| Backgrounds:Hepatocellular carcinoma (HCC) is a common solid malignant tumor which leads to the third largest cause of death compared to other cancers and has become a threat to all mankind as a public healthy problem. Genetic and environmental factors are involved in the pathogenesis of HCC. A number of researches have reported that the abnormalities of the pathways and cytokines involves in HCC are very important. Epidermal growth factor (EGF) and epidermal growth factor receptor (EGFR) can stimulate the proliferation of epidermal and epithelial cells. The EGF signal pathway has a relationship with the growth of the embryo, tissue repairing, and tumorigenesis. EGFR is a kind of membrane receptor, and it can be detected to be over-expressed in a variety of malignant tumors. EGFR can accelerate the propagation of tumor cells, promote tumor angiogenesis, accelerated tumor metastasis, and inhibit tumor apoptosis when activated. In the present study, we used a case-control study to investigate the association between the susceptibility of HBV-related disease and the host genetic factors with EGF and EGFR as candidate genes.Methods:In this study,416patients with HBV-related HCC and765patients with chronic hepatitis B as control I and645individuals who had never been infected with HBV of Chinese Han population as control â…¡ were enrolled. Eight single nucleotide polymorphisms (SNPs) whose minor allele frequency (MAF)>20%in the EGF and EGFR genes were genotyped to examine their associations with hepatocarcinogenesis. Genotyping experiments were carried out using TaqMan method.Results:There were significant differences in genotype distributions (.P=0.005) and allele frequencies (P=0.001, OR=1.43,95%CI=1.15-1.79) of rs11569017in EGF gene between HCC and control II groups. After binary logistic regression to determine independent factors for susceptibility to HCC under additive model, rs11569017was still independently associated with the susceptibility to HCC (P=0.021, OR=1.48,95%CI=1.06-2.07), but no significant differences in other SNPs were found. Additionally, the haplotype T-G constructed by rsl1569017and rs4444903of EGF gene might increase the risk of HBV-related HCC (P=0.002, OR=1.44,95%CI=1.15-1.82).Conclusion:The rs11569017T allele was associated with susceptibility to HBV-related HCC. Background:Varieties of factors impact the development of tumor, among which transcription factor located in the nuclear plays a key role. SP1(Specificity Protein1) transcription factor is a transcription factor expresses ubiquitously in mammal, some studies found that SP1is widely regarded as a cancer-promoting factor which is highly expressing in lung cancer, breast cancer, liver cancer, pancreatic cancer and other malignancies. Researches had demonstrated clearly that SP1could promote the growth of vascular endothelial and apoptosis, and it could also adjust the degree of phosphorylation itself, moreover it may also modulate the transcriptional activity of its downstream genes. Based on the important role in the regulation of tumor-related transcriptional activity of SP1protein, there were important implications to purified SP1protein for studying the way in the transcriptional regulation of genes related to tumorigenesis. As SP1protein exists in a form of insoluble inclusion bodies (IBS), the processes were needed as denaturing, dissolved, and then purified by Ni-NTA column to get the purified protein. Finally, the purified protein was dissolved in8M urea, then the urea should be removed or it would cause the degeneration of the protein. However, the removal of urea will lead to a protein re-precipitation, so we need to carry out refolding experiments to find suitable conditions to re-folded the correct natural structure as soluble SP1protein.Objective:1, to clone and express the full-length SP1protein to obtain the purified recombinant protein.2, to make use of the active and purified protein SP1to investigate the combination between SP1and initiation region of transcription and translation on transcriptional level.Methods:1, to get human SP1cDNA sequence from the extraction of total RNA from cultured HeLa cells by RT-PCR.2, to clone the full-length SP1to the expression vector, named SP1-pET28a, which was identified by sequencing.3, the recombinant plasmids were transformed into BL21(DE3) competent cells, IPTG was used to induce expression.4, the bacteria was broken by ultrasound, and purified the inclusion bodies under denaturing conditions. We dialyzed SP1protein and concentrated it by ultrafiltration. The purity of the protein was identified by the method of SDS-PAGE and Western blot, and the concentration of the purified protein was detected by Bradford. We used EMSA to detect the activation of SP1protein in vitro.Results:1, the SP1expression vector was constructed successfully, which has been sequenced to ensure the right clone.2, the SP1protein with biological activity in vitro was purified.Conclusion:1.The expression of full-length SP1protein can be relatively simple to get using pET expression system.2. Adding Zn2+to refold the inclusion bodies is the most important content to dialysate. |
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