Construction And Identification Of RNA Interference Expressing Vectors Targeting Human Epidermal Growth Factor

RNA interference(RNAi) can effectively silence the expression of specific gene on post-transcriptional level, which is one of the most important signal channels in Genetic control network and is widely used in cancer therapy. Epidermal growth factor receptor overexpressed in most tumors and involved in the multiple process of tumor formation and development, is an important target for the treatment of cancer. In this study, in view of the many advantages of RNAi and the potential application of EGFR as a tumor target, RNAi is used as a mean to reduce hEGFR gene expression to study its inhibition impact on human liver cancer cells SMMC-7721.The single-gene silencing vectors targeting hEGFR gene are named pRS-EGFR-sh vectors, and the negative control is Rsl. The vectors above were transiently transfected to human hepatoma cells, whose hEGFR gene expression level is detected by RT-PCR. The result showed that, compared with the negative control, hEGFR gene expression of hepatoma cells transfected with positive plasmids did not change significantly. At the same time, the stable expressing cells were established for two weeks, whose RT-PCR and MTT test were carried out. The result for RT-PCR suggested hEGFR gene was not inhibited; the growth of liver cancer cells were not affected by MTT. These results show that the shRNA designed by RNAi principle did not show the expected effects. This prompted us the factor of RNAi is multi-faceted and unknown. With the development of molecular biology technology, RNAi needs more research and deeply improvement.Cancer incidence is related to multiple single pathways. They are interrelated and interact to form a molecular network system, caused great distress to single-target treatment. Therefore, multi-target treatment of cancer is imperative to meet the law and treat requirements tumors. As the limited components of telomerase, hTERT is the ideal anti-tumor target, the expression of which was significantly enhanced in a variety of tumor cells. Are reported in the literature, EGFR overexpression can activate telomerase activity, blocking EGFR signaling pathways associated with a decreased telomerase activity and decreased TERT expression. In this study, under the close links between hTERT and hEGFR, also the importance of multi-targets therapy for tumor, the shRNA of hTERT and hEGFR were constructed in the same pRS vector to achieve multi-directional, low resistance, high strength anti-tumor purposes.The RNA interference vectors targeting hTERT are respectively named shRNA-T vectors, which were digested by EcoRI,then obtained with a large fragment of hTERT-shRNA. The shRNA-G were double-digested by EcoRI and Hindâ…¢recovered hEGFR-shRNA sequences included a small fragment.Then the double-gene RNA interference plasmids were constructed. Polyacrylamide gel electrophoresis results shows the expected DNA bands, indicating two-gene RNA interference vectors constructed successfully.