Study On Variation Of Radiosensitivity Of Tumor Cells By Short Interferencing RNA Blockage Of STAT3 Gene

Radiotherapy is an important way of clinical treatments of tumors. The radiation sensitivity of tumors is closely associated with many most fundamental activities of life such as DNA injury and repair, signal transduction, cell proliferation, apoptosis and cell cycle regulation. It has been a hot spot of study in recent years in which modern molecular biological techniques were applied to introduce, block and modify key molecules of signal pathways so as to promote radiation induced death and apoptosis of tumor cells and consequently elevate curative effects of radiotherapy while at the same time injury of normal tissues and cells is limited as much as possible.Signal transducer and activator of transcription 3(STAT3), a double-functional key protein exists in cytoplasm, is coupled with signal transduction pathways of tyrosine phosphorylation. Upon activation, it forms homologous or heterogenous dimer, translocates into nucleolus where it recognizes and combines with response elements of target DNAs specifically and consequently induces the expression of genes associated with cell proliferation, differentiation and apoptosis. Current evidences indicate that STAT3 plays an important role in formation and development of tumors.Here we investigate the variation of radiosensitivity of tumor cells by short interferencing RNA (siRNA) blockage of STAT3 gene.First of all, we investigated STAT3 gene expression and phospharalation of STAT3 protein in cultured human turner cell line (HepG-2) which has a high STAT3 gene expression in vitro.Secondly, we had a reported siRNA sequence targeting STAT3 mRNA synthesized.Thirdly, we investigated the influences of STAT3 siRNA on mRNA transcription and protein expression of STAT3 gene.Lastly, we observed the variation of radiosensitivity of tumor cells after siRNA transfection and discuss its possible mechanisms.MethodsIn our experiments, we evaluated the STAT3 gene expression and its encoded protein phosphoralation before and after lipid coated siRNA transfection by Real-time RT-PCR and Western Blot. We observed the variation of radiosensitivity of human hepatic cancer cell line HepG-2 after siRNA blockage of STAT3 gene expression.By adopting CCK-8 kit evaluation and morphologic observation of apoptosis by Hoechst33258, we studied the synergistic effects of STAT3 siRNA and radiation on the proliferation and apoptosis of HepG-2 cell.Statistic analysis was then carried out.ResultsIn contrast to human skin fibroblast cell line HSF, human hepatic cancer cell line HepG-2 has a high STAT3 mRNA transcription, protein expression and its phosphoralation.After siRNA transfection of certain concentration, HepG-2 cells with STAT3 siRNA transfection other than negative control siRNA transfection demonstrated a decrease of STAT3 mRNA transcription as well as STAT3 protein expression and its phosphoralation.Radiosensitivity of HepG-2 cell was dramatically enhanced after siRNA knockdown of STAT3 gene expression.Compared with control group, siRNA transfection can inhibit the proliferation of tumor cells.When we conbined siRNA transfection and radiation, proliferation of tumor cells were even further inhibited.Hoechst33258 staining indicated that although soly siRNA transfection did not elicited obvious increased morphologic demonstration of apoptosis, the frequency of apoptotic cells is dramatically increased when we combined siRNA transfection with radiation.We did not observed such obvious changes in HSFcells.DiscussionIn signal transduction of normal cells,STAT3 activation is quick and transient,whereas continuous STAT3 activation is linked to oncogenesis.STAT3 is the converging point of several oncogenetic protein tyrosine kinase pathways including EGFR,IL-6/JAK and Src.lt is over activated in many tumor cells and tumor tissues.We have previously detected abnormally high STAT3 expression and activation in such tumor cell lines as B16,SMMC-7721,HepG-2,A549 and Hela.STAT3 Activation induces expression of many genes which are closely associated with cell proliferation,differentiation,survival and apoptosis, consequently mediated cell proliferation,transformation,angiogenesis,oncogenesis and resistance to apoptosis induced by radiotherapy, chemotherapy and other cytotoxic treatments.We found that siRNA targeting STAT3 downregulated STAT3 protein expression and activation specifically and effectively.When we combined siRNA transfection with radiation, we observed more obvious proliferation inhibition as well as apoptotic morphology.We didn't observe comparably enhanced radiosensitivity in HSF cells.Our results indicated that siRNA knockdown of STAT3 might be an effective way of enhancing tumor radiosensitivity and thus of clinic value.Previous studies indicated that activation of STAT3 upregulated the expression of VEGF. When STAT3 signal pathway was blocked, its downstream genes(such as Bcl-xL* Mcl-K c-Myc? cyclin Dl),which are linked to tumor cell proliferation,resistance to apoptosis and decreased sensitivity to radiation, were also downregulated. STAT3 siRNA can block STAT3 expression and activation in HepG-2 cell and thus inhibit its proliferation and promote its apoptosis effectively. Mechanism of such a specific function is probably that knockdown of STAT3 causes varied expression levels of genes associated with cell cycle regulation, proliferation and apoptosis such as Bcl-xL> Mcl-K c-Myc. cyclin Dl > VEGF and P53, and consequently inhibits tumor proliferation and induces apoptosis. It also elevated radiosensitivity of tumor cells. Results from some researches indicated that compared with normal cell lines, many tumor cell lines seem to be more dependent on continuous and high expression and activation of STAT3. This is consistent with our results. Tumor cells' dependency on STAT3 provides STAT3 targeting treatments with certain tumor cell specificity. Thus it is possible that we could find a way of enhancing tumor radiosensitivity without increased normal tissue injury after radiation.