lncRNA-MALAT1 targets and downregulates miR-570-3p expression to promote proliferation of gastric cancer cells

AIM:To investigate whether long non-coding RNA MALAT1 (lncRNA-MALAT1) targets and down-regulates microRNA-570-3p (miR-570-3p) expression to further promote the proliferation of gastric cancer cells. METHODS:Gastric cancer cell line SGC7901 was cultured in vitro and divided into 3 groups:blank control, si-MALAT1 and si-MALAT1 NC. The si-MALAT1 and si-MALAT1 NC groups were transfected with MALAT1 siRNA and its negative control, respectively. The cell proliferation was evaluated by MTS assay. The expression of miR-570-3p was detected at different time points in the pure SGC7901 gastric cancer cell line, and the expression of lncRNA-MALAT1 and miR-570-3p in different groups was detected by RT-qPCR. The potential complementary binding sites of lncRNA-MALAT1 and miR-570-3p were predicted by RegRNA. The MALAT1 gene and its mutant fragment were cloned into luciferase reporter vector psiCHECK-2. Restriction enzyme analysis and sequencing were used to identify whether the recombinant plasmids carrying MALAT1 or MALAT1-Mut were successfully constructed. miR-570-3p mimic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control were co-transfected into the 293T cells with the luciferase repor-ters containing MALAT1 or MALAT1-Mut. Dual-luciferase reporter assay was performed to detect luciferase activity in different groups in order to verify the relationship between lncRNA-MALAT1 and miR-570-3p. RESULTS:Compared with blank control group and si-MALAT1 NC group, the A₄₉₀ value in si-MALAT1 group was significantly decreased (P<0.01). The expression of miR-570-3p presented an obvious declining trend over time. The expression of lncRNA-MALAT1 in si-MALAT1 group was remarkably decreased, whereas the expression of miR-570-3p was obviously increased. The dual-luciferase reporter assay indicated that the MALAT1 reporter luciferase activity decreased significantly in miR-570-3p mimic group compared with mimic negative control (P<0.01), and the luciferase activity of MALAT1 reporter was obviously up-regulated in miR-570-3p inhibitor group compared with miR-570-3p mimic group (P<0.01). However, miR-570-3p mi-mic, miR-570-3p inhibitor, miR-570-3p mimic negative control and miR-570-3p inhibitor negative control showed no effect on the luciferase activity of MALAT1-Mut reporter. CONCLUSION:lncRNA-MALAT1 targets and down-regulates miR-570-3p expression to further promote the proliferation of gastric cancer cells.     

Effect of metformin combined with paclitaxel on apoptosis and AMPK signaling in human breast cancer cells

AIM:To investigate the effect of metformin combined with paclitaxel on the viability and apoptosis of breast cancer cell line MCF-7 and its possible mechanism. METHODS:MCF-7 cells were treated with metformin at different concentrations (2, 5, 10, 20, 40 and 80 mmol/L) and in vitro cultured. The viability of MCF-7 cells was measured by MTT assay. Metformin at 2 mmol/L or paclitaxel at 2.4 mg/L alone or in combination was used to treat the cells, and compound C, an inhibitor of adenosine monophosphate-activated protein kinase (AMPK) signaling transduction pathway, was also used. The cells were divided into control group, metformin group, paclitaxel group, combination group, and combination +compound C group. The apoptosis of the cells was analyzed by flow cytometry. The expression of Bax, Bcl-2 and caspase-3 at mRNA and protein levels was determined by RT-qPCR and Western blot. The protein levels of AMPK and P21 were examined by Western blot. RESULTS:Metformin at different concentrations (2, 5, 10, 20, 40 and 80 mmol/L) significantly inhibited the cell viability in a concentration-dependent manner (P<0.05). Compared with control group, treatment with metformin at 2 mmol/L or paclitaxel at 2.4 mg/L alone or in combination significantly inhibited the cell viability, induced apoptosis (P<0.05), decreased the level of Bcl-2 (P<0.05), increased the levels of Bax and caspase-3 (P<0.05), and promoted the protein expression of AMPK and P21 (P<0.05). The effects of metformin and paclitaxel in combination were better than those of single drug treatment, while AMPK inhibitor weaken these effects. CONCLUSION:Metformin combined with paclitaxel inhibits the viability and induces the apoptosis of breast cancer MCF-7 cells by activating AMPK signaling pathway and regulating apoptosis signaling pathway.     

Effect of ixazomib on apoptosis and NF-κB signaling pathway in pancreatic cancer cells

AIM:To investigate the effects of ixazomib on the apoptosis and NF-κB signaling pathway in pancreatic cancer cells. METHODS:Human pancreatic cancer cell lines CFPAC-1 and PANC-1 were cultured, and the cells were treated with ixazomib at 0, 10, 20, 30 and 40 nmol/L for 12, 18, 24 and 48 h. The expression of NF-κB p65, IκB kinase (IKK), Bax and caspase-3 in the cells at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by CCK-8 assay. The apoptosis was analyzed by flow cytometry. RESULTS:Treatment with ixazomib at 10~40 nmol/L inhibited the viability of PANC-1 cells and CFPAC-1 cells, and the inhibitory rate was increased significantly with the increases in the concentration and time (P<0.05). Compared with the control cells, treatment with ixazomib significantly increased the apoptotic rates of PANC-1 cells and CFPAC-1 cells in a dose- dependent manner (P<0.05), and significantly decreased the mRNA expression levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), while the mRNA expression levels of apoptotic factors Bax and caspase-3 in the PANC-1 cells and CFPAC-1 cells were significantly increased (P<0.05). The results of Western blot showed that treatment with ixazomib significantly decreased the protein levels of NF-κB p65 and IKK in the PANC-1 cells and CFPAC-1 cells (P<0.05), which was consistent with the results of mRNA expression. The protein levels of apoptosis factors Bax and caspase-3 in the CFPAC-1 cells were significantly increased (P<0.05), and the protein level of caspase-3 in the PANC-1 cells was increased significantly (P<0.05). However, Bax protein did not increase significantly in 10 nmol/L ixazomib group. CONCLUSION:Ixazomib, a proteasome inhibitor, inhibits the viability of pancreatic cancer cells and promotes apoptosis by inhibiting the activation of NF-κB signaling pathway in a time- and dose-dependent manner.     

Clinical significance of miRNA-326 expression in gastric cancer and effect of up-regulation of its expression on viability and apoptosis of gastric cancer cells

AIM: To investigate the clinical significance of microRNA-326 (miRNA-326) expression in gastric carcinoma and the effect of up-regulation of its expression on the viability and apoptosis of gastric cancer cells. METHODS: The expression of miRNA-326 in 55 tissue samples of gastric cancer was detected by RT-qPCR, and the relationship between the expression and the clinicopathological features was analyzed. The expression of miRNA-326 in gastric cancer BGC-823 cells was detected by RT-qPCR. The BGC-823 cells were transfected by liposome method, and randomly divided into normal control group (untransfected), mimic-NC group (transfected with negative control mimic) and miRNA-326 mimic group (transfected with miRNA-326 mimic). After up-regulation of miRNA-326 expression, the cell viability was measured by CCK-8 assay, and the apoptosis of the cells was analyzed by flow cytometry. The protein levels of matrix metalloprotein 9 (MMP-9), p21, cyclin D1, Bcl-2 and cleaved caspase-3 were determined by Western blot, and the mRNA expression of cyclin D1 was detected by RT-qPCR. Whether CCND1 (the gene of cyclin D1) was the target gene of miRNA-326 was evaluated by dual-luciferase reporter assay. RESULTS: The expression of miRNA-326 in the gastric cancer tissues was significantly lower than that in the adjacent tissues (P<0.05). The miRNA-326 expression had a significant correlation with the tumor size, lymph node metastasis, differentiation, and clinical stages (P<0.05), but it had no correlation with the age and sex of the patients. Moreover, the expression of miRNA-326 was also closely related to the survival rate of the patients (P<0.05). The expression of miRNA-326 in the BGC-823 cells was significantly lower than that in the normal gastric mucosa GES-1 cells (P<0.05). Compared with normal control group, the expression of miRNA-326 in mimic-NC group did not change significantly, while that in miRNA-326 mimic group was increased significantly (P<0.05). Compared with normal control group, the cell viability in miRNA-326 mimic group was significantly decreased, and the apoptosis was increased (P<0.05). In addition, compared with normal control group, the protein levels of MMP-9, cyclin D1 and Bcl-2, and the mRNA expression of cyclin D1 in miRNA-326 mimic group were decreased, while the protein levels of p21 and cleaved caspase-3 were increased (P<0.05). However, no significant difference of above protein and mRNA levels between mimic-NC group and normal control group was observed. Compared with mimic-NC+miR-326 mimic group, the activity of luciferase in the cells transfected with pmiR-CCND1-WT plasmid was significantly decreased (P<0.05), but that in the cells transfected with pmiR-CCND1-Mut plasmid did not change significantly. CONCLUSION: The expression level of miRNA-326 in gastric cancer tissues is low, and it may promote cell viability and inhibit cell apoptosis by targeting CCND1.     

Effect of miR-23b-3p on viability and apoptosis of rheumatoid arthritis synovial fibroblasts by targeting XIAP

AIM:To investigate the effect of microRNA-23b-3p (miR-23b-3p) on the viability and apoptosis of rheumatoid arthritis synovial fibroblasts by targeting X-linked inhibitor of apoptosis protein (XIAP). METHODS:The expression of miR-23b-3p and XIAP was detected by RT-qPCR. The TargetScan was used to predict the targeting regulatory relation between miR-23b-3p and XIAP, and then the regulatory relation was confirmed by dual-luciferase reporter assay. After the miR-23b-3p mimic and inhibitor were transfected into the cells, the expression of miR-23b-3p and XIAP was detect by RT-qPCR. The effect of miR-23b-3p on the viability and apoptosis was measured by CCK-8 assay and flow cytometry. The protein expression levels of Ki67 and Bcl-2 were determined by Western blot. RESULTS:The expression level of miR-23b-3p was down-regulated significantly (P<0.05), and XIAP was up-regulated significantly in rheumatoid arthritis synovial fibroblasts (P<0.05). The miR-23b-3p mimic significantly inhibited XIAP expression and the cell viability, promoted the apoptosis, and down-regulated the expression of Ki67 and Bcl-2 (P<0.05). The effects of miR-23b-3p inhibitor were the opposite. CONCLUSION:miR-23b-3p inhibits the viability and promotes apoptosis of rheumatoid arthritis synovial fibroblasts by targeting XIAP.     

Metformin sensitizes human breast cancer MDA-MB-231 cells to tamo-xifen through down-regulation of c-Myc

AIM: To investigate whether metformin enhances the sensitivity of human breast cancer MDA-MB-231 cells to tamoxifen by down-regulating c-Myc. METHODS: The cell viability, colony formation, apoptosis, and migration and invasion abilities of MDA-MB-231 cells were detected by CCK-8 assay, colony formation experiment, flow cytometry and Transwell assay. The expression level of c-Myc was quantified by Western blot and immunohistochemical analysis. The antitumor effects of metformin and tamoxifen were investigated in vivo in a MDA-MB-231 triple-negative breast cancer xenograft model in the SCID mice. RESULTS: Metformin in combination with tamoxifen exerted synergistic effects on inhibition of the viability, colony formation, migration and invasion, and induced the apoptosis compared with the controls and either agent treatment alone in the MDA-MB-231 cells. The levels of c-Myc was down-regulated in vitro by treatment with metformin and/or tamoxifen (P<0.01). Moreover, metformin or in combination with tamoxifen also reduced the growth of MDA-MB-231 breast cancer tumors in the SCID mice by down-regulation of c-Myc in vivo. CONCLUSION: Metformin in combination with tamoxifen exerts synergistic effects on inhibition of the proliferation, migration, invasion and tumor growth of human triple-negative breast cancer MDA-MB-231 cells by down-regulating c-Myc expression, suggesting that metformin in combination with tamoxifen merits further evaluation as a target.     

Role of HMGA2 in epithelial-mesenchymal transition of gastric cancer cells

AIM:To investigate the role of HMGA2 in the epithelial-mesenchymal transition (EMT) in gastric cancer cells. METHODS:The expression of HMGA2 in human gastric cancer cell lines with different degrees of differen-tiation (MKN45, MKN28 and SGC7901) and immortalized human gastric epithelial cell line GES-1 was determined by Western blot and RT-qPCR. pcDNA3.0-HMGA2 plasmid was transfected into the MKN28 cells by liposome method. Transfection of si-HMGA2 interference fragments into MKN45 cells was also performed. The transfection efficiency was evaluated by Western blot and RT-qPCR. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the cell viability were measured by CCK-8 assay. The effects of HMGA2 over-expression in the MKN28 cells on the cell migration and invasion abilities were detected by wound healing and Transwell invasion assays. The effects of HMGA2 over-expression in the MKN28 cells and knock-down in the MKN45 cells on the expression of EMT-related markers E-cadherin, N-cadherin, vimentin at mRNA and protein levels were determined by RT-qPCR and Western blot. The changes of Wnt/β-catenin signaling pathway-related molecules in the MKN28 cells with HMGA2 over-expression were also determined by RT-qPCR. RESULTS:The expression levels of HMGA2 were quite different in different differentiation levels of gastric cancer cells (P<0.05). The increased expression level of HMGA2 in MKN28 cells inhibited the cell viability (P<0.05), while the decreased expression level of HMGA2 in MKN45 cells promoted the cell viability (P<0.05). The increased expression level of HMGA2 in MKN28 cells promoted cell migration and invasion (P<0.05), changed the expression of EMT-related markers (P<0.05), while the decreased expression level of HMGA2 in the MKN45 cells changed the expression of EMT-related markers (P<0.05). The increased expression level of HMGA2 in the MKN28 cells significantly increased the mRNA levels of β-catenin in the Wnt/β-catenin pathway and the downstream molecules c-Myc and cyclin D1 (P<0.05). CONCLUSION:HMGA2 is closely related to the migration and invasion abilities of gastric cancer cells. Moreover, it promotes the EMT process of gastric cancer cells by activating Wnt/β-catenin pathway.     

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.     

TPX2 induces apoptosis of rectal cancer HR-8348 cells through p38 MAPK signaling pathway

AIM: To study the effect of targeting protein for Xenopus kinesin-like protein 2 (TPX2) expression knockdown on the apoptosis of rectal cancer HR-8348 cells.METHODS: The HR-8348 cells transfected with TPX2 small interfering RNA (siRNA) served as TPX2 siRNA group. The non-transfected cells were used as control group. The cells transfected with siRNA negative control (siRNA-NC) were used as siRNA-NC group. The TPX2 siRNA-transfected cells exposed to p38 MAPK inhibitor SB203580 served as TPX2 siRNA+SB203580 group. The expression of TPX2 at mRNA and protein levels was determined by RT-qPCR and Western blot. The cell viability was measured by MTT assay, the apoptosis was analyzed by flow cytometry. The protein levels of p38 MAPK, p-p38 MAPK, cleaved caspase-3 and Bcl-2 in the HR-8348 cells were determined by Western blot.RESULTS: After transfection, the expression of TPX2 at mRNA and protein levels was decreased in TPX2 siRNA-transfected cells (P<0.05). Transfection with siRNA-NC had no effect on TPX2 mRNA and protein levels in the cells. After knockdown of TPX2 expression, the viability of rectal cancer HR-8348 cells and the expression of Bcl-2 were decreased, while the apoptotic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK were increased significantly reduced (P<0.05). Compared with TPX2 siRNA group, the apopto-tic rate and the protein levels of cleaved caspase-3 and p-p38 MAPK/p38 MAPK in TPX2 siRNA+SB203580 group were significantly decreased, while the viability was significantly increased (P<0.05).CONCLUSION: Knockdown of TPX2 expression promotes apoptosis of rectal cancer HR-8348 cells by activating p38 MAPK signaling pathway.     

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.     

miR-140-3p inhibits viability,invasion and migration of non-small-cell lung cancer A549 cells by targeting PD-L1

AIM: To explore the target relationship between microRNA-140-3p (miR-140-3p) and programmed cell death ligand 1 (PD-L1) and their effect on the viability, migration and invasion of non-small-cell lung cancer A549 cells.METHODS: RT-qPCR was used to detect the miR-140-3p expression in HLF-1, A549 and H1299 cells, and then the A549 cells with the most significant difference were selected as the subsequent research object. TargetScan software and dual-luciferase reporter assay were performed to predict and confirm the target relationship between miR-140-3p and PD-L1. RT-qPCR and Western blot were used to determine the effects of miR-140-3p mimic and inhibitor on PD-L1 expression level. MTT assay was used to detect the viability of A549 cells. Transwell assay was performed to detect the migration and invasion abilities of the A549 cells.RESULTS: miR-140-3p was significantly down-regulated in the A549 cells and H1299 cells (P<0.05). Transfection with miR-140-3p mimic decreased the expression of PD-L1 and inhibited the viability, migration and invasion of the A549 cells. Transfection with pcDNA3.0-PD-L1 reversed the inhibitory effect of miR-140-3p on the viability, migration and invasion of the A549 cells.CONCLUSION: miR-140-3p inhibits the viability, migration and invasion of A549 cells by targeting PD-L1.     

Effects of NOB1 gene silencing on drug sensitivity and invasion and migration abilities of human colon cancer SW480 cells

AIM:To investigate the effect of NOB1 gene expression knock-down by transfection of small interfering RNA (siRNA) on the viability, drug sensitivity, apoptosis, cell cycle distribution, and invasion and migration abilities of human colon cancer SW480 cells. METHODS:NOB1 siRNA was transfected into SW480 cells using Lipofectamine 3000. The mRNA and protein levels of NOB1 in the SW480 cells were determined by real-time PCR and Western blot. The cell viability and sensitivity to different chemotherapeutic drugs (cisplatin, 5-fluorouracil, oxaliplatin and capecitabine) were detected by MTT assay after knock-down of NOB1 gene expression in the SW480 cells. The apoptosis and cell cycle distribution of SW480 cells were analyzed by flow cytometry. The invasion and migration abilities of SW480 cells were detected by Transwell assay. RESULTS:After transfection with NOB1 siRNA, the mRNA and protein levels of NOB1 in the SW480 cells were significantly decreased (P<0.05). Compared with control group and control siRNA group, the viability of SW480 cells in NOB1 siRNA group was significantly decreased at 24~72 h. The half maximal inhibitory concentrations of the chemotherapy drugs cisplatin, 5-fluorouracil, oxaliplatin and capecitabine were significantly decreased. The apoptotic rate was significantly increased and the cell cycle were blocked. The cell invasion and migration abilities were significantly reduced (P<0.05). CONCLUSION:Knock-down of NOB1 gene expression inhibits the viability and invasion and migration abilities of colon cancer SW480 cells, and promotes drug sensitivity and apoptosis. NOB1 may be a new target for diagnosis and treatment of colon cancer.     

Effect of 27nt-miRNA on regulation of SM22α expression in vascular smooth muscle cells and its effect on cell viability,migration and phenotypic changes

AIM:To investigate the effect of 27nt-microRNA (27nt-miRNA) on the expression of smooth muscle 22α protein (SM22α) and the cell viability, migration and phenotypic changes of vascular smooth muscle cells (VSMCs). METHODS:The highly expression plasmids of 27nt-miRNA, and anti-27nt-miRNA and negative control plasmids were constructed, packaged with lentivirus and transfected into the rat primary VSMCs. Platelet-derived growth factor BB (PDGF-BB) was added to induce VSMCs phenotype conversion. The cell viability was measured by MTT assay. The migration ability was detected by scratch assay. The mRNA and protein expression of SM22α was determined by RT-PCR, immunocytochemical staining and Western blot. RESULTS:Compared with normal group, the cell viability in PDGF-BB group was increased (P<0.05), the migration ability was increased (P<0.05) and the expression of SM22α at mRNA and protein level was decreased (P<0.05). Compared with negative control lentiviral group, the cell viability in 27nt-miRNA over-expression group was decreased (P<0.05), the migration ability was decreased (P<0.05), and the mRNA and protein expression of SM22α was increased (P<0.05). While in anti-27nt-miRNA group, the cell viability was increased(P<0.05), the migration ability was increased (P<0.05), and the mRNA and protein expression of SM22α was decreased (P<0.05). CONCLUSION:27nt-miRNA significantly increases the expression of SM22α, while inhibits the viability and migration ability of VSMCs, and inhibits its phenotypic shift from contractile to synthetic.     

Knockdown of HMGA2 gene inhibits TGF-β1-induced human embryonic lung fibroblast growth and collagen synthesis

AIM: To investigate the effects of high mobility group A2(HMGA2) gene knockdown on the cell viability, apoptosis, collagen synthesis and oxidative stress of human embryonic lung fibroblast (HELF) induced by transforming growth factor-β1 (TGF-β1). METHODS: The HELF were divided into blank group, TGF-β1 group,negative control (NC) group and HMGA2 siRNA(si-HMGA2) group. The protein levels of HMGA2, AKT and p-AKT were determined by Western blot. The cell viability and apoptotic rate was analyzed by MTT assay and flow cytometry,respectively. The mRNA expression of collagen I (COL-Ⅰ) and COL-Ⅲ was detected by RT-qPCR. DCFH-DA was used to detect the content of reactive oxygen species (ROS). RESULTS: Compared with blank group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in TGF-β1 group were significantly increased, but the apoptotic rate and ROS level were significantly decreased (P<0.05). Compared with TGF-β1 group, the protein levels of HMGA2 and p-AKT, the cell viability, the mRNA expression of COL-Ⅰ and COL-Ⅲ in si-HMGA2 group were significantly decreased, but the apoptotic rate and ROS level were significantly increased (P<0.05). CONCLUSION: Knockdown of HMGA2 gene expression decreases the viability and collagen synthesis, and promotes apoptosis and ROS production of human embryonic lung fibroblasts induced by TGF-β1. The mechanism may be related to down-regulation of PI3K/AKT 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.     

IL-17 promotes viability and migration ability of endometrial carcinoma cells by inhibiting miR-195-5p expression

AIM:To investigate the potential mechanism of interleukin-17 (IL-17) promoting the viability, migration and invasion of human endometrial carcinoma cells. METHODS:The expression of IL-17 and microRNA-195-5p (miR-195-5p) in the human endometrial carcinoma and benign uterine lesion samples were detected by RT-qPCR. The expression of miR-195-5p in human endometrial carcinoma HEC-1-B cells after treatment with IL-17 at different concentrations for 48 h was detected by RT-qPCR. The viability, migration and invasion of HEC-1-B cells after treatment with IL-17 at 100 μg/L or transfection of miR-195-5p mimics were detected by MTT assay and Transwell assays. The viability, migration and invasion of HEC-1-B cells after over-expression of miR-195-5p combined with 100 μg/L IL-17 intervention were also observed. RESULTS:The expression of IL-17 was increased while the expression of miR-195-5p was decreased in the human endometrial carcinoma samples (P<0.05). The expression of miR-195-5p in the HEC-1-B cells after treatment with IL-17 at 10,100 and 300 μg/L for 48 h was significantly decreased (P<0.05). The results of MTT assay and Transwell experiments indicated that IL-17 at 100 μg/L enhanced the viability, migration and invasion of HEC-1-B cells, while over-expression of miR-195-5p resulted in the opposite effect. CONCLUSION:Over-expression of miR-195-5p inhibits the enhancing effects of IL-17 on the viability, invasion and migration of HEC-1-B cells.     

Effects of lutein on viability of breast cancer cells

AIM:To investigate the effect of lutein on the viability of breast cancer cells and its possible mechanism. METHODS:The human breast cancer T47D cells were divided into control group and lutein (6.25, 12.5, 25, 50 mg/L) treatment groups. The effect of lutein on the viability of T47D cells was measured by MTT assay. The mRNA expression of nuclear factor erythroid 2-related factor 2 (Nrf2), glutathione peroxidase 1 (GPx1) and superoxide dismutase 2(SOD2) was detected by RT-qPCR. Fluorescent probes DCFH-DA was used to determine the production of reactive oxygen species (ROS). The protein expression of Nrf2 and p65 was determined by Western blot. RESULTS:The MTT results showed that lutein inhibited T47D breast cancer cell viability in a dose-and time-dependent manner. The RT-qPCR results showed that the mRNA levels of Nrf2, GPx1 and SOD2 were higher in lutein treatment groups than those in the control group (P<0.05), and with the increased concentrations and extension of intervention time of lutein, the relative mRNA levels were all increased. The ROS levels were significantly decreased in the lutein-treated groups (P<0.05). The results of Western blot demonstrated that the protein expression of Nrf2 was significantly increased (P<0.05), and p65 protein was decreased (P<0.05) in a dose-dependent manner with lutein treatment for 48 h. CONCLUSION:Lutein significantly inhibits the viability of breast cancer cells, and the inhibition roles may be related to up-regulation of the expression of Nrf2, antioxidant enzymes GPx1 and SOD2 mRNA expression and down-regulation of oxidative stress, thus blocking the NF-κB signaling pathway.     

miR-556-3p regulates viability,migration and invasion of endometrial cancer cells by targeting SASH1

AIM To investigate whether microRNA-556-3p （miR-556-3p） regulates the viability, migration and invasion of endometrial cancer cells by targeting SASH1 gene. METHODS The expression of miR-556-3p, and the mRNA and protein levels of SASH1 in endometrial cancer tissues were detected by RT-qPCR and Western blot. Anti-miR-556-3p or pcDNA-SASH1 was transfected into endometrial cancer Ishikawa cells. The cell viability was detected by MTT assay, the migration and invasion abilities of the cells were detected by Transwell chamber method, and the protein expression levels of cyclin D1, p21, matrix metalloproteinase-2 （MMP-2） and MMP-9 were detected by Western blot. StarBase prediction and dual-luciferase reporter experiments were used to analyze the targeting relationship between miR-556-3p and SASH1. Anti-miR-556-3p and si-SASH1 were co-transfected into the Ishikawa cells, and their effects on cell viability, migration and invasion were examined by the methods described above. RESULTS Compared with adjacent tissues, the expression of miR-556-3p in endometrial cancer tissues was increased significantly, and the expression of SASH1 at mRNA and protein levels was decreased significantly （P<0.05）. Inhibition of miR-556-3p expression or induction of SASH1 over-expression obviously reduced the viability of Ishikawa cells, the number of migratory cells, the number of invasive cells and the protein levels of cyclin D1, MMP-2 and MMP-9, and dramatically increased the protein level of p21 （P<0.05）. miR-556-3p targeted SASH1 and negatively regulated its expression. Knock-down of SASH1 expression reversed the inhibitory effect of miR-556-3p expression inhibition on the viability, migration and invasion of Ishikawa cells. CONCLUSION Inhibition of miR-556-3p expression suppresses the viability, migration and invasion of endometrial cancer cells. The mechanism is related to the regulation of its target gene SASH1.     

Effects of metformin on nuclear factor-κB expression and serum high sensitivity C-reactive protein level in atherosclerosis model of rabbit

AIM: To investigate the effects of metformin on nuclear factor-κB (NF-κB),its inhibitor IκB,and the level of serum high sensitivity C-reactive protein (hs-CRP) in rabbits.METHODS: 24 New Zealand male rabbits were randomly divided into control group,atherosclerosis (AS) group and metformin (Met) group.AS group and Met group were made as models by cholesterolenriched diets feeding and vascular intimal immunologic injury.The AS model was confirmed by high frequency ultrasound.Met group were given metformin 150 mg·kg-1·d-1 for 8 weeks.At the end of experiment,serum hs-CRP and serum lipids in all three groups were detected.Immunohistochemistry and Western blotting technique were applied to detect the expression of nucleus NF-κB p65 and cytoplasma IκBα in aorta in all three groups.RESULTS: Compared to normal control group,the level of serum hs-CRP was elevated (1.27±0.43 vs 3.96±0.63,P<0.01),the expression of nucleus NF-κB p65 increased significantly (P<0.01) while the expression of IκBα reduced significantly (P<0.01).Compared to AS group,metformin significantly reduced the level of serum hs-CRP (2.79±0.40 vs 3.96±0.63,P<0.05) and the expression of nucleus NF-κB p65 (P<0.01),and increased the expression of IκBα (P<0.05).CONCLUSION: Metformin inhibits the activation of NF-κB p65 and the degradation of IκBα,and decreases the levels of serum hs-CRP in AS rabbits.These results suggest that metformin exerts direct vascular anti-inflammatory effects.It may be one important mechanism of metformins antiatherogenic properties.