miR-105 inhibits cell proliferation,migration and invasion abilities in non-small-cell lung cancer cells by targeting and negative regulation of KIFC1

AIM:To study the effects of microRNA-105(miR-105) on the cell proliferation, migration and invasion abilities of non-small-cell lung cancer (NSCLC) H460 cells, and further to explore its mechanism. METHODS:The expression of miR-105 and kinesin family member C1 (KIFC1) mRNA in the NSCLC tissues and adjacent tissues and cells was detected by RT-qPCR. The protein expression of KIFC1 in the NSCLC tissues, adjacent normal tissues and cells was determined by Western blot. The H460 cells were divided into miR-105 group (transfection with miR-105 mimics), miR-negative control (NC) group (transfection with miR-NC), inhibitor-NC group (transfection with NC of inhibitor), inhibitor-miR-105 group (transfection with miR-105 inhibitor), si-NC group (transfection with NC siRNA), si-KIFC1 group (transfection with KIFC1 siRNA), miR-105+vector group (miR-105 mimics and pcDNA 3.1 co-transfection) and miR-105+KIFC1 group (miR-105 mimics and pcDNA 3.1-KIFC1 co-transfection). The cell proliferation was measured by MTT assay and colony formation assay. The migration and invasion abilities were detected by Transwell methods. The relative luciferase acitivity was evaluated by double luciferase reporter assay. RESULTS:Compared with the adjacent tissues, the expression of miR-105 was significantly decreased and the expression of KIFC1 was significantly increased in NSCLC tissues (P<0.05). Compared with human normal embryonic lung fibroblasts MRC-5, the expression of miR-105 in the H460 cells was significantly decreased, and the expression of KIFC1 was significantly increased (P<0.05). miR-105 inhibited the relative luciferase activity of H460 cells with wild-type KIFC1 and negatively regulated the protein expression of KIFC1. Over-expression of miR-105 and knockdown of KIFC1 expression significantly inhibited the proliferation, migration and invasion abilities of H460 cells. Over-expression of KIFC1 reversed the inhibitory effect of miR-105 on the cell proliferation, migration and invasion abilities of H460 cells. CONCLUSION:miR-105 inhibits the proliferation, migration and invasion abilities of NSCLC cells. The mechanism may be related to targeting and negatively regulating expression of KIFC1.     

Expression of Wip1 in non small cell lung cancer tissue and cells

AIM: To detect the expression level of wip1 in lung cancer tissue and three lung cancer cell lines, and to explore the relations between the expression level of Wip1 in lung cancer and various clinical and pathological features. METHODS: Real-time PCR was employed to detect the expression of Wip1 mRNA in 44 specimens of non small cell lung cancer tissues and normal tissues. The relations between the expression of Wip1 mRNA and various clinical and pathological features were analyzed. Real-time PCR was also employed to detect the expression of Wip1 mRNA in A549, NCI-1299, NCI-H460 and HBE for relative quantitative analysis.RESULTS: In the 44 specimens, the expressions of Wip1 mRNA in both cancer tissues and normal lung tissues were positive. Wip1 gene was over-expressed in 17 specimens in 44 non small cell lung cancer specimens. The rate was 38.6%. The relative level of Wip1 mRNA in NSCLC tissues was significantly higher than that in normal lung tissues (ratio=2.1644±1.3940, P<0.01). The expression of Wip1 mRNA was also correlated with pathological grading (P<0.05). The relative level of Wip1 mRNA in three kinds of lung cancer cells was significantly higher than that in HBE cells. The difference was statistically significant (P<0.05).CONCLUSION: The Wip1 mRNA is over-expressed in non small cell lung cancer, indicating that Wip1 is related to the tumorigenesis and may become the new target of non small cell lung cancer gene therapy. The expression of Wip1 mRNA is related to tumor cell differentiation and may use for the molecular biological reference index to estimate the malignant degree of cancer.     

Effect of PAK4-LIMK1-cofilin signaling on non-small-cell lung cancer migration and invasion

AIM: To explore the mechanism of p21-activated kinase 4 (PAK4) on non-small-cell lung cancer (NSCLC) migration and invasion.METHODS: After A549 and NCI-H520 cell lines were transfected with PAK4-siRNA or negative control, the expression of PAK4 at mRNA and protein levels was detected by real-time PCR and Western blot, respectively. The invasion and migration of A549 cells and NCI-H520 cells were measured by Matrigel invasion assay and Transwell migration assay. LIMK1, cofilin, and their respective phosphorylation were examined by Western blot. The interaction of PAK4 and LIMK1 was investigated by co-immunoprecipitation assay. The relationship between PAK4 and LIMK1 phosphorylation was examined by a protein kinase assay in the A549 cells and NCI-H520 cells. The expression of PAK4 and p-LIMK1 in 10 human NSCLC tissues was examined by Western blot. A549 cells and NCI-H520 cells were individually or commonly transfected with PAK4-siRNA or LIMK1 plasmid in order to observe the cell migration and invasion. RESULTS: After A549 cells and NCI-H520 cells were transfected with PAK4-siRNA for 48 h, the expression of PAK4 at mRNA and protein levels, and the numbers of invasion and migration cells in PAK4-siRNA group were lower than those in control group. Compared with control group, the phosphorylation of LIMK1 and cofilin was lower in PAK4-siRNA group, whereas the total expression levels of LIMK1 and cofilin did not change. The results of co-immunoprecipitation assays showed that PAK4 specifically interacted with LIMK1 in A549 and NCI-H520 cells. LIMK1 phosphorylation in the presence of PAK4 (K350M) was significantly lower than that in the presence of PAK4 (WT) or PAK4 (S445N) in the protein kinase assay. The PAK4 upregulation was positively correlated with the level of p-LIMK1 (P<0.05). After A549 cells and NCI-H520 cells were co-transfected with PAK4-siRNA and LIMK1 plasmid, the migration and invasion cell numbers in co-transfection group were higher than those in PAK4-siRNA transfection group. CONCLUSION: PAK4 promotes the invasive and migratory abilities of NSCLC, which is mediated by LIMK1 phosphorylation.     

Expression and clinical significance of PAK4 in non-small cell lung cancer

AIM: To investigate the expression and clinical significance of PAK4 in the cell lines and tissues of non-small cell lung cancer (NSCLC). METHODS: PAK4 expression in human bronchial epithelial (HBE) cells, NSCLC cell lines, NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry, real-time PCR and Western blot. Prognostic value of PAK4 expression was evaluated by Kaplan-Meier analysis and Cox regression. RESULTS: PAK4 was over-expressed in the NSCLC cell lines at both mRNA and protein levels compared with HBE cells (P<0.05). PAK4 was over-expressed in the NSCLC tissues at both mRNA and protein levels compared with adjacent non-tumor tissues (P<0.05). PAK4 was over-expressed in the metastatic NSCLC tissues compared with the primary NSCLC tissues (P<0.05). Higher PAK4 staining scores were positively correlated with differentiation, lymph node metastasis, distant metastasis, and clinical stage. Kaplan-Meier analysis and log-rank test showed that overall survival was significantly different between the patients with up-regulated PAK4 and the patients with down-regulated PAK4(P<0.05). PAK4 over-expression was associated with NSCLC progression.CONCLUSION: Increased PAK4 expression was associated with tumor invasion, metastasis and prognosis in the patients with NSCLC. PAK4 is an important prognostic marker and potential therapeutic target in NSCLC.     

lncRNA TTN-AS1 regulates the viability and invasion of lung adenocarcinoma A549 cell via miR-519d-3p/MMP2 pathway

AIM To investigate the expression level of long noncoding RNA （lncRNA） TTN antisense RNA 1 （TTN-AS1） in lung adenocarcinoma tissues and the effects of TTN-AS1 silencing on the viability and invasion of lung adenocarcinoma A549 cells. METHODS RT-qPCR was used to detect the expression of TTN-AS1, microRNA-519d-3p （miR-519d-3p） and matrix metalloproteinase 2 （MMP2） mRNA in 32 cases of lung adenocarcinoma and adjacent normal tissues. The untransfected A549 cells were divided into blank group, si-NC group （with si-NC transfection） and si-lncRNA group （with silencing of lncRNA TTN-AS1 expression）, with n=5 in each group. The effects of TTN-AS1 silencing on the viability and invasion of A549 cells were detected by CCK8 and Transwell methods. The targeting regulatory effects of TTN-AS1 on miR-519d-3p and miR-519d-3p on MMP2 were determined by dual-luciferase reporter assay, RNA immunoprecipitation test, RT-qPCR and Western blot. RESULTS The expression level of TTN-AS1 in 32 cases of lung adenocarcinoma tissues is notably higher than that in the adjacent normal tissues （P<0.05）. Silencing of TTN-AS1 in A549 cells significantly suppressed the cell viability and invasion. TTN-AS1 negatively regulated the expression of miR-519d-3p via sponging and absorbing miR-519d-3p. MMP2 is the target gene of miR-519d-3p and can be negatively regulated by miR-519d-3p. Overexpression of MMP2 partially reversed the inhibitory effect of TTN-AS1 silencing and miR-519d-3p overexpression on the invasion of A549 cells. CONCLUSION The lncRNA TTN-AS1 is overexpressed in lung adenocarcinoma tissues, and it regulates lung adenocarcinoma A549 cell viability and invasion via miR-519d-3p/MMP2 pathway.     

Relationship between FRAS1 protein and brain metastases of NSCLC

AIM: To explore the relationship between FRAS1 protein and brain metastases of non-small cell lung cancer (NSCLC).METHODS: The mRNA expression of FRAS1 in the brain metastatic tumor tissues and primary tumor tissues of NSCLC was detected by qPCR. The protein expression of FRAS1 in the tumor tissues and normal tissues adjacent to tumor tissues of NSCLC was measured by SP method of immunohistochemistry. The protein expression of FRAS1 in NSCLC primary tumor tissues with or without brain metastases was also determined.RESULTS: The mRNA expression of FRAS1 in the brain metastatic zone was nearly 10 times higher than that in the primary tumor tissues, and there was significant difference between the 2 groups (P<0.05). FRAS1 protein was expressed in the NSCLC primary tumor tissues, but was not found in the normal tissues adjacent to primary tumor tissues. The protein expression of FRAS1 in the NSCLC with brain metastases was significantly higher than that without brain metastases (P<0.01).CONCLUSION: FRAS1 protein may be associated with the occurrence of NSCLC. The over-expression of FRAS1 protein may be related to brain metastases with NSCLC.     

DHA induced cytotoxicity in NSCLC cells via inhibiting PI3K pathway activation and GLUT2 expression

AIM: To investigate the molecular mechanisms of cytotoxicity induced by dihydroartemisinin (DHA) in non-small cell lung cancer (NSCLC) cells. METHODS: NSCLC cell lines A549 and NCI-H1650 were treated with various concentrations of DHA for indicated time. Subsequently, the effects of DHA on the cell activity, colony formation ability and apoptosis were determined by MTT assay, colony formation assay, Annexin V-FITC/PI staining and flow cytometry, respectively. At the same time, the effects of DHA on glucose, ATP and lactate levels were assessed, and the PI3K pathway activation and glucose transporter 2(GLUT2) expression were detected by Western blot in the A549 cells and NCI-H1650 cells. Overexpression of GLUT2 and Rheb was established in A549 and NCI-H1650 cells by transfection with GST-GLUT2 and GST-Rheb plasmids, respectively, and the effects of DHA on cell activity, apoptosis, glucose level, ATP content and PI3K pathway activation were analyzed in A549 cells and NCI-H1650 cells. The effect of glucose deprivation on the cytotoxicity triggered by DHA in NSCLC cells was also determined. RESULTS: Compared with control group, DHA significantly inhibited cell activity and colony formation ability, and induced remarkable cell apoptosis in the A549 cells and NCI-H1650 cells. At the same time, DHA reduced ATP and lactate contents, and hindered glucose uptake in a time-and dose-dependent manner in A549 cells and NCI-H1650 cells. The activity of PI3K pathway and GLUT2 expression were downregulated, while upregulated GLUT2 expression and activated PI3K pathway reduced the cytotoxicity induced by DHA in NSCLC cells. Glucose deprivation increased DHA-mediated cytotoxicity in NSCLC cells. On the contrary, high levels of glucose inhibited DHA-mediated cytotoxicity in NSCLC cells. CONCLUSION: DHA restrains cell activity and colony formation, and induces apoptosis. DHA induces cytotoxicity via inhibiting PI3K pathway activation and GLUT2 expression, leading to inhibit glycolytic metabolism in NSCLC cells.     

Effect of inhibiting lncRNA LINC01503 on viability,migration and invasion of lung cancer cells by targeted regulation of miR-335-5p

AIM To investigate the effects of long non-coding RNA (lncRNA) LINC01503 on the viability, migration and invasion of lung cancer cells and its mechanism. METHODS Human lung carcinoma H1299 cells were divided into si-NC group (transfected with si-NC), si-LINC01503 group (transfected with si-LINC01503), pcDNA group (transfected with pcDNA), pcDNA-LINC01503 group (transfected with pcDNA-LINC01503), miR-NC group (transfected with miR-NC), miR-335-5p group (transfected with miR-335-5p mimics), si-LINC01503+anti-miR-NC group (co-transfected with si-LINC01503 and anti-miR-NC), si-LINC01503+anti-miR-335-5p group (co-transfected with si-LINC01503 and anti-miR-335-5p), miR-NC+WT-LINC01503 group (co-transfected with miR-NC and WT-LINC01503), miR-NC+MUT-LINC01503 group (co-transfected with miR-NC and MUT-LINC01503), miR-335-5p+WT-LINC01503 group (co-transfected with miR-335-5p and WT-LINC01503) and miR-335-5p+MUT-LINC01503 group (co-transfected with miR-335-5p and MUT-LINC01503). The expression of miR-335-5p and LINC01503 was detected by RT-qPCR. Western blot was used to detect protein expression. MTT assay was used to detect cell viability. Transwell assay was used to detect the migration and invasion abilities. Dual-luciferase reporter assay was used to confirm the targeted relationship between LINC01503 and miR-335-5p. RESULTS Compared with normal tissues, the expression of LINC01503 was significantly increased in the lung cancer tissues, and the expression of miR-335-5p was significantly decreased (P<0.05). Compared with stage I/II , the expression level of LINC01503 in the lung cancer tissues of stage III/IV was significantly increased, and the expression of miR-335-5p was significantly decreased (P<0.05). The patients with high expression of LINC01503 had lower short-term survival rates than those with low expression of LINC01503 (P<0.05). Compared with normal human bronchial epithelial cell line BEAS-2B, the expression of miR-335-5p in lung cancer cell lines H1299, A549 and SPC-A-1 were significantly decreased, and the expression of LINC01503 was significantly increased (P<0.05). Over-expression of miR-335-5p and inhibition of LINC01503 expression inhibited the viability, migration and invasion of H1299 cells, and inhibited the protein expression of cyclin D1, matrix metalloproteinase-2 （MMP-2） and MMP-9 (P<0.05). LINC01503 targeted and regulated miR-335-5p expression, and interfering with miR-335-5p expression reversed the inhibitory effect of inhibiting LINC01503 expression on the viability, migration and invasion of H1299 cells. CONCLUSION Inhibition of lncRNA LINC01503 inhibits the viability, migration and invasion of lung cancer cells. The mechanism may be related to the targeted regulation of miR-335-5p.     

Expression and function of circular RNA_0000231 in non-small-cell lung cancer

AIM: To investigate the expression and function of circular RNA_0000231 (circ_0000231) in non-small-cell lung cancer (NSCLC). METHODS: RT-qPCR was used to detect the expression of circ_0000231 in the NSCLC tissues and cell lines. circ_0000231 small interfering RNA (si-circ_0000231) or negative control siRNA of circ_0000231 (NC) was transfected into the NSCLC cells. The proliferation and apoptosis of the NSCLC cells were detected by CCK-8 assay, colony formation assay and flow cytometry, respectively. The expression of cyclin D1 (CCND1) and anti-apoptotic protein Bcl-2 were determined by RT-qPCR and Western blot. RESULTS: The expression of circ_0000231 in the NSCLC tissues and cell lines was significantly up-regulated compared with precancerous tissues and lung epithelial cells BEAS-2B (P<0.05). After transfection of NSCLC cells with si-circ_0000231, the cell viability, colony formation numbers were significantly decreased, and the apoptotic rate in si-circ_0000231 group was significantly increased as compared with NC group (P<0.01). In addition, the results of RT-qPCR and Western blot showed that transfection of si-circ_0000231 inhibited the expression of CCND1 and Bcl-2 (P<0.01). CONCLUSION: The expression of circ_0000231 is significantly increased in the NSCLC tissues and cells. Knock-down of circ_0000231 expression significantly inhibits the proliferation of NSCLC cells.     

Silencing IDH-2 gene by siRNA-IDH-2 inhibits human small cell lung carcinoma growth

AIM：To investigate the effect of silencing isocitrate dehydrogenase 2 (IDH-2) gene by small interfering RNA (siRNA) on the biological characteristics of human small cell lung cancer cell line NCI-H446. METHODS：IDH-2 expression was knocked down in human small cell lung cancer cell line NCI-H446 by siRNA-IDH-2. The expression level of IDH-2 was determined by real-time PCR and Western blotting. The cell proliferation was measured by CCK-8 assay, the protein expression of MAPK p42 was detected by Western blotting, and the cell cycle was analyzed by flow cytometry. The migration was observed using Transwell cell migration system. BALB/c nude mice were subcutaneously injected on the back with NCI-H446 cells transfected with siRNA-IDH-2/negative control siRNA or non-transfected cells to study the tumor growth. RESULTS：siRNA-IDH-2 remarkably down-regulated the expression of IDH-2 and MAPK p42 in the NCI-H446 cells. siRNA-IDH-2 inhibited both the proliferation and migration abilities of NCI-H446 cells, and the cell cycle was arrested in S phase as compared with negative control group. Additionally, the volume of xenograft tumors in siRNA-IDH-2 group was significantly decreased as compared with control group. CONCLUSION：siRNA-IDH-2 down-regulates the expression of IDH-2 in NCI-H446 cells, reduces the cell migration efficiency and inhibits the tumor growth in vitro and in vivo.     

Effects of AGR2 on proliferation and apoptosis of NCI-H460 cells induced by thapsigargin

AIM:To observe the effects of anterior gradient 2 (AGR2) gene on the proliferation and apoptosis of NCI-H460 cells induced by thapsigargin, an endoplasmic reticulum (ER) stress inducer.METHODS:A stable cell line with knockdown of endogenously expressed AGR2 mediated by lentiviral shRNA was established. The proliferation abi-lity of stable NCI-H460 cells in the presence of thapsigargin was measured by colony formation assay, while the effect of AGR2 gene silencing on apoptosis in the presence of thapsigargin was analyzed by flow cytometry. RESULTS:Lentivirus-mediated AGR2 knockdown stable NCI-H460 cells was successfully constructed and the expression of AGR2 was markedly diminished as revealed by Western blot (P<0.01). Knockdown of AGR2 didn't significantly affect the colony formation rate and apoptosis of NCI-H460 cells without thapsigargin treatment. Colony formation rate of the cells with AGR2 gene knockdown was far lower than that of the control cells after 24 h of thapsigargin treatment (P<0.05). The results of flow cytometry showed that knockdown of AGR2 significantly increased the apoptosis of NCI-H460 cells induced by thapsigargin. CONCLUSION:Knockdown of AGR2 doesn't remarkably affect the proliferation and apoptosis of NCI-H460 cells in the absence of thapsigargin, but significantly decreases cell viability and increases apoptosis after thapsigargin treatment. This study reveals that AGR2 might promote the proliferation and reduce the apoptosis of lung cancer cells under ER stress, and lays the foundation for further study of the AGR2 role in initiation, development and drug resistance of lung cancer.     

Effect of oridonin on invasion and migration of human lung cancer NCI-H460 cells

AIM：To investigate the effect of oridonin on the invasion and migration of human lung cancer NCI-H460 cells. METHODS：NCI-H460 cells were divided into high-dose (HD), middle-dose (MD) and low-dose (LD) oridonin groups (cultured with 40, 20 and 10 μmol/L of oridonin, respectively, as experimental groups), and normal (N) group (treated without oridonin as control). The cell growth was observed. The cell proliferation was detected by MTT assay. Boyden chamber was used to determine the cell invasive capacity. The cell migration was also measured. The levels of MMP-2 and MMP-9 were assayed by Western blotting. RESULTS：The cell counts in the experimental groups were lower than that in N group. The cell proliferation was inhibited as the inhibitory rates were 48.94%, 36.17% and 19.15% for HD group, MD group and LD group, respectively. The numbers of the invasive cells were 26.67±5.16 for HD group, 36.17±5.08 for MD group, and 44.33±5.50 for LD group. The migration rates in the experimental groups were lower than that in N group. The expression of MMP-2 and MMP-9 decreased dependent on the oridonin dose as follows: HD group < MD group < LD group < N group. CONCLUSION：Oridonin inhibits the invasion and migration of NCI-H460 lung cancer cells, and reduces the expression of MMP-2 and MMP-9.     

NET-1 promotes metastasis of lung squamous-cell carcinoma by activating EMT

AIM: To explore the effect of neuroepithelial cell transforming gene-1 (NET-1) expression on the metastasis of lung squamous-cell carcinoma (LSC) and the underlying molecular mechanism. METHODS: Immunohistochemistry was used to detect the expression of NET-1 protein in 53 cases of lung squamous-cell carcinoma (LSC group), 24 cases of normal lung epithelium (NLE group) and 27 cases of lung squamous intraepithelial lesions (SIL group). The correlation of clinical and pathological factors was analyzed. The protein expression of NET-1 in human lung squamous-cell carcinoma cell lines H226, H1703, H2170, SK-MES-1, H520 and YTMLC-90 was determined by Western blot. The RNA interference recombinant adenovirus against NET-1 gene (Ad-NET-siRNA) and Ad-control with control sequence were constructed and infected with human lung squamous cell carcinoma cell YTMLC-90 to silence the expression of NET-1 gene. The protein expression of NET-1, E-cadherin, vimentin and Snail1 in the BEAS-2B cells and the YTMLC-90 cells was determined by Western blot. The mRNA expression of E-cadherin and vimentin in each group of the cells was detected by qPCR. The invasive ability of the cells in each group was detected by Transwell chamber assay. RESULTS: The positive expression rate of NET-1 in LSC group was significantly higher than that in NLE group and SIL group(P<0.05). The distribution of NET-1 protein positive expression population was correlated with histological grade, lymph node metastasis, and TNM stage. The NET-1 expression rate of LSC with lymph node metastasis was significantly higher than that without lymph node metastasis. Over-expression of NET-1 protein in YTMLC-90 cells was observed. The expression of E-cadherin was decreased, and the protein expression of vimentin and Snail1 was increased in YTMLC-90 cells. Knock-down of NET-1 expression increased the expression of E-cadherin, and decreased the expression of vimentin and Snail1 in the YTMLC-90 cells. CONCLUSION: The expression of NET-1 promotes the lymphatic metastasis of lung squamous-cell carcinoma. This promotion may be achieved through the activation of epithelial-mesenchymal transition (EMT) by NET-1 expression.     

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.     

Effects of down-regulation of AEG-1 expression on cell cycle and invasion of human cervical carcinoma cells

AIM: To investigate the effects of down-regulation of astrocyte elevated gene-1 (AEG-1) expression on cell cycle and invasion of human cervical carcinoma SiHa cells.METHODS: The protein expression of AEG-1 was detected by Western blotting in normal cervical tissues, cervical squamous cell carcinoma tissues, HeLa cells, SiHa cells and CaSki cells. Control siRNA or AEG-1 siRNA was transfected into SiHa cells, and the protein expression of AEG-1 in SiHa cells was detected by Western blotting. The changes of cell cycle distribution and cell invasion were determined by flow cytometry and Boyden chamber, respectively. The protein levels of cyclin D1, cyclin-dependent kinase 2(CDK2) and matrix metalloproteinase-9 (MMP-9) were analyzed by Western blotting.RESULTS: The protein expression of AEG-1 in cervical squamous cell carcinoma tissues was significantly higher than that in normal cervical tissues (P<0.05). Meanwhile, the protein expression of AEG-1 in the 3 cervical carcinoma cell lines was obviously higher than that in normal cervical tissues, in which SiHa cells displayed the highest AEG-1 protein level (P<0.05). In addition, AEG-1 siRNA effectively down-regulated the protein expression of AEG-1 in SiHa cells, which led to increase the percentage at G0/G1 phase and reduced the invasion of SiHa cells. Furthermore, the protein levels of cyclin D1, CDK2 and MMP-9 in AEG-1 siRNA group were markedly lower than those in non-treatment group and control siRNA group (P<0.05).CONCLUSION: Over-expression of AEG-1 may be closely associated with the occurrence and development of cervical carcinoma, and the AEG-1 down-regulation-mediated cell cycle arrest and attenuation of invasion may be tightly related to the down-regulations of cyclin D1, CDK2 and MMP-9 at protein levels.     

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.     

Sphingosine kinase 1 enhances invasion and migration of non-small-cell lung cancer cells by ERK1/2 signaling pathway

AIM To explore the effects of sphingosine kinase 1 (SphK1) on the migration and invasion of non-small-cell lung cancer (NSCLC) cells and its mechanism. METHODS Thirty-one tumor specimens, which were surgically resected and routinely histologically confirmed as NSCLC, and matched adjacent lung tissues were selected. Immunohistochemical staining and RT-qPCR were used to detect the expression of SphK1. The pcDNA3.1-SphK1 vector (SphK1 group), empty pcDNA3.1 vector control (NC group), SphK1 siRNA (siSphK1 group) or control siRNA (siNC group) was transfected into human lung adenocarcinoma A549 cells, and the protein levels of SphK1, E-cadherin, fibronectin and p-ERK1/2 were determined by Western blot. The effects of over-expression of SphK1 and inhibition of ERK1/2 on migration and invasion of A549 cells were evaluated by Transwell assays. RESULTS SphK1 was highly expressed in the NSCLC tissues and was associated with tumor stage. SphK1 over-expression significantly promoted the migration and invasion of A549 cells, increased the protein levels of p-ERK1/2 and fibronectin, and decreased the protein expression of E-cadherin (P<0.05), but the opposite result was observed after SphK1 interference. The ERK1/2 inhibitor U0126 significantly inhibited the up-regulation of p-ERK1/2 and fibronectin levels and the down-regulation of E-cadherin expression induced by SphK1 over-expression, and also inhibited the invasion and migration of A549 cells promoted by SphK1 over-expression (P<0.05). CONCLUSION SphK1 may reduce E-cadherin protein levels, increase fibronectin protein levels, and promote the invasion and migration of NSCLC cells through ERK1/2 signaling pathway.     

Effects of antihistamine treatment on mast cell infiltration and c-Kit and SCF expression in liver tissues of rats with experimental hepatitis

AIM：To study the infiltration of mast cells and the expression of c-Kit and stem cell factor (SCF) in liver tissues of rats with experimental hepatitis and their changes after antihistamine (AH) treatment. METHODS：Thirty Wistar rats were divided into 3 groups at random: normal control (NC) group, chronic hepatitis (CH) group and AH group. The rat model of CH was established by composite factors (subcutaneous injection of carbon tetrachloride, accompanied by a diet containing high cholesterol, high alcohol, low protein and low choline). The rats in AH group were treated with ketotifen based on CH. At the end of the 4th week, blood samples were taken to determine plasma tryptase (TS) and histamine (HA) levels. Liver tissues were taken to detect HA content, observe the histological changes with HE staining and count the number of mast cells with toluidine blue (TB) staining. The mRNA and protein expression of c-Kit and SCF in liver tissues was detected by RT-PCR and immunohistochemistry. RESULTS：(1) The plasma TS and HA levels and liver HA content in CH group were significantly increased compared with NC group (P<0.05), while those in AH group were obviously decreased compared with CH group (P<0.05). (2) Fatty degeneration and fibrosis were observed in CH group under light microscope, but the hepatic injury was obviously attenuated in AH group. TB staining showed there were many degranulating and degranulated mast cells filled with purple granules around liver blood vessels and in fiber interval in CH group, and there were few purple granules in the cytoplasm of mast cells in AH group. The number of mast cells in CH group was increased compared with NC group (P<0.05), and that in AH group was reduced compared with CH group (P<0.05). (3) The results of RT-PCR showed that AH down-regulated the expression of c-Kit and SCF mRNA (P<0.05). The expression of c-Kit and SCF proteins in liver tissues increased in CH rats (P<0.05 vs NC group), decreased after AH treatment (P<0.05 vs CH group) and was positively correlated with liver HA content (P<0.05). CONCLUSION：These data suggest that an inflammatory pathway mediated by mast cell activation is involved in experimental hepatitis. Ketotifen can reduce mast cell degranulation by down-regulating the expression of mast cell membrane receptor c-Kit and its ligand SCF, thereby attenuating the liver inflammation.