Identification Of Proteins Regulated By P28^GANK Based On RNA Interference And ITRAQ Technology

Hepatocellular carcinoma(HCC) is one of the most frequent cancers in China.Its development and progression is a complexed process involving many genes and several transforming steps in liver.P28^GANK plays a major role in the development of HCC,latter studing has confirmed that this gene was unexpressed,under expressed,overexpressed in normal,hepatocirrhosis,and hepatocellular carcinoma respectively.Our previous results showed that the significant down-regulation of p28^GANK expression through RNA interference in HCC cells inhibits cell growth in vitro and in vivo,and induces apoptosis. However,by which passways it performs this actions and how many interacting proteins with P28^GANK participate in this process was not solved.Today,protein-protein interaction analysis and mapping have become the focus of proteomics and promoted the development of relative techniques.Proteome analysis may allow the more ready identification of differences in protein expression during the activation,proliferation and differentiation of hepatic cells.The sophisticated mass spectrometric method with isobaric tags for relative and absolute quantitation(iTRAQ) technology allowed the relative quantification of hundreds of proteins in 4,or even 8 and 16,samples simultaneously.Now,it is also a powerful tool available for proteomics research.This new technology will continue to be developed as it is becoming extraordinary valuable to study protein-protein interaction.Further studies are needed to elucidate the novel gene biological function.Current research has successfully showed the wide application of RNAi in the silence of some specific oncogenes,shedding a light in the gene function study and in the prevention and treatment of human cancers.The adenoviral vector expressing small interfering RNA (siRNA) is highly effective in mammalian cells.In this project,we applied the adenovirus-delivered siRNA(AdSiRNA) to inhibit a hepatocellular carcinoma(HCC) oncogene,p28^GANK,in HCC cell lines and applied the iTRAQ technology coupled with electrospray ionisation tandem mass spectrometry(ESI-MS-MS) to discuss the protein expression alterations after 28^GANK was down-regulated..HepG2 cells were infected with the p28^GANK RNAi-containing adenovirus.Cell lystaes from HepG2 cells infected with p28^GANK RNAi adenovirus at Oh,48h and 72h were labeled using iTRAQ.The samples were further analyzed by Mass spectrometer to measure the quantity of proteins.Proteome alterations were observed using elctrospray ionization tadem mass spectrometry.About 700 proteins were identified,among which 436 proteins were significantly altered(over 1.5 fold or less than 0.62),including cellular metabolic proteins,cellular component organization and biogenesis,biosynthetic process, translation,catabolic and cellular localization ect.This study indicated that iTRAQ is a useful technologic method in proteomic alterations study of cultured cell.The proteins revealed in this experiment can be used in the future for studies of p28^GANK downstream regulating molecular mechanism.