Experimental Research On Radiosensitivity Of Esophageal Carcinoma By RNAi On HIF-1α

Objective:To study the expression of HIF-1αand VEGF in esophageal squamous cell carcinoma (ESCC), the relationship between them, and their relationship with pathological features and radiotheraputic response. To study the changes of HIF-1αand VEGF after RNA interference, its effect on proliferation and cell cycle. To study the change of radiosensitivity of KYSE-150 cell after RNA interference in vivo and in vitro, explore the mechanisms of radiosensitization and provide a new target for enhancing radiosensitivity and gene therapy on esophageal cancer.Methods:To choose 42 fresh specimens from patients performed resection for ESCC, detect the expression of HIF-1αand VEGF by immunohistochemistry and real-time PCR, analysis the corelation between them and their relationship with pathological features. To choose 60 specimens from patients received radiotherapy for ESCC, detect the expression of HIF-1αand VEGF by immunohistochemistry, and analysis their relationship with radiotheraputic response and prognosis. To design three short hairpin sequences targeting HIF-1αand construct recombinant plasmids expressing HIF-1αshRNA, transfect them into KYSE-150 cell, screen stable transfected cells with Hygromycin. To detect the expression of HIF-1αand validate the effect of RNAi by real-time PCR and Western-blot, detect the expression of VEGF by real-time PCR and Western-blot. To analyse the proliferation and cell cycle distribution of KYSE-150 cells after transfection by MTT assay and flow cytometry. Irradiated with different doses of X-rays, the survival fraction of KYSE-150 cells were examined by MTT and clonogenic assays. The subcutaneous xenograft models in nude mice were established with KYSE-150 and KYSE-150/HIF-1α(-) cell lines and irradiated with single doses of 5Gy,10Gy and 20Gy. To observe the tumor inhibitions by radiotherapy between two models, analyse the improvement of radiosensitivity on KYSE-150 cell after HIF-1αsiliencing by tumor growth delay and radiation dose. To detect the expression of HIF-1α, Ki67 and CD34 by immunohistochemistry between two models. Results:The positive expression rates of HIF-la and VEGF were 50.0% and 76.2% in ESCC respectively, while 14.3% and 33.3% in normal tissues. The expression of HIF-1αwas positively correlated with VEGF in ESCC with a very high degree, while a very weak positive relationship was found in normal tissues. HIF-1αcan be seen in the cytoplasm and nucleus of ESCC, which in the nucleus it is stronger than in the cytoplasm. The expression of HIF-1αin normal esophageal tissues is unstable and low. The expression of HIF-1αin ESCC was related with lymphatic metastasis and histological differentiation, the expression rate of HIF-1αin the cancers with lymphatic metastasis and worse differentiation was higher than that in the cancers with no lymphatic metastasis and better differentiation. The expression of HIF-1αand VEGF were related with radiosensitization and prognosis of ESCC after radiotherapy, those with negative expression had good radiosensitization and prognosis. Three HIF-1αshRNA were constructed and linked to pGCsi-Hl/Hygro/GFP plasmids, which were transfected into KYSE-150 cell. The stable transfected cells RNAi were screened. Real-time PCR and Western-blot confirmed that shRNA3 can silence HIF-1αand inhibit the expression of VEGF, MTT assay and flow cytometry showed that the proliferation attenuated, the ratio of G0/G1 stage cell decreased, and apoptosis increased in shRNA3 group. MTT and clonogenic assays showed the survival fraction in shRNA3 group after irradiation was lower than control group, which showed that the radiosensitivity was improved. The tumor xenografts derived from KYSE-150/HIF-1α(-) cell grew slowly than that from KYSE-150 cell. The result of immunohistochemistry showed the staining of HIF-1α, Ki67 and CD34 in KYSE-150/HIF-1α(-) tumors was weaker than in KYSE-150 tumors. Irradiation can inhibit the growth of both tumors, and decrease the volume of the tumors. The tumor growth delay was more obvious in KYSE-150/HIF-1α(-) group than KYSE-150 group, its sensitivity enhancement ratio was larger than 1.Conclusion:The expression rates of HIF-1αand VEGF were both high in ESCC, there was a strong correlation between them. HIF-la can be seen in the cytoplasm and nucleus of ESCC, while its expression is stronger and more characteristic. The expression of HIF-1αin ESCC was related with lymphatic metastasis and histological differentiation, the cancers with higher expression rate of HIF-1αhad more lymphatic metastasis and worse differentiation. The expression of HIF-1αand VEGF were related with radiosensitization and prognosis of ESCC received simple radiotherapy, those with negative expression had good radiosensitization and prognosis. In KYSE-150 cell the RNAi can silence the expression of HIF-1α, inhibit the expression of VEGF, attenuate the proliferation, increase apoptosis and change the distribution of cell cycle in hypoxia. RNAi on HIF-1αcan inhibit the proliferation and angiogenesis of tumor in vitro and in vivo. It can also enhance radiosensitization of KYSE-150 cell in vitro and in vivo, which mechanisms may be related with the inhibition on proliferation and angiogenesis.