miR-221 changes resistance of lung cancer cells to gefitinib by regulating PTEN

AIM: To explore the effect of microRNA-221 (miR-221) on resistance of lung cancer cells to gefitinib, and to investigate its related mechanism. METHODS: RT-qPCR was used to detect the levels of miR-221 expression between gefitinib-sensitive cell line PC9 and gefitinib-resistant cell line PC9/GR. The PC9/GR cells were transfected with miR-221 inhibitor by Lipofectamine 2000. The drug sensitivity of these cells to gefitinib was determined by CCK-8 assay. The protein expression level of phosphatase and tensin homologue deleted on chromosome ten (PTEN) was determined by Western blot. The 3'-UTR of PTEN was cloned into luciferase reporter vector and its luciferase activity was detected to verify whether miR-221 targets PTEN. RESULTS: The expression level of miR-221 in the PC9/GR cells was significantly higher than that in the PC9 cells (P<0.05). The protein expression level of PTEN in the PC9/GR cells was lower than that in the PC9 cells (P<0.05). The IC₅₀ of gefitinib was significantly reduced in the PC9/GR cells after transfection with miR-221 inhibitor (P<0.05). The protein expression level of PTEN in the cells transfected with miR-221 inhibitor was increased as compared with control group and blank group (P<0.05). Inhibition of miR-221 expression enhanced the enzymatic activity of luciferase reporter vector of PTEN. CONCLUSION: miR-221 enhances the resistance of lung cancer cells to gefitinib by down-regulating the protein expression of PTEN.     

Targeted inhibition of microRNA-21 with antisense oligonucleotide and their effects on human leukemic K562 cells

AIM: To explore the inhibitory effect of anti-miRNA-21 oligonucleotide (AMO-miRNA-21) on human leukemic K562 cells. METHODS: K562 cells were transfected with AMO-miRNA-21, which was complementary to the miRNA-21 in a sequence-specific manner. Viability of K562 cells was measured by MTT assay and the optimal concentration for transfection was determined. The inhibitory effect of AMO on the K562 cell growth was examined by trypan blue dye exclusion assay at 24 h, 48 h and 72 h after transfection. Giemsas staining was used to detect morphologic changes of the transfected cells. The cell apoptosis and cell cycle progression were assayed by flow cytometry. Expression of microRNA-21 in the cells was measured by real-time PCR. RESULTS: The growth of cells treated with AMO-miRNA-21 was obviously inhibited compared with that in control groups (P<0.05). Very low cytotoxic and high inhibitory effects of AMO-miRNA-21 were found at concentration of 0.6 μmol/L. The inhibitory effect lasted for 72 h. Apoptotic cells were increased in AMO group and typical morphologic changes were conformed by Giemsas staining. One visible hypodiploid peak was detected in the histogram. However, the cell cycle progression was not inhibited evidently. The expression of microRNA-21 in the transfected cells was down-regulated significantly. CONCLUSION: Targeted inhibition of microRNA-21 with antisense oligonucleotide effectively suppresses leukemic K562 cells growth by inducing apoptosis. miRNA-21 might be a potential target for leukemia therapy.     

miR-221 promotes proliferation of lung cancer A549 cells by down-regulating PTEN

AIM: To explore the effect of microRNA-221 (miR-221) on the proliferation of lung cancer A549 cells, and to investigate its mechanism. METHODS: The A549 cells were transfected with miR-221 mimics by Lipofectamine 2000. The expression of miR-221 was detected by RT-qPCR. The expression of PTEN at mRNA and protein le-vels was detected by RT-qPCR and Western blot, respectively. The cell proliferation was examined by CCK-8 assay and colony formation assay. The 3'-UTR of PTEN was cloned into luciferase reporter vector and its enzymatic activity was detected to verify whether miR-221 targeted to PTEN. RESULTS: The expression level of miR-221 in the A549 cells was significantly increased after transfection with miR-221 mimics as compared with negative control group and blank group (P<0.01). The mRNA and protein levels of PTEN were significantly down-regulated compared with control group and blank group (P<0.05). In addition, miR-221 over-expression significantly promoted the proliferation of A549 cells (P<0.05). Moreover, miR-221 inhibited the enzymatic activity of luciferase reporter vector of PTEN. CONCLUSION: Over-expression of miR-221 significantly promotes the proliferation ability of human lung cancer A549 cells by down-regulation of PTEN.     

Construction of eukaryotic expression vector miRNA let-7a1 and its effect on the proliferation of lung cancer A549 cells

AIM: To construct a recombinant eukaryotic expression vector, pSilencer 4.1-let-7a1 and to express it in lung cancer A549 cells for detecting its effect on the proliferation of A549 cells. METHODS: The pre-let-7a1 sequence was amplified by RT-PCR using RNA from human lung cancer A549 cells, and then inserted into pSilencer 4.1-CMV neo vector to generate pSilencer 4.1-let-7a1 which was transfected into lung cancer A549 cells. The expression of miRNA let-7a1 was verified by RT-PCR. Its activity in A549 cells was determined by luciferase reporter assay after cotransfection of let-7a1 target sequence-reporter gene plasmid with pMIR-report let-7a1T, which was constructed by inserting let-7a1 target sequence into the luciferase reporter 3’UTR of pMIR-report luciferase vector. The effect of pSilencer 4.1-let-7a1 transfection on A549 cell proliferation was detected by MTT method. RESULTS: The sequences of cloned pre-let-7a1 were correct. RT-PCR results indicated that pSilencer 4.1-let-7a1 was effectively expressed in the transfected A549 cells. The relative luciferase activity was decreased significantly after A549 cells were co-transfected with pSilencer 4.1-let-7a1 and pMIR-report let-7a1T, indicating that let-7a1 was expressed effectively and had biologic activity in A549 cells that were transfected with pSilencer 4.1-let-7a1. MTT results showed that miRNA let-7a1 gene overexpression in A549 inhibited cell proliferation. CONCLUSION: The eukaryotic expression vector pSilencer 4.1-let-7a1 is successfully constructed and effectively expresses in A549 cell. The overexpression of miRNAlet-7a1 gene inhibits lung cancer A549 cell proliferation.     

miR-486 targeted regulation of PTEN on LPS-induced apoptosis of alveolar epithelial cell A549

AIM: To investigate the effect of microRNA-486 (miR-486) on lipopolysaccharide (LPS)-induced apoptosis of alveolar epithelial cell A549. METHODS: A549 cells were treated with LPS, and the expression of miR-486 was detected by RT-qPCR. miR-486 mimics were transfected into LPS-induced A549 cells, and RT-qPCR was used to detect the up-regulation effect. The apoptotic rate was analyzed by flow cytometry and the protein levels of cleaved caspase-3 (C-caspase-3) and C-caspase-9 were determined by Western blot. The target gene prediction software was used to predict the target gene PTEN of miR-486. Luciferase reporter vector was used to identify the target relationship. pcDNA 3.1-PTEN and miR-486 mimics were co-transfected into A549 cells to detect the effect of PTEN up-regulation on apoptosis of miR-486 mimics transfected A549 cells stimulated with LPS. RESULTS: After LPS treatment, the expression of miR-486 in A549 cells was significantly decreased (P<0.05). Transfection of miR-486 mimics significantly up-regulated the expression of miR-486 in A549 cells stimulated with LPS (P<0.05). The apoptotic rate of A549 cells and the protein levels of C-caspase-3 and C-caspase-9 were significantly increased after LPS treatment (P<0.05). Up-regulation of miR-486 significantly down-regulated LPS-induced apoptosis of A549 cells (P<0.05). The expression of PTEN was negatively regulated by miR-486. Transfection of pcDNA 3.1-PTEN significantly increased the expression of PTEN, promoted the apoptosis and increased the protein levels of C-caspase-3 and C-caspase-9 in A549 cells stimulated with LPS after co-transfection with miR-486 mimics(P<0.05). CONCLUSION: miR-486 inhibits PTEN expression and reduces LPS-induced apoptosis of A549 cells.     

Lentivirus-mediated RNA interference targeting vascular endothelial growth factor gene enhances the sensitivity of K562 cells to STI571

AIM: To investigate the effects of RNA interference (RNAi) inhibiting the expression of vascular endothelial growth factor (VEGF) gene mediated by lentiviral vector on the proliferation and apoptosis of K562 leukemic cell line. METHODS: A lentiviral vector containing short hairpin RNA (shRNA) targeting VEGF was constructed and cotransfected with the packaging plasmids mixture into 293T cells by Lipofectamine 2000. K562 cells were infected with the packaged lentivirus. The levels of VEGF mRNA and protein were detected by real-time quantitative RT- PCR, Western blotting and ELISA. Cellular proliferation was determined by trypan blue dye exclusion and MTT assay. STI571 (imatinib mesylate)-induced apoptosis was analyzed by flow cytometry. RESULTS: The lentiviral shRNA vector targeting VEGF was successfully constructed and transfected into K562 cells. The expressions of VEGF mRNA and protein in K562-shVEGF cells transfected with pRNAT-shRNA were significantly inhibited when compared with those of K562 and K562-con cells (mock transduction). The proliferation rate of K562-shVEGF cells slowed down. After STI571 treatment, the percentages of apoptotic cells in K562-shVEGF cells increased more significantly than those of K562 and K562-con cells (P<0.05). CONCLUSION: Inhibition of VEGF by lentivirus-mediated RNAi effectively inhibits proliferation and increases the sensitivity of K562 cells to STI571.     

Wild-type PTEN gene enhances the inhibitory effect of artesunate on K562 cell growth

AIM: To investigate the effect of wild-type PTEN transfection on the sensitivity of human leukemia K562 cells to artesunate (ART) and its molecular mechanism. METHODS: The adenovirus containing wild-type PTEN (Ad-WT-PTEN) or empty vectors (Ad) were transfected into K562 cells[with multiplicity of infection (MOI)=200]. The untransfected cells served as normal control. The effect of wild-type PTEN on the inhibition of K562 cell growth by ART was observed. The sensitizing ratio of PTEN combined with ART based on IC₅₀ was calculated. The viability of K562 cells was detected by MTT assay. The apoptosis was analyzed by flow cytometry. The mRNA level of PTEN was assessed by real-time PCR. The protein expression of PTEN, p-Akt and Akt was detected by Western blot. The activity of caspase-3/7 was measured by caspase activity kits. RESULTS: The sensitivity of K562 cells to ART was significantly increased by 2.25 folds after transfected with PTEN based on the IC₅₀. The cell viability in Ad-WT-PTEN+ART group was significantly lower than that in Ad+ART group after transfection for 3 d (P<0.01). The apoptotic rate in Ad-WT-PTEN+ART group was significantly higher than that in Ad+ART group (P<0.01). The expression of PTEN at mRNA and protein levels in the K562 cells after transfection with PTEN was significantly increased, and the protein level of p-Akt and caspase-3/7 activity were down-regulated, particularly in PTEN combined with ART group. CONCLUSION: The wild-type PTEN gene enhances the sensitivity of the K562 cells to ART by down-regulating the level of p-Akt and up-regulating the caspase-3/7 activity.     

Effects of miR-92a and miR-19b on insulin gene expression in mouse pancreatic β-cells

AIM To investigate the effect of microRNA-92a （miR-92a） and microRNA-19b （miR-19b） on the insulin expression in mouse pancreatic β-cells. METHODS The relative expression levels of endogenous miR-92a and miR-19b in mouse insulinoma MIN6 cells were detected by qPCR. The MIN6 cells were divided into control group， and experimental groups I and II， with 3 samples in each group， and transfected with negative control miRNA （NC）， miR-92a and miR-19b， respectively. The over-expression of the miRNAs was detected by qPCR. The morphological changes and viability of the cells were detected by optical microscopy and CCK8 assay， respectively. The expression of insulin was detected by qPCR， Western blot and immunofluorescence. The possible mechanisms of miR-92a and miR-19b regulating insulin expression were analyzed by bioinformatics prediction， dual-luciferase reporter assay， and Western blot. RESULTS Compared with the adult pancreatic progenitor cells， the expression of endogenous miR-92a and miR-19b in the MIN6 cells was decreased significantly （P<0.05）. Over-expression of miR-92a and miR-19b had no effect on the viability of MIN6 cells， but inhibited the expression of insulin at mRNA and protein levels. miR-19b significantly inhibited the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1 （P<0.05）. miR-92a had a fine-tuning effect on the luciferase activity of NeuroD1 3′UTR and the protein expression of NeuroD1. CONCLUSION miR-92a and miR-19b inhibit the insulin expression in mouse pancreatic β-cells.     

Hsa-miR-218 inhibits growth of cervical cancer HeLa cells by targeting to LASP1

AIM: To study the effect of hsa-miR-218 on cervical cancer HeLa cell growth and the underlying molecular mechanism.METHODS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed. pmiR-218 was transfected into HeLa cells. The number of viable HeLa cells was counted by the method of Trypan blue exclusion. The inhibitory rate of cell activity was detected by WST-8 assay. The expression of LIM and SH3 protein 1(LASP1) at mRNA and protein levels was determined by real-time PCR and Western blot. The interaction between miR-218 and LASP1 was examined using a luciferase reporter assay.RESULTS: The lentivirus expression vector pmiR-218 targeting to hsa-miR-218 was constructed successfully and confirmed by DNA sequencing. Over-expression of miR-218 inhibited the activity of HeLa cells with the inhibitory rates of 15%, 26% and 65% at 24 h, 48 h and 72 h, respectively. The difference between transfection group and blank control/negative control group was statistically significant. The luciferase activity was reduced when co-transfection with miR-218 mimics and LASP1-3,UTR plasmid. The relative expression of miR-218 was increased after transfection with pmiR-218. Over-expression of miR-218 down-regulated the LASP1 expression at mRNA and protein levels by 25% and 75% respectively. Compared with blank control group and negative control group, the difference was statistically significant(P<0.05).CONCLUSION: pmiR-218 effectively inhibits the growth of HeLa cells in a time-dependent manner. miR-218 targets to the 3,UTR of LASP1, thus down-regulating the expression of LASP1 in HeLa cells.     

miR-137 regulates invasion,migration and apoptosis of breast cancer MDA-MB-231 cells through TWIST1

AIM: To investigate the effect and its molecular mechanism of microRNA-137(miR-137) on the invasion, migration abilities and apoptosis of breast cancer cells. METHODS: miR-137 mimimics were transfected into the breast cancer MDA-MB-231 cells. The expression of miR-137 was detected by RT-qPCR. Apoptosis was analyzed by flow cytometry. The invasion and migration abilities were detected by Transwell assays. The protein levels of matrix metalloproteinase 9 (MMP-9), cleaved caspase-3 (C-caspase-3) and Bax were determined by Western blot. Bioinformatics software was used to predict that TWIST1 might be the target gene of miR-137 and then it was conformed by luciferase reporter gene identification. The effect of miR-137 mimics on TWIST1 protein expression was evaluated by Western blot. TWIST1 over-expression vector and miR-137 mimics were co-transfected into the MDA-MB-231 cells, and then the apoptosis, invasion, migration abilities and the protein levels of MMP-9, C-caspase-3 and Bax were determined. RESULTS: In the miR-137 mimics transfected MDA-MB-231 cells, the expression level of miR-137 and the apoptosis rate were increased, the cell invasion and migration abilities were decreased, the protein levels of C-caspase-3 and Bax were increased, the protein expression of MMP-9 was decreased (P<0.05). In addition, the target regulation of TWIST1 by miR-137 was identified by luciferase reporter assay. Moreover, the expression of TWIST1 in the MDA-MB-231 cells was inhibited by miR-137 mimics. Compared with the MDA-MB-231 cells co-transfected with negative control vector and miR-137 mimics, the protein expression levels of TWIST1 and MMP-9 in the MDA-MB-231 cells co-transfected with TWIST1 over-expression vector and miR-137 mimics were increased, the protein levels of C-caspase-3 and Bax and the apoptosis rate were decreased, the cell invasion and migration abilities were increased. CONCLUSION: miR-137 inhibits the invasion, migration abilities and induces apoptosis of breast cancer cells through targeting TWIST1.     

miR-499 attenuates myocardial injury in rats by targeting PTEN

AIM:To investigate the effect of microRNA-499 (miR-499) on myocardial injury in rats and its mechanism. METHODS:The rat model of ischemia-reperfusion (I/R) injury was established and the myocardial cells were primarily cultured. The expression level of miR-499 was detected by RT-qPCR. After hydrogen peroxide stimulation, CCK-8 assay and LDH kit were used to detect the viability and LDH release of the cells transfected with miR-499 mimic and siR-PTEN. The targeting relationship between miR-499 and PTEN was predicted by TargetScan and confirmed by luciferase test. RT-qPCR and Western blot were used to detect the effect of miR-499 mimic and inhibitor on the mRNA and protein expression levels of PTEN. After miR-499 inhibitor and siR-PTEN were co-transfected into the cells, CCK-8 assay and LDH kit were used to detect the viability and LDH release of the myocardial cells induced by hydrogen peroxide. RESULTS:The expression level of miR-499 in the I/R rats was increased rapidly, and then was decreased gradually in a time-dependent manner (P<0.05). miR-499 mimic and siR-PTEN significantly promoted the viability and decreased the LDH release of cardiomyocytes induced by hydrogen peroxide (P<0.05). miR-499 and PTEN had a targeting relationship. The expression of PTEN was significantly down-regulated by miR-499 mimic, and up-regulated by miR-499 inhibitor (P<0.05). Transfection with siR-PTEN reversed the inhibitory effect of miR-499 inhibitor on the cells. CONCLUSION:miR-499 attenuates the myocardial injury in rats by targeting PTEN.     

Roles of microRNA-29a in expression of Bcl-2 and Mcl-1 in rat cardiomyocytes

AIM: To investigate the regulatory mechanisms of microRNA-29a (miR-29a) on the expression of Bcl-2 and Mcl-1 in rat cardiomyocytes (CM cells). METHODS: The CM cells were isolated from the hearts of newborn rats and transfected with miR-29a mimic (100 nmol/L) by Lipofectamine RNAiMAX. The expression of Bcl-2 and Mcl-1 at mRNA and protein levels was detected by real-time fluorescence quantitative PCR and Western blotting. The luciferase assay was performed in HEK293T cells and CM cells, which were co-transfected with plasmid DNA and miRNA using Lipofectamine 2000. RESULTS: Transfection of miR-29a mimics significantly reduced the expression levels of Bcl-2 and Mcl-1 in CM cells as compared with the control cells (P<0.05). In addition, HEK293T cells co-transfected with miR-29a mimic and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid significantly reduced the luciferase activity as compared with control group (P<0.05). While CM cells transfected with miR-29a inhibitor and Bcl-2-3’UTR-WT or Mcl-1-3’UTR-WT plasmid in succession, the luciferase activity was increased inversely (P<0.05). CONCLUSION: miR-29a may regulate apoptosis by targeting the bcl-2 and mcl-1 genes.     

MicroRNA-9 inhibits epithelial-mesenchymal transition in gastric cancer SGC-7901 cells by NRP1

AIM:To investigate the inhibitory effect of microRNA-9(miR-9) on epithelial-mesenchymal transition (EMT) in the gastric cancer SGC-7901 cells and its mechanism.METHODS:The gastric cancer cell line SGC-7901 was transfected with miR-9 mimics or negative control mimic (NCM),as miR-9 or NCM group,respectively.The SGC-7901 cells without transfection were used as control group.The expression level of miR-9 in each group was detected by RT-qPCR.The migration and invasion abilities of the SGC-7901 cells in the 3 groups were detected by Transwell assay.The protein expression of N-cadherin,E-cadherin,α-catenin and neuropilin-1(NRP1) was determined by Western blot.Antagonistic effect of NRP1 over-expression on miR-9 inhibition of EMT was detected by Western blot.The relationship between miR-9 and NRP1 was analyzed by dual luciferase assay.RESULTS:The expression level of miR-9 in miR-9 group was significantly up-regulated,which was 538 times higher than that in control group (P<0.05).The number of migratory cells in miR-9 group was significantly lower than that in control group (P<0.05).Compared with control group,the protein expression of N-cadherin and NRP1 in miR-9 group was significantly decreased,while the protein expression of E-cadherin and α-catenin protein was significantly increased.Over-expression of NRP1 resulted in the increase in the protein expression of N-cadherin in the gastric cancer cells of miR-9 group,and the decrease in the protein expression of E-cadherin and α-catenin significantly.The result of dual luciferase assay showed that NRP1 was a downstream target gene of miR-9(P<0.05).CONCLUSION:miR-9 may inhibit the expression of EMT-related proteins through the downstream target gene NRP1,thus inhibiting the EMT of gastric cancer SGC-7901 cells.     

PAK6 signaling attenuates migratory and invasive abilities of non-small cell lung cancer cells

AIM：To investigate the roles of p21-activated kinase 6 (PAK6) and its target miRNA on the migratory and invasive abilities of non-small cell lung cancer cells. METHODS：miRNA candidates targeting PAK6 were predicted by a target prediction program. The expression of PAK6 was measured by real-time PCR and Western blotting after A549 cells were transfected with miR-23a mimics or inhibitory oligonucleotides. Luciferase reporter assay was used to determine whether PAK6 was the direct target of miR-23a. The abilities of cell migration and invasion were detected by Matrigel invasion assay and Transwell migration assay. The expression of PAK6 and matrix metalloproteinase 9 (MMP-9) was analyzed by Western blotting after A549 cells were transfected with siPAK6 or miR-23a mimics. RESULTS：miR-23a was identified by a target prediction program. Exogenetic over-expression of miR-23a resulted in a remarkable decrease in PAK6 expression (69%), whereas miR-23a inhibitory oligonucleotides induced pronounced increase in PAK6 expression (52%). The luciferase activity was significantly inhibited by 52% in wild-type PAK6 group, while there was no significant difference in the mutation group. The mRNA level of PAK6 had no change as detected by real-time PCR. Matrigel invasion assay and Transwell migration assay demonstrated there exogenetic over-expression of miR-23a markedly reduced the migration and invasion of PC-3 cells (73% and 59%, respectively). The MMP-9 expression remarkably decreased by 85％ and 76％ in the A549 cells transfected with siPAK6 and miR-23a mimics, respectively. CONCLUSION：miR-23a inhibits the migration and invasion of non-small cell lung cancer cells by repressing PAK6-MMP-9 signaling pathway.     

Up-regulation of Dicer1 gene expression by prostate-specific transcriptional factor NKX3.1

AIM: To investigate the effect of NKX3.1 on the Dicer1 gene expression.METHODS: The NKX3.1 eukaryotic expression plasmid was transfected into PC3 cells. The stable clones were isolated using cloning cylinders and grew continuously under G418 selection. The gene expression profile in PC3 (+) cells induced by NKX3.1 was analyzed by cDNA microarray. The effect of NKX3.1 on the Dicer1 expression was further investigated by RT-PCR and Western blotting in PC3 and PC3 (+) cells according to the results of gene chip. To determine if the increase in Dicer1 promotes the mature of microRNA, the pMIR-report luciferase expression plasmid of miRNA let-7a1 target sequence (pMIR-report-let7a1T) was constructed and transfected into PC3 and PC3 (+) cells. The effect of the miRNA let-7a-1 on its target sequence was determined by luciferase reporter assay.RESULTS: The result of gene chip showed that the expression level of Dicer1 gene was higher in PC3 (+) cells than that in PC3 cells. The results of RT-PCR and Western blotting indicated that the expression of Dicer1 gene was much higher in PC3 (+) cells than that in PC3 cells. The relative luciferase activity was much lower in PC3 (+) cells than that in PC3 cells when the cells were transfected with the pMIR-report-let7a1T vector.CONCLUSION: Up-regulation of Dicer1 expression induced by NKX3.1 promotes the mature and functions of microRNAs in prostate cancer PC3 cells.     

miR-21 inhibits apoptosis of Schwann cells following peripheral nerve injury

AIM: To explore the relationship and molecular mechanism between microRNA-21(miR-21) and Schwann cells (SC) following peripheral nerve injury. METHODS: The mRNA expression of miR-21 and phosphatase and tensin homologue deleted on chromosome ten (PTEN) in animal model were detected by real-time PCR. The over-expression of miR-21 and inhibition of miR-21 expression in the Schwann cells according to transfection of lentiviral vectors were performed, the nonspecific miRNA was used as a negative control (NC). The cell apoptosis was measured by flow cytometry. The mRNA expression of miR-21 and PTEN in the cells was detected by real-time PCR. The protein expression of PTEN and cleaved caspase-3 was determined by Western blot. RESULTS: The level of miR-21 was significantly higher and the mRNA level of PTEN was significantly lower in the model of nerve injury than those in control group. miR-21 over-expression decreased the number of apoptotic Schwann cells compared with NC-SC. The mRNA expression of PTEN was down-regulated by over-expression of miR-21. The protein expression of PTEN and cleaved caspase-3 was down-regulated by over-expression of miR-21(P<0.05). CONCLUSION: miR-21 may play an important role in the peripheral nerve injury through inhibiting apoptosis of Schwann cells by down-regulating the expression of PTEN.     

miR-21 enhances resistance of glioma cells to carmustine by down-re-gulating PTEN

AIM:To explore the function of miR-21 in human glioma cells resistant to carmustine and to elucidate its related mechanism. METHODS:SWOZ2 cells were transfected with miR-21 mimics(SWOZ2-miR-21mimics) or miRNA mimics negative control(control group) by the method of jetPRIME. The real-time fluorescence quantitative PCR was used to detect and compare the levels of miR-21 expression between BCNU-resistant cell line SWOZ2-BCNU and BCNU-sensitive cell line SWOZ2, or between SWOZ2-miR-21 mimic group and control group. The drug sensitivity of these cells to BCNU was determined by CCK-8 assay. The protein expression of phosphatase and tensin homology deleted on chromosome 10(PTEN), phosphorylated protein kinase B(p-Akt) and P-glycoprotein(P-gp) in these cells were also detected by Western blotting. RESULTS:The expression level of miR-21 was remarkably higher in SWOZ2-BCNU cells than that in SWOZ2 cells. The expression level of miR-21 was significantly higher in SWOZ2-miR-21 mimics group than that in control group. The half-maximal inhibitory concentration(IC₅₀) of BCNU was obviously higher for SWOZ2-BCNU cells than that for SWOZ2 cells. The IC₅₀ of BCNU was markedly higher in SWOZ2-miR-21 mimics group than that in control group. PTEN protein expression was remarkably lower, but p-Akt and P-gp protein expression levels were markedly higher in SWOZ2-BCNU cells than those in SWOZ2 cells. The protein level of PTEN was significantly lower, but the protein levels of p-Akt or P-gp were distinctly higher in SWOZ2-miR-21 mimics group than those in control group. CONCLUSION:miR-21 enhances the resistance of human glioma cells to BCNU by down-regulating the expression of PTEN protein.