关键词: 小干扰 RNA, TrKA, 乳腺癌MCF-7细胞

Abstract: BACKGROUND AND AIM: To construct the expression vector of TrKA small interfering RNA,and to observe its effect on cell proliferation induced by nerve growth factor(NGF) and cell cycle. MATERIALS AND METHODS: Using the mRNA complete sequence of TrKA gene provided by Genbank, DNA sequence which could transcribe short hairpin RNAs was selected and designed by software,and was connected with the vector of PsilencerTM 4.1-CMV neo .Then it was transfected into MCF-7 cells after confirmed by sequencing. The stable cell line expressing TrKA small interfering RNA were selected by G418. The mRNA and protein levels of TrKA were tested by real-time PCR, Western blot, and immunohistochemistry. The alteration of cell proliferation induced by NGF was assessed by MTT assay after TrKA interference and cell cycle was measured by flow cytometry. RESULTS: The expression vector of TrKA siRNA was successfully constructed . The level of TrKA mRNA and protein was decreased by 74.7％ (P<0.01) and 80.5％,respectively. Immunohistochemistry also showed that TrKA was down-regulated.The expression of GAPDH gene was decreased by 85.0％ (P<0.05). The density value of NGF group was higher than the group of NGF＋siRNA,siRNA and control each time(P<0.05),but the density value of NGF＋siRNA group was lower than other group(P<0.05) . The result indicated that TrKA could effectively inhibit the proliferation of breast cancer cells MCF-7 induced by NGF.The cell cycle showed that compared with control, G0/ G1 period of NGF＋siRNA was higher and the S cell of siRNA was lower (P<0.05), cell cycle was arrested by G0/G1. CONCLUSION: The expression vector of TrKA siRNA could decrease the expression of TrKA in MCF-7 cells effectively,and inhibit the proliferation of MCF-7 induced by NGF.

Key words: small interfering RNA, TrKA, breast cancer cell MCF-7