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.     

miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

MicroPNA-21 promotes proliferation of rat Schwann cells following nerve injury

AIM: To investigate the molecular mechanism of microRNA-21 (miR-21) in the regulation of Schwann cell proliferation following nerve injury. METHODS: The expression of miR-2l was detected by real-time PCR. Synthetic miR-21 mimic and its control were transfected into rat Schwann cells. CCK-8 assay was performed to evaluate the influence of miR-21 on the proliferation of Schwann cells. The cell cycle distribution was determined by flow cytometry. The expression of transforming growth factor β-induced protein (TGFBI) and cyclin D1 were detected by Western blotting. RESULTS: The expression of miR-21 in model group was 7.87±0.75 and 7.75±0.80 times higher than that in sham operation group and blank group respectively. After transfected with miR-21 mimic, the expression of miR-21 in experimental group was 2.21±0.14 and 2.29±0.21 times higher than that in negative control group and blank group respectively. Moreover, the A₄₅₀ value of CCK-8 assay in experimental group at 48 h was higher than that in negative control group and blank group. The proliferation index in experimental group was higher than that in negative control group and blank group. At the same time, the expression of TGFBI obviously decreased and the cyclin D1 increased in the Schwann cells 48 h after transfection with miR-21. CONCLUSION: miR-21 promotes the proliferation activity of Schwann cells by down-regulating TGFBI expression.     

Effect of microRNA-132 transfection on lipopolysaccharide-induced inflammation in rat alveolar macrophages

AIM: To investigate the effect of microRNA-132 (miR-132) transfection on the lipopolysaccharide (LPS)-induced inflammation in rat alveolar macrophages. METHODS: The rat alveolar macrophage NR8383 cultured without pyrogen in vitrowere divided into blank control group, negative control group and transfected group. The cells in the 3 groups were transfected with phosphate buffer solution (PBS), Lipofectamine 2000 and synthesized miR-132 mimic respectively. The cell proliferation was detected by Cell Counting Kit-8 (CCK-8) assay. Real-time PCR was used to detect the expression of miR-132 in the cells. After NR8383 cells were stimulated with LPS for 6 h, the NF-κB DNA-binding activity was measured by electrophoretic mobility shift assay (EMSA). The expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in NR8383 cells was assayed by Western blotting.RESULTS: After transfection, the expression of miR-132 was significantly higher than that in blank control group and negative control group. The growth of NR8383 cells in transfected group was significantly inhibited compared with blank control group and negative control group (P<0.05). After the cells were stimulated with LPS, the productions of NF-κB, TNF-α and IL-6 in transfected NR8383 cells were decreased compared with blank control group and negative control group (P<0.05).CONCLUSION: Transfection of alveolar macrophages with miR-132 significantly suppresses the cell growth, and inhibits inflammatory responses induced by LPS.     

Overexpression of miR-132 attenuates Alzheimer disease-like learning and memory impairment

AIM: To obtain miR-132 lentivirus and to detect the effect of miR-132 overexpression on the improvement of learning and memory impairment in Tg2576 mice. METHODS: The pri-miR-132 gene was purified from mouse brain and cloned into lentivirus vector pLenti7.3-miR-132. The miR-132 lentivirus was packaged by co-transfected pLenti7.3-miR-132 with ViraPower Packaging Mix into 293FT cells. The miR-132 lentivirus was purified 72 h later from the supernatant by ultracentrifugation and poly(ethylene glycol) method. The efficiency of miR-132 lentivirus was identified by transfection into N2A cells and mouse hippocampus region. Tg2576 mice were injected with miR-132 virus in the hippocampus region and the expression of learning- and memory-related proteins and functions were determined. RESULTS: miR-132 lentivirus expression vector was constructed successfully. The purified virus was obtained with transfection activity. Tg2576 mice with overexpression of miR-132 had a significant improvement in learning and memory abilities. CONCLUSION: miR-132 lentivirus with biological activity was obtained. Overexpression of miR-132 in the hippocampus region of Tg2576 mice attenuates the learning and memory impairment.     

Proctective effect of microRNA-30a on regulating beclin-1 expression in hypoxia-reoxygenated neonatal rat cardiomyocytes

AIM: To explore the potential mechanism of microRNA-30a (miR-30a) overexpression in neonatal rat cardiomyocytes during hypoxia/reoxygenation (H/R). METHODS: The miR-30a overexpression was induced in primary neonatal rat cardiomyocytes by lentivirus transfection. The cardiomyocytes were divided into 5 groups: normal group, H/R group, LV-GFP+H/R group, LV-GFP-miR-30a+H/R group and 3-methyladenine(3-MA)+H/R group. The expression level of miR-30a after lentivirus transfection and H/R was determined by real-time PCR, while the protein levels of LC3 and Beclin-1 after H/R and lentivirus transfection were detected by Western blotting. The cardiomyocyte death after H/R were measured by TUNEL and PI staining. RESULTS: Compared with LV-GFP group, significant down-regulation of Beclin-1 protein level was observed in cardiomyocytes with miR-30a overexpression, while the protein levels of Beclin-1 and LC3 in the cardiomyocytes with miR-30a overexpression were down-regulated after H/R, and apoptosis of these cells were significantly decreased after H/R. CONCLUSION: The protein level of Beclin-1 is down-regulated in cardiomyocytes with miR-30a overexpression. Inhibition of autophagy decreases the cardiomyocyte death after H/R.     

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.     

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.     

Effect of over-expression of transcription factor CDX2 on proliferation and cell cycle of human gastric cancer cell line SGC-7901

AIM: To study the effect and the molecular mechanism of CDX2 over-expression on the proliferation, growth and cell cycle of human gastric cancer cell line SGC-7901. METHODS: The SGC-7901 cells in LV-CDX2-GFP group were transfected with the recombinant lentivirus vector LV-CDX2-GFP, the cells in LV-GFP group were transfected with the negative control lentiviral vector for the negative control, and the cells in blank control group were without any treatment. The cell proliferation was detected by CCK-8 assay. The cell cycle distribution was analyzed by flow cytometry. The expression of CDX2, Bax, Bcl-2, cyclin D1 and survivin was determined by semi-quantitative RT-PCR and Wes-tern blotting. RESULTS: Compared with LV-GFP group and blank control group, the proliferation activity of the SGC-7901 cells was significantly lower (P<0.05), the G0/G1 phase proportion increased (P<0.05), the mRNA and protein levels of Bcl-2, cyclin D1 and survivin were reduced (P<0.05), and the mRNA and protein levels of Bax were up-regulated (P<0.05) in LV-CDX2-GFP group. No statistically significant difference of the above indexes was observed (P>0.05) between LV-GFP group and blank control group. CONCLUSION: Over-expression of CDX2 mediated by lentivirus inhibits the proliferation and growth of human gastric cancer SGC-7901 cells and arrestes the cell cycle at G0/G1 phase, which may be related to down-regulation of Bcl-2, cyclin D1 and survivin and up-regulation of Bax.     

Effect of SMYD3 over-expression on miR-124 expression and proliferation ability in human intrahepatic cholangiocarcinoma cells

AIM：To explore the effect of SET and MYND domain-containing protein 3 (SMYD3) over-expression on miR-124 expression and proliferation ability of human intrahepatic cholangiocarcinoma cells. METHODS：Transient transfection of SMYD3 eukaryotic expression plasmid into human intrahepatic cholangiocarcinoma cell line HCCC-9810 were performed. The expression of SMYD3 at mRNA and protein levels was measured by qRT-PCR and Western blotting, respectively. The expression of miR-124 was detected by qRT-PCR, and the methylation status of miR-124 gene was determined by methylation-specific PCR. Cell proliferation was examined by CCK-8 assay and colony formation experiment. RESULTS：After transfected with SMYD3 eukaryotic expression plasmid, the over-expression of SMYD3 in HCCC-9810 cells was observed. Compared with the blank cells, the expression level of miR-124 was significantly decreased and miR-124 gene promoter methylation was significantly increased. In addition, SMYD3 over-expression significantly promoted the proliferation of HCCC-9810 cells. CONCLUSION：The transient transfection of SMYD3 plasmid increases the methylation of miR-124 gene promoter and induces under-expression of miR-124. Over-expression of SMYD3 promotes the proliferation of cholangiocarcinoma cells.     

MicroRNA-146a promotes apoptosis of gastric cancer SGC-7901 cells by targeting TAK1

AIM: To explore the effect of microRNA-146a (miR-146a) on apoptosis of human gastric cancer SGC-7901 cells and the underluing mechanism. METHODS: miR-146a mimic (up-regulated miR-146a expression) and miR-146a inhibitor (down-regulated miR-146a expression) were transfected into the SGC-7901 cells by liposome method. At the same time, miRNA nonsense sequence transfection group as the negative control group (NC group) was set up. RT-qPCR was used to evaluate the levels of miR-146a in the SGC-7901 cells after transfection. The effects of miR-146a on the cell apoptosis and growth were assessed by flow cytometry analysis and CCK-8 assay, respectively. The effect of over-expression or knockdown of miR-146a on transforming growth factor-β-activated kinase 1 (TAK1)/nuclear factor-kappa B (NF-κB) signaling was evaluated by RT-qPCR and Western blot. RESULTS: miR-146a modulated apoptosis of SGC-7901 cells. Over-expression of miR-146a significantly increased apoptosis, whereas knockdown of miR-146a inhibited the apoptosis of SGC-7901 cells. The expression of TAK1 at mRNA and protein levels was significantly decreased when miR-146a mimic was transfected into the SGC-7901 cells (P<0.05). On the contrast, the expression of TAK1 at mRNA and protein were significantly higher in miR-146a inhibitor transfection group than that in NC group (P<0.05), suggesting that miR-146a negatively regulated TAK1 expression. Moreover, knockdown of TAK1 enhanced the apoptosis of SGC-7901 cells (P<0.01), while over-expression of TAK1 inhibited the apoptosis of SGC-7901 cells(P<0.01). Additionally, both over-expression of miR-146a and knockdown of TAK1 led to a prominent increase in the expression of NF-κB inhibitor protein alpha (IκBα) and a significat decrease in B cell lymphoma-2 (Bcl-2) level in the SGC-7901 cells. CONCLUSION: miR-146a significantly promotes apoptosis of SGC-7901 cells by inhibition of NF-κB pathway via targeting TAK1.     

Effect of microRNA-100 on proliferation activity and cell cycle of hepatocarcinoma cells

AIM：To investigate the effect of microRNA-100 (miR-100) on the proliferation activity and cell cycle of hepatocarcinoma cells. METHODS：Synthetic miR-100 mimic and its negative control were transfected into human hepatocarcinoma HepG2 cells by liposome method. After transfection, the cell counting kit-8 (CCK-8) was used to measure the cell proliferation activity. The cell cycle distribution was determined by flow cytometry. The expression of Polo-like kinase 1 (Plk1) at mRNA and protein levels was detected by quantitative real-time PCR (qRT-PCR) and Western blotting. RESULTS：The transfection efficiency mediated by cationic liposome was greater than 85%. The inhibitory rates of cell proliferation in HepG2 cells were (43.5±12.2)%, (46.5±3.7)% and (52.1±0.2)% at 24 h, 48 h and 72 h after transfected with miR-100 mimic, respectively, which were significantly increased as compared with the control cells. Moreover, the cell proliferation index in experimental group (35.8 ± 1.4) was higher than that in negative control group (39.2 ± 1.0) and simple liposome group (40.7 ± 2.0) at 72 h. At the same time, the mRNA and protein expression levels of Plk1 obviously decreased in HepG2 cells transfected with miR-100 at 72 h after transfection. CONCLUSION：miR-100 suppresses the proliferation activity of hepatocarcinoma cells by down-regulating Plk1 gene expression.     

Effect of abnormal expression of miR-141 on malignant biological behaviors of human hepatocarcinoma cells

AIM: To investigate the expression of microRNA-141 (miR-141) in human hepatocellular carcinoma (HCC) cell line SMMC-7721 and normal hepatocyte line HL-7702, and to analyze the effect of abnormal expression of miR-141 on the malignant biological behaviors of human hepatocarcinoma cells. METHODS: The RNA from SMMC-7721 cells and HL-7702 cells was extracted. SYBR Green real-time PCR was performed to detect the expression of miR-141. Synthetic miR-141 mimic and its negative control were transfected into the SMMC-7721 cells, and miR-141 inhibitor and its negative control were transfected into the HL-7702 cells by the method of Lipofectamine. After transfection, MTS assay and BrdU-ELISA were employed to evaluate the effect of miR-141 on the cell proliferation. Flow cytometry was used to detect cell cycle and apoptosis. The changes of migration ability were investigated by Transwell invasion assay. RESULTS: The expression of miR-141 in the SMMC-7721 cells was significantly lower than that in the HL-7702 cells (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of the SMMC-7721 cells transfected with 25 nmol/L miR-141 mimic was significantly inhibited in a time-dependent manner (P < 0.05). The percentages of G₁ phase cells and early apoptotic rate were significantly increased when miR-141 was up-regulated, but the migration ability was inhibited (P < 0.05). Compared with blank group, Lipofectamine group and negative control group, the proliferation of HL-7702 cells transfected with 50 nmol/L miR-141 inhibitor was significantly increased in a time-dependent manner (P < 0.05). When miR-141 was down-regulated, the percentages of G₁ phase cells and early apoptotic rate were significantly decreased, but the migration ability was enhanced (P < 0.05). CONCLUSION: miR-141 is down-regulated in human hepatocarcinoma cell line. Up-regulation of miR-141 will not only inhibit cell proliferation and migration ability, but also affect the cell cycle and apoptosis of SMMC-7721 cells. miR-141 may function as a tumor suppressor gene during HCC development.     

miR-193b enhances cytotoxicity of doxorubicin by targeting Mcl-1 in breast cancer

AIM: To investigate the effect of microRNA(miR)-193b on doxorubicin therapy in breast cancer in vitro.METHODS: miR-193b level in plasma was detected by real-time PCR in the patients with breast cancer or the healthy controls. MTT assay was performed to measure the inhibitory effect of miR-193b plus doxorubicin on the growth of MDA-MB-231 cells. Bioinformatics, real-time PCR and Western blot were performed to determine whether the expression of Mcl-1 was regulated by miR-193b. Mcl-1 expression vector was constructed, and the role of Mcl-1 vector toward miR-193b plus doxorubicin-induced cytotoxicity in MDA-MB-231 cells was observed by MTT assay.RESULTS: Down-regulation of miR-193b was found in breast cancer patients. The miR-193b plus doxorubicin group showed a higher growth inhibition than cisplation group in MDA-MB-231 cells. The expression of Mcl-1 at both mRNA and protein levels was down-regulated after miR-193b transfection. The growth inhibition of MDA-MB-231 cells treated with miR-193b plus doxorubicin was significantly decreased after the transfection of Mcl-1 expression vector.CONCLUSION: miR-193b sensitizes doxorubicin-induced cytotoxicity by targeting Mcl-1 in breast cancer.     

Mechanism of miR-30c over-expression inhibiting malignant phenotypes of cervical cancer cells

AIM:To investigate the molecule mechanism of microRNA (miR)-30c over-expression inhibiting malignant phenotypes of cervical cancer cells. METHODS:Cervical cancer cell lines C33A, HeLa, SiHa and CaSki were transfected with pGenesil-1-miR-30c plasmid using Lipofectamine 2000 kit, and the expression of miR-30c was determined by TaqMan real-time PCR. The cell viability inhibition rate, colony formation ability, migration rate and apoptotic rate were measured by MTT assay, colony formation assay, Transwell experiment, and flow cytometry with Annexin V-FITC staining. The protein expression of Bax, Bcl-2, matrix metalloproteinase (MMP)-9, MMP-13 and tissue inhibitor of metalloprotei-nase-1 (TIMP-1) was detected by Western blot. RESULTS:The expression levels of miRNA-30c in the cervical cancer cell lines transfected with pGenesil-1-miR-30c plasmid were significantly higher than those in negative control groups (cell lines transfected with pGenesil-1 plasmid) (P<0.01). Significantly increased cell viability inhibition rate, and decreased colony formation ability and migration rate were found in the cervical cancer cell lines over-expressing miR-30c as compared with negative control groups (P<0.05). The apoptotic rate in the cervical cancer cell lines over-expressing miR-30c was dramatically higher than that in control groups (P<0.05). Over-expression of miR-30c in cervical cancer cells promoted the protein expression of Bax and TIMP-1, and decreased the protein expression of Bcl-2 and MMP-13 (P<0.05 or P<0.01). CONCLUSION:Over-expression of miR-30c significantly inhibits the viability and migration, and induces apoptosis of cervical cancer cells. The mechanism may be related to activating apoptosis pathway and inhibiting MMP-13 protein expression.     

Overexpression of SHP-1 mediated by lentivirus restrains atherosclerotic progression in mice

AIM: To investigate the role of SH2-domain-containing protein-tyrosine phosphatase-1 (SHP-1) lentivirus in atherosclerotic mice. METHODS: ApoE knock-out mice were randomly assigned to 3 groups: control group, GFP transfection group and SHP-1 transfection group. All mice were placed with carotid collars on the right common carotid arteries near its bifurcation, following feeding with high-fat diet for 8 weeks and then transfected with GFP blank vector or SHP-1 lentivirus (SHP-1-LV). The fluorescence density of the plaques, body weight, the levels of plasma total cholesterol (TC) and triglyceride (TG) were determined at 1st, 2nd, and 6th week after lentivirus transfection. Furthermore, the mRNA and protein expression of SHP-1, interleukin-6 (IL-6), tumor necrosis factor-α (TNF-α), matrix metalloproteinase (MMP)-2 and MMP-9 were analyzed by real-time PCR and Western blot. Additionally, pathological analysis of the plaques was also performed by HE and oil red O staining. RESULTS: The fluorescence of the plaques was observed at 1st, 2nd, and 6th week after lentivirus transfection, with a highest density at 2nd week. The body weight and the levels of TC and TG in the mice were not influenced by lentivirus transfection. Moreover, SHP-1-LV transfection significantly upregulated the expression of SHP-1 at mRNA and protein levels, but inhibited the expression of IL-6, TNF-α, MMP-2 and MMP-9. In addition, SHP-1-LV transfection also decreased the plaque size ratio and lipid content in right common carotid arteries. CONCLUSION: SHP-1 overexpression accelerates the regression of atherosclerotic plaque, thus emerging SHP-1 as a target for prevention and treatment of atherosclerosis.     

Mechanism of microRNA-138-5p inhibiting proliferation,migration and invasion of lung cancer cells

AIM: To investigate the mechanism of microRNA-138-5p (miR-138-5p) inhibiting the proliferation, migration and invasion abilities of lung cancer cells.METHODS: The lung cancer A549 and H460 cells were transfected with miR-NC (control group) or miR-138-5p (experimental group). The bioinformatic analysis was performed to predict the target genes of miR-138-5p.The expression levels of miR-138-5p, forkhead box protein C1 (FOXC1) mRNA and vimentin mRNA were detected by RT-qPCR. The protein expression of FOXC1, vimentin, E-cadherin, N-cadherin and β-catenin was determined by Western blot. MTS method and colony formation assay were used to detect cell viability and proliferation ability. Wound healing assay and Transwell assay were used to detect cell migration and invasion ability.RESULTS: Over-expression of miR-138-5p significantly reduced the expression of FOXC1 and vimentin at mRNA and protein levels (P<0.05). The expression of E-cadherin and β-catenin were up-regulated and the expression of N-cadherin was down-regulated. The proliferation, migration and invasion abilities of the lung cancer cells were inhibited by the over-expression of miR-138-5p.CONCLUSION: miR-138-5p inhibits the proliferation, migration and invasion abilities of lung cancer cells by targeting FOXC1 and vimentin. It may be a potential target for lung cancer gene therapy.     

Effect of miR-21 over-expression on proliferation,migration and diffe-rentiation of c-Kit+ cardiac stem cells

AIM: To explore the effect of microRNA (miR)-21 on proliferation, migration and differentiation abilities of c-Kit⁺ cardiac stem cells (CSCs). METHODS: c-Kit⁺ CSCs were cultured and selected by the methods of enzyme digestion and magnetic bead separation. miR-21 mimics (50 nmol/L) and mimics negative control (MNC) were transfected into c-Kit⁺ CSCs with Lipofectamine^® 2000. The cells was divided into 3 groups:control group:c-Kit⁺ CSCs without any pretreatment; MNC group:the cells were transfected with MNC for 48 h; mimics group:the cells were transfected with miR-21 mimics for 48 h. qPCR was used to assess the expression of miR-21 in each group. CCK-8 and EdU assays were used to determine the cell proliferation. qPCR and immunofluorescence were used to detect the differentiation in each group. Scratch assay was adopted to explore the migration ability of the cells. RESULTS: The expression of c-Kit in the c-Kit⁺ CSCs were 90.8%, with 0.6% of CD45 and 0.5% of CD34. A significant increase in miR-21 expression was observed when the cells were transfected with miR-21 mimics for 48 h (P<0.05). CCK-8 and EdU assays showed that miR-21 significantly increased cell proliferation as compared with MNC group and control group (P<0.05). No difference in the expression of Nkx2.5, CD31 and α-SMA at mRNA and protein levels was observed, and no difference of the migration ability in 3 groups of the c-Kit⁺ CSCs was found. CONCLUSION: Over-expression of miR-21 significantly promotes the proliferation of c-Kit⁺ CSCs, without any effect on the cell migration and differentiation.     

Effect of specific hTERT-siRNA on growth of human tongue cancer cells in vivo and in vitro

AIM: To investigate the effect and mechanisms of siRNA-hTERT-induced inhibition of Tca8113 tongue cancer cells in vitro and in vivo. METHODS: A small interference RNA (siRNA) targeting to hTERT mRNA (siRNA-hTERT1) was constructed. The siRNA was transfected into Tca8113 tongue cancer cells in vivo and in vitro with cationic liposome. A non-specific siRNA (siRNA-hTERT2) and non-treatment were used as negative control group and blank group. The cell growth in vitro was detected by MTT method. The cell apoptosis in vitro was analyzed by flow cytometry. The effect of siRNA-hTERT1 on xenografts in nude mice was observed by determining the tumor size. The cell apoptosis in xenografts was analyzed by Hoechst staining. The expressions of hTERT mRNA in vitro and in vivo were detected by RT- PCR. RESULTS: The inhibition rates of cell growth in vitro 72 h after siRNA-hTERT1 treatment was 47.2％, significantly higher than that in siRNA-hTERT2 treatment group (2.6％, P<0.01). The cell apoptosis rate was 27.30%±0.18% in vitro, significantly increased at 48 h after transfection of siRNA-hTERT1, compared to negative control group and blank group (P<0.01). The size of xenografts in siRNA-hTERT1 treatment group was (298.8±138.7)mm3, significantly smaller than that in siRNA-hTERT2 treatment group and blank group (495.1±151.6)mm3 and (506.8±207.4)mm3, the inhibition rate was 40.0% (P<0.01). The numbers of apoptotic cells in xenografts significantly increased after transfection of siRNA-hTERT1, compared to negative control group and blank group (P<0.01). Compared to negative control group and blank group, the expression of hTERT mRNA in Tca8113 tongue cancer cells in vitro and in vivo was inhibited by siRNA-hTERT1. CONCLUSION: siRNA-hTERT1 powerfully inhibits the growth of Tca8113 tongue cancer cells in vitro and in vivo. The specific inhibition of hTERT mRNA expression and cell apoptosis may be its main mechanisms.     

Expression and significance of miR-193b in cervical cancer

AIM: To investigate the expression of microRNA-193b(miR-193b) in the cervical tissues, and further to explore the effect of silencing miR-193b on diamminedichloroplatinum(DDP)-treated HeLa cell viability. METHODS: The expression levels of miR-193b in different cervical tissues were examined by qPCR. After transfection of miR-193b-inhibitor, the cell migration was determined by Transwell assay, the sensitivity of HeLa cells to DDP was measured by MTT assay, the protein levels of phosphate and tension homology deleted on chromsome ten(PTEN), protein kinase B(Akt), p-Akt and p-glycoprotein(P-gp) were determined by Western blot. RESULTS: The mRNA level of miR-193b was significantly increased in the cervical cancer tissues compared with normal cervical tissues(P<0.05). Knockdown of miR-193b obviously inhibited migration and enhanced sensitivity to DDP of HeLa cells(P<0.05). Additionally, after transfection of miR-193b-inhibitor, the expression of PTEN was increased, whereas the protein levels of p-Akt and P-gp were decreased(P<0.05). CONCLUSION: miR-193b is highly expressed in the cervical cancer tissues. Inhibition of miR-193b augments the sensitivity to DDP of HeLa cells, at least in part, through PTEN-PI3K/Akt signaling pathway.