| Head and neck squamous cell carcimoma (HNSCC) is a common malignant tumor in respiratory system of human body, and it′s incidence is increasing intensly year by year. Carcinogenesis is a complicate staging process. From the point of view in molecular biology, major pathogenesy is reinforcement of oncogene activity or inactivation of anti-oncogene in human body interacting with various extent and internal factors, the proto-oncogene coding protein mainly include growth factor and its receptor and associated protein molecule about signal transduction, genetic transcription regulatory factor and generation of cycle modulin and so on. Signal Transducer and Activator of Transcription 3 (STAT3) is important factor of signal transduction pathway, and it is constantly activated in great quantities of human tumor cell. It links closly with the occurrence, development and evolution of human malignant tumor. So gene therapy with target for STAT3 generated vitality for tumorous treatment, recently. People constantly pay close attention to RNA interference with high performance and high degree of specificity from it′s discovery, it is a hot spot in pathogence genomics and functional genomics investigation which develops gradually to become a criticality technique for defining gene function.A great quantity of studies indicate that HNSCC exhibits rapid growth and strong invasiveness clinically, combined radiotherapy and chemotherapy with operation has become a chief method and tendency for this disease, especially for patient with recurrence and metabasis. Because of low sensitivity and strong resistance to radiotherapy and chemotherapy in HNSCC, it is a new direction of oncotherapy to combined orthodox radiotherapy and chemotherapy with gene therapy. Through such a process we can study and choose effective, low toxic, enforcible cure medicine and method for oncotherapy. This experiment was separated into 3 parts:Part One Construction of STAT3 eukaryotic expression vector and it effceton laryngeal carcinoma cells.Objective: To construct signal transduction and activators of transcription 3 (STAT3) small interference RNA (siRNA) expression vector and to select one that was effect on inhibiting STAT3 expression.Method:We constructed and identifiy small interference RNA (siRNA) expression vector. The expression vectors designed on STAT3 different target sites are packaged with lipids, then transfected into Hep-2 cells to study the effcet of eukaryotic expression vector for laryngeal carcinoma cells. MTT was used for detecting the hyperplasia condition in different plasmid transfected groups after 24h, 48h and 72h. Flow cytometry was used for detecting the cell cycle condition in different plasmid transfected groups after 24h, 48h and 72h and the protein expression level of STAT3 after 72h. RT-PCR was used for detecting mRNA expression level of STAT3 mRNA. Western blotting was used for detecting the protein expression level of STAT3 after 72h.Results:Enzyme cutting results indicate that positive recombination vector can be cleaved by BamH I, can not be cleaved by EcoR I. Then chosen clones were sequenced and identified, we found that sequencing results of 3 recombination plasmids STAT3-siRNA-A, B and C were all correct in structure.we observed different transfected cells showing green fluorescence in cell. The cell multiplication level decreased obviously in Hep-2 cells transfected with recombination plasmid, especially in STAT3-siRNA-C group. After transfection Hep-2 cells with recombination plasmid for 72h, the translation from G1 phase to S phase in the hep-2 was inhibited. After transfection Hep-2 cells with recombination plasmid for 72h, flow cytometry analysis showed that in the STAT3-siRNA treaded cells the apoptosis rate was significant higher than that in the control group, whereas the apotosis rate in STAT3-siRNA-C is higher. Treatment of Hep-2 with recombination plasmid resulted in a significant decrease of STAT3 expression at mRNA level, which was more prominent in STAT3-siRNA-C group. The results of Flow cytometry (FCM) experiment: After transfection with STAT3-siRNA, the expression of STAT3 protein in Hep-2 cells were inhibited, their expressions in STAT3-siRNA group were significantly lower in statistic than that in other 2 control groups (P<0.05). Results of Westernblot showed that treatment of Hep-2 with recombination plasmid lead to significant decrease of STAT3 expression at protein level compared with the control groups, which the lower STAT3 expression at protein level was in STAT3-siRNA-C group.Part two The study of enhancement of chemosensitivity of the laryngeal carcinoma cells by STAT3 RNA silencingObjective: After blocking activated STAT3 signal pathway by STAT3 RNA silencing STAT3, we investigate the possibility of reducing laryngocarcinoma chemotherapy resistance and enhancing its sensitivity to DDP with its impact on the hyperplasia and apoptosis of laryngeal squamous carcinoma cell with interact technique.Methods:STAT3-siRNA plasmid were packaged with lipid body and transfected into laryngocarcinoma cells. Experiments were divided into: group①(Negative control group); group②(LP2000 for control); group③(DDP alone); group④ (STAT3-siRNA transfection alone); group⑤(Negative control group). MTT was used for detecting the hyperplasia condition of Hep-2 cells in different groups after treatment for 24h, 48h and 72h. Flow cytometry was used for detecting the cell cycle condition of Hep-2 in different groups for different time and apoptosis rate of Hep-2 in different group for different time. Flow cytometry was also used for detecting expression of STAT3, p-STAT3, Clyclin and Bcl-2 in protein level after 72h.Results:1. Study about STAT3-siRNA combined chemotherapy in regulation of the hyperplasia and apoptosis of laryngeal carcinoma cells. STAT3-siRNA had enhanced the inhibition effect of hyperplasy level in Hep-2 cell by DDP. After Hep-2 cells were treated with STAT3-siRNA and DDP, the hyperplasy level was decreased obviously, which was significantly lower in⑤group than that in control (P<0.05). STAT3-siRNA had enhanced the blocking effect by DDP on cell cycle of Hep-2 cell. When Hep-2 cells were treated with STAT3-siRNA and DDP for 72h, G1 phase cell ratio increasd gradually with times, S phase cell ratio decrease gradually with times. By combined STAT3-siRNA and DDP treatment on Hep-2 cells for 72h, the apoptosis rates in group STAT3- siRNA+DDP was different with group DDP, group STAT3-siRNA, group lipid body and group control. Statistical analysis results showed that apoptosis of Hep-2 cells increased after treated with STAT3-siRNA and DDP .2. The effect of STAT3 and correlate protein after tearment with STAT3- siRNA and DDPSTAT3-siRNA and DDP could down-regulate the activity and expression of STAT3 signal transduction pathway members. The expression and activity of STAT3, p-STAT3 protein and its target gene product Bcl-2 and CyclinD1 decreased after treating with STAT3-siRNA and DDP in Hep-2 laryngeal carcinoma cells. 3. The effect of STAT3 mRNA after tearment with STAT3-siRNA and DDPTreatment of Hep-2 with recombination plasmid and DDP after 72h, resulted in a significant decrease of STAT3 expression at mRNA level.Part three The study of enhancement of chemosensitivity of the model of Human Laryngeal squamous cacinoma in nude mice by STAT3 RNA silencingObjective:A human Laryngeal cancer model in nude mice was established to study the effect of applying RNAi to silence STAT3 gene combined chemotherapy in vivo.Method:Model of Human Laryngeal squamous cacinoma in nude mice was established. 30Balb/c nude mice were selected. Experiment group: 0.2ml 2×106 Hep-2 cell was injected into nude mice in the back. When tumor reaches 8-10 mm in size, they were used experiment. Inhibitiont effect of applying RNAi to silence STAT3 gene combined chemotherapy applying RNAi to silence STAT3 gene combined chemotherapy. Thirty nude mice were divided into five groups: group①(Negative control); group②(DDP); group③(STAT3-siRNA transfection); group④(STAT3-siRNA transfection with DDP), 20μg DNA was injected in the③and④group every 2 days. The weight and tumor's volume were determinded every three days. The nude mice was killed after 21days. Flow cytometry (FCM) was used to detect cell apoptosis. The expression of STAT3, p-STAT3, Clyclin and Bcl-2 protein was detected by immunocytochemical method of PV and flow cytometry.Results1. Formation of tumor begins from 48h after injection of o.2ml 2×106 Hep-2 cells, it reaches 10mm at 4 weeks. The rate of tumor formation was 96.25%. It was reported squamous cell cacinoma by pathology.2. Effect of applying RNAi to silence STAT3 gene combined chemotherapy on nude mice tumors.Tumor weight of group④(STAT3-siRNA transfection with DDP) was the smallest in all groups. Tumor inhibition rate of group④were significantly higher than that in other 3 group (P<0.05). The average proliferation index (28.96±2.28%) were significantly lower than that in other 3 groups (P<0.05), and apoptosis rate (37.61±2.09%) of group④were significantly higher than that in other 3 groups (P<0.05). The results of electron micrscope: In group④(STAT3-siRNA trans- fection with DDP) the membrane of cells sticked out at angle, chromosome was concentrated to nucleus membrane, some were like moons. Mitochondria had vacuole, and rough endoplasmic reticulum was degranulated. The enlarged nucleus could be seen in group①.3. The expression of STAT3, p-STAT3 and correlated gene at protein levelThe results of immunocytochemical experiment: STAT3 protein were observed in nuclear in most laryngeal squamous cells in all groups, p-STAT3 protein protein were seen in cytoplasm in laryngeal squamous cells in all groups. Group④(STAT3-SiRNA transfection with DDP) were significantly lower than that in any other 3 groups. The results of Flow cytometry (FCM) experiment: After tearment with STAT3-siRNA and DDP, the expressions of STAT3, p-STAT3, Bcl-2, Clyclin D1 protein in Hep-2 cells were reduced, their expressions in Hep-2 cells were significantly lower than that in any other 3 groups (P<0.05)Conclusion:1. Applying RNAi to silence STAT3 gene could down-regulate the express- on of STAT3 and inhibite effect of hyperplasy level in Hep-2 cell. It could promoted the apoptosis of laryngeal carcinoma cells.2. RNAi silencing STAT3 could not completely inhibite the hyperplasy of tumor cells, and the apoptotic-inducing effect of STAT3-siRNA was limited. STAT3-siRNA could coordinate with therapeutical effect by DDP. STAT3-siRNA and DDP could down-regulate the activity and expression of STAT3 signal transduction pathway members, it could enhance the inhibition effect of hyperplasy level in Hep-2 cell and promoted the apoptosis of laryngeal carcinoma cells. which indicates RNAi technique targeting STAT3 might become a new strategy of gene therapy in laryngocarcinoma.3. It is demonstrated that applying RNAi to silence STAT3 gene can enhance chemsensitivity by studying therapeutical effect of human laryngeal squamous cacinoma in nude mice. This has not been reported in the literature. Our results may provide a new clue for exploring a new method of treatment for laryngeal carcinama. |
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