Preliminary Screening And Verification Of Protein Interacting With HSPC238

Background and ObjectivesPrimary hepatocellular carcinoma (HCC) is a clinical common malignant tumor, in the recent years, HCC incidence rate was increased gradually with the increasing infection rate of hepatitis B and C virus in the world, there are reports that the new cases of HCC each year are more than 700 thousand. At present, the HCC is the fifth most common malignant tumors in the world, and ranked the third in the death rate of cancer. Only about 15% of the HCC have the opportunity to be cured by surgery (such as, liver resection or transplantation) or minimally invasive local treatment (such as, radiofrequency ablation). With the improvement of medical imaging technology, the early diagnosis and treatment of HCC have made great progress, but the long-term prognosis is still not satisfactory, some scholars have reported that the average survival rate of patients with HCC is only 8.9%.Most HCC were caused by the development of chronic liver disease (such as hepatitis B, hepatitis C, alcoholic liver disease, etc.), but there are still 15-50% of new cases of the etiology is not clear. The early metastasis of HCC is an important factor which affecting the clinical prognosis, in this complex process, involving multiple genes and proteins. At present, the research of the mechanism on tumor occurrence development are mostly focus on the role and function of some single or multiple moleculars, such as oncogene, anti cancer genes and small RNA molecules, while the protein as the coding product of gene is the final executor of life activities, its expression in the tumor, and its role in the tumor development are remained to be elucidated. With the development of molecular biology and proteomic technology, the proteins which perform biological functions in the cell have been emphasized again, the interactions between protein molecules as one of the main content studies of proteome has become the research hotspot in the recent years. The interaction between proteins can not only provide information of protein function, and can provide information in the metabolic regulation, signal transduction and the role in the proteins complex, it brings new hope to reveal the mechanism of tumor genesis and development.Zinc finger protein have a finger like structure, it often appears in DNA binding protein, and it belongs to a family of eukaryotic transcription factors. Zinc finger protein domain is composed of cysteine, histidine residues and zinc ion. According to the combination way between the zinc ion and the two kinds of amino acids, zinc finger protein can be divided into C2H2, C3H, C4 and some other types. Many zinc finger proteins are involved in protein RNA and protein protein interactions in addition to the identification of specific DNA sequences. In this study, the HSPC238 protein, which genome length was 716 bp, encoding 153 amino acids, its 40 to 60 amino acid residues can form a special RING finger domain, so it is also a kind of C3HC4 type zinc finger protein.In the previous studies, we found that HSPC238 protein is a new tumor suppressor protein. We carried out immunohistochemical detection on 139 cases of HCC tissue samples which have complete clinical and follow-up data. The results showed that, the expression level of HSPC238 in normal liver tissue was significantly higher than that the HCC tissue, which have no correlation with the patient’s age, sex and clinical stage, but have a negative correlation with the degree of differentiation of HCC; we changed the expression of HSPC238 in SMMC7721 cell lines in vitro, and found that upregulated the expression of HSPC238 can inhibit the proliferation and colony formation of SMMC7721 cell lines, down regulated the expression of HSPC238 can promote the proliferation and clone formation of SMMC7721 cell lines. Further through the tumor formation experiment by nude mice in vivo, we confirmed that upregulated the expression of HSPC238 can inhibit the proliferation of tumor cells in nude mice, and disturbed the expression of HSPC238 can promote the proliferation of tumors in the nude mice. Further study found that, the HSPC238 exerts its inhibitory effect on HCC by ERK/MAPK pathway. These preliminary results provide a new theoretical basis and research targets for the exploration of the mechanism of tumor suppression and the searching of new potential biomarkers for HCC diagnosis.In addition to the above results, there was no report on the function of HSPC238 protein in HCC at home and abroad, for further research on the tumor suppressor mechanism of HSPC238 in HCC, this study was aimed to screen the target proteins that interaction with HSPC238 by yeast two hybrid system, and further to validate the screened target proteins by laser scanning confocal microscopy technology, co-immunoprecipitation and pull-down assay, so as to lay a foundation for the comprehensive study of the function of HSPC238 and its mechanism in HCC.Method:1. Construction of recombinant bait plasmid vector, yeast transformation and self activation detection.1.1 construction of recombinant bait plasmid vectorThe plasmid vector (plasmid pUC19-HSPC238. Constructed and saved in the previous studies) which have loaded the artificial synthetic gene HSPC238 was transformed in Escherichia coli TOP 10 competent cells for amplification and plasmid extraction. In order to obtain a sufficient amount of the target gene fragments. The obtained plasmid (pUC19-HSPC238) which passed electrophoresis and DNA sequencing analysis was connected to the yeast two hybrid bait plasmid pGBKT7 after double enzyme digestion, we obtained the recombinant vector pGBKT7-HSPC238 of bait protein (HSPC238) initially, the connection system transformed in Escherichia coli TOP 10 once again to amplify the recombinant plasmid pGBKT7-HSPC238, three transformants which randomly selected were inoculated to liquid LB medium with the kanamycin resistance for overnight, then the plasmid was extracted and identified by electrophoresis, further to identify the inserted target fragment (HSPC238) by the enzyme digestion.1.2 Yeast transformation and self activation detection of bait vectorThe pGBKT7-HSPC238 vector which passed identification were transformed into yeast AH 109 cells with pGADT7 vector by Lithium acetate method, and set a positive and negative control at the same time according to the kit instructions, coated with different nutritional defect plate respectively, through the blue white spot screening and nutritional defect screening, to judge whether the bait protein HSPC238 has toxicity and self activation activity to yeast AH 109.2. Screening of human fetal liver cDNA Library2.1 Amplification, extraction and transformation of library plasmidCoated the LB+Amp plate for amplified culture with the library bacteria after diluted, then extracted the library plasmid, and to detect the concentration and quality of the library plasmid. The library plasmid was transferred to the yeast AH 109 which contained the correct pGBKT7-HSPC238 vector, and then observe the results.2.2 Identification of positive clones and Plasmid ExtractionThe screen plates (SD-Trp-Leu-His) were cleared by flannelette to eliminate the influence of colony growth background, the transformants grew again were selected to screen by SD-Trp-Leu-His+3AT, SD-Trp-Leu-His-Ade and blue white spot experiment in turning, the positive clones which passed the three reporter genes (LacZ, HIS3 and ADE2) simultaneously were inoculated to liquid SD-Leu, then extracted the oscillation yeast plasmid after culture overnight, further translated to Escherichia coli TOP 10 for proliferation culture, transformants were transfered to LB liquid which containing Amp for culture after the extraction of plasmid.2.3 BLAST comparison analysis and rotation verification of positive clonesThe plasmids were used for DNA sequencing and BLAST comparison, and combined the bioinformatics analysis, to confirm the gene name of the target protein which contained in the positive clones. The plasmid which extracted from the positive clones and the bait plasmid (pGBKT7-HSPC238) were co-transformed to a yeast cells respectively, the activation of 3 reporter genes (LacZ, HIS3, and ADE2) in co-transformed yeast cells of each pair proteins was tested again. Among them, the longest ORF sequences were selected in the repeated positive clones. Finally, according to the literature analysis, five possible target proteins that may interact with HSPC238 are used for the follow-up studies.3. Identification of the interaction between HSPC238 and target proteins3.1 Detection by the laser confocal localizationThe over expression plasmids of HSPC238 and the target proteins were constructed, among them the HSPC238 protein carried a Flag tag (pcDNA3.1- HSPC238-Flag), and the target proteins carried a His tags (pcDNA3.1-target protein-His). PcDNA3.1-HSPC238-Flag plasmid and the pcDNA3.1-target protein-His plasmid were transiently co-transfected to SMMC7721 and 293T cells respectively, added the primary antibodies (rabbit origin anti-HSPC238, mouse origin anti-target protein), and secondary antibodies (Goat-anti-Rabbit-CY3, Goat-anti-mouse-FITC) in turn, and then incubated. The co localization of HSPC238 and target proteins in 293T and SMMC7721 cells was observed by laser scanning confocal microscope, in order to confirm whether the target protein and HSPC238 protein could meet in the cell.3.2 Co-immunoprecipitation analysis whether there is an interaction between HSPC238 and target protein in vivoThe HSPC238 overexpression plasmid (pcDNA3.1-HSPC238-Flag) and the target protein overexpression plasmid (pcDNA3.1-target protein-His) were co-transfected to SMMC7721 and 293T cell lines respectively, splitted the cells and gathered the protein supernatant, the anti-Flag antibody was used for co-immunoprecipitation to obtain the coprecipitate, the anti-His and anti-Flag, anti-HSPC238, anti-target protein were used for Western blotting, compared with the cell lysis solution, to confirm whether there is interaction between the HSPC28 and the target proteins.3.3 Pull-Down analysis whether there is an interaction between HSPC238 and target protein in vivoThe HSPC238 overexpression plasmid (pcDNA3.1-HSPC238-Flag) and the target protein overexpression plasmid (pcDNA3.1-target protein-His) were co-transfected to SMMC7721 and 293T cell lines respectively, splitted the cells and gathered the protein supernatant, nickel column was used for the purification of the target protein which carried His tag, the concentration of the eluted liquid was measured by the BCA method, then the highest concentration was selected for point sample, the anti-His and anti-Flag, anti-HSPC238, anti-target protein were used for Western blotting, compared with the cell lysis solution, to confirm whether there is interaction between the HSPC28 and the target proteins.Results:1. Construction of recombinant bait plasmid vector, yeast transformation and self activation detection.After amplification, the pUC19-HSPC238 plasmid and the bait plasmid pGBKT7 which digested by the double enzyme were connected successfully by the T4 DNA ligase, the connection system translated to the Escherichia coli TOP 10 for amplification and then extracted plasmids for electrophoresis and sequenced. The results showed that:No mutations occurred in the all sequence including the sfiI enzyme cleavage site, and the recombinant plasmid pGBKT7-HSPC238 was successfully constructed; further transformed into AH109 yeast cells, the three reporter genes (lacZ and his3 and ade2) were not activated through the blue white spot screening and nutritional deficiencies screening, so the bait protein HSPC238 does not exist toxicity and self activation on the AH 109 yeast.2. Screening of human fetal liver cDNA LibraryAfter dilution, the library was amplified and extracted, and the concentration of plasmid was 600ng/ul finally. Coated the LB+Amp resistance plate and then the single clones were picked for Plasmid Extraction and detected by agarose gel electrophoresis, all the gained library plasmids were inserted into the target genes of different bands, that is, the library quality was qualified.The library plasmid were transfected into the yeast AH 109 which containing the correct pGBKT7-HSPC238 vector, cleared the screen plates (SD-Trp-Leu-His) by flannelette, continue to cultivate for two weeks, we obtained 124 transformant colonies preliminary. Transfered to SD-Trp-Leu-His+3AT plate, the 11 false positive colonies were excluded due to the His3 gene seepage in the AH 109 bacterial strain. Transfered to the SD-Trp-Leu-His-Ade plate once again, the 7 false positive colonies which could not activate the HIS3 and ADE2 reporter genes were excluded. Finally, we detected the LacZ reporter gene, through the blue and white spot screening, the 42 false positive colonies that could not become blue were excluded once again, and finally we got 64 suspicious colonies which may presence proteins that interacted with HSPC238.The 64 suspicious positive clones which passed 3 reporter genes (LacZ, HIS3 and ADE2) were transferred to the liquid SD-Leu medium, after amplification by cultivation, we extracted the yeast plasmid, then we transformed into Escherichia coli Top 10 for amplification. The transformants were transferred into LB liquid medium which containing Amp for amplification and then extracted the plamids, the plasmids were used for DNA sequencing and blast comparison combined with bioinformatics analysis, and showed that they encode 19 different proteins. And the positive colonies which coded the 19 protein were verified by rotary verification, finally, we reviewed the literature and selected five kinds of protein (HMOX1, MT2A, RPS27A, UBB, RPL5) which may most likely have the interaction with HSPC238 for the follow-up study.3. Identification of the interaction between HSPC238 and target proteinsThe results of laser confocal microscopy showed that, HSPC238 protein and the target proteins (HMOX1, MT2A, RPS27A, UBB, RPL5) are distributed in the cytoplasm in 293T and SMMC7721 cells, they have a height coexistence in the cytoplasmic, that is they have the possibility of interaction in the cytoplasm.In the preliminary experiments of co-immunoprecipitation, the plasmids of pCDNA3.1-RPL5-His were transfected into 293 T cells transiently, the anti-His was used for many times, but still unable to detect the expression of RPL5 protein, so the RPL5 protein was excluded, so the HMOX1, MT2A, RPS27A and UBB were used as the research object in the subsequent experiments only.The results of co-immunoprecipitation experiment showed that, we used the anti-Flag antibody to obtain the subsidence from the supernatant protein that collected from the cell lysates of the co-transfection group, then the anti-His, anti-Flag, anti-HSPC238 and anti-target protein (HMOX1, RPS27A, UBB, MT2A) were used for Western blotting, all of them can detect the correct size of the bands, and the expression location are the same as in the cell lysis solution, and thus we hypothesized that HSPC238 and the target protein (HMOX1, RPS27A, UBB, MT2A) had a combination in 293T and SMMC7721 cell lines.The results of pull-down assay showed that, we used the Ni column to purificate the protein complexes which carried His tag from the supernatant protein that collected from the cell lysates of the co-transfection group, the group of the highest concentration of elution was selected for sample points, then the anti-His, anti-Flag, anti-HSPC238 and anti-target protein (HMOX1, RPS27A, UBB, MT2A) were used for Western blotting, the results showed that the expected target bands could be detected in all the elution solution, and the location of the electrophoretic bands was consistent with the pyrolysis liquid group. So we presume that there is an interaction between the HSPC238 and HMOX1、RPS27A, UBB and MT2A.Conclusion:The eukaryotic expression vector of HSPC238 was successfully constructed in this study, the four target proteins (HMOX1, RPS27A, UBB, MT2A) which may interact with HSPC238 was screened by yeast two hybrid technique from the human fetal liver cDNA library, several specific proteins were locked preliminary after literature analysis, and then the interaction between them with HSPC238 was confirmed by laser confocal, co-immunoprecipitation and Pull-Down experiments in the cells. This study laid a foundation for further study on the function of HSPC238 and the mechanism of action in HCC.