| Pancreatic cancer is one of the most aggressive tumor of digestive tract. Although the technology of diagnosis and therapy of pancreatic cancer was greatly improved in recent years, the survival rate of five year is still pressimistic, which is mainly due to the high degree of malignancy, lack of clinical features, accompanying with distant metastasis, and the complicated pathologic mechanism. Furthermore, tumorigenesis of pancreatic cancer is attributed to the dysregulation of multiple genes. Double bromodomain-containing protein 4 (BRD4) belongs to the BET family. BRD4 plays an important role in the process of DNA transcription, cell cycle progression, tumor genesis and progression. Previous studies have showed that BRD4 serves a critical role in tumor proliferation and growth in melanoma, neuroblastoma, glioblastoma, malignant peripheral nerve sheath tumors, lymphoblastic leukemia and lung adenocarcinoma and hepatic cancer. Inhibition of BRD4 can downregulate lots of oncogenes that promote the caner progression. However, the mechanism by which BRD4 takes part in the regulation of PDAC cell proliferation remains elusive. Therefore, there is a vital necessity to illustrate the function and mechanism of BRD4 in regards to PDAC.In this study, we illustrated the function of BRD4 on pancreatic cancer from five aspects. Firstly, we have compared the expression expression level of BRD4 in the PDAC cell lines COLO357, L3.6PL, SW1990,PANC-1, Capan-2 and MIAPaCa-2, as well as in the normal pancreatic epithelial cell line HPDEo。econdly, we construct the siRNA vector to downregulate the expression of BRD4 in pancreatic cancer cells. Thirdly, we revealed the function of BRD4 on the cell viability, proliferation, invasion and metastasis of pancreatic cancer cell lines. And then we illustrated the effects of BRD4 on chemosensitivity to gemcitabine in pancreatic caner cell lines. At last, we have showed that BRD4 promoted the Shh signaling pathway in PDAC cells in a ligand dependent or independent manner.Part 1:The different expression level of BRD4 in the PDAC cell lines and in the normal pancreatic epithelial cell line HPDE.Objective:to compare the expression of BRD4 PDAC cell lines with the normal pancreatic epithelial cell line HPDE.Method:Westernblot and real-time PCR were used to examined the BRD4 expression of PDAC cell lines COLO357, L3.6PL, SW1990, PANC-1, Capan-2 and MIAPaCa-2 and the normal pancreatic epithelial cell line HPDE.Results:Westernblot analyses showed that the protein expression levels of BRD4 were significantly higher in the PDAC cells than these levels in the HPDE cells. Similary, we have revealed that the mRNA expression levels of BRD4 were significantly higher in the PDAC cells than these levels in the HPDE cells. The highest expression levels of BRD4 were detected in the PANC-1 and MIAPaCa-2 cells.Conclusions:The increased level of BRD4 in PDAC suggests that BRD4 promotes pancreatic tumorigenesisPart 2:the construction of Phblv-u6-puro shBRD4 vector to downregulate the expression of BRD4 and trasfection the PDAC cell lines PANC-1 and MIAPaCa-2.Objective:To construct the Phblv-u6-puro shBRD4 vector containing the siRNA sequence which targeting the endogenous BRD4 and doweregulate the expression of BRD4 in pancreatic cancer cell line.Methods:firstly, we construct the Phblv-u6-puro shBRD4 vector. Secondly, the virus vector was packaged in 293T cell. Thirdly, Phblv-u6-puro shBRD4 vector was transfected into the pancreatic cancer cell lines PANC-1 and MIAPaCa-2. At last, we have studied the silencing efficiency of BRD4 in PANC-1 and MIAPaCa-2 by westernblot.Results:We successfully constructed the Phblv-u6-puro shBRD4 vector and transfected the virus vector into the pancreatic cancer cell line PANC-1 and MIAPaCa-2. The westernblot result showed that we expression level of BRD4 was significantly silenced in PANC-1 and MIAPaCa-2.Conclusions:We successfully constructed the Phblv-u6-puro shBRD4 vector and got the stably silencing BRD4 pancreatic cancer cell line.Part 3 the effects of BRD4 on the biological behaviors in pancreatic cancer cell linesObjective:To illustrate the function of BRD4 on the cell viability, proliferation, metastasis and invasion of pancreatic cancer cell lines.Methods:The function of BRD4 on the cell viability, proliferation, metastasis and invasion of pancreatic cancer cell lines was examined by CCK8 assay, colony formation assay, wound healing assay, Boyden chamber assay, tumor xenograft assay and immunohistochemistry.Results:BRD4 knockdown evidently inhibited the viability of both cell lines via CCK-8 assay (P<0.05). Furthermore, the colony formation assay showed that the proliferation of stable BRD4-knockdown cells was severely inhibited (P<0.05). The results of Boyden chamber assay and wound healing assay showed that BRD4 knockdown significantly inhibited the migration of PANC-1 and MIPACA-2. We also revealed that the invasion of shBRD4PANC-1 and shBRD4 MIAPaCa-2 remarkable decrease compared to shcontrolPANC-1 and shcontrol MIAPaCa-2 (P< 0.05). The effect of BRD4 on in vivo tumor growth was assessed by the subcutaneous injection of shBRD4PANC-1 and shcontrolPANC-1 for 21 days. The results showed that significantly decrease of tumor size was observed in the shBRD4PANC-1 group compared to that of the control group (P<0.05). At 20 days after cell implantation, the tumors were removed and weighted, the average tumor weight of the shBRD4PANC-1 group significantly light compared to that of the control group (P <0.05). Histologic analysis of tumor proliferation revealed that shBRD4PANC-1 tumors had significantly fewer Ki-67 positive cells than the shcontrol tumors (P< 0.05)Conclusions:downregulation of the expression of BRD4 inhibited the cell viability, proliferation, metastasis and invasion of pancreatic cancer cell lines.Part 4:the function of BRD4 on chemosensitivity to gemcitabine in pancreatic cancer cell lines.Objective:To illustrate the function of BRD4 on chemosensitivity to gemcitabine in pancreatic cancer cell lines.Method:PANC-1 and MIAPaCa-2 cells were treated with 2 μmol/1 gemcitabine and then examined the expression of BRD4. The real time PCR was used to examine the expression of the BRD4 mRNA. The westernblot was used to examine the expression of the BRD4 protein. Cell apoptosis induced by gemcitabine or BRD4 silencing or gemcitabine plus BRD4 silencing was assessed by flow cytometry.Results:Firstly, we showed that gemcitabine significantly increased the mRNA expression levels of BRD4 in the PANC-1 and MIAPaCa-2 cells via real time PCR (P <0.05). The westenblot illustrated that gemcitabine significantly increased the mRNA expression levels of BRD4 in the PANC-1 and MIAPaCa-2 cells. Thirdly, BRD4 knockdown alone induced cell apoptosis in the PANC-1 and MIAPaCa-2 cells (P<0.05); however, treatment with GEM significantly promoted cell apoptosis compared to the control and shBRD4 cell lines (P<0.05)Conclusions:BRD4 downregulation and GEM treatment synergistically influenced the chemotherapeutic efficacv in the PDAC cellsPart 5:BRD4 regulation the SHH signaling pathway in pancreatic cancer cell lineObjective:to explore the correlation between BRD4 and the SHH signaling pathway.Method:The real time PCR was used to examine the mRNA expression of the SHH signaling pathway target gene SHH, PTHC1, GLI1. The westerablot was was used to examine the protein expression of the SHH signaling pathway target gene SHH, PTHC1, GLI1.Results:The real time PCR showed that BRD4 knockdown decreased the mRNA level of Shh pathway-related genes, including SHH, PTCHl and GLIl. The westernblot revealed that BRD4 knockdown decreased the protein level of SHH pathway-related genes, including SHH, PTCHl and GLIl. We further investigated whether or not BRD4-induced Shh contributes to Shh signaling pathway activation. BRD4 knockdown in PANC-1 and MIAPaCa-2 cells followed by rhShh stimulation markedly inhibited Shh target gene PTCHl and GLIl expression both in mRNA and protein.Conclusions:BRD4 induced SHH pathway activation can be ligand depend or independent manner. |
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