Linarin inhibits migration and invasion abilities of human breast cancer MDA-MB-231 cells through IKK/NF-κB signaling pathway

AIM: To investigate the effect of linarin (LIN) on the migration and invasion abilities of human breast cancer MDA-MB-231 cells and its underlying mechanism. METHODS: MCF-7, MDA-MB-231 and MCF-10A cells were cultured in vitro and treated with LIN at 5, 10, 20, 40, 80 and 160 μmol/L for 24 h, and the cell proliferation was measured by CCK-8 assay and colony formation assay. The protein levels of Snail, E-cadherin, matrix metalloproteinase-9 (MMP-9), IκBα, p-IKKα/β and p-p65 were determined by Western blot. RESULTS: LIN remarkably reduced the viability of MDA-MB-231 cells in a dose-dependent manner (P<0.05), and the IC₅₀ was 55.89 μmol/L for 24 h. LIN decreased the colony formation rate of MDA-MB-231 cells at the concentration of 20 μmol/L (P<0.05). After exposed to LIN at 5 μmol/L and 10 μmol/L for 24 h, the migration and invasion abilities of the MDA-MB-231 cells were significantly reduced (P<0.05), the protein expression levels of E-cadherin and IκBα were up-regulated (P<0.05), the protein expression levels of Snail and MMP-9 were down-regulated (P<0.05), and the phosphorylation levels of IKKα/β and p65 were decreased (P<0.05) in comparison with the control group. Meanwhile, IKK-16 (IKKα/β inhibitor) and PDTC (NF-κB inhibitor) also down-regulated the protein expression levels of Snail and MMP-9 (P<0.05), and up-regulated the protein expression level of E-cadherin (P<0.05). CONCLUSION: LIN down-regulates the protein expression levels of Snail and MMP-9, and up-regulates the protein expression level of E-cadherin most likely through inhibiting IKK/NF-κB signaling pathway, and ultimately lead to decreases in the migration and invasion abilities of MDA-MB-231 cells.     

MicroRNA-200a affects malignant biological behaviors of breast cancer cells by inhibiting epithelial-mesenchymal transition

AIM: To investigate the effect of microRNA-200a (miR-200a) on the malignant biological beha-viors of breast cancer cells and its regulatory mechanism. METHODS: The expression of miR-200a in human breast can-cer cell lines MDA-MB-231, MDA-MB-468 and MCF-7, and normal human mammary epithelial cell line MCF-10A was detected by RT-qPCR. CCK-8 assay was used to detect the viability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. Flow cytometry method and Transwell assay were used to detect the apoptosis and invasive ability of MDA-MB-231 cells after transfection with miR-200a mimic or miR-200a inhibitor. The expression of SIP1, E-cadherin, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was determined by RT-qPCR and Western blot. RESULTS: Compared with MCF-10A cells, the lowest expression of miR-200a was observed in the MDA-MB-231 cells (P<0.05). Over-expression of miR-200a attenuated the viability of MDA-MB-231 cells (P<0.05), increased apoptosis (P<0.05) and decreased the invasion ability (P<0.05). The expression of SIP1, N-cadherin, Snail, Twist, ZEB1 and ZEB2 at mRNA and protein levels was also significantly down-regulated, while the mRNA and protein expression of E-cadherin was significantly increased (P<0.05). Transfection with miR-200a inhibitor reversed the above results. CONCLUSION: Up-regulation of miR-200a inhibits the viability and invasion ability of MDA-MB-231 cells and promotes the apoptosis of MDA-MB-231 cells. miR-200a may regulate the biological behaviors of breast cancer by inhibiting epithelial-mesenchymal transition.     

Effect of Akt/GSK-3β/Snail signaling pathway on EMT in A549/DDP cells mediated by TGF-β1

AIM: To observe the expression of Akt/GSK-3β/Snail signaling pathway-related molecules in cisplatin-resistant cell line A549/DDP mediated by transforming growth factor-β₁ (TGF-β₁), and to explore the association of Akt/GSK-3β/Snail signaling pathway with epithelial-mesenchymal transition (EMT). METHODS: The A549/DDP cells were divided into TGF-β₁ (+) group, TGF-β₁ (-) group and LY294002 group. The morphological changes of A549/DDP cells treated with TGF-β₁ were observed under microscope. The protein expression of E-cadherin and N-cadherin was determined by the methods of immumofluorescence and Western blot. The protein levels of Akt, p-Akt, GSK-3β, p-GSK-3β^Ser9 and Snail were also detected by Western blot. RESULTS: The A549/DDP cells in TGF-β₁ (+) group were dispersive, showed a spindle-like shape and developed pseudopodia. This transformation was conformed to classic EMT markers. Compared with TGF-β₁ (-) group, the protein expression of E-cadherin in TGF-β₁ (+) group was significantly decreased (P<0.05), and N-cadherin was significantly increased (P<0.05). The protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were also significantly increased (P<0.05). Compared with TGF-β₁ (+) group, the protein levels of p-Akt, p-GSK-3β^Ser9 and Snail were significantly decreased in LY294002 group (P<0.05). No difference of Akt and GSK-3β expression between TGF-β₁ (-) group and TGF-β₁ (+) group was observed. CONCLUSION: The mechanism of EMT in A549/DDP cells might be related to Akt/GSK-3β/Snail signaling pathway activated by TGF-β₁.     

Up-regulation of Snail1 expression in renal tubular epithelial cells through Akt/GSK-3β pathway under high-glucose condition

AIM: To examine the effects of high glucose (HG) on the expression of Snail1 and protein kinase B (Akt)/glycogen synthase kinase 3β (GSK-3β) in primary renal tubular epithelial cells (RTECs). METHODS: The primary RTECs were randomly treated with normal glucose, high glucose or D-mannitol for 30 min~72 h. RT-PCR and Western blotting were used to observe the expression of Snail1, Akt and GSK-3β at mRNA and protein levels in these cells. The primary cultured RTECs were pretreated with LY294002 (a PI3K inhibitor, 25 μmol/L) to observe the specific inhibitory effects of phosphatidylinositol 3-kinase (PI3K) on HG-induced expression of Snail1 protein. RESULTS: Treatment of RTECs with HG resulted in increased mRNA and protein levels of Snail1, Akt1, and phosphorylation of Akt and GSK-3β. LY294002 blocked the HG-induced up-regulation of p-Akt, p-GSK-3β and Snail1 expression at protein level, but no effect of LY294002 was seen on the total protein expression of Akt1 and GSK-3β. HG did not affect the expression of GSK-3β at mRNA and protein levels. CONCLUSION: HG-induced up-regulation of Snail1 may be regulated by Akt/GSK-3β pathway in RTECs.     

Mechanism of AKT1 negatively regulating breast cancer cell migration

AIM: To investigate the molecular mechanism and downstream signaling pathway by which AKT1 inhibition regulates breast cancer cell migration. METHODS: RNA interference was used to knockdown the expression of AKT1. Western blot assay was performed to examine the expression of AKT1 total protein, β-catenin total protein and β-catenin nuclear protein. Immunofluorescence was used to examine the cellular localization of β-catenin. Transwell assay was used to investigate whether β-catenin nuclear accumulation as an alternative pathway was responsible for breast cancer metastasis induced by AKT1 inhibition. RESULTS: The total protein expression of AKT1 was decreased in MCF-7 and MDA-MB-231 cells treated with AKT1 siRNA. A significant increase in the protein expression of β-catenin was observed in MCF-7 cells and MDA-MB-231 cells treated with AKT1 siRNA. Immunofluorescence staining showed that MCF-7 cells and MDA-MB-231 cells displayed strong β-catenin staining in the cytoplasm and nucleus after knockdown of AKT1 expression. The ability of tumor cell migration increased dramatically after treated with AKT1 specific siRNA in the breast cancer MCF-7 cells and MDA-MB-231 cells in Transwell assay. XAV-939 reversed breast cancer cell migration induced by knockdown of AKT1 expression. CONCLUSION: β-catenin nuclear accumulation contributes to AKT1 inhibition-mediated breast cancer cell migration.     

Effect of Wnt/β-catenin signaling pathway regulated by HDAC1 on apoptosis of breast cancer cells

AIM: To study the effect of histone deacetylase 1 (HDAC1) on the apoptosis of breast cancer cells.METHODS: The expression of HDAC1 at mRNA and protein levels in normal mammary epithelial cell line MCF-10A and breast cancer cell lines BT549, MCF-7 and MDA-MB-231 was measured by RT-qPCR and Western blot. HDAC1 siRNA was transfected into MDA-MB-231 cells, and then RT-qPCR and Western blot were used to determine the expression level of HDAC1. The cell viability was measured by MTT assay, and apoptosis was analyzed by flow cytometry. The protein levels of β-catenin, c-Myc, cyclin D1 and cleaved caspase-3 were determined by Western blot. Breast cancer cells with HDAC1 knockdown were treated with Wnt/β-catenin signaling pathway activator, and then the cell viability and apoptosis were measured.RESULTS: The expression of HDAC1 at mRNA and protein levels in BT549, MCF-7 and MDA-MB-231 cells was significantly higher than that in normal mammary epithelial cell line MCF-10A, and the highest expression level of HDAC1 was observed in MDA-MB-231 cells (P<0.05). HDAC1 siRNA reduced the expression of HDAC1 at mRNA and protein levels in the breast cancer cells. The viability of MDA-MB-231 cells was decreased after knockdown of HDAC1 expression, the apoptotic rate was increased, the protein level of cleaved caspase-3 in the cells was elevated, and the protein levels of β-catenin, c-Myc and cyclin D1 were decreased (P<0.05). Wnt/β-catenin signaling pathway activator reversed HDAC1 knockdown-induced apoptosis and decrease in viability of MDA-MB-231 cells, and reduced the protein level of cleaved caspase-3.CONCLUSION: Knockdown of HDAC1 expression induces apoptosis of breast cancer cells by inhibiting the activation of Wnt/β-catenin signaling pathway.     

Effect of DEC1 gene silencing on viability,invasion and migration of human breast cancer MDA-MB-231 cells under hypoxia

AIM: To investigate the effect of differentiated embryonic chondrocyte gene 1 (DEC1) expression silencing on viability, invasion and migration of human breast cancer MDA-MB-231 cells and its possible mechanism under hypoxia. METHODS: The expression of DEC1 was detected by RT-qPCR and Western blot in breast cancer MDA-MB-231 cells under normoxia and hypoxia. MDA-MB-231 cells were transfected with the siRNA targeting DEC1 and the protein levels of DEC1, Smad3 and phosphorylated Smad3 (p-Smad3) were examined under hypoxia. Subsequently, the changes in the viability, invasion and migration abilities of MDA-MB-231 cells were analyzed by CCK-8 assay, Transwell experiment and Scratch test, respectively. RESULTS: The expression of DEC1 in MDA-MB-231 cells under hypoxia was higher than that in the MDA-MB-231 cells under normoxia condition at both mRNA and protein levels (P<0.05). The viability, invasion and migration abilities of MDA-MB-231 cells in siRNA-DEC1 group were decreased significantly as compared with control group (P<0.01). Besides, the protein level of p-Smad3 in the MDA-MB-231 cells in siRNA-DEC1 group was lower than that in negative control group under hypoxia condition (P<0.05). CONCLUSION: Down-regulated DEC1 expression significantly decreases the viability, invasion and migration abilities of breast cancer MDA-MB-231 cells by blocking the TGF-β/Smad3 signaling pathway under hypoxia condition.     

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.     

Juglone inhibits epithelial-mesenchymal transition of prostate cancer cells by regulating Wnt/β-catenin/Snail signaling pathway

AIM: To investigate the mechanism of juglone on epithelial-mesenchymal transition in prostate cancer cells. METHODS: Human prostate cancer LNCaP cells were divided into control group (without juglone), 12.5 μmol/L juglone group and 25 μmol/L juglone group. LNCaP cells in the latter 2 groups were treated with juglone for 24 h. The invasion ability of the LNCaP cells was detected by Transwell assay. The protein expression of E-cadherin, vimentin, Snail and β-catenin was determined by Western blot. The LNCaP cells were treated with LiCl and juglone in combination for 24 h, and the protein expression of Snail and E-cadherin was detected by Western blot.RESULTS: The results of Trans-well invasion assay showed that the invasion ability in juglone groups was significantly decreased (P<0.01). The protein expression of E-cadherin in the LNCaP cells treated with juglone was increased, and the expression levels of vimentin and β-catenin were reduced (P<0.01). Treatment with LiCl significantly attenuated the inhibitory effect of juglone on Snail expression and subsequent down-regulation of E-cadherin expression. CONCLUSION: Juglone inhibits the epithelial-mesenchymal transition by inhibiting the Wnt/β-catenin/Snail signaling pathway in the LNCaP cells.     

Role of ILK siRNA on expression of GSK-3β and β-catenin in human tubular epithelial cells stimulated by high glucose

AIM：To investigate the effects of siRNA targeting integrin-linked kinase (ILK) on the expression of glycogen synthase kinase 3β (GSK-3β) and β-catenin during epithelial-mesenchymal transition (EMT) in human kidney proximal tubular epithelial cell line HKC induced by high glucose. METHODS：HKC cells were divided into 4 groups:normal glucose (NG) group, high glucose (HG) group, HG+HK (a vector containing the non-specific siRNA designed as negative control) group and HG＋ILK siRNA group. The inverted fluorescence microscope was used to examine the expression of green fluorescent protein (GFP). The expression of ILK at mRNA and protein levels was detected by RT-PCR and Western blotting. The expression of p-GSK-3β and β-catenin was observed by immunocytochemical staining. The protein expression of total GSK-3β, p-GSK-3β, nuclear β-catenin, total β-catenin, E-cadherin and α-smooth muscle actin (α-SMA) was measured by Western blotting. RESULTS：GFP was observed in HKC cells, indicating that the transfection was successful. Both the protein and mRNA of ILK were down-regulated in HG＋ILK siRNA group compared with HG group and HG+HK group, but still higher than those in NG group. Silencing of ILK down-regulated the expression of p-GSK-3β and nuclear β-catenin. No difference of total GSK-3β or total β-catenin was observed among the 4 groups. CONCLUSION:These data support a functional role of ILK, GSK-3β and β-catenin in tubular EMT induced by high glucose. ILK may promote tubular EMT by regulating the activity of GSK-3β and β-catenin, the downstream effectors of the Wnt/β-catenin pathway.     

Effect of HMGB1 expression knockdown on invasion ability of breast cancer cells induced by TNF-α

AIM:To investigate the effect of high-mobility group box-1 (HMGB1) expression knockdown on the invasion ability of breast cancer cells induced by tumor necrosis factor-α (TNF-α). METHODS:HMGB1 siRNA was used to transfect into the breast cancer MDA-MB-231 cells. The expression of HMGB1 at mRNA and protein levels was determined by RT-qPCR and Western blot. After the MDA-MB-231 cells with HMGB1 expression knockdown were treated with TNF-α, the apoptosis rate was analyzed by flow cytometry, the cell invasion ability was measured by Transwell assay, and the cell migration ability was detected by cell scratch test. The protein expression of E-cadherin, MMP-2, N-cadherin, MMP-9 and Bax was determined by Western blot. RESULTS:The expression of HMGB1 at mRNA and protein levels in the MDA-MB-231 cells transfected with HMGB1 siRNA was significantly lower than that in the non-transfected cells (P<0.05). The apoptosis rate in the cells was increased after TNF-α treatment, and the cell invasion and migration abilities were also increased. The protein level of E-cadherin in the cells was decreased, the protein level of N-cadherin was increased, and the protein levels of MMP-2, MMP-9 and Bax were also increased (P<0.05). After the MDA-MB-231 cells with HMGB1 expression knockdown were induced by TNF-α, the apoptotic rate was increased, the invasion and migration abilities were decreased, the protein levels of E-cadherin and Bax were increased, and the protein levels of N-cadherin, MMP-2 and MMP-9 were decreased, as compared with the cells only induced by TNF-α without knockdown of HMGB1 expression (P<0.05). CONCLUSION:Knockdown of HMGB1 expression enhances the apoptosis of breast cancer cells induced by TNF-α, and inhibited the cell invasion, migration and epithelial-mesenchymal transition induced by TNF-α. The mechanism may be related with the changes of protein expression of MMP-2, MMP-9 and Bax.     

miR-125a-5p reverses cisplatin resistance of non-small-cell lung cancer A549/DDP cells by targeting LIMK1

AIM:To explore the effect of microRNA-125a-5p (miR-125a-5p) on cisplatin (DDP) resistance of non-small-cell lung cancer A549/DDP cells and its related mechanisms. METHODS:The expression levels of miR-125a-5p and LIM kinase 1 (LIMK1) in non-small-cell lung cancer tissues, A549 cells and A549/DDP cells were detected by RT-qPCR. The A549/DDP cell viability, apoptotic rate and expression of drug resistance-related proteins after over-expression or knockdown of miR-125a-5p and/or LIMK1 expression were detected by MTT assay, flow cytometry and Western blot, respectively. The targeting relationship between miR-125a-5p and LIMK1 was verified by TargetScan online prediction and dual-luciferase reporter system. The cell viability, apoptotic rate and expression of drug resistance-related proteins after co-expression of miR-125a-5p and LIMK1 were also determined. RESULTS:The expression level of miR-125a-5p was down-regulated and LIMK1 expression was up-regulated in non-small-cell lung cancer tissues and cell lines (P<0.05). The results of dual-luciferase assay indicated that miR-125a-5p negatively regulated the expression of LIMK1. The expression of drug resistance-related proteins and the viability of A549/DDP cells were inhibited after over-expression of miR-125a-5p or knockdown of LIMK1, while the apoptosis was enhanced. Over-expression of LIMK1 attenuated the inhibitory effect of miR-125a-5p on A549/DDP cell viability and drug resistance-related protein expression (P<0.05). CONCLUSION:miR-125a-5p reverses the resistance of A549/DDP cells to DDP by inhibiting the expression of LIMK1 and drug resistance-related proteins.     

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 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.     

Hypoxia promotes apoptosis of neural stem cells and down-regulates miR-26a

AIM: To investigate the effect of cobalt chloride (CoCl₂) on the apoptosis of neural stem cells (NSCs) and the expression of microRNA-26a (miR-26a) in vitro, and to explore the mechanisms of NSC apoptosis induced by CoCl₂. METHODS: NSCs were exposed to CoCl₂ at different doses (200~600 μmol/L) for 24 h. The cell viability and apoptosis were measured by CCK-8 assay and TUNEL method. The expression of miR-26a-3p, miR-26a-5p, GSK-3β, caspase-3, Bcl-2 and Bax was examined by real-time PCR. The protein levels of Bcl-2 and Bax were detected by Western blotting. RESULTS: The cell viability was inhibited and the apoptosis of NSCs was increased significantly by CoCl₂ in a dose-dependent manner (P<0.05). CoCl₂ at concentration of 400 μmol/L for 24 h was used to induce apoptosis and the expression of miR-26a was down-regulated compared with control (P<0.05). Exposure to CoCl₂ at concentration of 400 μmol/L up-regulated the expression of GSK-3β, caspase-3 and Bax, down-regulated the expression of Bcl-2 and Bcl-2/Bax (P<0.05). CONCLUSION: CoCl₂ at concentration of 400 μmol/L induces the apoptosis of NSCs obviously. CoCl₂ may induce the NSC apoptosis by mitochondrial apoptotic pathway. Declining miR-26a may be related to NSC apoptosis.     

Effect of reversing methylation of SARI gene by 5-azacytidine on proliferation and invasion of human breast cancer cells

AIM: To observe whether reversing methylation of SARI (suppressor of activator protein-1 regulated by interferon) gene by 5-azacytidine (5-Aza) inhibits the proliferation and invasion of human breast cancer MDA-MB-231 cells. METHODS: MDA-MB-231 cells were treated with 5-Aza at 5 and 10 μmol/L. The methylation of SARI gene promoter was detected by methylation-specific PCR (MSP), and the mRNA expression of SARI was detected by RT-PCR. The protein expression of SARI was determined by Western blot. The cell growth was detected by MTT assay and colony formation assay. The cell invasion ability was detected by Transwell invasion assay. RESULTS: The results of MSP detection showed that the methylation levels of SARI promoter were significantly decreased after 5-Aza treatment (P<0.01). The results of RT-PCR and Western blot showed that the expression of SARI were significantly increased after 5-Aza treatment (P<0.05). The results of MTT and colony formation assays showed that the cell proliferation was significantly decreased after 5-Aza treatment (P<0.05). The results of Transwell invasion test showed that the invasive ability of the cells was significantly decreased after 5-Aza treatment (P<0.05). CONCLUSION: 5-Aza reverses the methylation status of SARI promoter in MDA-MB-231 cells, up-regulates the expression of SARI, and restores its ability to inhibit tumor cell growth and invasion.     

ER-α36 and miR-143 involved in invasion of gastric cancer cells

AIM: To investigate the relationship between the expression of estrogen receptor α36 (ER-α36) and the invasion of gastric cancer cells. METHODS: SGC7901 cells were treated with 17β-estradiol at high or low concentration. The invasion of gastric cancer cells and the expression of ER-α36 were detected. The SGC7901 cells with the characteristics of stable low expression and high expression of ER-α36 were constructed. The invasion ability and microRNA sequences were determined in above-mentioned recombinant cells. RESULTS: The decreased invasion ability and ER-α36 expression were detected in SGC7901 cells treated with low concentration of 17β-estradiol. The situation was the opposite in the cells treated with high concentration of 17β-estradiol. The expression of miR-143 was significantly decreased in the SGC7901 cells with stable high expression of ER-α36 and was increased in the SGC7901 cells with stable low expression of ER-α36. CONCLUSION: The expression of ER-α36 is positively related to the invasion of gastric cancer cells. It is possible that miR-143 plays an important role in the regulation of gastric cancer invasion.     

Effects of GSK-3β knockdown by RNA interference on formation of keloid in vitro

AIM: To study the suppressive effect of glycogen synthase kinase-3β (GSK-3β) knockdown by RNA interference on the formation of keloid. METHODS: Human keloid fibroblasts (KFB) in vitro were transfected with 3 pairs of specific GSK-3β small interfering RNA (siRNA). The best siRNA to inhibit the GSK-3β expression in human KFB was screen by RT-PCR and Western blot. The expression of GSK-3β and related proteins at mRNA and protein levels in the KFB was determined by RT-PCR and Western blot.RESULTS: The GSK-3β siRNA1434 remarkably inhibited the expression of GSK-3β at mRNA and proteins levels in the human KFB. After transfection with GSK-3β siRNA, the protein levels of β-catenin, p-GSK-3β, Wnt2 and cyclin D1 were all decreased. KFB growth became slow. With the extension of time, the inhibition of cell growth increased, and the cell doubling time was significantly delayed. CONCLUSION: siRNA targeting GSK-3β efficiently knocks down the expression of GSK-3β in the human KFB, and inhibits the activation of Wnt signaling pathway, thus inhibiting the growth of keloid. GSK-3β may be a potential therapeutic target for keloid.     

Differential microRNA expression profile in laryngeal cancer and effect of miR-125a-5p on proliferation of laryngeal cancer cell line

AIM：To investigate the differential microRNA expression profiles between laryngeal cancer and adjacent normal laryngeal mucosa. METHODS：Forty two pairs of laryngeal cancer tissue and adjacent normal laryngeal mucosa tissue were collected. Ten pairs of samples were used for determining microRNA expression by the method of miRNA microarray chip. Data analysis was performed to find out the significant differential microRNA expression profile in laryngeal cancer, and the difference was verified by quantitative real-time PCR (qRT-PCR) analysis on another 32 pairs of samples. Methyl thiazolyl tetrazolium (MTT) assay and colony-forming assay were used to analyze the proliferation of Hep2 cells induced by miR-125a-5p. RESULTS：Both miRNA microarray and qRT-PCR showed that the expression of let-7f-5p, miR-10a-5p, miR-125a-5p, miR-144-3p, miR-195-5p and miR-203 was down-regulated in laryngeal cancer tissues. miR-125a-5p suppressed the proliferation of Hep2 cells. CONCLUSION：The results of microarray are accordant with those of qRT-PCR. Significant difference of miRNA expression profiles between laryngeal cancer and adjacent normal laryngeal mucosa indicates that miRNAs may play a role in carcinogenesis and progression of laryngeal cancer. miR-125a-5p inhibits the proliferation of Hep2 cell, indicating a novel therapeutic target against laryngeal cancer.