These results further demonstrate that both JNK and JAK STAT signal ing pathways are able to activate the ISL 1 transcription effectively. To selleck chemicals Rapamycin confirm whether p STAT3 and p c Jun bind to the ISL 1 regulatory region, a set of primers covering the ISL 1 promoter region between ?994 and ?216 were designed for real time PCR in ChIP assay. The ChIP analysis showed that p STAT3 was recruited to the region of ISL 1 promoter covered by primer 2 by appro imately 12 folds, and p c Jun was recruited to the region of ISL 1 promoter covered by primer 4 by about 6 folds, respectively, as compared with primer 1 as the control. Interestingly, we also observed magnificent enrichment of p STAT3 at the p c Jun binding region, p c Jun at the p STAT3 binding region, and both p STAT3 and p c Jun at the primer 3 covered region.
Therefore, we suppose that p STAT3 possibly cooperate with p c Jun and synergistically regulate ISL 1 e pression in NHL cells. According to previous reports, p STAT3 could interact with p c Jun to regulate MMP 1, MMP 7 or other genes e pression in human cancers. Meanwhile, the cooperation and co localizations between p STAT3 and ISL 1, p c Jun and ISL 1 are also authen ticated in different genes transcription. These evidences promote us to hypothesize that p STAT3, p c Jun and ISL 1 may form a transcriptional activation comple that regulates the e pression of ISL 1 by direct binding to the ISL 1 promoter. To verify this hypothesis, co immunoprecipitation and ChIP re IP were performed to analyze whether p STAT3, p c Jun and ISL 1 could form a comple and bind directly on the ISL 1 promoter.
Co IP results demonstrate that one component of the presumptive comple could co immunoprecipitate with all of the other components, supporting the e istence of this comple . Furthermore, ChIP re IP analysis confirmed that p STAT3, p c Jun and ISL 1 indeed e isted in the same protein comple and co localized on the primer 2 and primer 4 covered region of ISL 1 promoter. These results reveal that p STAT3, p c Jun and ISL 1 could form a transcriptional activation comple on the ISL 1 promoter, which further indicates that there might be a positive feedback loop to contribute to ISL 1 up regulated e pression in NHL cells. To determine whether ISL 1 is involved in the positive feedback loop on the ISL 1 transcription, luciferase assay was performed with ISL 1 luc.
As shown in Figure 8F, ISL 1 luc activity was increased in a dose dependent manner in ISL 1 overe pressing Ly3 cells, indicating, for the first time, that ISL 1 could promote its own e pression in NHL cells and therefore to form a positive feedback. Collectively, these results indicate that ISL 1 may have a positive feedback Batimastat regulation p STAT3 and p c Jun up regulate ISL 1 e pression, then ISL 1 form a comple with p STAT3 and p c Jun to participate ISL 1 overe pression. The consequence is to promote the proliferation of NHL cells.