RNAi Suppresses PC-1 Gene Expression In NIH3T3 And C4-2 Cells

Objective: The gene PC-1(GeneBank accession no. AF202897)was firstly identified and cloned by Zhou Jian-Guang et al, and the expression of PC-1 gene varied obviously among the prostate cancer cell lines on different progression stages. Our previous study showed elevated expression of PC-1 correlated with prostate cancer. For a better understanding of its role in prostate cancer development and progression, we selected the most appreciate RNA interferencing target by suppressing the expression of extraneous PC-1 gene in NIH3T3 cells, then stably suppressed the expression of internal PC-1 gene in the advanced prostate cancer cell line C4-2 by RNA interference technology and observed the changes of the biological action of prostate cancer cell line C4-2.Methods: According to the of basic principle of RNA interference, five candidate targets were designed in the PC-1 cDNA encoding region. Recombinant plasmids of expressing short hairpin RNA corresponding to PC-1 coding region and expressing PC-1-EGFP were respectively constructed by DNArecombmant technology. Plasmid expressing short hairpin RNA was cotransfected into NIH3T3 cells via liposome with PC-1-EGFP express plasmid respectively. The most appreciate RNA interferencing target was selected from 5 candidates by observing the expression of EGFP using inverted fluorescence microscope 48hs after transfection. The effect of RNA interference was confirmed by reverse transcriptase polymerase chain reaction and western-blotting analysis.Then the C4-2 cells were transfected with recombinant plasmid expressing short hairpin RNA corresponding to the selected target and negative recombinant plasmid respecively and selected for stable integrants by using G418 for 8 weeks. The stable integrant of extraneous DNA was identified by PCR analysis.The expression of PC-1 gene were analyzed by RT-PCR and Western blotting...