Structural And Functional Prediction And Molecular Function Study And Clinical Diagnosis Application Of P62/IGF2BP2

Backgroundp62/IGF2BP2was identified as tumor associated antigen, named as insulin-like growth factor2mRNA binding protein2(IGF2BP2) by using cDNA expression library immune-screening approach. Since molecular size of this protein is62kDa, this protein was also called p62/IGF2BP2.The tumor antigen is referred to when the process of cancerous cells, the emergence of some of the new antigens and excessive expression of antigenic material collectively. Numerous studies showed that the serum samples from cancer patients contain antibodies against some cellular proteins, and these antibodies are called autoantibodies, some of them also called anti-TAA antibodies. The detection of anti-TAA antibodies in the sera from patients with cancer might be used for early diagnosis and prognosis of this disease. Many researchers have considered using a panel of multiple TAAs to enhance anti-TAA antibody detection and further to evaluate their significance in tumorigenesis.Objective1. The prediction on secondary structure and protein property of protein p62/IGF2BP2and proteins interacted with it, aims to provide the initial data for designing protein crystal and third structure prediction and useful guidance for its molecular function and signaling pathway;2. To investigate the effects of short hairpin RNA(shRNA) of p62gene on the cell growth by using RNAi technology and provide experimental evidence for clinically immune diagnosis of cancer;3. To evaluate the diagnostic value of p62with other TAAs and develop a valid and reliable method on breast cancer.Method1. Web servers of PSIPRED2.0, SSpro, Jpred, PDB, PROF-predictprotein and software of DNASTAR were used to predict the secondary structure(helix, strand, coil) and function of the p62protein by inputting its protein sequence;2. String web server and DNASTAR software were used to predict protein property and proteins interacted with p62;3. The hMGFP-p62-shRNA interference plasmid was built. After stable transfection into SNU-449cells by Lipofectamine2000, fluorescence microscopy was used to observe transfection efficiency, Western-blot examined p62protein expression and flow cytometry tested cell cycle of the transfected cells.4. The antibodies against Impl,p62,Koc, p53, c-myc, survivin, p16, cyclin B1, cyclinDl, CDK2in sera with patients in breast cancer and normal human serum were detected by enzyme-like immunosorbent assay(ELISA).Results1. The results show that the p62protein has a higher epitope index. The percentage of helix, strand, coil might be suspectively21%,28%,51%.2. The nine proteins were predicted to interact with p62, which are IGF2(insulin-like growth factor2), CDKAL1:(CDK5regulatory subunit associated protein1-like1), SLC30A8(solute carrier family30, member8), HHEX (hematopoietically expressed homeobox), TCF7L2(transcription factor7-like2), FTO (fat mass and obesity associated), TCF7(transcription factor7), WFS1(Wolfram syndrome1), KCNJ11(potassium inwardly-rectifying channel, subfamily J, member11).The prediction of protein interacted with p62may indicate that the p62protein not only play a role on IGF2expression, but also can be a part of activities about Zn(+) transportation, adjusting Ca(2+) concentration in cell and transcription activation of T lymphocyte differentiation.3. After the four p62RNA interference plasmids (hMGFP-p62-shRNA) with different target sequence (211-231,1102-1122,1126-1146,1369-1389) was transfected to SNU-449cells, results showed that the amount of p62protein with target sequence (1369-1389) expression obviously decreased, which suggests the p62RNA interference plasmid with target sequence(1369-1389) can silence the expression of p62/IGF2BP2in SNU-449cells. Whereas, the proliferation of SNU-449cells was significantly inhibited, which indicates p62promote cell growth of SNU-449cells.4. The antibody frequency to the individual TAAs in breast cancer was ranged between7.3and22.0%. This relatively low sensitivity using one individual antiTAA antibody as a diagnostic marker does not meet the requirements of clinical early diagnosis of breast cancer. With the successive addition of TAAs to a total of eight antigens, there was a stepwise increase in positive antibody reactions, reaching a sensitivity of61.0%and a specificity of89.0%. The positive and negative likelihood ratios were5.545and0.438, respectively, which showed that the clinical diagnostic value of a parallel assay of eight TAAs was high. The PPVs and NPVs were73.5and82.0%, respectively, indicating that the parallel assay of eight TAAs raised the diagnostic precision significantly. The agreement rate and K-value were79.7%and0.52, respectively, which indicated that the observed value of this assay had a middle range coincidence with the actual value.Conclusion1. The p62protein is a flexible soluble protein with relatively smooth surface.2. The p62may have a significant effect on stable and concentration of ions involved in tumor cells so as to affect tumor growth and differration.3. The most effective target sequence silencing p62gene expression is1369-1389. The p62protein expression can be decreased using RNAi in SNU-449cells; the hMGFP-p62-shRNA interference plasmid can silence p62gene expression; The p62protein promote the proliferation of human liver cancer cells.4. It was demonstrated that a mini-array of eight TAAs(c-myc, survivin, cyclinBl, cyclinDl, p62,p53, p16,CDK2) could increase the sensitivity and specificity for diagnosis of breast cancer and the approach could be applicated to screen breast cancer patients from population at high risk in breast cancer, and be used as a routine test in clinical practice.