Effecting On Proliferation, Differentiation And Apoptosis Of Human Glioblastoma Stem Cells After Inhibition Of STAT3 Expression By SiRNA

Background and objectiveIt is now considered that cancer stem cells(CSCs) are tumor-initiating cells of glioblastoma and may result in relapse and metastasis of glioblastoma. The cancer cells that had been differentiated from CSCs only have limited capacity of proliferation and lost the ability to form tumors. Targeting of the CSCs is more likely to be successful in the therapy of glioblastoma. Evidence shows that CSCs were more resistant to chemotherapy and radiotherapy compared with other differentiated tumor cells. CSCs of glioblastoma lacked in specific markers and had little variability in marker expression compared with neural stem cells. It is now difficult to kill CSCs of glioblastoma by targeted therapy. Stat3 is required for maintenance of multipotency to embryonic stem cells in embryogenesis. It was known as oncogene because of its relation to the generation and development of diverse tumor. Constitutively activation of STAT3 results in oncogenic transformation of cell line and induces expression of genes against apoptosis and promotes proliferation of cells. We had obtained cue by the ways that the CSCs of glioblastoma were cultured in vitro. The CSCs of human glioblastoma in vitro were cultured in serum-free neural stem cell medium with LIF. LIF can active JAK/STAT3 pathways. We hypothesize that STAT3 gene play a key role in regulation of proliferation and differentiation in CSCs of human glioblastoma. Therefore, we are going to isolate and identify the CSC within human gliomas tissue in vitro and inhibit STAT3 expressing in CSCs of human glioblastoma by RNA interference, observe the effect on proliferation, differentiating and apoptosis in CSCs of human glioblastoma.Methods1. Isolation and identification of CSCs of glioblastoma: switching primary human glioblastoma cells into a defined serum-free neural stem cells medium, isolating glioblastoma spheres. Examining the percentage of CSCs for glioblastoma cells and self-renewing capacity of CSCs by limiting dilution assay, secondary spheres forming assay and flow cytometry assay. Differentiation of CSCs were observed using immunocytochemistry after cultured in medium with 10% FBS for 10 days. Observing the ability of tumor initiation after CSCs were implanted into nude mice. Assaying cell proliferation of CSCs. Expression of GFAP, MAP2, MBP and CD133 in CSCs and implanted tumor tissue were examined with immunocytochemistry or immunohistochemistry.2. Preparation of vector virus that was able to express siRNA targeting human STAT3 gene: the lentivirus siRNA vector targeting human STAT3 gene was constructed. The pSC-STAT3,pCMV-dR and pMD2G were cotransfected into 293T incasing cells by calcium acid phosphate. Harvesting vector virus that was able to express siRNA targeting human STAT3 gene. Detecting efficiency that vector virus infected CSCs by flow cytometry assay and inhibited expression of STAT3 in CSCs of human glioblastoma by realtime PCR and Westernblot.3. Observing the proliferation and differentiation of CSCs of human glioblastoma after infected pSC-LT vector virus: growth curves of CSCs infected vector virus were surveyed by cell proliferation assaying kits. Assaying distribution of cell cycle to CSCs infected vector virus by flow cytometry assay. Evaluating CD133+ cell in CSCs of human glioblastoma by flow cytometry assay and immunofluorescence and capability of initiating tumor after CSCs were implanted into nude mice.4. Assaying apoptosis in CSCs of human glioblastoma infected pSC-LT vector virus by flow cytometry assay. The expression of Bcl-2, Bax and Caspase-3 gene were evaluated by realtime PCR and Westernblot.ResultsGlioblastoma spheres consisted by CSCs expressing CD133 but no GFAP and MAP2, a few CSCs express MBP. The percentage of CD133+ cell in 3 cases of primary cultured glioma cells were 7.4%, 2.8% and 4.2%. About 1% cells in primary cultured glioma cells generate free-floating glioblastoma spheres and 100 cells of primary tumor spheres can generate 6.07±2.15 secondary glioma spheres expressing CD133. Most cells produced of CSCs spheres express GFAP, a few express MAP2 and MBP. Glioma CSCs cultured in serum-free neural stem cells medium show a lower degree of cell proliferation than primary glioma cells cultured in medium with 10% FBS. CSCs have stronger capable of tumor initiation in nude mice than primary cultured glioma cells.The efficiency that pSC-LT and pSC-LC vector virus infected CSCs of human glioblastoma were 98.5% and 98.6%. The STAT3 mRNA and protein and pStat3 in CSCs of human glioblastoma infected pSC-LT vector virus decreased significantly compared with cell infected control vector virus and no infection(P<0.01). The inhibition ratio of pSC-LT to Stat3 and pStat3 protein in CSCs of human glioblastoma was 81.5%. and 98.5%.The CSCs of human glioblastoma knockdown of STAT3 expression by RNAi grew adherence and were inhibited the growth by inducing G1 cell-cycle arrest. The rate of CD133+ cell decreased from 92.2% to 5.7% and capability of initiating tumor in nude mice decreased after CSCs infected pSC-LT vector virus. Inhibition of STAT3 expression by RNAi in CSCs of human glioblastoma induces apoptosis by the reduced expression of Bcl-2.Conclusion1. We isolated the CSCs of human glioblastoma by culturing in neural stem cells medium and identify the pluripotency and self-renewal of CSCs. 2. The lentivirus siRNA vector (pSC-LT ) targeting STAT3 gene was constructed. It can infect the CSCs of human glioblastoma in highly efficiency and inhibit significantly the expression of STAT3 gene. 3. Knockdown of STAT3 expression by RNAi suppresses cell growth and the capacity of initiating tumor and promotes differentiation in CSCs of human glioblastoma. The STAT3 gene may play a key role in regulation of proliferation and differentiation in CSCs of human glioblastoma. 4. Inhibition of STAT3 expression induces apoptosis in CSCs of human glioblastoma. It may be relative to decreasing of Bcl-2 expression level in CSCs.