Inhibitory Effects Of RNA Interference On The Expression Of IL-8 Oncogene In Cervical Cancer Cell Line CaSki

Carcinoma of the uterine cervix that we are usually to see is the second commonest female cancer in the world.The prevalence lies in first place in our country and become one of the main disease which severely threaten health of women in our country.Expression of the CXC chemokine,interleukin-8(IL-8),in many cancer cells has been shown to correlate with the angiogenesis,tumorigenicity,and metastasis of experimental cancer,our previous study have been selected the differential expression gene by cDNA gene chip and found many genes correlated strongly with lymph node metastasis of early squamous cervical cancer.In these genes, the expression of IL-8 in early squamous cervical cancer with lymph node metastased was higer 3.4 times than with no metastatic lymph node.our previous study also have been shown that IL-8 might enhance early cervical cancer to invase and metastases by stimulate directly cervical cancer cells proliferation,migration and enhance tumor-endothelial adhesion in vitro experiment. RNA interference is an effective method of blocking gene expression at the post-transcriptional level.The aim of the current study was to construct expressing vector of RNAi and investigate its inhibi-tory efect on IL-8 expression in human cervical cancer,and to research the effect on biological behaviour of CaSki cell,and to explore the possibility of RNAi in treatment of cervical cancer.PURPOSE:To construct expressing vector of RNAi and investigate its inhibitory efect on IL-8 expression in human cervical cancer and its inhibitory effects on angiogenesis, tumorigenicity,and metastasis of CaSki cell.METHODS:The first,Two oligonucleotides IL-8 cDNA and one negative control oligonucleotide were designed referring to that of GenBank then double-stranded RNA was derived through annealing,finally cloned into PGenesil-1 vector directed orientation.The PGenesil-1 vector has been signed by EGFE The second,the vectors were transfected into CaSki cell by liposome.we observed its transfection efficiency by fluorescence microscope.The expression levels of IL-8 mRNA were assayed by RT-PCR.Confirmation of IL-8 protein expression was performed by ELISA and Immunocytochemistry.The last,the cell proliferation and chemotactic activity were observed by MTT assay,cloning test and Boyden chamber.RESULTS:PCR analysis and DNA sequencing confirmed that the PGenesil-1 IL-8 shRNA vectors were constructed successfully.After transient transfection 72 hours,we observed that transfection efficiency of cell was 60.37%±0.68%.IL-8 mRNA expression could be respectively inhibited 63.90%and 71.14%.IL-8 protein expression could be respectively inhibited 60.80% and 72.13%.So the inhibition effect of IL-8shRNA2 vector was more effective.After transfected by vector IL-gshRNA2,the ImmunocytoChemistry confirmed that IL-8 was weakly positive expressed,and the control group was strong positive expressed.After observed by MTT assay cloning test and Boyden chamber,we concluded that IL-8shRNA2 vector was obviouse inhibitory effects on tumorigenicity and metastasis of CaSki cell.CONCLUSIONS:The expression of IL-8 gene and protein in CaSki cell can be inhibited specifically and high efficiently by IL-8shRNA eukaryotic vectors.The cloning efficiency, proliferation and metastasis of CaSki cell was also reduced.So it might be one of the ideal strategies for gene therapy of cervical cancer.