ABSTRACT
10.3969/j.issn.1000-8179.2013.12.004
ABSTRACT
Objective To construct an eukaryotic vector expressing short hairpin RNA(shRNA) of HIF-1α,and to observe its effects on location and metastasis of HeLa cells under hypoxic condition.Methods shRNA templates was developed based on HIF-1α gene sequence and then cloned into pSilencer2.1-U6-neo vector.The resultant plasmid was transfected into HeLa cells with Lipofectamine 2000.The cells were incubated in hypoxic condition.The HIF-1α protein and mRNA were detected by Western blot and RT-PCR.The colony formation assay and Transwell cabin assay were performed to measure the colony formation and metastasis.Results The plasmid pSilencer2.1-U6-neo-HIF-1α was successfully constructed and transfected into HeLa cells.The expression of HIF-1α in HeLa cells decreased,and the number of colony formation in soft agar and cells penetrating matrigel also decreased under hypoxic condition.Conclusion The shRNA expressing plasmid targeting at HIF-1α may suppress the location and metastasis of cervical carcinoma cells under hypoxic condition.
ABSTRACT
Background and purpose:The signal transducers factor and activator in transcription 3(STAT3) is a recently studied gene from a variety of solid tumors such as breast, stomach, and ovarian cancer, in which the increase of abnormal expression and activity are accompanied.The aim of this study was to investigate the effects of eukaryotic vectors that express short hairpin RNA(shRNA) in signal transducers and activators of STAT3 on chemosensitivity of human ovarian cancer SKOV3 cells.Methods:Vectors containing shRNA targeting STAT3(pSTAT3-siRNA) were constructed and transfected into SKOV3 cells.These vectors were then divided into 3 groups:SKOV3, SKOV3NS, and SKOV3siRNA.The mRNA and protein expressions of STAT3 were determined by RT-PCR and Western blot, respectively.The growth inhibition and apoptosis rates of the different group cells under chemotherapeutic agents such as cisplatin(20 ?mol/L) were measured by MTT assay and flow cytometry(FCM).Results:MTT assay growth inhibition rates in the tumor cells of the SKOV3 group, SKOV3NS group and SKOV3siRNA group had inhibition rates of 0.46?0.13, 0.44?0.11 and 0.71?0.12.Compared with the SKOV3, SKOV3NS and SKOV3siRNA group, there was a marked increase of SKOV3siRNA group in inhibition rate of cells.The differences were also statistically significant(P0.05).The SKOV3 group, SKOV3NS group and SKOV3siRNA group's cell apoptosis rates were 18?4, 18?3 and 35?4, respectively.However, the SKOV3 group, SKOV3NS group, and SKOV3siRNA group cell apoptosis were significantly increased and the differences were statistically significant(P0.05).The results for the SKOV3 group, SKOV3NS group and SKOV3siRNA in cell STAT3 mRNA were 0.50?0.08, 0.48?0.07 and 0.31?0.09.With the SKOV3 group, SKOV3NS group, SKOV3siRNA group of cells in STAT3 mRNA, its expression in the lungs were significantly lower and the differences were statistically significant(P0.05).SKOV3 group, SKOV3NS group and SKOV3siRNA group of cells checked the results of STAT3 protein were 0.54?0.09, 0.56?0.08 and 0.32?0.09, respectively.The SKOV3 group, SKOV3NS group, and SKOV3siRNA group in STAT3 protein expression was significantly lower and the differences were statistically significant(P0.05).Conclusion:The STAT3 specific shRNA expression vector could effectively suppress the expression level of STAT3 gene in SKOV3 cells as well as enhance their sensitivity to cisplatin.