Primary Study Of Transcriptional Regulatory Mechanism Of Fascin Gene In Esophageal Carcinoma Cells

Cancer has become one of three killers which threaten human health. Many researches had demonstrated that, on the one hand, tumor invasion and metastasis are responsible for the poor prognosis and the failure of therapy, and on the other hand, the complicated processes are closely related to the aberrant reconstitution of cytoskeleton. Esophageal carcinoma is one of the most common malignant tumors in China, but up to now, its metastatic mechanism still remains unclear. Fascin, a cytoskeleton bundling protein, can modulate cell adhesion and motility by combining to the filament actin (F-actin). Many researches had reported that the expression of Fascin was up-regulated in many kinds of tumor cell lines which originated from epithelium. Studies from Department of Biochemistry and Molecular Biology, Medical College of Shantou University had found that expression of Fascin was up-regulated in malignant esophageal carcinoma cells, and that fascin gene not only participated in division and proliferation of tumor cells, but also was closely related to some biological behaviours such as invasion, metastasis and adhesion of carcinoma cells. So Fascin was considered to be an important biomarker for tumor metastasis. Up to now, the up-regulation mechanism of its overexpression in carcinoma cells remains unclear. To approach this issue, we amplified series length of DNA fragments from 5' flanking region of fascin gene with genomic DNAs of esophageal carcinoma cell line EC109 as template, and had constructed series of firefly luciferase reporter gene expression vectors, transfected these recombinant vectors into EC109 cells, tested the luciferase activity of EC109 cells using Dual-Luciferase Reporter Assay System (DLR). We had identified the core promoter region of fascin gene in EC109 cells, and had also found the key regulatory element which may play important roles in transcriptional regulation of fascin gene in esophageal carcinoma cells. Our research will form a base for further research on transcriptional regulatory mechanism of fascin gene in esophageal carcinoma cells, and also provide a new way for tumor biotherapy in which the transcription factor be taken as molecular targets.Materials and methods: 1. To extract genomic DNAs from esophageal carcinoma cell line EC109, amplified the 3 kb upstream fragment from 5' flanking region of fascin gene using the genomic DNAs as template, and then cloned the fragment into T vector, finally constructed plasmid pTF-2900 (transcription initiation site of fascin gene was defined as+1, -2900 means that the fragment inserted into the vector located 2900bp upstream of transcription initiation site, the designation following are as the same), sequencing.2. Four serial deletion fragments which truncated 500bp one by one were amplified by PCR using pTF-2900 as template and ligased into T vector, verified by sequencing.3. Then sub-cloned those five amplified fragments into pGL3-Basic Vector (pGLB) using DNA recombination method, and firefly luciferase reporter gene expression vector pBF-290～-436 were constructed, and verified by sequencing.4. With vacant vector (pGLB) as control, those five recombinant plasmids (pBF-2900～436) and internal control pRL-TK were cotransfected into esophageal carcinoma cell line EC109. 48h after transfection, EC109 cells were lysed and harvested to test the luciferase activity using Dual-Luciferase Reporter Assay System (DLR).5. Nested deletion were then carried out using pBF-1435 as template, series of recombinant plasmids were constructed and sequenced. Also control vector (pGLB) and those recombinant plasmids with pRL-TK as internal control were cotransfected into esophageal carcinoma cell line EC109. 48h after transfection, those EC109 cells were lysed and the luciferase activity were detected using DLR.6. Potential regulatory elements within -386 bp (386bp upstream of transcription initiation site)were predicted by using bioinformatics method.7. Plasmids which contained regulatory elements (binding site of transcription factor SP1andCREB, TATA box) deletion were constructed by using PCR and enzymatic digesting methods with pBF-387 as the template, verified by sequencing.8. Also control vector (pGLB) and those regulatory elements deleted plasmids were cotransfected into esophageal carcinoma cell line EC109, with pRL-TK as internal control. 48h after transfection, cells were lysed and collected, then luciferase activity were analyzed by using DLR.Results and conclusions:1. The 3kb fragment amplified from genomic DNAs of EC109 was successfully obtained by PCR amplification, verified by sequencing and BlastN over NCBI. The results showed that the amplified DNA fragment shared high similarity with 5' flanking region of human fascin gene, the identity is 99.9%, with exception of four base alteration. The recombinant plasmid pTF-2900 was successfully constructed.2. The four fragments which deleted 500bp one by one were amplified by PCR with pTF-2900 as the template. DNA sequencing results of these fragments coincided completely with the expectation.3. Firefly luciferase reporter gene expression vectors pBF-2900～-436 were successfully constructed.4. Luciferase activity detected by Dual-luciferase Reporter Assay System (DLR) revealed that pBF-2900～-436 had distinct promoter activity in EC109 cells and the promoter region located within 436bp upstream of transcriptional initiation site.5. Firefly luciferase reporter gene expression vectors pBF-1435～+82 which were generated by nested deletion experiment were successfully constructed.6. Luciferase activity of pBF-1435～+82 in EC109 cells detected by DLR indicated that the core promoter region of fascin gene located within the region between 316bp to 22bp upstream of transcriptional initiation site.7. Prediction of potential transcription factor binding site within -387bp in 5' flanking region of fascin gene using software AliBaba2.1 revealed 56 regulatory elements, there are many SP1 binding site, near the TATA box there is a region which can bind many transcription factors at one time, such as CREB, C/EBP alpha, ATF, CRE-BP1, c-Jun and so on.8. Firefly luciferase reporter gene expression vectors pBF-255～-40 and pBF(-44～- 47)～(-107～-14) which contained different regulatory elements deletion were successfully constructed.9. Luciferase activity driven by pBF-255～-40 and pBF(-44～-47)～(-107～-14) in EC109 cells detected by using DLR revealed that SP1 (binding site is -74～-59) played the most important roles in control of fascin gene expression. So Sp1 may be the key factor in transcriptional regulation of fascin gene in EC109 cells. Even so, futher studied, such as western blot, EMSA and RNAi, are needed to eventually verify the mechanism that how Sp1 control the expression of fascin gene in esophageal carcinoma cells.In conclusion, the present studies had identified the core promoter region of fascin gene in esophageal carcinoma cell line EC109, ans for the first time in the world demonstrated that transcription factor Sp1 located at position -74～-59bp upstream of transcriptional initiation site was the key factor for fascin gene expression control in EC109 cells. These finding may then shed light on the understanding of esophageal carcinoma invasion and metastasis, and pave the way for the exploration of targeted biotherapy against esophageal carcinoma.