| Objective:To explore the characteristics maintenance of Yes-associated protein(YAP)in Lung cancer stem-like cells(LCSLCs)and the association between YAP and telomerase.The differences in the expression of YAP and human telomerase reverse transcriptase(hTERT)in benign and malignant pleural fluid were compared in order to provide a new method for the stem-like cells characteristics regulation of lung malignant tumor cells and the diagnosis and treatment of benign and malignant pleural effusion.Method:(One)Cell experiment1.Human lung adenocarcinoma cells A549 was induced to form A549 cell spheres with Fetal bovine serum(FBS),and the expression of tumor stem cell markers(EpCAM?CD44?CD133?OCT4?SOX2)was detected by qRT-PCR.2.A549 cells and A549 cell spheres were transfected with YAP plasmid to up-regulate the expression of YAP protein and mRNA of A549 cells and A549 cell spheres.3.A549 cells and A549 cell spheres were treated with YAP inhibitor verteporfin(VP).4.Western blot was used to detect the expression of YAP protein in A549 cells and A549 cell spheres after transfection with YAP and addition of VP.5.The expression of YAP and hTERT mRNA in A549 cells and A549 cell spheres after transfection with YAP and addition of VP was detected by qRT-PCR.6.CCK8 was used to detect the proliferation of A549 cells and A549 cell spheres after transfection with YAP and addition of VP.(Two)Clinical study1.Collect pleural effusion cells from 25 patients with pathologically confirmed lung malignant tumors,and collect pleural effusion cells from 7 patients with benign disease as controls.2.qRT-PCR was used to detect the expression of YAP,hTERT and stem cell markers(EpCAM,CD44,CD133,OCT4,SOX2)in benign and malignant pleural effusion cells to compare their differences in benign and malignant pleural effusion and their association with distant metastasis of malignant tumor.3.The activity of telomerase in benign and malignant pleural effusion cells was detected by PCR-ELISA.Result:(One)Cell experiment1.qRT-PCR showed that the expressions of CD44,CD133,OCT4,and SOX2 in A549 cell spheres were higher than A549 cells,and the difference was statistically significant(?<0.05),while the difference of EpCAM between them was not statistically significant(p>0.05).2.Western Blot and qRT-PCR detected that YAP protein and mRNA expression in A549 cells were lower than those in A549 cell spheres,and the differences were statistically significant(p<0.05).3.Western Blot and qRT-PCR detected that YAP protein and mRNA expression of A549 cells and A549 cell spheres were up-regulated after YAP WT transfection.The differences were statistically significant(p<0.05).4.Western Blot detected that after the addition of VP,the expression of YAP protein in A549 cells and A549 cell spheres was reduced,the difference was statistically significant(p<0.05),but about qRT-PCR,A549 cells and A549 cell spheres were treated with VP,there was no statistically significant difference in YAP mRNA expression(p>0.05).5.qRT-PCR showed that hTERT mRNA in A549 cell spheres was higher than that in A549 cells,and the difference was statistically significant(p<0.05);After transfection of A549 cells and A549 cell spheres with YAP WT,the expression of hTERT mRNA increased in A549 cells,the difference was statistically significant(p<0.05),but no statistically significant difference in A549 cell spheres(p>0.05).After VP was added to A549 cells and A549 cell spheres,there was no significant change in hTERT mRNA expression(p>0.05).6.CCK8 detected that the cell proliferation ability of A549 cell spheres was higher than that of A549 cells,and the difference was statistically significant(p<0.05);After transfection of A549 cells and A549 cell spheres with YAP WT,the cell proliferation ability increased in A549 cells,the difference was statistically significant(p<0.05),but no statistically significant difference in A549 cell spheres(p>0.05);After adding VP,the cell proliferation ability of A549 cells and A549 cell spheres decreased,the difference was statistically significant(p<0.05).(Two)Clinical study1.qRT-PCR showed that the expressions of YAP,hTERT and stem cell markers(CD44,CD 133,OCT4,and SOX2)in malignant pleural effusion cells were higher than benign pleural effusion cells,and the difference was statistically significant(p<0.05),while the difference of EpCAM between benign and malignant pleural effusion cells was not statistically significant(p>0.05).2.The expression of YAP,hTERT and CD 133 in pleural effusion cells of lung malignant tumors with distant metastasis were higher than that of pleural effusion cells of lung malignant tumors without distant metastasis(p<0.05),while the expression differences of CD44?OCT4?SOX2 in the presence or absence of distant metastases were not statistically significant(p>0.05).3.PCR-ELISA showed that the activity of telomerase in malignant pleural effusion cells was higher than that in benign pleural effusion cells,and the difference was statistically significant.(p<0.05).Conclusion:1.In A549 cell spheres induced from A549 cells and pleural effusion cells,we find that the expression of YAP and hTERT mRNA is positively correlated,which are involved in the maintenance of lung cancer stem-like cell characteristics.2.Higher YAP and hTERT mRNA expression are associated with distant metastasis of lung cancer.3.The expression of YAP,hTERT,tumor stem cell markers(CD44,CD133,OCT4,SOX2)in pleural effusion cells are of certain value in the differentiation of benign and malignant pleural effusion. |
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