Construction Of Induced RNAi System And Investigation Of Anti-tumor Immunity Change Induced By Apoptosis Of Cancer Stem Like Cells

Host anti-tumor immunity has been proved to play a key role during tumor initiation and progression. However, a large numbers of previous studies indicate that tumor cells can escape from the host immune surveillance. Several mechanisms have been identified to affect this progression, such as the secretion of suppressor cytokines, inhibition of dendritic cells (DC) maturation, infiltration of CD4+CD25+regulatory T cells (Treg), which can all suppress effector T cells function and tumor-specific T-cell response in tumor site. In this regard, recovery of anti-tumor immunity and protection of immune cells from tumor-induced suppression may be a helpful procedure for tumor therapy. Although there are numerous investigations indicating that effective anti-tumor immunity was developed by depleting Treg, few ones focus on the role of cancer stem cell-like cells (CSCLCs) in tumor immunity, which is the main purpose of our current study.CSCLCs, a small subgroup of cancer cells, which have the same functional properties with normal stem cells as following:self-renewal, pluripotency, and extensive proliferation potential, have been found to initiate tumor and decrease the effectiveness of widely applied treatment used to eliminate tumor. It has been proved that current clinical therapies are unable to eliminate CSCLCs. In fact, because of the resistance to chemotherapy and radiotherapy like normal stem cells, CSCLCs can not be killed by widely used therapies, which function against the bulk population of tumor cells and surely shrink tumors, effectively. So, it is absolutely important to find out effective therapeutic strategies against CSCLCs. And there are already several researches focusing on CSCLCs special treatment.Oct4, a member of the POU family of transcription factors, first reported to have an important role in keeping pluripotency and proliferation potential of stem cells, is also detected in various tumors. Because of the ability to keep proliferation of CSCLCs, Oct4 seem to be an important target to deplete CSCLCs. Our previously research indicates that expression of Oct4 turned off by Oct4 small interfering RNA cause CSCLCs apoptosis and tumor growth inhibition in vivo. We also found Oct4/Tcll/Aktl signal pathway, involving in ES proliferation by inhibiting the apoptosis of ES cells, also affects tumor cells apoptosis. Those studies provided an effective procedure to reduce CSCLCs in vivo.Aiming to demonstrate the anti-tumor immunity recovery after reduction of CSCLCs, in the present study, we employed Oct4 small interfering RNA to induce CSCLCs apoptosis and then investigated the immunity changes happening in vitro and in vivo.RNAi is a specific, potent, and highly successful approach for loss-of-function studies in virtually all eukaryotic organisms in vivo or in vitro. There are several appropriate tools to induce RNAi-including chemically synthesized siRNA,shRNA or siRNA-encoding plasmid or viral vectors. However, it is impossible to tightly regulate gene expression by this way. So, variety of controlled gene expression systems were developed, among which Tetracycline (Tet)-inducible expression system is one of the most prominent and widely-accepted inducible systems so far. The ideal controlled system should permit the investigators to rapidly and reversibly switch the transgene expression on and off, exclusively in the desired cells or tissue(s) at any time point during development.Based on pTet-On-Advanced vector,pTRE-tight vector pEGFP-N1 (mutation in AgeⅠsite) and pLKO.1-puro, pEGFP-Tet-on-TRE-sh, a new tet-on plasmid which could express siRNA targeted Oct4 induced by DOX was constructed.Then, we investigated if anti-tumor immunity in tumor site recovery, when Oct4 siRNA was injected into tumor. Firstly, immunosuppressive cytokines, such as IL4,IL10 and TGF-β, were tested in mouse hepatoma cell line hepal-6(in vitro) and mouse tumor model(in vivo), before and after cells(or tumors) were treated with Oct4 siRNA. We found reduction of cytokines when Oct4 siRNA was applied. The same phenomenon was found from experiments in vitro carried out in human hepatoma cell line Huh7. We also found that mature DC,activated CD4+T cells and CD8+T cells infiltrated in tumor issue increased after Oct4 siRNA treatment. Our results indicate that Oct4 RNA interference could enhance immunoreactions in situ and this procedure may be applied in tumor therapy.