Objective: The aim of the study was to explore the effects of the same target (si-10) on lung cancer cells with different expression levels of cyclooxygenase-2 (COX-2) protein by RNAi and malignant proliferation of th...Objective: The aim of the study was to explore the effects of the same target (si-10) on lung cancer cells with different expression levels of cyclooxygenase-2 (COX-2) protein by RNAi and malignant proliferation of these cells. Methods: COX-2 was selected as the target and one siRNA expression vector with the best effect was selected and thought as the subject from three COX-2 siRNA expression vectors with human U6 promoter. The siRNA expression vector (psi-10) and the vacant vector (pEGFP) were transfected into these cells with different COX-2 expression states (801D, A549 and LTEP-A2) with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve and clonogenic assay. Results: The siRNA and U6 promoter were validated by PCR, restriction endonucleases identification and DNA sequencing and BLAST alignment and cloned into the pEGFP vector. The cell strains transfected that 801D was used as maternal line were named as 801D-p and 801D-10 respectively. The cell strains transfected that A549 was used as maternal line were named as A549-p and A549-10 respectively. The cell strains transfected that LTEP-A2 was used as maternal line were named as LTEP-A2-p and LTEP-A2-10 respectively. These cells transfected pEGFP (801D-p, A549-p and LTEP-A2-p) had the expression of GFP and 801D-10, A549-10 and LTEP-A2-10 cells had not in 24, 48 and 72 hours after transfected. The results of RT-PCR and Western blot showed the siRNA expression vector produced marked effects in two cells (A549 and LTEP-A2) expressing COX-2 and the expression of COX-2 was inhibited. But the inhibited effects were differ- ent and the expression of COX-2 was more inhibited obviously in LTEP-A2 cells than in A549 cells though the expression of COX-2 was also inhibited obviously in A549 cells. In contract to their maternal line, the levels of COX-2 mRNA of LTEP-A2-10 and A549-10 cells reduced 64.2% and 61.2% respectively; the levels of COX-2 protein reduced 60.2% and 56.2% respectively. But the levels of COX-2 mRNA and protein had not change in 801D cells not expressing COX-2. The results of cell growth curve and clonogenic assay showed the growth of LTEP-A2-10 cells slowed and the clonal formation rate reduced and the size of the colonies became small; the growth of A549-10 cells showed slow and more obviously in the cell growth curve especially. But the growth of 801D-10 cells had not obvious change. Conclusion: The si-10 target of COX-2 has different inhibition effects on lung cancer cells with different COX-2 expression levels and the different inhibition effects have different effects on cells malignant proliferation.展开更多
文摘Objective: The aim of the study was to explore the effects of the same target (si-10) on lung cancer cells with different expression levels of cyclooxygenase-2 (COX-2) protein by RNAi and malignant proliferation of these cells. Methods: COX-2 was selected as the target and one siRNA expression vector with the best effect was selected and thought as the subject from three COX-2 siRNA expression vectors with human U6 promoter. The siRNA expression vector (psi-10) and the vacant vector (pEGFP) were transfected into these cells with different COX-2 expression states (801D, A549 and LTEP-A2) with lipofectamine respectively and the transfected cell strains were constructed. The change of COX-2 expression levels was examined by Western blot and RT-PCR. The effects on the proliferation of lung cancer cells were studied by cell growth curve and clonogenic assay. Results: The siRNA and U6 promoter were validated by PCR, restriction endonucleases identification and DNA sequencing and BLAST alignment and cloned into the pEGFP vector. The cell strains transfected that 801D was used as maternal line were named as 801D-p and 801D-10 respectively. The cell strains transfected that A549 was used as maternal line were named as A549-p and A549-10 respectively. The cell strains transfected that LTEP-A2 was used as maternal line were named as LTEP-A2-p and LTEP-A2-10 respectively. These cells transfected pEGFP (801D-p, A549-p and LTEP-A2-p) had the expression of GFP and 801D-10, A549-10 and LTEP-A2-10 cells had not in 24, 48 and 72 hours after transfected. The results of RT-PCR and Western blot showed the siRNA expression vector produced marked effects in two cells (A549 and LTEP-A2) expressing COX-2 and the expression of COX-2 was inhibited. But the inhibited effects were differ- ent and the expression of COX-2 was more inhibited obviously in LTEP-A2 cells than in A549 cells though the expression of COX-2 was also inhibited obviously in A549 cells. In contract to their maternal line, the levels of COX-2 mRNA of LTEP-A2-10 and A549-10 cells reduced 64.2% and 61.2% respectively; the levels of COX-2 protein reduced 60.2% and 56.2% respectively. But the levels of COX-2 mRNA and protein had not change in 801D cells not expressing COX-2. The results of cell growth curve and clonogenic assay showed the growth of LTEP-A2-10 cells slowed and the clonal formation rate reduced and the size of the colonies became small; the growth of A549-10 cells showed slow and more obviously in the cell growth curve especially. But the growth of 801D-10 cells had not obvious change. Conclusion: The si-10 target of COX-2 has different inhibition effects on lung cancer cells with different COX-2 expression levels and the different inhibition effects have different effects on cells malignant proliferation.
