Panning And Preliminary Functional Analysis Of Proteins Bound By Hepatocarcinoma Homing Peptides

Human hepatocellular carcinoma (HCC) is one of the most common and deadly cancers in the world. China is one of the most widespread countries of HCC. At least 120,000 people in China were dead from it every year. However, there are no efficient clinical methods for radical cure of HCC, the reason of which is that we still know little about the mechanisms of HCC carcinogenesis and development and that we should do further studies on the special biological characteristics and molecular mechanisms of HCC. Therefore, it is of high theoretical and practical value to strengthen researches in HCC basic theory and to improve efficient technologies in early diagnosis and therapy.The preceding work of this research is the panning of hepatocarcinoma specific homing peptide by phage display technology. During the preceding research, several panning methods had been established, especially the in vivo panning technology based on nude mice animal model, and a series of identification technologies for detecting the in vitro and in vivo target effect of hepatocarcinoma homing peptide. So far, several specific hepatocarcinoma homing peptides have been gained, and several Discovery Patents of China for them had been obtained. Based on the above work, in this paper, several specific hepatocarcinoma homing peptides we had got (A54, WP05and JY96) were used to screen peptide-binding proteins which is on the HCC cells and can interact with those homing peptides, by several methods such as cDNA library screening and protein-affinity isolation. Then the correlation between those proteins and hepatocarcinogenesis as well as proteins and HCC development was analyzed.This paper can be divided into three parts: A. Identification of hepatocarcinoma homing peptide binding activity in vitro.A series of phage peptides specifically recognizing HCC cells had been obtained during the preceding research. However, it should be further verificated whether the chemically synthesized peptides keeps the homing characteristics. The peptides WP05, JY96, A54 were chemically synthesized and marked with FAM. The specific binding of homing peptides to HCC cells was verificated via the in vivo binding activity of them. Through verification, we found that chemically synthesized peptides WP05, JY96, A54 indeed bind specifically to HCC cells in vivo, which through the theoretical foundation for isolating HCC cell proteins bound by homing peptides.B. Construction of a hepatocarcinoma cDNA expression library and panning of proteins bound by specific HCC homing peptides.Proteins bound by hepatocarcinoma homing peptides WP05, JY96 and A54 were isolated by the strategy of screen of cDNA expression library. A hepatocarcinoma cDNA expression library was constructed using human transplantation tumor bear by nude mice. The titer of original library is 6.1 ×10⁷pfu/ml, recombination rate is 99.3%. While after amplification, the titer grows to 1.4×10¹¹ pfu/ml, which reaches the required criteria for constructing a library to screen and isolate target genes. The cDNA library was screened by homing peptides as probes. Every peptide was used for two kinds of screening, which were solid-phase bio-screening and immunology screening. Gene SNX27 was obtained by screening with WP05 and gene RBX1 was obtained by screening with JY96.C. Effects of SNX27 gene expression inhibition by siRNA on hepatocellular carcinoma.SNX27 is a gene obtained through screening based on homing peptide WP05 and is up-regulation in hepatocarcinoma. The correlation between SNX27 expression and hepatocarcinogenesis as well as HCC development was expected to be analyzed by RNAi. The target sequence of SNX27 siRNA was designed by online software and the eukaryotic expression vector of SNX27 siRNA named pAS/SNX27i was constructed. pAS/SNX27i was transfected into BEL-7402 cells with transfection reagent Sofast^TM and the inhibition effects of RNAi was analyzed. The proliferation, adhesion and migration of cancer cells after SNX27 expression being inhibited were detected. Results show that after transfection, the inhibition rate of SNX27 gene expression reached to 29.5%, cell proliferation was significantly inhibited; cell adhesion was significantly inhibited, different from that of control with P=0.00084<0.005; cell migration was significantly inhibited, different from control with 0.05>P=0.0016>0.001.D. Effects of RBX1 gene expression inhibition by siRNA on hepatocellular carcinoma.RBX1 is a gene obtained through screening based on homing peptide JY96. The correlation between RBX1 expression and hepatocarcinogenesis as well as HCC development was expected to be analyzed by RNAi. The sequence of RBX1 siRNA was designed by online software and the eukaryotic expression vector of RBX1 siRNA named pAS/RBX1i was constructed. pAS/RBX1i was transfected into BEL-7402 cells with transfection reagent Sofast^TM and the inhibition effects of RNAi was analyzed. The proliferation, adhesion and migration of cancer cells after RBX1 expression being inhibited were detected. Results show that after transfection, the inhibition rate of RBX1 gene expression reached 68.6%, the speed of cell proliferation almost totally inhibited; cell adhesion was significantly inhibited, different from that of control with P= 0.0003<0.005 ; cell migration was significantly inhibited too, different from control with P= 0.0007<0.005. E. The study on isolation of proteins bound by specific HCC-binding peptides.To obtain the protein bound by specific hepatocarcinoma homing peptides A54, the proteins from HCC tissue were screened by affinity chromatography. The tumor tissue from nude mice model was used for the extraction of HCC tissue proteins. With biotin-A54 as affinity mediator, proteins, which might interact with A54, were isolated from the protein extraction of HCC tumor tissue. The 67kDa eluate was found to be a mixture of several proteins including a potential tumor membrane marker HSC71. By bioinformatics analysis, we found protein HSC71 has a three-dimensional structure which supporting its interaction with A54. From all the above, we can conclude that:â… . Hepatocarcinoma homing peptide WP05, JY96 and A54 were identified indeed bind specifically to HCC cells in vivo.â…¡. A full-length cDNA expression library for human hepatocarcinoma has been successfully constructed by SMART technology. It reaches the required criteria for constructing a library to screen and isolate target genes.â…¢. A gene SNX27 was obtained by screening full-length cDNA expression library with Biotin-WP05 peptide.â…£. A gene RBX1 was obtained by screening full-length cDNA expression library with Biotin-JY96 peptide.â…¤. siRNA eukaryotic expression vectors pAS/SNX27i had been successfully constructed. The inhibition rate of gene expression reached 29.5%. The proliferation, adhesion and migration of hepatocarcinoma cell were inhibited after transfection.â…¥. siRNA eukaryotic expression vectors pAS/RBX1i had been successfully constructed. The inhibition rate of gene expression reached 68.6%. The proliferation, adhesion and migration of hepatocarcinoma cell were inhibited after transfection.â…¦. Affinity chromatography was tried to isolate proteins bound by hepatocarcinoma homing peptide A54. A mixture of 67kDa protein was obtained, including HSC71.HSC71 has a dimensional structure via which HSC71 may interact with A54.