: Compared with the healthy control group, the expression of miR-132 in the plasma was decreased in other groups(<i>P</i><0.05)except in the background of diabetic retinopathy, however, there was no significant difference between the NPDR groups and the PDR groups.<p>CONCLUSION: The expression of miR-132 in the plasma of patients with DR was slightly lower in the non-proliferative and proliferative stage than in healthy subjects and background diabetic retinopathy. Furthermore, miR-132 may be a new biomarker for DR.
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Objective To study the effect of exosomes ( EXs) released from high expression of miR-132-3p mesenchymal stem cells (MSCs) on hypoxia/reoxygenation (H/R) injured endothelial cell function. Methods MSCs extracted from bone marrow of C57BL/6 mice were cultured primarily. MSCmiR-132-3p was obtained from MSCs infected with lentivirus loaded with miR-132-3p vector. At the same time,MSCNC was obtained by infecting MSCs with control lentivirus loaded with scramble sequence. EXs released from MSCNCand MSCmiR-132-3pwas isolated,and MSC-EXs and MSC-EXsmiR-132-3pwere obtained respectively. The obtained EXs and H/R damaged mouse brain microvascular endothelial cells (bend3) were co-cultured. According to culture conditions,the cells were divided into normal culture group (normal cell culture),H/R group (making a H/R model),MSC-EXs group (MSC-EXs co-culture),MSC-EXsmiR-132-3p group (MSC-EXsmiR-132-3pco-culture), and MSC-EXsmiR-132-3p+ LY294002 group ( before the cells and MSC-EXsmiR-132-3pwere co-cultured,treated by adding phosphatidyl alcohol 3 kinase [ PI3K] signaling pathway blocker LY294002 [20 μmol/L]). Quantitative real-time quantitative polymerase chain reaction was used to detect the expression of miR-132-3p in MSCs,MSC-EXs,and bend3 cells. Angiogenesis kit was used to detect angiogenic ability of bend3 cells,and 3-(4,5-dimethylthiazole-2)-2,5-diphenyltetrazolium bromide (MTT) assay was used to detect the proliferative capacity of bend3 cells. Scratch test was used to detect the migration ability of bend3 cells. hochest33258 staining showed cell apoptosis. Western blot was used to detect the phosphorylation level of protein kinase B ( Akt) . Results Compared with the H/R group, the MSC-EXs treatment group significantly improved the angiogenesis,proliferation,migration abilities, and Akt phosphorylation level of bend 3 cell damage induced by H/R (The H/R group were 3 ± 1,0. 275 ± 0. 020,147 ± 8 μm,and 0. 89 ± 0. 12,respectively;the MSC-EXs treatment group were 8 ± 3,0. 358 ± 0. 030,218 ± 10 μm, and 1. 37 ± 0. 25 μm,respectively;all P<0. 01). Apoptosis was significantly reduced (47 ± 2% vs. 63 ± 2%,all P<0. 01). Compared with the MSC-EXs treatment group,the angiogenesis,proliferation,migration abilities,and Akt phosphorylation level of bend 3 cells in the MSC-EXsmiR-132-3ptreatment group were increased (14 ±3,0. 444 ± 0.050,357±10μm,and1.67±0.23,respectively,all P<0.01).Apoptosis was significantly reduced (34±1%,all P<0. 01) . Compared with the MSC-EXsmiR-132-3ptreatment group, cell proliferation, migration, angiogenesis abilities,and Akt phosphorylation level in the MSC-EXsmiR-132-3p+LY294002 group were significantly reduced (5 ± 2,0. 304 ± 0. 050,175 ± 8 μm and 0. 95 ± 0. 11,respectively,all P<0. 01). Conclusion MSC-EXs with high expression of miR-132-3p may improve many physiological functions of H/R-induced damaged cerebrovascular endothelial cells by activating PI3K/Akt signaling pathway.
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PURPOSE: Long non-coding RNA taurine upregulated gene 1 (TUG1) is reported to be a vital regulator of the progression of various cancers. This study aimed to explore the exact roles and molecular mechanisms of TUG1 in osteosarcoma (OS) development. MATERIALS AND METHODS: Real-time quantitative PCR was applied to detect the expressions of TUG1 and microRNA-132-3p (miR-132-3p) in OS tissues and cells. Western blot was performed to measure protein levels of sex determining region Y-box 4 (SOX4). Cell viability was assessed using XTT assay. Cell apoptosis was evaluated using flow cytometry and caspase-3 activity detection assays. Bioinformatics analysis and luciferase reporter experiments were employed to confirm relationships among TUG1, miR-132-3p, and SOX4. RESULTS: TUG1 was highly expressed in human OS tissues, OS cell lines, and primary OS cells. TUG1 knockdown hindered proliferation and induced apoptosis in human OS cell lines and primary OS cells. Moreover, TUG1 inhibited miR-132-3p expression by direct interaction, and introduction of miR-132-3p inhibitor partly abrogated the effect of TUG1 knockdown on the proliferation and apoptosis of OS cells. Furthermore, SOX4 was validated as a target of miR-132-3p. Further functional analyses revealed that miR-132-3p inhibited proliferation and induced apoptosis of OS cells, while this effect was greatly abated following SOX4 overexpression. Moreover, TUG1 knockdown suppressed proliferation and promoted apoptosis by upregulating miR-132-3p and downregulating SOX4 in primary OS cells. CONCLUSION: TUG1 facilitated proliferation and suppressed apoptosis by regulating the miR-132-3p/SOX4 axis in human OS cell lines and primary OS cells. This finding provides a potential target for OS therapy.
Subject(s)
Humans , Apoptosis/genetics , Biomarkers, Tumor , Bone Neoplasms/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic/genetics , Gene Knockdown Techniques , MicroRNAs/genetics , Osteosarcoma/genetics , RNA, Long Noncoding/genetics , Real-Time Polymerase Chain Reaction , Reverse Transcriptase Polymerase Chain Reaction , SOXC Transcription Factors/genetics , Transcriptional Activation , Tumor Cells, Cultured , Up-RegulationABSTRACT
Objective To investigate the miR-132 expression in different segments of the nervous system through mechanical damage induced neuropathic pain rat model. Methods A total of 45 male SD rats(250~300 g)were randomly divided into Native,Shame and BCCI groups,with 15 rats in each group. The mechanical with-drawal threshold(MWT)was measured 1 day before BCCI,and 1 d,7 d,14 d after BCCI. The expression levels of miR-132 in Hippocampus(HP),L4~6 spinal dorsal horn(SDH)and dorsal root ganglia(DRG)were measured indi-vidually by reverse transcription-polymerase chain reaction(RT-PCR). Results Compared with Shame group and Native group,MWT decreased significantly in BCCI group 7 d and 14 d after operation(P<0.01). Compared with the Native group,the expression of miR-132 in DRG decreased in BCCI group and Shame group 7 d after operation (P < 0.05). Rats in shame group recovered by 14 d while BCCI was still decreased significantly(P < 0.05). The miR-132 expression of SDH was upregulated significantly in BCCI group at day 7(P<0.05). On the 14 day after operation,the expression of miR-132 was downregulated significantly in BCCI group and Shame group(P<0.05). BCCI group decreased significantly at 14 d(P < 0.01). Conclusions MiR-132 was involved in the regulation of neuropathic pain. The expression of miR-132 has its own characteristics in different segments and time points.
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Objective To investigate the miR-132 expression in different segments of the nervous system through mechanical damage induced neuropathic pain rat model. Methods A total of 45 male SD rats(250~300 g)were randomly divided into Native,Shame and BCCI groups,with 15 rats in each group. The mechanical with-drawal threshold(MWT)was measured 1 day before BCCI,and 1 d,7 d,14 d after BCCI. The expression levels of miR-132 in Hippocampus(HP),L4~6 spinal dorsal horn(SDH)and dorsal root ganglia(DRG)were measured indi-vidually by reverse transcription-polymerase chain reaction(RT-PCR). Results Compared with Shame group and Native group,MWT decreased significantly in BCCI group 7 d and 14 d after operation(P<0.01). Compared with the Native group,the expression of miR-132 in DRG decreased in BCCI group and Shame group 7 d after operation (P < 0.05). Rats in shame group recovered by 14 d while BCCI was still decreased significantly(P < 0.05). The miR-132 expression of SDH was upregulated significantly in BCCI group at day 7(P<0.05). On the 14 day after operation,the expression of miR-132 was downregulated significantly in BCCI group and Shame group(P<0.05). BCCI group decreased significantly at 14 d(P < 0.01). Conclusions MiR-132 was involved in the regulation of neuropathic pain. The expression of miR-132 has its own characteristics in different segments and time points.
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Objective:To explore the biological function of miR-132 in ovarian cancer and the target. Methods: 22 cases ovarian cancer tissue and non-tumor tissue adjacent were collected,the expression of miR-132 in tumor tissue and non-tumor tissue, normal ovarian epithelial cells and ovarian cancer cell were detected by RT-PCR. The normal ovarian epithelial cells which the expression of miR-132 maximum or minimum were chosen, and they were divided into two groups, respectively with transfection of negative control plasmid ( NC) and miR-132 mimic plasmid. The expression of miR-132 after transfection was detected by RT-PCR,the cell proliferation and cell apoptosis were detected by CCK-8 method and flow cytometry instrument respectively,the expression of Ezrin protein was detected by Western blot. Results:The expression of miR-132 in tumor tissue was significantly lower than the tumor tissue adjacent,the expression of miR-132 in ovarian cancer cell lines was significantly lower than normal ovarian epithelial cells, the differences were statistically significant (P<0. 05). The SKOV3 cell lines was chosed for gene transfection,compared with NC group, transfection with miR-132 mimic plasmid could significantly reduce cell proliferation, increase cell apoptosis, the difference had statistical significance ( P<0. 05 ) . Western blot results showed that up-regulation miR-132 significantly increased the Ezrin protein expression in ovarian cancer SKOV3 cells ( P<0. 05 ) . Conclusion: In ovarian cancer, miR-132;inhibits proliferation and induces apoptosis of ovarian cancer via Ezrin,it may be a tumor suppressor gene.
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Objective:To investigate the expression of miR-132 in peripheral blood mononuclear ceils(PBMCs) and plasma of patients with oral lichen planus (OLP) and the relationship of miR-132 with clinical forms of OLP.Methods:40 OLP patients (24 nonerosive OLP,16 erosive OLP) and 40 healthy controls were included.The relative expression of miR132 in peripheral blood mononuclear cells (PBMCs) and plasma was examined by quantitative real-time PC R.Results:The expression of miR-132 in both PBMCs and plasma of OLP patients was significantly higher than those in healthy controls(P ＜ 0.01).miR-132 expression of PBMCs and plasma in erosive OLP group was significantly higher than that in non-erosive OLP group and healthy control(P ＜ 0.01).However,the expression of miR-132 in PBMCs and plasma showed no significant difference between non-erosive OLP group and healthy control (P ＞0.05).Conclusion:miR-132 might be involved in the abnormal immune response of OLP patients,and miR-132 can be utilized as an assistant biomarker for the evaluation of the severity of OLP.
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Objective To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7. Methods Hepatocellular carcinoma Huh7 was cultured in vitro and lipidosome was used to transfect miR-520c-3p and miR-132, respectively or together. The effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry, and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime PCR. Results Compared with the control group, the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly, and the apoptosis ratio increased distinctly (P < 0.05). Besides, the effect of the co-transfection group was better than that of the single transfection group. The protein levels of GPC3 (Glypican-3) and YAP (Yes-associated protein), the target genes transfected only by miR-520c-3p and miR-132, respectively, reduced obviously (P < 0.05), which was similar with the co-infected cells, but cells transfected by miR-132 only showed a decrease of YAP. Conclusions The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP, enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.
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<p><b>Objective</b>To explore the expression of miR-132 in prostate cancer and its effects on the growth and invasiveness of prostate cancer cells and the influence of hypoxia on the level of miR-132 and biological behavior of prostate cancer cells.</p><p><b>METHODS</b>Real time PCR was used to measure the expression level of miR-132 in the prostate cancer tissue, analyze its relationship with the clinical stage and Gleason score of prostate cancer, and determine the influence of hypoxia on the miR-132 level in the human prostate cancer PC3 cell line in vitro. Sulfor-hodamine B chromatometry and Matrigel invasion assay were employed to detect the effects of hypoxia and miR-132 mimic plasmid transfection on the viability and invasiveness of PC3 cells in vitro.</p><p><b>RESULTS</b>The miR-132 level in the prostate cancer was significantly declined to 52.38% (in T1－T2 stages) and 21.59% (in T3－T4 stages) of that in the cancer-adjacent tissue (both P<0.01). In hypoxia, the expression of miR-132 was significantly decreased in the PC3 cells (P<0.01). After 48 and 72 hours of transfection with miR-132 mimic plasmid, the viability of the PC3 cells was markedly reduced (P<0.05 or P<0.01), and their invasiveness decreased by 57.5% after 48 hours (P<0.01). However, there was no significant difference in the viability or invasiveness of the PC3 cells transfected with miR-132 mimic plasmid between normoxia and hypoxia.</p><p><b>CONCLUSIONS</b>The reduced expression of miR-132 is closely related to the clinical stage and Gleason score of prostate cancer. Hypoxia increases the viability and invasiveness of prostate cancer cells in vitro by down-regulating the expression of miR-132 and consequently may promote the growth and metastasis of prostate cancer.</p>
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OBJECTIVE@#To investigate the effects of co-transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of hepatocellular carcinoma Huh7.@*METHODS@#Hepatocellular carcinoma Huh7 was cultured in vitro and lipidosome was used to transfect miR-520c-3p and miR-132, respectively or together. The effects of transfection of miR-520c-3p and miR-132 on proliferation and apoptosis of Huh7 were detected by CCK8 and Annexin V staining and flow cytometry, and the expression level of the targeted gene of over-expressed miR-520c-3p and miR-132 was determined by Western blot and realtime PCR.@*RESULTS@#Compared with the control group, the proliferation ability of Huh7 of the single transfected and co-transfected miR-520c-3p and miR-132 decreased significantly, and the apoptosis ratio increased distinctly (P < 0.05). Besides, the effect of the co-transfection group was better than that of the single transfection group. The protein levels of GPC3 (Glypican-3) and YAP (Yes-associated protein), the target genes transfected only by miR-520c-3p and miR-132, respectively, reduced obviously (P < 0.05), which was similar with the co-infected cells, but cells transfected by miR-132 only showed a decrease of YAP.@*CONCLUSIONS@#The co-transfection of miR-520c-3p and miR-132 can target-regulate the expression of GPC3 and YAP, enhance the exhibition effect on proliferation of hepatocellular carcinoma Huh7 and induce cell apoptosis synergistically.