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 toosendanin on invasion and migration of human ovarian cancer cells

AIM: To investigate the effect of toosendanin (TSN) on invasion and migration abilities of human ovarian cancer cells and the related mechanism. METHODS: The human ovarian cancer cell lines CAVO-3 and SKVO-3 were treated with TSN at different concentrations. The cell viabilty at 12, 24, 48, 72 and 96 h after TSN treatment was measured by CCK-8 assay. Scratch wound healing assay and Transwell assay were employed to measure the invasion and migration abilities of CAVO-3 cells. The protein expression of nuclear factor-κB (NF-κB) p65, E-cadherin, N-cadherin, vimentin and Snail was determined by Western blot. RESULTS: TSN significantly inhibited the viability of CAVO-3 and SKVO-3 cells (P<0.05). Compared with control group, the migration and invasion abilities of CAVO-3 cells in TSN group decreased significantly (P<0.05). In addition, the expression of NF-κB p65 and E-cadherin protein increased notably, followed with N-cadherin, vimentin and Snail protein decreased significantly (P<0.05). However, the inhibitor of NF-κB BAY11-7082 reversed the impact above. Compared with TSN group, the migration and invasion abilities in TSN+BAY11-7082 group increased significantly (P<0.05). The protein expression of E-cadherin also decreased notably, followed with the protein expression of N-cadherin, vimentin and Snail increased significantly (P<0.05). CONCLUSION: TSN inhibits the invasion and migration abilities of human ovarian cancer cells, which is related to the inhibition of epithelial-mesenchymal transition process mediated by NF-κB/Snail signaling pathway.     

Effect of formononetin on viability,migration and invasion of ovarian cancer cells in vitro

AIM To observe the effect of formononetin on the viability, migration and invasion of ovarian cancer cells, and to explore its mechanism. METHODS Human ovarian serous cystadenocarcinoma SKOV-3 cells were cultured in vitro. The cells were treated with formononetin at 0, 25, 50 and 100 μmol/L for 48 h. The cell viability was measured by MTS assay. The migration and invasion abilities of the SKOV-3 cells were detected by scratch wound assay and Transwell assay. RT-qPCR and Western blot were used to detect the mRNA and protein levels of E-cadherin and matrix metalloproteinase-9 (MMP-9). RESULTS The viability of SKOV-3 cells was decreased with the increase in the formononetin concentration compared with control group (P<0.01). The wound migration distance of the cells in 50 μmol/L formononetin group was less than that in control group (P<0.01). The number of invasive SKOV-3 cells across the Transwell sub-compartment was significantly decreased in 50 μmol/L formononetin group compared with control group (P<0.01). The mRNA and protein levels of E-cadherin in 50 μmol/L formononetin group were significantly higher than those in control group (P<0.01), while the mRNA and protein levels of MMP-9 in 50 μmol/L formononetin group were significantly lower than those in control group (P<0.01). CONCLUSION Formononetin inhibits the migration and invasion abilities of ovarian cancer SKOV-3 cells by increasing expression of E-cadherin and decreasing expression of MMP-9.     

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.     

Gene silencing of indoleamine 2,3-dioxygenase 2 influences proliferation,migration and invasion of B16-BL6 melanoma cells

AIM: To investigate the effects of indoleamine 2, 3-dioxygenase 2 (IDO2) silencing on proliferation, migration and invasion of B16-BL6 melanoma cells.METHODS: IDO2-siRNA was transfected into the B16-BL6 melanoma cells in vitro. The expression of IDO2 or IDO1 at mRNA and protein levels was detected by real-time PCR and Western blot. Colony formation assay was performed to analyze the proliferation of IDO2-silencing tumor cells. The migration ability of B16-BL6 cells after silencing of IDO2 was measured by wound healing assay and Transwell cell migration assay. The invasion ability of the tumor cells was detected by Transwell cell invasion assay.RESULTS: IDO2-siRNA signi-ficantly down-regulated IDO2 expression in B16-BL6 melanoma cells, and did not affect IDO1 expression. Compared with control group, the colony formation ability, the migratory distance measured by wound healing assay, and the migration and the invasion cell numbers detected by Transwell assay all remarkably decreased in the IDO2-silencing cells.CONCLUSION: IDO2 silencing affects the proliferation, migration and invasion abilities of the B16-BL6 melanoma cells.     

Role of endoplasmic reticulum stress and autophagy mediated by endocannabinoid N-arachidonic acid aminoethanol in ovarian cancer cells

AIM To study the effect of endocannabinoid N-arachidonic acid aminoethanol （AEA） on ovarian cancer and its mechanism. METHODS The serum levels of AEA in healthy control group and ovarian cancer group were analyzed by ELISA, and the diagnostic value of AEA in ovarian cancer patients was evaluated by ROC curve. The effects of AEA on the viability, migration and invasion abilities of the ovarian cancer cells were detected by CCK-8 assay, Transwell cell invasion test and Scratch test. The effect of AEA on ovarian cancer was further verified by the measurement of tumor volume, tumor weight and visual map of tumor tissue. Meanwhile, flow cytometry and Western blot were used to determine the effect of AEA on the apoptosis of ovarian cancer cells, so as to explore the mechanism of AEA promoting the apoptosis of ovarian cancer cells through detecting the endoplasmic reticulum stress and autophagy related proteins by Western blot. RESULTS The serum levels of N-arachidonic aminoethanol in the patients with ovarian cancer were significantly decreased. ROC results suggested that AEA was a sensitive biological marker to distinguish the patients with ovarian cancer from healthy dedividuals. In addition, AEA inhibited the cell viability, migration and invasion abilities of ovarian cancer cells, and inhibited the growth of ovarian cancer tissues. CONCLUSION By promoting endoplasmic reticulum stress and affecting autophagy of ovarian cancer cells, AEA promotes apoptosis of ovarian cancer cells.     

β-estradiol promotes invasion and migration of lung cancer A549 cells via ERβ-mediated ERK1/2 membrane-initiated steroid signaling

AIM: To investigate the regulatory mechanism of β-estradiol in the invasion and migration of lung cancer A549 cells. METHODS: Breast cancer MCF-7 cells and lung cancer A549 cells were cultured in vitro. The MCF-7 cells were used as the estrogen receptor (ER) positive expression cell model. Real-time PCR and immunofluorescence were employed to measure the expression level and the localization of ER in A549 cells. The phosphorylation of ERK1/2 upon β-estradiol stimulation was quantified by Western blot. The invasion and migration abilities of A549 cells upon β-estradiol stimulation with or without ERK1/2 inhibitor PD98059 were measured by Transwell and Cell-IQ assays. RESULTS: ERβ was the dominant ER subtype in the A549 cells and primarily comprised of ERβ2 and ERβ5. Immunofluorescence revealed that ERβ expression was mainly localized in the cytoplasm. β-estradiol induced phosphorylation of ERK1/2 and promoted the invasion and migration of the cells. Inhibition of ERK1/2 signaling reversed β-estradiol-promoted invasion and migration of A549 cells. CONCLUSION: ERβ-mediated membrane-initiated steroid signaling is involved in the process of β-estradiol-promoted invasion and migration of A549 cells, through which ERK1/2 signaling plays a pivotal role.     

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.     

Effect of ABCE 1 gene silencing on proliferation and migration of human bladder cancer cell line T24

AIM: To investigate the effect of small interfering RNA (siRNA)-mediated ABCE1 knockdown on the survival, cell cycle and invasion of human bladder cancer cell line T24. METHODS: The siRNA against ABCE1 was constructed and transfected into the T24 cells with Lipofectamine^TM 2000. The expression of ABCE1 was detected by RT-PCR and Western blot. Flow cytometry was used to detect the cell cycle. The effects of ABCE1 gene silencing on proliferation, migration and invasion of T24 cells were evaluated by CCK-8 assay, wound-healing assay and Transwell invasion assay, respectively. RESULTS: Compared with control group and blank group, the mRNA and protein levels of ABCE1 were significantly decreased in experimental group after transfected with ABCE1 siRNA. The cell cycle was arrested at G₀/G₁ phase and the cell number in S phase was decreased in the T24 cells. Compared with control group and blank group, the proliferation of T24 cells in experimental group was inhibited significantly, and the migration and invasion abilities of T24 cells in experimental group decreased significantly. CONCLUSION: Knockdown of ABCE1 gene may decrease migration, invasion and proliferation abilities in T24 cells.     

Value of endonuclease domain containing 1 in progression of prostate cancer

AIM: To analyze the difference of endonuclease domain containing 1 (ENDOD1) expression between benign prostatic hyperplasia (BPH) tissues and prostate cancer (PCa) tissues and to investigate the effect of ENDOD1 on the biological function of human prostate cancer cells. METHODS: The BPH samples (n=20) and PCa samples (n=21) were processed and analyzed according to the instruction of immunohistochemical (IHC) staining. The mRNA and protein levels of ENDOD1 in the normal prostate epithelial cells and prostate cancer cells were evaluated by RT-qPCR and Western blot, respectively. The recombinant plasmids pCMV-N-Flag-ENDOD1 was constructed and was transfected into the human prostate cancer cells. The proliferation, apoptosis, migration and invasion abilities of the prostate cancer cells were evaluated by MTT assay, flow cytometry, Transwell migration and Matrigel invasion assays, respectively. RESULTS: The analysis of variance of the immunoreactivity score showed that PCa tissues with high Gleason score displayed significantly lower ENDOD1expression than that with low Gleason score and BPH (P<0.05). The expression of ENDOD1 at mRNA and protein levels in PC3 cells and DU145 cells was significantly lower than that in the LNCap cells (P<0.05). The proliferation of DU145 transfected with ENDOD1 was inhibited. The flow cytometry indicated that ENDOD1 over-expression in the DU145 cells resulted in a notable increase in G₀/G₁ phase arrest (P<0.05), but the apoptotic rates showed no statistical difference. The results of Transwell assay showed that migration and invasion abilities of the cells were also inhibited after transfection with over-expressing ENDOD1 plasmid (P<0.05). CONCLUSION: The expression of ENDOD1 significantly decreased in prostate cancer with high Gleaon score. Meanwhile, the ENDOD1 is specifically down-regulated in androgen independent prostate cancer (AIPC) cell lines. Over-expression of ENDOD1 remarkably inhibits the proliferation, migration and invasion abilities of AIPC.     

Effects of lentivirus-mediated siRNA targeting IGF-1R on migration and invasion abilities of endometrial carcinoma cells

AIM:To investigate the down-regulation of insulin-like growth factor tgpe 1 receptor(IGF-1R) on the migration and invasion abilities of human endometrial cancer cell HEC-1B. METHODS:The siRNAs targeting IGF-1R gene were synthesized, cloned into a lentivirus expression vector and transfected into endometrial cancer HEC-1B cells(HEC-1B-KD group). The control cells(without virus transfection, HEC-1B-CON group) and negative virus transfection control cells(HEC-1B-NC group) were also set up. The gene silencing effect of siRNA targeting IGF-1R was determined by real-time PCR and Western blotting at mRNA and protein levels,respectively. The proliferation rate was detected by colony formation assay. The cell migration and invasion abilities were determined by Transwell experiment. The mRNA levels of matrix metalloproteinase(MMP)-2 and MMP-9 were measured by real-time PCR. RESULTS:The mRNA and protein levels of IGF-1R in HEC-1B-KD cells were significantly reduced by 81% and 91.5%, respectively(P<0.05). In anchorage-dependent growth by colony formation assay, HEC-1B-KD cells showed much less colonies than HEC-1B-CON cells and HEC-1B-NC cells. Compared with the control cells, knockdown of IGF-1R in HEC-1B cells resulted in significant reduction of cell motility. Down-regulation of IGF-1R in HEC-1B cells also significantly reduced the invasion potential(P<0.05). Down-regulation of IGF-1R substantially reduced the expression of MMP-2 and MMP-9 compared with the control cells. CONCLUSION:Knockdown of IGF-1R reduces the migration and invasion abilities of human endometrial cancer cells in vitro accompanied with a decrease in MMP-2 and MMP-9 expression.     

Proton-sensing receptor OGR1 mediates inhibitory effects of acidic environment on viability and tube formation ability of endothelial progenitor cells

AIM: To analyze the expression of ovarian cancer G-protein-coupled receptor 1 (OGR1), a proton-sensing receptor, in the endothelial progenitor cells (EPCs), and to explore the role of OGR1 in acid-regulated viability and tube formation ability of EPCs. METHODS: The method of FITC-UEA-I and DiI-Ac-LDL double staining was used to identify the EPCs. The expression of OGR1 in mouse EPCs was analyzed by real-time-PCR and Western blot. After treatment with different pH media, the OGR1 expression was analyzed in EPCs. The viability and cell cycle distribution of the EPCs were analyzed by CCK-8 and flow cytometry assay. The migration and vascularization of EPCs were measured by scratch test, Transwell migration assay and tube formation experiments after silencing OGR1 using small interfering RNA(siRNA). RESULTS: The isolated endothelial progenitor cells were well differentiated, and FITC-UEA-I and DiI-Ac-LDL staining was positive. The expression of OGR1 at mRNA and protein levels was observed in mouse EPCs. The expression of OGR1 was increased gradually with the decrease of pH value of the medium, while the expression of OGR1 was the highest in the medium of pH 6.4 (P<0.05). pH 6.4 medium inhibited the viability of the EPCs and induced cell cycle arrest at G₀/G₁ phase. Knock-down of OGR1 expression by siRNA partially reversed the effect of acidic environment on the EPCs (P<0.05). The abilities of migration and tube formation of EPCs were inhibited by pH 6.4 medium, while transfection of siRNA to silence OGR1 expression partially reversed the effect (P<0.05). CONCLUSION: The expression of OGR1 in EPCs is positive, and OGR1 mediates the effects of acid on the viability, migration and tube formation ability of EPCs.     

Effects of marrow stromal cell-mediated microenvironment on human hepatocellular carcinoma cell line SMMC-7721

AIM: To investigate the effects of marrow stromal HS-5 cells on hepatocellular carcinoma SMMC-7721 cells in the tumor microenvironment. METHODS: The effects of HS-5 cell-conditioned medium (HS-5-CM) on the proliferation, migration and invasion abilities of SMMC-7721 cells were detected by MTT, wound-healing and Transwell assays. After co-culture of SMMC-7721 cells with HS-5 cells in the Transwell chamber, the expression of chemokine CCL5 and its receptor CCR5 at mRNA and protein levels in SMMC-7721 cells was examined by quantitative real-time PCR (qRT-PCR), ELISA or Western blotting. Akt and p-Akt473 protein levels in SMMC-7721 cells treated with PI3K inhibitor LY294002 were observed by Western blotting. RESULTS: HS-5-CM promoted the proliferation, migration and invasion abilities of SMMC-7721 cells. The expression of CCL5 and CCR5 at mRNA and protein levels in SMMC-7721 cells was increased after co-cultured with HS-5 cells. PI3K inhibitor LY294002 inhibited the activation of PI3K-Akt signaling pathway and the secretion of CCL5 in SMMC-7721 cells after co-cultured with HS-5 cells. CONCLUSION: HS-5 cells significantly promote the proliferation, migration and invasion abilities of SMMC-7721 cells. Co-culture of SMMC-7721 cells with HS-5 cells activates PI3K-Akt signaling pathway to increase the secretion of CCL5 in SMMC-7721 cells.     

Toll-like receptor 4 promotes migration and invasion abilities of human non-small-cell lung cancer cells

AIM:To evaluate the expression and biological role of Toll-like receptor 4 (TLR4) in human non-small-cell lung cancer (NSCLC) cells. METHODS:The mRNA and protein levels of TLR4 in NSCLC tissue were exa-mined by RT-qPCR, Western blot, and immunohistochemistry. After treating the A549 cells and SPC-A-1 cells with TLR4 stimulator lipopolysaccharide (LPS) and inhibitor TAK-242, RT-qPCR, Western blot and flow cytometry were performed to detect the expression of TLR4. The migration and invasion abilities were detected by Transwell assay, and the mRNA expression of matrix metalloproteinase (MMP)-2, MMP-9, and vascular endothelial growth factor (VEGF) was also detected. RESULTS:The mRNA and protein levels of TLR4 were higher in the NSCLC tissue than those in the noncancerous tissue (P<0.01). LPS stimulation significantly increased the mRNA and protein expression levels of TLR4 in the NSCLC cell lines A549 and SPC-A-1 (P<0.01). The LPS-induced TLR4 activation enhanced the migration and invasion abilities of A549 cells and SPC-A-1 cells (P<0.01). LPS increased the expression levels of MMP-2, MMP-9 and VEGF in the A549 cells and SPC-A-1 cells (P<0.01). Moreover, the expression levels of TLR4, MMP-2, MMP-9 and VEGF, as well as the migration and invasion abilities of the cells were blocked by TAK-242 (P<0.01). CONCLUSION:TLR4 might be involved in the migration and invasion of NSCLC cells, and TLR4 inhibition might be considered as a therapeutic target for treatment of NSCLC.     

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 BMP9 over-expression on migration and invasion of human lung squamous-cell carcinoma NCI-H520 cells

AIM: To investigate the effect of bone morphogenetic proteins 9(BMP9) on the migration and invasion abilities of human lung squamous-cell carcinoma NCI-H520 cells and its mechanism. METHODS: The expression of BMP9 at mRNA and protein levels in the NCI-H520 cells and human bronchial epithelial (HBE) cells was detected by RT-PCR and Western blot. The NCI-H520 cells were transfected with the recombinant adenovirus AdBMP9 and the expression of BMP9 at mRNA and protein levels was validated by RT-PCR and Western blot. The migration and invasion abilities of the NCI-H520 cells were determined by wound-healing and Transwell assays. The mRNA and protein levels of the migration-related factor matrix metalloproteinase 2(MMP2) were detected by RT-PCR and Western blot. The level of phosphorylated Smad1/5(p-Smad1/5) was detected by Western blot. Meanwhile, NCI-H520 cells were treated with BMP specific antagonist AdNoggin and AdBMP9. The level of p-Smad1/5 and the cell migration ability were measured by Western blot, wound-healing and Transwell assays. RESULTS: The expression of BMP9 at mRNA and protein levels was lower in NCI-H520 cells than that in HBE cells. After AdBMP9 was stably transfected into the NCI-H520 cells, the expression of BMP9 at mRNA and protein levels was significantly up-regulated, cell migration and invasion abilities were significantly decreased, and the mRNA and protein levels of MMP2 were decreased. Meanwhile, the level of p-Smad1/5 was increased. Noggin reversed BMP9-caused the increase in p-Smad1/5 and the decrease in cell migration ability. CONCLUSION: Over-expression of BMP9 inhibits the migration and invasion abilities of lung squamous-cell carcinoma NCI-H520 cells. The activation of BMP-Smad signaling pathway may be involved in this inhibitory process.     

Effects of TRPC1 on TGF-β1-induced migration of human bronchial epithelial cells

AIM: To investigate the role of canonical transient receptor potential channel 1 (TRPC1) in the migration of human bronchial epithelial cells (16HBE) induced by transforming growth factor-β1 (TGF-β1). METHODS: Silencing of TRPC1 gene expression was performed by siRNA. The cell activity and apoptosis were measured by CCK-8 assay and flow cytometry, respectively. The migration and invasion abilities of the 16HBE cells were detected by wound- healing assay and Transwell assay. The protein expression of E-cadherin and vimentin was determined by Western blot. RESULTS: TGF-β1 treatment significantly enhanced the cell migration distance compared with control groups (P < 0.01). The results of CCK-8 assay and flow cytometry indicated that there were no significant difference in proliferation and apoptosis among TRPC1 siRNA group, TGF-β1 group and control group (P > 0.05). The results of wound-healing and Transwell assays showed that migration and invasion abilities in TRPC1 siRNA + TGF-β1 group were markedly suppressed compared with TGF-β1 group (P < 0.01). The protein expression of E-cadherin and vimentin induced by TGF-β1 was inhibited by TRPC1 silencing compared with TGF-β1 group (P < 0.05). CONCLUSION: TRPC1 is involved in the migration of human bronchial epithelial cells induced by TGF-β1 through regulating the protein expression of E-cadherin and vimentin.