Screening And Identification Of Protein Interacting With Tiam1and Its Preliminary Functional Studies

Background and ObjectiveHepatocellular carcinoma (HCC), one of the most common cancers in the world, is the third leading cause of cancer-related deaths worldwide and the second in our country. Population-based studies show an increasing incidence and mortality. Although early diagnosis, surgery and drug therapy have been improved steadily, the five-year survival rate remains low. There are approximately54.1%-61.5%of HCC patient relapse with radical excision. Metastasis and recurrence are the two major factors affecting the outcome. It is an urgent task to work out the metastasis-associated factors and find out the preventive and therapeutic methods so that to decrease mortality and improve overall survial.The development and progression of malignant is a complex multistep process, involving the tumor cells shed from the primary tumor, invade into the blood vessels or lymphatic vessels, migrate and adhere to appropriate site, induce tumor angiogenesis, resistant to antitumor immunity and ultimately form metastases in the distance. There are multiple genes involved in this complex process. At present, most of the researchs associated with the tumor pathogenesis focuse on the function of some single or multiple molecules such as oncogenes, anti-oncogene, small RNA. As gene encoding product, protein acts as the final execution of life activities and plays important roles in tumorigenesis. It remains to elucidate the expression and role of protein in tumor pathogenesis. The proteomics technology carried out in recent years has brought new hope for revealing the mechanisms of tumor development, and research on the biological function of the protein has also become a current research focus.Tiaml protein is a novel metastasis-related protein for HCC found in our previous study. We previously examined the expression of Tiaml in213primary HCC tissue samples with5-year complete clinical follow-up data by IHC. As the result shows, the Tiam1protein expression was strong in HCC tissues. Tiaml is considered to be correlated to the metastasis and overall survial of HCC patients, thus could be served as a biomarker for predicting outcome for HCC patients. Tiaml expression in HCC cell lines was significantly higher than the normal human liver cell line. Focused on the high and low metastastic HCC cell lines (HCCLM3, MHCC97L), we determined that Tiaml overexpression could promote cell proliferation, invasion and metastasis by using gene transfection and RNAi technology. On the contrary, Tiaml knockdown results the reverse. Our study should clarify the molecular mechanism for HCC metastasis and provides a theoretical basis for potential markers that for predicting HCC metastasis.Searching for Tiaml interacting protein is an important way to understand Tiaml protein function. Based on our previous study, we attend to screen Tiaml interacting protein by using yeast two-hybrid, thus to clarify the potential mechanism for HCC development and provide important theoretical basis for early diagnosis and therapeutic targets.Methods1. Screening of protein interacting with Tiaml by yeast two-hybrid1.1Construction of a yeast two-hybrid expression vector with bait protein A recombinant plasmid pGBKT7-Tiaml/C1199for Tiaml expressing in yeast cells was constructed by recombinant gene technique. C1199code domain sequence of Tiaml was amplified from a commercial Tiaml cDNA clone which was then cloned into pGBKT7vector and confirmed by sequencing. Yeast strain AH109cells were transiently transformed with pGBKT7-Tiaml/C1199plasmid. Western blot was performed to confirm that if Tiaml protein could be normally expressed in Saccharomyces cerevisiae without toxicity and autoactivation(Strain AH109).1.2Screening of protein interacting with Tiaml in cDNA library.We used pGBKT7-Tiaml/C1199as a bait to screen the "Universal Human Mate&Plate" Library by performing the classic PEG/LiAc yeast transformation method, and large-scale conversion technologies. Partial positive clones were selected for plasmid extraction, separation library plasmid (AD-X) and bait plasmids, rotary hybrid verification. Some true positive clones were obtained. These positive clones were sequenced and analyzed in the NCBI database.2. Crosslinking immunoprecipitation was applied to validate the interaction between candidate proteins and TiamlBy recombinant DNA technology, we constructed some different epitopelabel (Flag and HA) candidate protein and Tiaml recombinant expression vectors. The recombinant plasmid with different epitopelabel and Tiaml-C1199recombinant plasmids were co-transfected into human embryonic kidney cells HEK293T. Cells were fixed by10ml of1%formaldehyde in PBS at room temperature for10min. sonicated for total cellular protein. Protein, antibody and Protein G were incubated together. Anti-HA and anti-Flag antibodies were used respectively for the recipitation of immune-complexes. And then the immune-complexes were detected by anti-Flag and anti-HA antibody in western blot assay, so that to confirm whether Tiaml is really interect with SETDB1and ZNF307protein. 3. Detection of SETDB1expression in HCC tissues and its clinical significanceSETDB1expression were detected by IHC in87HCC patients’ tissue samples which with complete clinical follow-up data, and was analysed to explore its role in HCC development.4. Lentivirus-mediated silencing of SETDB1and the influence on human HCC cell line.Protein and transcriptional expression of SETDB1were detected in several HCC cell lines by real time PCR and western blot. Four short hairpin RNAs that specific targeted against SETDB1were designed by Designer3.0software (Genepharma).The oligonucleotides were annealed into double-stranded DNA, and then cloned into linearized lentiviral vector to construct an SETDB1interference lentiviral vector. The lentiviral vector plasmid and the helper plasmids were co-transfected into HEK293cells. The viral supernatant was collected and titered.HCC cell lines which is endogenous SETDB1high expressed were transfected with lentiviral containing SETDB1-shRNA. Flow cytometry were used for cell selecting with EGFP gene reported. Real time PCR and western blot were performed to test the RNA interference efficiency of SETDB1. The transfected cell line which exhibited the higest RNA interference efficiency was selected for further study.We employed CCK8assay, plate colony formation and flow cytometry to detect the effect of SETDB1on cell growth and proliferation. As well as we used wound-healing assay and Transwell inserts to compare the migration and invasion ability between the treat and control groups.5. Statistical analysisDatas are analyzed by SPSS13.0software packet. The χ2test (chi-square test) was used to analyzed the relationship between SETDB1expression level and clinicopathological parameters, while Kaplan-Meier survival analysis and Cox proportional-hazard model were applied for analysis of the correlationship between SETDB1expression/other clinicopathological parameters and survival time.The in vitroproliferation and migration ability of different treated cells were compared by factorial analysis and repeated-measures analysis respectively.The results of quantitative PCR, colony formation assay, cell cycle assay, invasion assay were analyzed by One-way ANOVA analysis, with LSD method and Dunnett’s T3method were used according to the homogeneity of variance test. P<0.05are considered to be statistically significant different.Results1. Tiaml-interacting protein by yeast two-hybrid screening1.1A bait protein, pGBKT7-Tiaml/C1199, was successfully constructed.The Tiaml/C1199truncated protein was expressed well without either toxicity nor auto-activating ability, when pGBKT7-Tiam1/C1199plasmid was transduced into AH109yeast strain. So that this bait could be used for the yeast two-hybrid screening.1.2Tiaml-interacting protein was screened by yeast two-hybridWe used pGBKT7-Tiaml/C1199as a bait to screen the "Universal Human Mate&Plate" Library. And finally we got a total of24positive clones, those were were sequenced and analyzed in the NCBI database, exactly match to6known proteins, including OSBPL1A, ZKSCAN4,(also known as ZNF307), FNDC3B, SRSF5, SYCP1and SETDB1. After literature mining and bio informatics analysis, we select SETDB1and ZNF307for further verification study.2. Identification of Tiaml-interacting proteinCrosslinking immunoprecipitation was applied to validate the interaction in v/vobetween candidate proteins (SETDB1and ZNF307) and Tiaml.The results showed that Tiaml and Flag-SETDB1protein could be detected by corresponding antibody when we do the IP by anti-Flag antibody. This result could prove a real interaction between Tiaml and SETDB1. However, we couldn’t get positive result when IP with anti-HA antibody in other direction. As far as now, we haven’t gotten a real positive result that could confirm the interaction of ZNF307and Tiaml.3. Expression of SETDB1in HCC and its clinical significanceImmunohistochemical results showed that SETDB1protein positive signals mainly localized in the cytoplasm, interstitial expression found only in isolated cases. We observed that SETDB1protein in tumor tissues showed lower expression level (positive to strongly positive,49in54) than their adjacent tissue (negative to weak positive). Results showed that SETDB1expression does not correlate with any of the clinicopathological parameters such as sex, age, tumor size, HBsAg, serum AFP, metastasis and recurrence (P value are0.140,0.090,0.927,0.245,0.091,0.102,0.245,0.068, respectively), whereas correlate with the tumor differentiation and cirrhosis of the liver.Our Kaplan-Meier survival analysis revealed that patients with SETDB1overexpression exist a longer median survival time (48m versus19m), which was significantly different (Log Rank=4.106, P=0.043<0.05). By univariate analysis of Cox proportional-hazard model, SETDB1low expression is closely related to the poor prognosis of the patients. Age, HBsAg, serum AFP, recurrence, and metastasis are associated with the survival of HCC (P value are0.009,0.010,0.018,<0.001,0.010, respectively). However, SETDB1expression could not server as an independent predictor for prognosis as the results showed in Cox proportional-hazard model.4. SETDB1expression in HCC cell lines were detected by real time PCR and western blotWe found that SETDB1expressed differently in8primary liver cancer cell lines. SETDB1mRNA expression in MHCC97L and Bel-7402are lower than that in SMMC-7721and HCCLM3. The result of western blot is quite the same with real time PCR. Finally, we selected HCCLM3, which is SETDB1endogenous high expressed for further study.5. Effects of SETDB1gene silencing on the biological features of HCC cells.We employed CCK8assay to detect the effects of SETDB1on cell growth. By comparing thegrowth curves of M3/SET-and its controls (M3parental and M3/m ock). As time passed by, M3/SET-was found to significantly suppress cell growth, there are significantly different among the three groups (different groups:F=424.808, P<0.001; different time piont:F=1100.419, P<0.001, cross action:F=44.516, P<0.001).Similarly,SETDB1silencing significantly reduces colony formation ability of HCCLM3cells relative to parental cells and cells transfected with empty vector (F=198.358,P<0.001).Flow cytometry revealed that endogenous SETDB1silencing could arrest Gl phase of cell cycle, which directly result in a reduction in proliferation. In a wound-healingassay, evident suppression in the wound closure rate wasobserved in suppression cells at72hr compared with the controls (cross action:F=44.516, P<0.001). The number of cells that passed through Matrigel after incubated for42h was as nearly six-fold lower than those in the mock and parental cell groups (F=721.0, P <0.001). All of these reveal that SETDB1silencing could reduce cells’ proliferation, migrate and invasive ability in vitro.Conclusions1. We successfully construct bait plasmid pGBKT7-Tiaml/C1199, by using traditional PEG/LiAc method and large tlassic PEG/LiAc yeast transformation method, and large-scale conversion technologies, we screened the "Universal Human Mate&Plate" library, and confirmed by reverse hybrid assay, and we finally obtained6positive potein interacting with Tiaml. 2. With plasmids co-transfected HEK293T cells, we indentified the interaction between Tiaml and SETDB1intracellular through Crosslinking IP.3. IHC assay and statictic analysis were performed to explore the relationship between SETDB1and HCC. By univariate analysis of Cox proportional-hazard model, SETDB1low expression is closely related to the poor prognosis of the patients. Age, HBsAg, serum AFP, recurrence, and metastasis are associated with the survival of HCC patients. Multivariate analyses revealed that Age, HBsAg, recurrence, and metastasis but SETDB1expression may play a role in predicting the overall survival in HCC patients. However, SETDB1expression could not server as an independent predictor for prognosis in this study.4. SETDB1silencing could suppress HCC cells proliferation, migration and invasion ability.Innovations1. We screened and identified SETDB1interacte with metastasis-related protein Tiaml by using the yeast two-hybrid system combined with the Cross-linked immune precipitation technology. It provides new opportunities for researching the molecular mechanisms of metastasis of HCC.2. We successfully constructed a cell line stably express low SETDB1, and confirmed the function of SETDB1in HCC, which should provide an new insight for clarifying the molecular mechanism of Tiaml-SETDB1in the pathogenesis of HCC.Weakpoints1. All of our work are not sufficient for elucidating the direct or indirect interaction between Tiaml and SETDB1. GST pull down and Immunofluorescent techniques could compensate it.2. This study is still unable to confirm the value that expression level of SETDB1serves as a prognosis independent predictor of HCC, which need a further validation in a larger set of sample.3. The biological function of SETDB1gene in liver cancer is required in vivo further experimental verification.