ABSTRACT
Objective:To silence ERCC2 (excision repair cross-complementing rodent repair deficiency,complementatin group 2,ERCC2) expression in esophageal cancer KYSE150 cells by small interfering RNA (siRNA) and observe the altered sensitivity of KYSE150 cells to paclitaxel (PTX) and elucidate the mechanism underlying the reversion of the PTX resistance of KYSE150 cells. Methods:ERCC2-targeted siRNA was synthesized in vitro and transiently transfected into ERCC2 overexpressing KYSE150 cells via Lipofectamine mediation. The mRNA and protein expression levels of ERCC2 were determined by using RT-PCR and FCM method, respectively. The sensitivity of KYSE150 cells to PTX was measured by MTT assay before and after siRNA transfection. Results:RT-PCR results suggested that the specific bands of ERCC2 mRNA were not detected in si-ERCC2 group at 24, 48 and 72 h post transfection. FCM results indicated that the expression levels of ERCC2 protein gradually decreased by 31.2%, 51.6% and 60.0% at 24, 48 and 72 h respectively(P<0.01). The IC_(50) value of PTX for ERCC2-silencing KYSE150 cells was (6.32±0.87) μg/mL, lower than that for control cells (P<0.01). Conclusion:siRNA successfully silenced the expressions of target gene ERCC2 at both the transcription and translation levels. Silencing ERCC2 expression partly reversed the resistance of KYSE150 cells to PTX.
ABSTRACT
Background and purpose: ERCC2 gene silence by siRNA interference was observed in esophageal cancer cell line and it could change cell sensitivity to taxol. This study was to investigate the biological mechanism of paclitaxel-resistance in esophageal cancer cells. Methods: ERCC2 targeting siRNA (si-ERCC2) has been synthesized. The constructor were transfected into ERCC2 cell lines high-KYSE150(bigh expression of ERCC2) through lipofectamine. RT-PCR and flow cytometry (FCM) were used to detect the ERCC2 mRNA and protein expression levels. MTr assay was used to estimate the paclitaxel sensitivity of the cells. Results: In si-ERCC2 group, ERCC2 could not be detected and ERCC2 protein expression were reduced by 31.2%, 51.6% and 60.0%, respectively, 24,48,72 b after transfection. Paclitaxel IC_(50) value for si-ERCC2 group was 6.32±0.87 μg/mL, lower than the control group(49%). Conclusion: siRNA could successfully silence the target gene ERCC2 at the level of transcription and translation of the gene, the reduction of ERCC2 expression may reverse taxol resistance of the cells.
ABSTRACT
Most of the CHD patients reveal string pulse, mainly due to damage of heart function, lowering of arterial compliance and increase of total peripheral resistance. The common pulse in patients of blood diseases reveal frequent, tiny, string and slippery characteristic, mostly due to the increase of compensatory pumping action of the heart, shortening of ejection time of the left heart, with better vessel compliance and hemorheology, low total peripheral resistance.