ABSTRACT
@#[Abstract] Objective: To investigate the effect of miR-93/EphA4 (Eph receptor A4) axis on the proliferation and migration of nonsmall cell lung cancer (NSCLC) H460 and H1299 cells via regulating extracellular regulated protein kinases (ERK) pathway. Methods: The expression levels of miR-93 in H460 and H1299 cells was detected by qPCR. miR-93 mimics and EphA4 overexpression plasmids were transfected into H460 cells and miR-93 inhibitor was transfected into H1299 cells respectively, after which MTT assay and Transwell assay were used to detect the effects of miR-93 on proliferation and migration of transfected cells. The targeted regulatory relationship betweenmiR-93andEphA4wasverifiedbyDual-luciferasereportergeneassay.Theexpression levels of PCNA(proliferating cell nuclear antigen), EphA4, ERK and p-ERK were detected by Westernblotting.The effects of simultaneous overexpression of miR-93 and EphA4 on proliferation and migration of H460 cells were detected by MTT assay and Transwell assay. Results: The expression of miR-93 in H1299 cells was higher than that in H460 cells (P<0.01). Overexpression of miR-93 promoted proliferation and migration of H460 cells (all P<0.01), and knockdown of miR-93 inhibited proliferation and migration of H1299 cells (all P<0.01). The Dualluciferase reporter gene assay confirmed that miR-93 could target EphA4. Overexpression of miR-93 down-regulated the mRNA and protein expression levels of EphA4(allP<0.05), and promoted proliferation and migration of H460 cells through targeted regulation of EphA4 and activation of ERK pathway (all P<0.01). Conclusion: miR-93 promotes the proliferation and migration of NSCLC cells, and its mechanism may be related to the targeted regulation of EphA4 and activation of the ERK pathway.
ABSTRACT
To investigate the proliferation and apoptosis of lung adenocarcinoma cells line HI299 by the lentiviral vector mediated RNA binding protein quaking-5 ( QKI-5). Methods GV358 ( up-regulation ) and GV248 ( down-regulation) vectors were used to construct the lentiviral QKI-5 up-regulation vector and down-regulation vector, respectively. The vectors were transfected into 293T cells for lentiviral packaging and viral titer were then determined. Gene sequencing was performed to screen the sequence of vectors. Then Real-time PCR was used to evaluate the expression of QKI-5 mRNA and the proliferation of H1299 cells was examined by colony forming assay after transfection. The apoptosis of HI299 cells was determined by the detection of the expression membrane protein V (annexin V ) and propyl iodide ingot (PI) by flow cytometry. Pro-apoptotic protein Caspae-3 and Caspase-8 were evaluated by Western blotting. Results QKI-5 up-regulation and down-regulation lentiviral vectors were constructed successfully. Compared with the controls, the expression of QKI-5 mRNA of HI299 cells was up-regulated, the cell colony formation was decreased, and the early apoptosis of HI299 cells was increased with the over-expression of Caspase-8 after transfected with up-regulated vector, whereas transfecting with QKI-5 down-regulated vector had opposite effect. Conclusion Lenviral vector mediated QKI-5 could inhibit proliferation and promote apoptosis of lung adenocarcinoma cells through Caspase-8.
ABSTRACT
Objective: To identify the relationship between the expression of protein tyrosine phosphatase non-receptor type 12 (PTPN12) and radiotherapy effect in non-small cell lung cancer (NSCLC) tissues and to determine whether PTPN12 deficiency can sensi-tize lung cancer cells to irradiation. Methods: From September 2013 to October 2014, 92 NSCLC patients undergoing radiotherapy with or without platinum-based combination chemotherapy were analyzed retrospectively. Before the treatment, PTPN12 expression was detected through immunohistochemistry. After the completion of radiotherapy, the patients' responses were assessed and radio-therapeutic efficacy analyzed. The human NSCLC cell line H1299 was infected with shPTPN12 knockdown, and colony survival assay was analyzed after irradiation. Chi-square test was used to examine the correlation between PTPN12 expression and clinicopathologi-cal characteristics. Univariate analyses and Logistic regression test were used to analyze the relationship between clinicopathological characteristics and radiotherapeutic response. Results: Patients with low PTPN12 expression were more sensitive to radiotherapy than those with high PTPN12 expression (80.0%vs. 57.1%, P=0.018). Multivariate analysis showed that PTPN12 expression was the on-ly independent predictor of radiotherapeutic response in NSCLC. The H1299-shPTPN12-knockdown cells were sensitive to irradiation. Conclusions:The results of the study indicated that downregulation of PTPN12 improved the radiosensitivity of NSCLC cells.
ABSTRACT
OBJECTIVE:To explore the effects of recombinant adenovirus vector Adxsi-GFP-VP3 carrying apoptin gene VP3 on the apoptosis of human lung squamous carcinoma SK-MES-1 cell lines and human lung adenocarcinoma NCI-H1299 cell lines. METHODS:The exponential phase SK-MES-1 and NCI-H1299 cell lines were respectively divided into a recombinant adenovirus (Adxsi-GFP-VP3) group,a empty virus (Adxsi-GFP) group and a cell control (phosphate buffer) group,which were marked as group A,B and C respectively. Reverse transcription polymerase chain reaction and Western blot method were used to detect the ex-pressions of VP3 mRNA and Apoptin in the cells of groups A and B 48 and 72 h after transfection. The change in the ultrastructure of the cells in group A was observed under transmission electron microscope 72 h thereafter. MTT method was adopted to detect the cell proliferation activities of three groups 24,48,72 and 96 h thereafter and flow cytometry to determine the apoptosis rates and cell cycle changes 24,48 and 72 h thereafter. RESULTS:Compared to group B,group A demonstrated the expression of VP3 mRNA in SK-MES-1 and NCI-H1299 cell lines 48 h after transfection,and Apoptin expression and ultrastructure change for apopto-sis of SK-MES-1 and NCI-H1299 cell lines 72 h thereafter. Compared to groups B and C,group A showed lower proliferation activ-ities and higher apoptosis rates of SK-MES-1 and NCI-H1299 cell lines,which had a positive correlation with transfection time;and in the group A,there was a decrease in the proportion of the SK-MES-1 and NCI-H1299 cell lines in S phase and an increase in the proportion of those in G2/M phase,72 h after transfection. There was statistically difference (P<0.05). CONCLUSIONS:Adxsi-GFP-VP3 can effectively induce the apoptosis of SK-MES-1 and NCI-H1299 cell lines.
ABSTRACT
ObjectiveTo explore the expression and clinical significance ofaquaporin (AQP) 1 and AQP 4 in human pulmonary adenocarcinoma H1299 cell line.MethodsH1299 cell line in human pulmonary adenocarcinoma(pulmonary adenocarcinoma group) were obtained,the expressions of AQP1 and AQP4 in mRNA level and their locations were determined in H1299 cell line respectively by semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis.The migration of tumor cells were observed by Matrigel invasion assay.Then normal tissues adjacent of pulmonary adenocarcinoma (above cancer line 3 cm,no tumor cell with pathological proven) were as control group.ResultsThe results of RT-PCR showed that AQP1,AQP4 mRNA was 1.030 ± 0.070 and 1.140 ± 0.190 in conlrol group,which were lower than those in pulmonary adenocarcinoma group (2.021 ± 0.250 and 2.180 ±0.180)(P＜0.05 ).The results of Western blot showed AQP1,AQP4 located on the membrane of H1299 cell.Both AQPI and AQP4 mRNA expressed very high in pulmonary adenocarcinoma group,while expressed very low in control group (P＜0.05).Matrigel invasion assay showed that the invasion was positively related to AQP1,AQP4(r =0.351,P ＜ 0.05 ).ConclusionAQP1,AQP4 significantly over express in H1299 cell line,both of them phy important roles in the growth of tumor tissue and cell migration.
ABSTRACT
Background and purpose: Protein kinase P1k3 could increase the transcriptional activity of p53. However, the effect of P1k3 on the transcriptional activity of p73 is still unknown. Our study was to investigate the effect of P1k3 on the transcriptional activity of p73. Methods: Luciferase reporter assay, RT-PCR and colony formation assay were adopted to study the effect of P1k3 and kinase-deficient P1k3 (K52R) on the transcriptional activity of p73 in p53-deficient human lung carcinoma H1299 cells. Results: Luciferase reporter assay showed that p73 increased the luciferase activities induced by p21~(WAF1) and Bax promoters (P<0.05). After co-transfection with p73 and P1k3, the luciferase activities induced by p21~(WAF1) and Bax promotors were significantly decreased in a dose-dependent manner as compared with the group that transfected p73 only (P<0.05). However, kinase-deficient Plk3 (K52R) had no significant effect on the luciferase activities induced by p21~(WAF1) and Bax promoters (P>0.05). RT-PCR showed that p73 increased the mRNA expressions of p21~(WAF1) and BAX (P<0.05). P1k3 decreased the expressions of p21~(WAF1) and Bax induced by p73 (P<0.05). Kinase-deficient P1k3 (K52R) had no significant effect on the expressions of p21~(WAF1) and Bax induced by p73 (P>0.05). Colony formation assay revealed that p73 decreased the colony formation of H1299 cells (P<0.05). P1k3 decreased the inhibitory effect of p73 on the colony formation of H1299 cells (P<0.05). Kinase-deficient P1k3 (K52R) had no significant effect on the inhibitory effect of p73 on the colony formation of H1299 cells (P>0.05).Conclusion: P1k3 can inhibit the transcriptional activity of p73, where as kinase-deficient P1k3 has no effect on the transcriptional activity of p73.