Search

Aim: To observe the expression of let-7 in breast cancer stem cells and explore the role of let-7 in maintaining the characteristics of breast cancer stem cells., Methods: We separated breast cancer stem cells (SP and NSP) from MCF-7 cell line using SP sorting, and observed the expression of let-7a/b/c on SP and NSP cells using quantitative real-time PCR and the expressions of Ras and ERK using Western blotting to study the mechanism by which let-7 maintains the characteristics of breast cancer stem cells., Results: The SP cells accounted for 3.3% in MCF-7 cells, however, the rate dropped to 0.4% when verapamil was added into the process of seperation. The level of Let-7a/b/c in SP cells were lower than that in NSP cells, and among let-7 miRNAs, let-7b/c showed the most obvious difference. The expressions of t-Ras and t-ERK showed no difference between SP and NSP cells, nevertheless, the expressions of p-Ras, p-ERK were higher in SP cells than in NSP cells., Conclusion: SP sorting is an effective method to separate cancer stem cells. There do exist cancer stem cells in MCF-7 breast cancer cell line. Let-7 is down-regulated in SP cells, and the down-regulation makes let-7 lose the opportunity to restrain Ras mRNA, finally, p-Ras and p-ERK are activated. They play an important role in maintaining the characteristics of breast cancer stem cells.

Aim: To explore the expression of caspase-8 in non-small cell lung cancer (NSCLC) tissue, and its role in invasion and metastasis of lung cancer A549 cells., Methods: The expression of caspase-8 in the lung cancer tissues was detected by immunohistochemistry. RNA interference technology was used to silence the expression of caspase-8 in lung cancer A549 cells to explore the function of caspase-8 protein in invasion and metastasis of cancer cells by means of Western blot, Transwell and scratch migration assays., Results: The expression of caspase-8 was found in 38/52 cancer tissues, 22/30 adjacent tissues and 34/52 normal tissues. The tissues number of caspase-8 positive-expression in different groups was similar, but the intensity of caspase-8 expression in different groups was statistically different(P<0.05). In cancer tissues, the expression of caspase-8 indicated more likely to have lymph node-metastasis and worse clinical stage(P<0.05). After RNA interference, we successfully silenced the expression of caspase-8 in the lung cancer A549 cell, and this was confirmed by Western blotting. Transwell and cell scratch test showed that without the expression of caspase-8 A549 cells had a lower invasion and metastasis abilities compared to the control cells (P<0.05)., Conclusion: Caspase-8 is not only an apoptosis protein, but has non-apoptosis function, such as promoting invasion and metastasis of lung cancer A549 cell. In NSCLC, it more likely locates in nucleus. And its expression in NSCLC may indicate a later clinical stage and a higher probability of lymph node metastasis.

Aim: To investigate the effects of Smac gene overexpression on chemotherapeutic sensitivity of esophageal cancer cell line Eca109 to cisplatin., Methods: pcDNA3.1-Smac with GFP and pcDNA3.1-Smac with GFP were transfected into esophageal cancer cell line Eca109 by liposome and incubated with G418 for subclone selection.The efficiency of transfection was observed under fluorescence microscope, cellular Smac gene expression were determined by Western blot. Cisplatin treated group (1, 5, 10 mg/L) and cisplatin untreated group were selected to treat untransfected and transfected esophageal cancer cell line Eca109. Apoptosis was determined by Annexin V/PI., Results: The subclone esophageal cancer cell line Eca109, stable expressing Smac+GFP and neo+GFP respectively, were successfully selected, named as Eca109/Smac, Eca109/neo. Compared with the Eca109/neo and Eca109, the Smac expression level of Eca109/Smac was significantly increased(P<0.05). In cisplatin untreated group, the apoptosis rate was not associated with Smac expression. In cisplatin treated group (1, 5, 10 mg/L), compared with Eca109/neo and Eca109, the apoptosis rate of the Eca109/Smac was significantly increased after the treatments of cisplatin, the difference were significant(P<0.05). In addition, the apoptosis rate of Eca109/Smac was significantly increased with the concentration of cispaltin increased(P<0.05). The difference between Eca109/neo and Eca109 were not significant., Conclusion: Smac gene didn't induce apoptosis in the cisplatin untreated Eca109 cells. Smac gene overexpression could increase chemotherapeutic sensitivity of esophageal cancer cell line Eca109 to cisplatin.

Aim: To construct the eukaryotic expression vector of human gene Smac pcDNA3.1-Smac and express it in the lung adenocarcinoma A549 cells., Methods: The Smac was amplified from human testis tissue by reverse transcriptase polymerase chain reaction (RT-PCR). Then recombined eukaryotic expression vector pcDNA3.1-Smac was constructed. After the reconbinant plasmid was proved to be constructed correctly by endonucleases digesting and DNA sequencing, we trasfected it into lung adenocarcinama cells A549 through liposome inducing. The expression of Smac in transfectant A549 was detected by RT-PCR and Western blot. And the cell growth inhibition ratio after trasfection was detected by MTT., Results: The amplified fragment by PCR was coincident with the anticipated result, and its sequence was in concordance with that published on GenBank.Therefore, the gene Smac was cloned successfully, and the recombinant plasmid pcDNA3.1-Smac was also constructed successfully. Both on the mRNA level and the protein level, the expression of Smac gene was increased obviously in the transfected A549 detected by RT-PCR and Western blot respectively. The cell growth inhibition ratio in the group transfected pcDNA3.1-Smac was significantly higher compared with the pcDNA3.1 group after 72 hours., Conclusion: The recombinant eukaryotic expression vector pcDNA3.1-Smac was constructed, and it could be obviously expressed in lung adenocarcinoma cells A549. It is also proven that Smac has the function of growth inhibition.