Effect Of Downregulated ZEB1 Expression On The B16F10 Cancer Stem Cell Tumorigenicity And Lung Metastasis In Mice

Malignant melanoma (MM) arising from melanophore is one of the most lethal skin cancer. The overall incidence rate of melanoma has changed a lot and has still been rising trend in the past 20 years. The skin appears to be the most frequent site of origin for these lesions and accounts for approximately seventy-five percent of skin cancer related death. Moreover, it can also affacts eyes, nasal cavity and so on. Locally invasion and distant metastasis are the basic characteristic of MM, which contribute to the major cause of death in patients with melanoma. Early it can metastasize to lung and brain, but later may spread to the whole body. However, studies has been shown that epithelial mesenchymal transition (EMT) plays a key role during this progress and is considered to be the dominating event of tumors’ invasion and metastasis. From the research we find melanoma not only can ecsape form the primary location,invading other tissues but also may move to father organs through blood or lymphatic pathway in the whole EMT. Besides, campared with the normal tumors, the MM with EMT phenotypes become more resistant, immunosuppressio, and stem like cell characteristic. E-cadherin, a crucial molecule to maintain the characteristic of epithelial cells, is the major regulating object during the EMT progress. Loss of E-cadherin is an significant characteristic of EMT. The cells with the decreased expression of E-cadherin have the characteristic of non-epithelial cells, such as loss of cell polarity that results in weakened adhesion ability, enhanced motility, and changed into metastatic mesenchymal cells. An other pivotal mark for regulating EMT is up-regulation of Vimentin expression. Thus, investigating the mechanism of EMT is capble to provide a new therapeutic target for curing cancers.Nowadays, with the highly rapid development of molecular biology, scientists found the unique subpopulation in the tumors, which can possess the ability to initiate tumor growth and sustain self-renewal as well as metastatic potential. They are non-sensitive to chemical and radical therapy. Thus, these cells have an extremely close realtion with the tumors’ process. They are defined as tumor stem cells (TSCs) or cancer stem cells (CSCs). In addition, the another important protein called zinc finger E-box-binding protein 1(ZEB1) should also be responsible for the tumor metastasis. ZEB1, the zinc finger motif transcription factor in zinc finger E-box-binding protein, both can both regulate the process of EMT and has a great regulatory function on the malignant tumor progression through regulating cell cycle, apoptosis, senescence, invasion, metastasis and mesenchymal angiogenesis, etc. In early work in our group, the lower capability of clonogenicity in flat plate, the cellular proliferation activity, the higher sensitivity to Epirubicin, the lower capability of metastasis and invasion in vitro, and the lower tumorigenicity in the mice were found in B16F10 cells transfected with ZEB1-shRNAs. But how it works in B16F10 CSCs is still unkonwn. Therefore, in this study, we isolated B16F10CD133+CD44+CSCs from the murine melanomas as mainly research object with immune magnetic beads by magnetic activated cell sorting system (MACS) to explore the effect of down regrlated ZEB1 expression on CD44+CD133+CSCs’ oncogenicity and lung metastasis.Objective:To investigate the effect of down regulated ZEB1 expression in melanoma B16F10 CSCs on the tumorigenicity and lung metastasis of B16F10 cells transfected with pSUPER-EGFP1-ZEB1-shRNA, and to provide the experiment data for exploring new therapeutic target in clinical treatment of melanoma.Methods:1. The constructed recombinant shRNA plasmids were transfected into melanoma B16F10 cells by using Lipofectamine TM 2000 reagent, and the stable transfected cells were selected by using G418 according to the characteristic of the vector.2. ZEB1-shRNA CD133+CD44+CSCs and scramble-pSUPER-EGFP CD44+CD133+ CSCs were isolated from the stable transfected cells with MACS. B16F10 CD133+ CD44+CSCs were isolated from B16F10 cells in the same way.3. The biological characteristics of B16F10CD44+CD133+CSCs with ZEB1 down-regulated were identified by analyzing the ability of colony forming in a common media, the ability of proliferation, migration and invasion as well as the ability of tumorigenicity along with lung metastasis in C57BL/6 mice.Results:1. The constructed recombinants shRNA plasmids were transfected into B16F10 cells respectively, and the stable transfected cells were selected according to the characteristics of resistance to G418.2. The ZEB1-shRNACD133+CD44+CSCs, scramble-pSUPER-EGFP CD133+CD44+ CSCs as well as B16F10 CD133+CD44+CSCs were successfully sorted from the stable transfected cells and B16F10 cells with MACS, respectively. After the expression of ZEB1 was inhibited in B16F10 CD133+CD44+CSCs transfected with the recombinant ZEB1-shRNA, the cellular proliferation activity and the capability of clonogenicity in flat were decreased in contrast to B16F10 CD133+CD44+CSCs transfected with the scramble-pSUPER-EGFP. The capability of migration and invasion of B16F10 CD133+CD44+CSCs transfected with the recombinant ZEB1-shRNA was also slower than that of B16F10 CD133+CD44+ CSCs transfected with the scramble-pSUPER-EGFPCD133+CD44+CSCs. Importantly, the tumorigenicity of B16F10 CD133+CD44+CSCs with ZEB1 knockdown was lower than that of B16F10 CD133+CD44+CSCs, and the differences was statistically significant (P<0.05).3. The results of qRT-PCR, Western blot and immunohistochemistry showed that the downregulation of ZEB1 resulted in a significant down-regulation of the Vimentin and N-cadherin expression as well as upregulation of the E-cadherin expression in ZEB1-shRNA CD44+CD133+CSCs. The lung metastasis apparently decreased compared with the B16F10 CD44+CD133+CSCs and scramble-pSUPER-EGFP CD44+CD133+CSCs, and the differences was statistically significant (P<0.05).Conclusions: These results showed that the downregulated ZEB1 expression inhibited the B16F10 CD44+CD133+CSC" tumorigenicity and metastasis, and that ZEBlmay serve as a new therapeutic target for treatment of melanoma by inhibition of EMT in B16F10 CD44+CD133+CSCs.