miR-339 suppresses proliferation of pulmonary arterial smooth muscle cells via regulating IGF2BP1

AIM: The present study aimed to investigate the role of microRNA-339 miR-339 in the proliferation of pulmonary artery smooth muscle cells (PASMCs). METHODS: After treating with angiotensin Ⅱ of different concentrations for 48 h, miR-339 mimic and miR-339 inhibitor were transfected into PASMCs, respectively. CCK-8 assay and viable cell counting were performed to determine cell proliferation. The expression levels of miR-339 and PCNA mRNA were measured by RT-qPCR. The protein levels were detected by Western blot. The interaction between miR-339 and insulin-like growth factor 2 mRNA-binding protein 1(IGF2BP1) was confirmed by luciferase reporter assay. RESULTS: Angiotensin Ⅱ concentration-dependently increased cell proliferation and mRNA expression of PCNA, and decreased miR-339 expression in the PASMCs. Over-expression of miR-339 inhibited cell proliferation and mRNA expression of PCNA in the PASMCs, while mutation of miR-339 promoted cell proliferation and mRNA expression of PCNA in the PASMCs. In addition, miR-339 inhibited cell proliferation in angiotensin Ⅱ-treated PASMCs. Bioinformatics analysis showed that miR-339 targeted the IGF2BP1 3'-UTR, which was confirmed by luciferase reporter assay, and miR-339 negatively regulated the expression of IGF2BP1 in the PASMCs. More importantly, over-expression of IGF2BP1 attenuated the inhibitory effects of miR-339 on cell proliferation and mRNA expression of PCNA in the PASMCs. miR-399 over-expression suppressed phosphorylated p38 protein level but not p38 protein level. CONCLUSION: miR-339 suppresses anti-proliferative effects in PASMCs partly via regulating IGF2BP1 and p38 MAPK signaling pathway.     

miR-24-3p targeting KLF6 gene affects viability and apoptosis of esophageal cancer cells via IL-6/STAT3 signaling pathway

AIM: To investigate the effect of microRNA-24-3p (miR-24-3p) on the viability and apoptosis of esophageal cancer cells. METHODS: The expression of miR-24-3p and KLF6 mRNA in the esophageal cancer cells TE11, Eca109 and EC9706 were detected by RT-qPCR. The protein expression of KLF6 was determined by Western blot. EC9706 cells were transfected with anti-miR-24-3p and KLF6 siRNA. The cell viability was measured by MTT assay, the apoptotic rate was analyzed by flow cytometry, and the proliferation, apoptosis and IL-6/STAT3 signaling pathways related proteins were determined by Western blot. The level of IL-6 was measured by ELISA. The dual luciferase reporter gene assay was used to verify the relationship between miR-24-3p and KLF6. RESULTS: The levels of miR-24-3p were up-regulated in the esophageal cancer cells TE11, Eca109 and EC9706 (P < 0.05), and the expression of KLF6 at mRNA and protein levels was down-regulated (P < 0.05). Knock-down of miR-24-3p expression inhibited the cell viability, induced apoptosis, and inhibited the protein levels of CDK4, cyclin D1, CDC25A, p-STAT3, Bcl-2 and IL-6, and promoted the protein expression of caspase-3 and Bax in EC9706 cells. CONCLUSION: miR-24-3p targets KLF6 gene to affect the viability and apoptosis of esophageal cancer cells by regulating IL-6/STAT3 signaling pathway.     

Effects of miRNA-25 on proliferation of human esophageal squamous-cell carcinoma cell line TE1

AIM: To investigate the effects and mechanisms of microRNA-25(miRNA-25) on the proliferation of human esophageal squamous-cell carcinoma cell line TE1. METHODS: The abundance of miRNA-25 in different tissues was measured by RT-PCR. After silencing or over-expression of miRNA-25 with mimics or inhibitor in TE1 cells, the cell proliferation, cell cycle distribution and the expression of cyclin E1 and cyclin-dependent kinase 2(CDK2) at mRNA and protein levels were measured by CCK-8 assay, BrdU detection, flow cytometry, RT-PCR and Western blot, respectively. RESULTS: miRNA-25 was prominent in esophageal mucosal tissue and highly expressed in TE1 cells (P<0.05). Over-expression of miRNA-25 increased TE1 cell proliferation, promoted the cell cycle progression and enhanced the entrance of the cells into S phase (P<0.05). Inverse results were obtained after down-regulation of miRNA-25(P<0.05). Furthermore, the expression of cyclin E1 and CDK2 at mRNA and protein levels was significantly increased after over-expression of miRNA-25, but decreased after down-regulation of miRNA-25(P<0.05). CONCLUSION: miRNA-25 enhances cell cycle transition by increasing the expression of cyclin E1 and CDK2, thus accelerating TE1 cell proliferation. This study provides a novel mechanism by which miRNA-25 increases the proliferation of human esophageal squamous-cell carcinoma cell line TE1, suggesting that down-regulation of miRNA-25 may be a potential new therapeutic strategy for treating esophageal squamous-cell carcinoma.     

Over-expression of SIRT1 gene inhibits high glucose-stimulated H9c2 cardiomyocyte apoptosis and ROS level through PI3K/AKT signaling pathway

AIM: To investigate the effects of silent information regulator 1 (SIRT1) over-expression on the apoptosis and the level of reactive oxygen species (ROS) in high glucose-induced H9c2 cardiomyocytes. METHODS: H9c2 cardiomyocytes were transfected with empty plasmid (pcDNA3.1-NC) and SIRT1 over-expression plasmid (pcDNA3.1-SIRT1), and then stimulated by high glucose. The H9c2 cells were divided into control group, high glucose group, high glucose + pcDNA3.1-NC group and high glucose + pcDNA3.1-SIRT1 group. The expression of SIRT1 at mRNA and protein levels in each group was determined by qPCR and Western blot. The viability of the cells was measured by MTT assay. The apoptotic rate was analyzed by flow cytometry. The protein levels of phosphatidylinositol 3-kinase (PI3K), phosphorylated PI3K, AKT and phosphorylated AKT were examined by Western blot. RESULTS: SIRT1 was significantly decreased in high glucose-induced H9c2 cardiomyocytes, the cell viability was significantly decreased compared with control group, while the ROS levels and apoptotic rate were increased, and the phosphorylated PI3K and AKT protein levels were down-regulated (P<0.05). Over-expression of SIRT1 significantly promoted the viability of H9c2 cardiomyocytes induced by high glucose, decreased the ROS levels and apoptotic rate, and up-regulated phosphorylated PI3K and AKT protein levels (P<0.05). CONCLUSION: SIRT1 over-expression reverses the decrease in the viability of high glucose-stimulated H9c2 cardiomyocytes, and the increases in apoptotic rate and oxidative stress by regulating PI3K/AKT signaling pathway.     

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.     

Rab11-FIP4 regulates migration and invasion of colorectal cancer cells through IGF1R

AIM To investigate the role, clinical implications and the underlying mechanisms of Rab11-family interacting protein 4 (Rab11-FIP4) in colorectal cancer (CRC). METHODS The expression levels of Rab11-FIP4 in CRC tissues and corresponding paracancerous tissues were compared by immunohistochemistry, Western blot and RT-qPCR. The above methods were also used to detect the expression levels of Rab11-FIP4 in CRC cells under normal environment and hypoxia. The patiens were divided into Rab11-FIP4 high expression group (n=61) and Rab11-FIP4 low expression group (n=39) according to the immunohistochemical staining score.The overall survival and recurrence time of the 2 groups were compared by Kaplan-Meier survival analysis. HCT116 and LoVo cells with stable over-expression of Rab11-FIP4 were constructed using a lentiviral system. The cytological characteristics effects of Rab11-FIP4 over-expression in CRC cells were examined by CCK-8 assay, clonogenic assay and the Transwell assay. The co-immunoprecipitation was used to detect the correlation between Rab11-FIP4 and insulin-like growth factor 1 receptor (IGF1R). Human phosphokinase array was performed to investigate the signaling pathhway affected by IGF1R in CRC cells with increased expression of Rab11-FIP4. The relationship between Rab11-FIP4 and hypoxia-inducible factor 1α (HIF-1α) was analyzed by tissue microarrays and dual-luciferase reporter assay. RESULTS Rab11-FIP4 expression was up-regulated in CRC tissues and high expression of Rab11-FIP4 was associated with poor prognosis of the patients with CRC (P<0.05). Over-expression of Rab11-FIP4 promoted the viability, migration and invasion of CRC cells (P<0.05). High expression of Rab11-FIP4 regulated ERK1/2 and AKT signaling pathway via IGF1R (P<0.05). Hypoxia promoted the activation of HIF-1α on the Rab11-FIP4 promoter, thereby up-regulating the expression of Rab11-FIP4 (P<0.05). CONCLUSION Rab11-FIP4 may act as an oncogene to regulate migration and invasion of colorectal cancer cells through IGF1R.     

Expression of TSTA3 in esophageal squamous-cell carcinoma and its significance

AIM To investigate the expression of TSTA3 in esophageal squamous cell carcinoma (ESCC) and the effects of TSTA3 on the proliferation, invasion and migration abilities of human ESCC cell lines KYSE150 and KYSE450. METHODS The expression of TSTA3 was detected by immunohistochemistry in 45 cases of ESCC and matched adjacent tissues. The mRNA expression levels of TSTA3 in ESCC cells were detected by qPCR. Over-expression of TSTA3 in KYSE150 cells and KYSE450 cells was carried out by lentivirus infection. The effects of TSTA3 on the viability and colony formation ability of ESCC cells were examined by MTT assay and colony formation assay. Transwell assays were performed to detect the effects of TSTA3 on migration and invasion abilities of ESCC cells. The effect of TSTA3 on core fucosylation modification of ESCC cells was analyzed by flow cytometry. RESULTS The expression of TSTA3 in the ESCC tissues was significantly higher than that in matched adjacent tissues (P<0.01). Over-expression of TSTA3 had no effect on the proliferation of ESCC cells (P>0.05), but promoted the migration and invasion of ESCC cells (P<0.01). Moreover, over-expression of TSTA3 increased the core fucosylation modification level of ESCC cells (P<0.01). CONCLUSION TSTA3 may promote the development and progression of ESCC via regualting fucosylation modification level, and serve as a biomarker for early diagnosis and treatment of ESCC.     

Effect of transcription factor BACH2 over-expression on viability and apoptosis of human acute lymphoblastic leukemia T lymphocytes

AIM To investigate the effect of over-expression of BTB and CNC homology 2 （BACH2） on the viability and apoptosis of human acute lymphoblastic leukemia T lymphocytes CCRF-CEM. METHODS CCRF-CEM cells were divided into 3 groups： control group, empty vector group, and BACH2 over-expression group. The BACH2 over-expression vector was transfected into CCRF-CEM cells of BACH2 over-expression group by liposome transfection method. The difference in mRNA expression of BACH2 between CCRF-CEM cells and peripheral blood mononuclear cell （PBMC） was detected by qPCR. CCK8 assay was performed to evaluate the viability of CCRF-CEM cells. Flow cytometry was used to analyzed the apoptosis of CCRF-CEM cells. The protein expression of BACH2 and cyclin D3 in the CCRF-CEM cells was observed by immunofluorescence staining. The protein expression of cyclin D3, Bcl-2 and caspase-3 was determined by Western blot. RESULTS The mRNA expression of BACH2 in CCRF-CEM cells was significantly lower than that in PBMC （P<0.05）. Compared with control group, BACH2 over-expression significantly suppressed the viability,increased the apoptotic rate and caspase-3 expression, and decreased the expression of cyclin D3 and Bcl-2 in CCRF-CEM cells （P<0.05）. CONCLUSION BACH2 expression is decreased in T lymphocytes of human acute lymphoblastic leukemia. Over-expression of BACH2 inhibited the viability of human acute lymphoblastic leukemia T lymphocyte and induced apoptosis.     

FAM3C activates Akt to promote viability of oral squamous-cell carcinoma cells

AIM: To investigate the expression and roles of family with sequence similarity 3, member C (FAM3C) in oral squamous-cell carcinoma cells. METHODS: The mRNA and protein expression levels of FAM3C in dysplastic oral keratinocyte (DOK) and oral squamous-cell carcinoma WSU-HN6 cells were detected by RT-qPCR and Western blot. The WSU-HN6 cells were treated with siFAM3C or FAM3C antibody. After 24, 48 and 72 h, the viability of WSU-HN6 cells was measured by CCK-8 assay, and the activation of protein kinase B (Akt) was detected by Western blot. Adenovirus was used to mediate over-expression of FAM3C in the DOK cells. The DOK cell viability was measured by CCK-8 assay after adenovirus infection for 24, 48 and 72 h, and the activation of Akt was detected by Western blot. RESULTS: Compared with the DOK cells, the mRNA and protein levels of FAM3C were significantly increased in the WSU-HN6 cells (P<0.05). The viability of WSU-HN6 cells transfected with siFAM3C was significantly inhibited at 48 h and 72 h (P<0.05). siFAM3C treatment inhibited the activation of Akt (P<0.05). FAM3C antibody treatment also suppressed the viability of the WSU-HN6 cells at 48 h and 72 h and the activation of Akt (P<0.05). Over-expression of FAM3C in the DOK cells promoted the cell viability at 48 h and 72 h and activated Akt (P<0.05). CONCLUSION: FAM3C might promote oral squamous-cell carcinoma cell growth by activating Akt.     

Effect of up-regulation of Shh-Gli1 signaling pathway on radioresistance and cell cycle in human esophageal cancer cell line Eca109

AIM:To investigate the relationship between Sonic Hedgehog (Shh) signaling pathway and cell cycle and radioresistance of esophageal cancer by up-regulating Gli1, a key factor in Shh signaling pathway. METHODS:The human esophageal cancer cell line Eca109 was transfected with plasmid to induce Gli1 over-expression, which served as Eca109-ox-Gli1 group. In addition, Eca109 cells transfected with empty plasmid served as negative control group and the untreated Eca109 cells were used as normal control group. The expression of Gli1 was confirmed by real-time PCR and Western blot. The radiosensitivity of the cells in the 3 groups was determined by colony formation assay. The effect of irradiation on the cell cycle was analyzed by flow cytometry. RESULTS:The expression of Gli1 in Eca109-ox-Gli1 group was higher than that in the other 2 groups (P<0.05). The survival fraction at dose of 2 Gy in Eca109-ox-Gli1 group was higher than that in normal control group, indicating that the radioresistance of the Eca109 cells transfected with Gli1 plasmid was increased. The cells in Eca109-ox-Gli1 group showed higher S phase proportion than that in normal control group and negative control group (P<0.01). After irradiation at dose of 6 Gy, all cells in the 3 groups found that the cell cycle was arrested at G₂/M phase, while the cells in normal control group showed higher G₂/M phase proportion than that in Eca109-ox-Gli1 group (P<0.01). CONCLUSION:The up-regulation of Gli1 may enhance the radioresistance of esophageal cancer by regulating the cell cycle.     

Antitumor effect of allicin on esophageal cancer EC109 cells compared with chemotherapeutics in vitro

AIM: To investigate the inhibitory effect of allicin on human esophageal cancer cell line EC109 and its possible mechanism by comparison with chemotherapeutic drugs. METHODS: The EC109 cells were treated with different concentrations of allicin, 5-fluorouracil and cisplatin. The cell viability was detected by CCK8 assay and the activity of lactic dehydrogenase (LDH) was analyzed at 6 h, 12 h, 24 h, 48 h and 72 h. The apoptosis of the EC109 cells induced by Z-VAD-FMK, allicin, allicin+Z-VAD-FMK, 5-fluorouracil and cisplatin was analyzed by flow cytometry with Annexin V-FITC and PI double staining. The enzyme activity changes of caspase-3, caspase-8 and caspase-9 were detected by spectrophotometry. RESULTS: Allicin had inhibitory effect on the growth of the EC109 cells and killed them in concentration-dependent and time-dependent manners. LDH activity was decreased compared with 5-fluorouracil and cisplatin. The increased activity of caspase-3 and caspase-8 in allicin-treated cells was statistically significant, but caspase-9 activity changed without statistical significance. CONCLUSION: Allicin inhibits the growth of EC109 cells in concentration-and time-dependent manners through extrinsic apoptosis pathways activated by caspase-8, and the side effects are weaker compared with 5-fluorouracil and cisplatin.     

Regulation of DNA polymerase β expression in esophageal carcinoma cell line EC9706

AIM: To investigate the influence of DNA polymerase β(DNA polβ) on the biological characteristics of esophageal carcinoma cells.METHODS: siRNA expression plasmid targeting DNA polβ was constructed and transfected into EC9706 cells. The positive colonies were screened by G418 selection. The proliferation of EC9706 cells was detected by flow cytometry and nude mice xenograft model. The expression of DNA polβ was detected by real-time PCR. RESULTS: Compared to empty vector group and control group, the expression of DNA polβ was significantly reduced in the cells transfected with siRNA expression plasmid pSINsi-hU6-siRNA polβ288(experimental group 1) and the cells transfected with siRNA expression plasmid pSINsi-hU6-siRNA polβ814(experimental group 2). The expression of DNA polβ was significantly increased in DNA polβ over-expression group(P＜0.05). The proportion of the cells in S phase increased significantly in experimental group 1, experimental group 2 and DNA polβ over-expression group(P＜0.05). The weight of tumors increased significantly(P＜0.05).CONCLUSION: The over-expression and extremely low expression of DNA polβ may increase genetic instability of the cells and play a role in the occurrence of esophageal carcinoma.     

APPL1 reduces injury of H9c2 cardiomyocytes induced by LPS

AIM:To study the effect of APPL1 (adaptor protein, phosphotyrosine interacting with PH domain and leucine zipper 1) on H9c2 cardiomyocyte injury induced by lipopolysaccharides (LPS). METHODS:The H9c2 cells were treated with LPS. RT-qPCR and Western blot were used to detect the expression of APPL1 in the H9c2 cells. The recombinant APPL1 lentiviral vector was used to transfect into the H9c2 cells. After LPS treatment, the over-expression efficiency was detected by RT-qPCR and Western blot. The viability was measured by MTT assay. The apoptosis was analyzed by flow cytometry. The protein level of activated caspase-3 in the H9c2 cells was determined by Western blot. The content of malonaldehyde (MDA) in the H9c2 cells and the level of lactate dehydrogenase (LDH) in the culture medium were detected. The activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px), and the level of reative oxygen species (ROS) in the H9c2 cells were also examined. RESULTS:The expression of APPL1 at mRNA and protein levels in LPS-treated H9c2 cells was decreased significantly (P<0.05). Over-expression of APPL1 by transfection of recombinant lentiviral vector significantly increased the level of APPL1 at mRNA and protein levels in the H9c2 cells with LPS treatment (P<0.05). LPS treatment reduced the viability, but increased the apoptotic rate of the H9c2 cells, the protein level of activated caspase-3, the content of MDA and the level of LDH in the culture medium. The activity of SOD and GSH-Px was reduced, while the level of ROS was increased as compared with control group (P<0.05). Over-expression of APPL1 elevated the viability of H9c2 cells treated with LPS, and the apoptotic rate and the protein level of activated caspase-3 were decreased. The content of MDA and the level of LDH in the culture medium were reduced, the activity of SOD and GSH-Px was elevated, and the level of ROS was reduced as compared with the H9c2 cells treated with LPS alone (P<0.05). CONCLUSION:Over-expression of APPL1 reduces oxidative damage and apoptosis of the H9c2 cells induced by LPS.     

Effects of Homer1a over-expression on apoptosis and AMPK protein expression in mechanically injured neurons

AIM:To investigate the effects of Homer1a over-expression on the apoptosis and AMP-activated protein kinase (AMPK) protein expression in mechanically injured neurons. METHODS:The rat cortical neurons were isolated and cultured in vitro, and then ramdomly divided into control group, model group, empty vector group, and Exp-Homer1a group. Neuron models with mechanical injury were constructed and infected with the Homer1a over-expression vector. The mRNA expression of Homer1a was detected by qPCR. The cell viability in each group was detected by MTT assay. The activity of lactate dehydrogenase (LDH) in the supernatant of each group was measured by LDH test kit. The apoptosis level was analyzed by flow cytometry. The protein levels of Hormer1a, cleaved caspase-3, Bax, Bcl-2, p-AMPKα and AMPKα were determined by Western blot. RESULTS:Compared with control group, the viability of mechanically injured neurons was significantly decreased, the LDH activity in the supernatant and neuronal apoptotic rate were significantly increased (P<0.05), and Homer1a expression at mRNA and protein levels was significantly increased (P<0.05). Compared with model group, the LDH activity in the supernatant and neuronal apoptotic rate in Exp-Homer1a group were significantly decreased, the protein levels of cleaved caspase-3 and Bax were significantly decreased (P<0.05), and the protein levels of Bcl-2 and p-AMPKα were significantly increased (P<0.05). CONCLUSION:Over-expression of Homer1a may increase the viability of mechanically injured neurons and inhibit their apoptosis by promoting the activation of AMPKα phosphorylation.     

Over-expression of Hsp75 in neural stem cells reduced Aβ-mediated neurotoxicity

AIM: To investigate the effect of heat shock protein 75 (Hsp75) over-expression on Aβ-induced neurotoxicity in the neural stem cells and to explore its mechanism. METHODS: An adenovirus-mediated Hsp75 over-expression vector was used in vitro. The mouse neural stem cell C17.2 was cultured in vitro and divided into control group, Aβ group, negative adenovirus vector transfection group and Hsp75 over-expression adenovirus vector transfection group. The transfection and cellular immune identification were detected by fluorescence microscopy. The cell morphology was observed under inverted phase-contrast microscope. The cell viability and apoptosis were detected by MTT assay and flow cytometry, respectively. Hsp75 over-expression and cleaved caspase-3 protein level were measured by Western blot. RESULTS: Observation by fluorescence microscopy indicated that C17.2 cells were successfully transfected and Hsp75 gene was effectively expressed in the neural stem cells after transfection. In addition, the morphology and viability of the cells did not change and these cells did not differentiate after transfection. As compared with control group, the cell viability in Aβ group and negative adenovirus vector transfection group was significantly decreased (P < 0.05), and the cell apoptotic rate and cleaved caspase-3 level (P < 0.05) were increased. As compared with Aβ group and negative adenovirus vector transfection group, Hsp75 over-expression significantly increased the cell viability, and decreased the cell apoptosis and cleaved caspase-3 level (P < 0.05). CONCLUSION: Hsp75 over-expression protects the neural stem cells against Aβ-induced injury. The mechanism may be related to inhibiting caspase-3 pathway-dependent apoptosis.     

Regulatory effects of DIS3 expression on colony formation ability in human myeloma cells and tube structure formation of endothelial cells

AIM:To investigate the effects of DIS3 expression on the colony formation ability of 3 kinds of human myeloma cells and tube structure formation of the endothelial cells. METHODS:Human myeloma cell lines NCI-H929, RPMI-8226 and U266 were selected as the study objects, and DIS3 gene over-expression vector and DIS3-siRNA were designed and constructed respectively. The cell experiments were divided into 5 groups:control group, siRNA negative control (siRNA-NC) group, siRNA-DIS3 group, empty vector group and DIS3 over-expression group. The colony formation ability was tested by the plate colony formation assay. Western blot was used to detect the protein expression of hypoxia inducible factor-1α (HIF-1α) and HIF-3α. The expression of angiogenesis-related molecules angiogenin 1 (Ang1), Ang2 and vascubar enelothelial growth factor-A(VEGF-A) at the mRNA and protein levels was determined by RT-qPCR and Western blot, respectively. Matrigel method was used to detect the effect of supernatant from each group of the cells on the tube structure formation of HUVECs. RESULTS:The trends of the following indexes in NCI-H929 cells, RPMI-8226 cells and U266 cells were similar. Compared with empty vector group, the colony formation ability of the cells in DIS3 over-expression group was significantly inhibited (P<0.05). Compared with siRNA-NC group, siRNA-DIS3 significantly enhanced the colony formation ability of the cells (P<0.05). DIS3 over-expression significantly reduced the expression of HIF-1α and HIF-3α (P<0.05), while knock-down of DIS3 expression significantly increased the protein levels of HIF-1α and HIF-3α (P<0.05). In addition, DIS3 over-expression significantly reduced the expression of Ang1, Ang2 and VEGF-A at mRNA and protein levels (P<0.05), while siRNA-DIS3 significantly promoted the expression of Ang1, Ang2 and VEGF-A (P<0.05). Compared with empty vector group, the supernatant from DIS3 over-expression group significantly inhibited the tube structure formation of HUVECs (P<0.01). Compared with siRNA-NC group, the supernatant from siRNA-DIS3 group significantly promoted the tube structure formation of HUVECs (P<0.05). CONCLUSION:DIS3 over-expression significantly inhibits the colony formation ability of human myeloma cells and tube structure formation of HUVECs, which may be closely related to the regulation of the expression of HIF-1α and HIF-3α.     

Effect of DEC1 gene over-expression on proliferation and invasion abilities of human esophageal cancer ECA109 cells

AIM: To investigate the effect of DEC1 gene over-expression on the proliferation and invasion abilities of human esophageal cancer ECA109 cells.METHODS: ECA109 cells were transfected with plasmid pcDNA3.1 (-)/DEC1 (DEC1 group) or pcDNA3.1 (-) (vector group). The mRNA and protein levels of DEC1, cyclin D1 and MMP-9 were evaluated by real-time PCR and Western blot, respectively. The effects of DEC1 over-expression on the proliferation and invasion abilities of the ECA109 cells were evaluated by CCK-8 assay, colony formation assay and Transwell test respectively.RESULTS: The DEC1 expression level in ECA109 cells in DEC1 group was significantly higher than that in vector group (P<0.01), but the levels of MMP9 and cyclin D1 expression were opposite (P<0.01). However, both the proliferation and invasion abilities of ECA109 cells in DEC1 groups decreased significantly as compared with those in vector group (P<0.05).CONCLUSION: The over-expression of DEC1 significantly inhibits the proliferation and invasion of ECA109 cells, which may be involved in the expression of cyclin D1 and MMP9.     

Effect of ARMCX1 on proliferation and apoptosis of cervical cancer SiHa cells

AIM: To observe the effect of Armadillo repeat-containing X-linked protein 1 (ARMCX1) on the proliferation and apoptosis of human cervical cancer SiHa cells by knock-down of ARMCX1 expression with small interfering RNA. METHODS: After knock-down of ARMCX1 expression, the cell cycle distribution and apoptosis of SiHa cells were detected by flow cytometry. The proliferation of SiHa cells was observed by plate colony formation assay after knock-down of ARMCX1 for 10 d. The protein levels of cell proliferation-and apoptosis-related molecules were determined by Western blot. RESULTS: After knock-down of ARMCX1 expression in the SiHa cells, the cell colony formation ability was significantly inhibited (P < 0.05), the cell cycle was arrested in S phase, and the protein levels of cyclin E and cell division cycle 25A (Cdc25A) in the SiHa cells were decreased. Meanwhile, knock-down of ARMCX1 expression promoted the apoptosis of SiHa cells, significantly reduced the protein expression of Bcl-2, and significantly increased the protein levels of Bax and active caspase-3 (P < 0.05). CONCLUSION: Knock-down of ARMCX1 expression inhibits the proliferation of SiHa cells and induces apoptosis.     

Growth inhibition of colorectal cancer HCT116 cells by lincRNA-p21 through STAT3 signaling pathway

AIM: To study the effect of long intergenic non-coding RNA-p21 (lincRNA-p21) on the growth inhibition of colorectal cancer HCT116 cells via STAT3 signaling pathway. METHODS: The human colorectal cancer cell line HCT116 was used to construct the cells with over-expression of lincRNA-p21 by transfection of pcDNA-lincRNA-p21, and negative control cells were also set up. After transfection, the expression level of lincRNA-p21 was detected by RT-qPCR. The cell viability and proliferation were examined by MTT assay and plate colony formation assay, respectively. The protein levels of STAT3 and phosphorylated STAT3 (p-STAT3) were determined by Western blot. After STAT3 signaling pathway activator SD19 was used to treat the colorectal cancer HCT116 cells with over-expression of lincRNA-p21, Western blot was used to detect the protein levels of STAT3 and p-STAT3, MTT assay was used to measure the viability of the cells, and flow cytometry analysis was used to determine the cell apoptosis. RESULTS: Compared with control group and pcDNA group, the expression of lincRNA-p21 in pcDNA-lincRNA-p21 group was significantly up-regulated, the cell proliferation was inhibited, and the protein levels of STAT3 and p-STAT3 were significantly decreased (P<0.05). After treatment with STAT3 activator SD19, the protein levels of STAT3 and p-STAT3 in pcDNA-lincRNA-p21+SD19 group were higher than those in pcDNA-lincRNA-p21 group, the cell viability was increased, and the apoptotic rate was decreased significantly (P<0.05). CONCLUSION: Over-expression of lincRNA-p21 inhibits the growth of colorectal cancer HCT116 cells. STAT3 signaling pathway activator abolishes the growth inhibitory effect of lincRNA-p21 over-expression. lincRNA-p21 inhibits the growth of colorectal cancer cells by inhibiting the activation of STAT3 signaling.