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.     

Effects of GSK-3β inhibitor on proliferation and apoptosis of SW480 cells

AIM: To investigate the mechanism and the effect of glycogen synthase kinase 3β (GSK-3β) inhibitor (2’Z,3'E)-6-bromoindirubin-3'-oxime (BIO) on the protein expression of β-catenin and Bcl-2, and proliferation and apoptosis in colon carcinoma SW480 cells.METHODS: The immunohistochemical staining and Western blotting were performed to detect the protein expression of β-catenin, cyclin D1 and Bcl-2. The cell cycle distribution and apoptotic rate were detected by flow cytometry. The morphologic features of SW480 cells before and 24 h after BIO exposure at different concentrations were observed under microscope with HE staining.RESULTS: Compared with the untreated SW480 cells, the protein expression of β-catenin significantly increased and some β-catenin positive nuclear staining positive cells appeared in BIO treated cells. and The cells exposed to BIO showed that the cyclin D1 protein and the cells in S stage and G₂/M stage moderately increased, the protein level of Bcl-2 moderately decreased, and the cell apoptosis rate was significantly lower than those in control cells. Furthermore, the morphological changes of the SW480 cells were observed 24 h after BIO treatment. CONCLUSION: Our results indicate that GSK-3β inhibitor BIO participates in the cellular processes of promoting proliferation and inhibiting apoptosis in colon carcinoma cells. The mechanisms are mainly associated with activating the β-catenin pathway and regulating the balance of Bcl-2 pathway, and the up-regulation of β-catenin is most likely the possible factor for SW480 cell regression.     

Effect of WT1 silencing by siRNA on expression of Wnt/β-catenin and nephrin in mouse podocytes

AIM: To study the effect of WT1 silencing by small interfering RNA (siRNA) on podocyte vitality and expression of Wnt/β-catenin and nephrin in mouse podocytes. METHODS: Conditionally immortalized mouse podocytes were cultured at 33 ℃ in RPMI-1640 medium for proliferation and induced for differentiation at 37 ℃. The podocytes were transfected with WT1 siRNA. The cell vitality was detected by MTT assay. The expression of WT1,Wnt1,β-catenin and nephrin at mRNA and protein levels was determined by real-time qRT-PCR and Western blotting. RESULTS: WT1 siRNA induced the increase in the expression of Wnt1 at mRNA and protein levels, inhibited the phosphorylation of β-catenin, and reduced the cell vitality. Meanwhile, the expression of nephrin at mRNA and protein levels was decreased. CONCLUSION: WT1 siRNA reduces the expression of nephrin in podocytes and the vitality of the cells by activating Wnt/β-catenin signaling pathway.     

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.     

Dickkopf-1 silencing inhibits invasion and epithelial-mesenchymal transition in gastric carcinoma cells by down-regulating β-catenin

AIM: To explore the expression of Dickkopf-1 (DKK1) in human gastric carcinoma cells, and the influences of DKK1 gene silencing on cell invasion. METHODS: The levels of DKK1 in the human gastric mucosa cell line GES-1 and gastric carcinoma cell lines MKN-45 and SGC-7901 were detected by real-time PCR and Western blot. DKK1 gene was silenced by RNA interference, which was verified by real-time PCR, Western blot and ELISA. The cell invasion ability was determined by Transwell assay, and the cell proliferation was inhibited by mitomycin C. The levels of E-cadherin, N-cadherin, vimentin and β-catenin were determined by real-time PCR and Western blot. RESULTS: The expression of DKK1 was significantly higher in MKN-45 cells and SGC-7901 cells than that in GES-1 cells, indicating that DKK1 expression was obviously increased in gastric carcinoma cells. After successful silencing of DKK1 gene in the MKN-45 cells and SGC-7901 cells, the cell invasion ability was markedly decreased in a time-dependent pattern with increased expression of E-cadherin and decreased expression of N-cadherin and vimentin, indicating that DKK1 silencing dramatically inhibited gastric carcinoma cell invasion and epithelial-mesenchymal transition (EMT). The introduction of exogenous recombinant DKK1 (rDKK1) demonstrated the promoting effect of DKK1 on gastric carcinoma cell invasion and EMT. In addition, the inhibitory effects of DKK1 silencing on gastric carcinoma cell invasion and EMT were fulfilled by down-regulating β-catenin. CONCLUSION: The expression of DKK1 is significantly increased in human gastric carcinoma cells. Silencing of DKK1 markedly inhibits gastric carcinoma cell invasion and EMT by down-regulating β-catenin.     

Expression changes of Wnt/β-catenin signaling pathway in diabetic ulcer

AIM: To observe the effect of Wnt/β-catenin signaling pathway on diabetic ulcer. METHODS: Diabetic animal model was established in the female Wistar rats by intraperitoneal injection of low-dose streptozotocin following high-fat diet feeding. A circular wound was made on the dorsum of the rats in both control group and diabetic group. The condition of wound healing was recorded and the structures of the wound tissues were observed by HE staining in the 2 groups at 3, 7 and 14 d after wounding. The expression of β-catenin, GSK-3β and Rspo-3 at mRNA and protein levels in the wound tissues was detected by RT-PCR and ELISA. RESULTS: In diabetic group, the wound healing rate was lower (P<0.05), and the inflammatory cells, fibroblast cells and new capillaries in the wound tissues were fewer than those in control group. The expression of β-catenin and Rspo-3 at mRNA and protein levels in the wound tissues in control group was significantly higher than those in diabetic group, and the expression of GSK-3β was exactly the opposite (P<0.05). CONCLUSION: The down-regulation of Wnt/β-catenin probably resultes from the decreased level of Rspo-3, which may be one of the reasons for delaying the diabetic ulcer healing.     

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.     

Over-expression of SCUBE2 suppresses epithelial-mesenchymal transition through Wnt/β-catenin signaling pathway in colorectal cancer cells

AIM: To study the effect of SCUBE2 on epithelial-mesenchymal transition (EMT) in colorectal cancer cells and its mechanism. METHODS: The expression of SCUBE2 in human colorectal cancer cell line HCT116 and normal colonic cell line FHC was detected by real-time PCR and Western blot. HCT116 cells were transfected with GV144-SCUBE2 to over-express SCUBE2, and then the cell viability, migration, and apoptosis were determined by MTT assay, Transwell assay and flow cytometry, respectively. The expression of EMT markers (E-cadherin, vimentin, and Snail), β-catenin, c-Myc and cyclin D1 in the HCT116 cells was analyzed by real-time PCR or Western blot after transfection with GV144-SCUBE2 for 6 h, followed by the stimulation of 10 μg/L recombinant TGF-β1 protein for 48 h. Additionally, the EMT process of HCT116 cells, which were stimulated by TGF-β1, over-expressed SCUBE2, and treated with Wnt/β-catenin pathway activator lithium chloride (LiCl) or inhibitor XAV93920, was analyzed by Western blot. RESULTS: Compared with FHC cells, the expression of SCUBE2 in the HCT116 cells was significantly decreased. The viability and migration ability of the HCT116 cells were suppressed by SCUBE2 over-expression, but the apoptosis was not markedly changed. Elevated expression of SCUBE2 increased E-cadherin expression, and decreased the expression of vimentin, Snail, β-catenin, c-Myc and cyclin D1 induced by TGF-β1. Treatment with LiCl blocked but treatment with XAV93920 enhanced the effects of SCUBE2 on EMT. CONCLUSION: Over-expression of SCUBE2 may inhibit the cell growth and migration, and suppress EMT through Wnt/β-catenin signaling pathway.     

PI3K/Akt/GSK-3β pathway mediates ABC transporter regulating multidrug resistance in human colon cancer HCT-15 cells

AIM: To investigate whether the PI3K/Akt signaling pathway regulates the expression of ABC transporter through the downstream glycogen synthase kinase-3β (GSK-3β) pathway and participates in the multidurg resistance of colorectal cancer (CRC) HCT-15 cells. METHODS: Colorectal cancer HCT-15 cells were cultured and then treated with GSK-3β inhibitor (HY-19807) and PI3K/Akt pathway inhibitor (HY-13898), respectively. The median inhibitory concentration (IC₅₀) of oxaliplatin for HCT-15 cells in each group was detected by CCK-8 assay, the inhibition rate and resistance index were also calculated. The protein levels of Akt, p-Akt, GSK-3β, p-GSK3β-Ser9 and ABC transporters P-glycoprotein (P-gp) and multidrug resistance-associated protein 2 (MRP-2) in the HCT-15 cells were determined by Western blot. The mRNA expression of ABC transporter in the HCT-15 cells was detected by RT-qPCR. The cell cycle distributions were analyzed by flow cytometry assasy. RESULTS: After GSK-3β inhibitor HY-19807 was used in the HCT-15 cells, the median inhibitory concentration of oxaliplatin was significantly increased, the protein levels of p-GSK3β-Ser9, P-gp and MRP-2 were up-regulated compared with control group (P<0.05), the changes of Akt and p-Akt were not obvious compared with control group (P>0.05). The results of RT-qPCR also showed that the mRNA levels of ABCB1 and ABCC2 were increased (P<0.01). Meanwhile, analysis of the cell cycle distribution showed that GSK-3β inhibitor HY-19807 promoted HCT-15 cell transition from G₁ phase to S phase, and cell proliferation was vigorous. After the PI3K/Akt pathway inhibitor HY-13898 was applied to HCT-15 cells, the IC₅₀ of oxaliplatin was decreased compared with control group (P<0.05). Moreover, the protein levels of p-Akt, p-GSK3β-Ser9, P-gp and MRP-2 were down-regulated (P<0.01). RT-qPCR results also showed that the mRNA expression of ABCB1 and ABCC2 was decreased (P<0.01). At the same time, G₁ phase was prolonged, which inhibited cell transition from G₁ phase to S phase, and inhibited cell proliferation. The protein expression of total GSK-3β was consistent in each group. CONCLUSION: The PI3K/Akt signaling pathway is involved in the proliferation and multidrug resistance of colorectal cancer HCT-15 cells by regulating the phosphorylation of GSK-3β and changing the expression of ABC transporter.     

miR-125a-5p suppresses epithelial-mesenchymal transition via GSK-3β/Snail signaling pathway in breast cancer cells

AIM: To investigate the function of microRNA-125a-5p (miR-125a-5p) on epithelial-mesenchymal transition (EMT) of breast cancer cells via GSK-3β/Snail signaling pathway.METHODS: The expression of miR-125a-5p in normal breast epithelial cells and breast cancer cells, as well as the transfection efficiency of miR-125a-5p plasmid in MDA-MB-231 cells was detected by RT-qPCR. The chemotaxis ability and invasion ability were detected by chemotaxis assay and Transwell invasion assay. The changes of EMT-related markers, the protein level of phosphorylated glycogen synthase kinase-3β (p-GSK-3β) and the nuclear translocation of Snail were determined by Western blot. RESULTS: The expression of miR-125a-5p in the breast cancer cells was significantly lower than that in the normal breast epithelial cells. The expression of miR-125a-5p was significantly higher in MDA-MB-231/miR-125a-5p cells than that in MDA-MB-231/NC cells. The ability of epithelial growth factor (EGF) at 10 μg/L to induce chemotaxis of MDA-MB-231 cells was the strongest. Compared with MDA-MB-231/NC group, stimulation of EGF decreased the invasion ability of MDA-MB-231/miR-125a-5p cells, and resulted in the increase in E-cadherin expression, while significantly decreased the protein levels of vimentin and p-GSK-3β. Meanwhile, the nuclear localization of Snail was significantly inhibited. The invasion capacity of MDA-MB-231/miR-125a-5p+GAB2 cells was significantly enhanced compared with MDA-MB-231/miR-125a-5p+Con cells, the expression of E-cadherin was decreased, and the protein levels of vimentin and p-GSK-3β were significantly increased, while the nuclear localization of Snail was promoted. CONCLUSION: miR-125a-5p suppresses EMT via GSK-3β/Snail signaling pathway, thus inhibiting the invasion ability of breast cancer cells.     

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.     

Overexpression of HBXIP in HepG2 cells induces cell migration and regulates expression of β-catenin

AIM: To investigate the effects of hepatitis B virus X-interacting protein(HBXIP) in hepatic cancer cells on the cell migration and expression of β-catenin. METHODS: Transwell assay was used to assess the cell migration. Gelatin zymography was used to observe the activity of matrix metalloproteinase 9 (MMP-9). The expression of MMP-9, glycogen synthase kinase 3β(GSK-3β), p-GSK3β, β-catenin and p-β-catenin in HepG2 cells was determined by Western blotting. RESULTS: HepG2 cells which stably overexpressed HBXIP (HepG2-HBXIP) exhibited higher migration ability than the control cells. The results of the gelatin zymography assay showed that HBXIP overexpression increased the activity of MMP-9 in HepG2 cells. The results of Western blotting indicated that HBXIP increased the expression of MMP-9 and β-catenin, inhibited the phosphorylation of β-catenin and promoted the phosphorylation of GSK-3β (Ser9). CONCLUSION: HBXIP regulates the GSK-3β/β-catenin signaling pathway, resulting in a significant improvement of hepatocellular carcinoma cell migration.     

Effects of ATP-competitive GSK-3 inhibitor on multidrug resistance of human esophageal squamous-cell carcinoma cells

AIM: To study the changes and correlations of β-catenin, multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp), Bcl-2 and the free ATP level in esophageal squamous-cell carcinoma (ESCC) cell line EC-109 induced by ATP-competitive glycogen synthase kinase(GSK-3) inhibitor 6-bromoindirubin-3'-oxime (BIO). METHODS: The methods of flow cytometry, immunofluorescence, cytochemistry and ATP assay were used to study the expression levels of MRP2, P-gp, β-catenin and Bcl-2, the efflux capability of ATP-binding cassette (ABC) transporters, and the free ATP level in ESCC cells. RESULTS: After induced by BIO, up-regulation of β-catenin and Bcl-2 expression in cytoplasm and accumulation of β-catenin in nucleus were found in ESCC cells. The expression of MRP2 was up-regulated in cytoplasm and cell membrane. On the contrary, the expression of P-gp was down-regulated in cytoplasm and cell membrane. The free ATP level and the efflux capability of ABC transporters increased in ESCC cells. CONCLUSION: The multidrug resistance of ESCC cells in enhanced by BIO treatment.     

Role of β-catenin in apoptosis of pancreatic acinar cells induced by cae-rulein

AIM: To study the role of β-catenin in the apoptosis of pancreatic acinar cells induced by cae-rulein. METHODS: Rat pancreatic acinar AR42J cells were treated with caerulein. The expression of β-catenin at mRNA and protein levels in the AR42J cells was determined by real-time PCR and Western blot. The β-catenin over-expression vector was transfected into AR42J cells. After treatment with caerulein, the over-expression effect was evaluated by real-time PCR and Western blot. The changes of cell viability were measured by MTT assay. The leakage rates of lactate dehydrogenase (LDH) and amylase (AMY) were measured by binitrophenyl hydrazine method and iodine starch colorimetry, respectively. The apoptosis was analyzed by flow cytometry. The protein levels of endoplasmic reticulum stress protein CHOP and cleaved caspase-12 in the AR42J cells were determined by Western blot. RESULTS: The expression of β-catenin at mRNA and protein levels in the AR42J cells was decreased after treatment with caerulein (P<0.05). The expression of β-catenin in the AR42J cells was significantly increased by transfection with β-catenin over-expression vector. The viability of AR42J cells after treatment with caerulein was reduced, while the leakage rates of LDH and AMY, the apoptotic rate and the protein levels of CHOP and cleaved caspase-12 in the cells were increased (P<0.05). Over-expression of β-catenin enhanced the viability of AR42J cells after treatment with caerulein, reduced the leakage rates of LDH and AMY, and decreased the apoptotic rate and the protein levels of CHOP and cleaved caspase-12 in the AR42J cells. CONCLUSION: β-Catenin significantly inhibits the apoptosis of pancreatic acinar cells induced by caerulein. The mechanism is related to the reduction of endoplasmic reticulum stress.     

Expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and its clinical significance

AIM: To evaluate the expression of Wnt/β-catenin signaling pathway-related proteins in breast cancer and the significance. METHODS: The patients with breast cancer (n=150) in our hospital from January 2015 to January 2017 were selected as study object. The tumor tissue samples of these patients were obtained from paraffin section of breast cancer by surgical resection with complete clinicopathological data. The corresponding paracancerous tissue sam-ples were taken from the non-tumor tissue samples from the above breast cancer patients, which were 0.5~1 cm away from the tumor tissue. The methods of real-time PCR and Western blot were performed to examine the expression of Wnt-1 and β-catenin at mRNA and protein levels. Human breat cancer MCF-7 cells were divided into 3 groups:control group (MCF-7 cells without treatment), agonist group[MCF-7 cells+Wnt3a (1 mg/L)] and antagonit group[MCF-7 cells+DKK1 (16 μmol/L)]. The expression of Wnt-1 and β-catenin at mRNA and protein levels was detected by real-time PCR and Western blot. RESULTS: Compared with the paracancerous tissues, the expression levels of Wnt-1 and β-catenin were higher in tumor tissues at mRNA and proteins levels (P<0.05). Notably, the positive expression rates of Wnt-1 and β-catenin were significantly higher in tumor tissues than that in the paracancerous tissues. Furthermore, Wnt-1 expression was associated with tumor metastasis (χ²=5.352, P=0.021), tumor stage (χ²=9.412, P=0.002) and tumor size (χ²=9.412, P=0.002). In addition, β-catenin expression was also associated with tumor metastasis (χ²=9.851, P=0.002) and tumor stage (χ²=5.661, P=0.017). Compared with control group, the expression of Wnt-1 and β-catenin at mRNA and protein levels in agonist group was increased (P<0.05),while that in antagonist group was decreased (P<0.05). CONCLUSION: The expression levels of Wnt-1 and β-catenin related with Wnt/β-catenin signaling pathway are increased in the breast cancer, which are closely related to the malignant state of the tumor.     

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.     

Effects of fluctuant high blood glucose on apoptosis in glomerular endothelial cells and renal tubular epithelial cells in diabetic rats

AIM:To explore the effects of fluctuant high blood glucose and stable high blood glucose on apoptosis and the expression of Bax and Bcl-2 in glomerular endothelial cells and renal tubular epithelial cells in diabetic rats. METHODS: 24 SD rats were divided into 3 groups: control group, stable high blood glucose group and fluctuant high blood glucose group. Diabetic rats were induced by intraperitoneal injection of STZ, and the fluctuant high blood glucose animal model was induced by intraperitoneal injection of aspart and glucose at different time points every day. Apoptosis was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL), and immunohistochemistry was used to detect apoptosis associated gene bax and bcl-2 expression in kidney. RESULTS:After 4 experimental weeks, a significant increase in cell apoptosis, up-regulation of Bax protein expression in kidney tubular epithelial cell and down-regulation of Bcl-2 in glomerular endothelial cell in fluctuant high blood glucose rats were observed compared with stable high blood glucose rats.CONCLUSION: Fluctuant high blood glucose induces more apoptosis in renal tubular epithelial cells than that in stable high blood glucose diabetic rats.     

miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells by repressing KLF10 expression

AIM:To explore the effect and the underlying mechanisms of microRNA-10b (miR-10b) on high glucose-stimulated epithelial-mesenchymal transition (EMT) of renal tubular epithelial cells. METHODS:The expression level of miR-10b was examined by RT-qPCR in the kidney tissues of the type 2 diabetes patients with kidney fibrosis. The EMT model of HK-2 cells was induced by high glucose stimulation and the miR-10b expression in the process was detected by RT-qPCR. The morphological changes of the HK-2 cells were observed using a microscope. EMT markers, such as fibronectin and N-cadherin, were examined by Western blot. The online database predicted that the 3'-UTR of KLF10 bound to miR-10b and their direct interaction was confirmed by dual luciferase report assay. RESULTS:Compared with the para-carcinoma normal tissues, the expression level of miR-10b was up-regulated in the tissues of type 2 diabetes patients with kidney fibrosis (P<0.01). In high glucose-stimulated HK-2 cells, the expression level of miR-10b was increased in a time-dependent manner (P<0.01). miR-10b inhibitor reversed the morphological changes and the increases expression of the EMT markers including fibronectin, SLUG, N-cadherin and SNAI1 induced by high glucose stimulation. Online database showed miR-10b was able to bind with the 3'-UTR in the promoter region of KLF10, thus negatively regulating its expression. Meanwhile, over-expression of KLF10 inhibited the EMT induced by high glucose. Inhibition of TGF-β/Smad3 activation was observed during the process of KLF10-repressed EMT. CONCLUSION:miR-10b promotes high glucose-stimulated epithelial-mesenchymal transition of renal tubular epithelial cells may through repressing KLF10 expression.