| Primary hepatocellular carcinoma is currently representing the third leading cause of male-related deaths worldwide.Hepatocellular carcinoma(HCC)is an aggressive and high mortality malignancy with the five-year survival rate less than 5%in China,HCC serious threat to human life and health and in urgent need of developing effective treatments.Many therapeutic methods have been tried in cancer including HCC,such as systemic therapy that combined multiple drugs、molecular target therapy and immune therapy(current research interests).Same as other cancer,the molecular targets of therapy is a good example of HCC but still making slow progress.Gab2 is a scaffold protein which is similar to the insulin receptor substrate,can integrate a variety of signals and play an important role in the signal network.In our previous work,Gab2 was found to be the key protein of fatty liver disease.Otherwise,Gab2 can also promote the proliferation,invasion and metastasis of HCC cells and promote the development of hepatocellular carcinoma through the integration of Jak2/Stat3 signal pathway.Therefore,Gab2 may serve as a key node for the integration of HCC signaling,the development of disease is promoted by the combination of various signals induced by disease factors.HCC often involves a complex mechanism,the therapies of single or a small amount of signal therapy are minimally effective,but the therapies of Gab2 could potentially change the signaling pathways of multiple diseases,effectively changing the direction of disease.Therefore,reducing the expression level of Gab2 may be an effective way to prevent and treat liver cancer.Unfortunately,the current mechanism of gene expression regulation of Gab2 gene is still unknown,restricting the prevention of liver cancer of Gab2.Therefore,our study focus on the regulation mechanisms of Gab2 gene transcription have been found,which provide the basis for the development of Gab2.We first amplified different length of DNA fragments in Gab2 gene promoter,inserted it into pGL6-Basic luciferase expression vector,tested the transcriptional activity of different length of DNA fragments in Gab2 promoter by using Dual-Luciferase assay,finally identified the core regions of Gab2 gene promoter in HepG2 cell.Based on this transcriptional active region,we then predicted the transcription factor binding site of Gab2 promoter region by using the bioinformatics software JASPER,Match and PROMO,and found multiple candidate transcription factors.Based on these transcription factors,we selected the most likely transcription factor,Foxd3,for further testing.To investigated whether Foxd3 indeed binds to the Gab2 promoter region,we using ChIP-qPCR and the result showed that the Gab2 promoter was specifically pulled-down by a Foxd3-specific antibody.The results suggest that the transcription factor Foxd3 do binds to the Gab2 promoter region.Then a single base mutation was performed on the possible binding site of the Foxd3 on Gab2 promoter,identified the core binding site of Foxd3.Transcription activity analysis also showed that Foxd3 inhibits the transcription expression of Gab2 genes.These results also confirmed that Foxd3 was a transcription factor for Gab2 gene expression,which was combined with the gene transcription of Gab2 on promoterIn conclusion,our study started with the transcription factor of Gab2 transcription regulation,preliminary revealed the Gab2 gene expression regulation mechanism,setted up a research technology system of Gab2 expression mechanism,and found some candidates of transcription factors,which laid a foundation for further research.In addition,we preliminary confirmed that Foxd3 is interacting transcription factor of Gab2 gene expression,which is not only prove that the system we have setted up is feasible,and also provide possible approach for prevention and treatment of liver cancer through regulating the expression of Gab2. |
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