Study On Targeting HIF-1α In Human Lung Cancer By RNAi And Its Effects On Tumor Growth And Radiosensitivity In Nude Mice

Objective To construct HIF-1αRNAi lentiviral vectors and HIF-1αgene specific silenced A549 cells.To establish a proper model of human lung carcinoma in nude mice with A549 cells and A549/HIF-1α(-)cells and study the ability of tumor formation,the survival state of nude mice with tumor,the growth curve of tumor, the expression of HIF-1α,angiogenesis and apoptosis of the tumors.To study the effects of different doses of radiotherapy on the lung carcinoma models and the radiosensitivity defference of the tumors.Methods(1)HIF-1αRNAi lentiviral vectors were constructed with BLOCK-iT Lentiviral RNAi Expression System.HIF-1αgene specific silenced A549 cells were constructed by virus transduction and blasticidin screening.HIF-1αmRNA level was determined by real time RT-PCR.HIF-1αprotein was analyzed by Western Blot.(2) Tumors were initiated by injection of 2×10~6 cells under the dorsal skin in the right flank of BALB/c nude mice.Tumor growth were observed.The tumors were pathologically examined by microscope.The expression of HIF-1αand microvessel density were detected by immunohistochemistry method.Tumor apoptosis was detected by TUNEL assay.(3)The human lung cancer model in nude mice were established with A549 and A549/HIF-1α(-)cell lines and were locally irradiated with single doses of 5Gy,10Gy,15Gy,20Gy radiation.The tumor inhibition by radiotherapy was observed.The tumor dose-response ralationship and therapeutic gain of HIF-1αsiliencing were analized by tumor growth delay and radiation dose.Results(1)HIF-1αRNAi lentiviral vectors were constructed successfully.HIF-1αgene silenced A549 cells were established and named A549/HIF-1α(-).The lentivirus transduction efficiency was 1.2×10~6TU/ml.A549/HIF-1α(-)cells were 85.2%and 87.3%decreases of HIF-1αmRNA transcription and 97.2%and 88.4% decreases of protein expressions in normoxia and hypoxia respectively.(2)The size of tumor xenograft derived from A549/HIF-1α(-)cells was significantly reduced when compared to the xenograft derived from A549 cells.The morphological feature,cell arrangement and differentation tumorgenic mass had the characteristic feature of human lung cancer.Tumors from A549/HIF-1α(-)mice showed a dramatic decrease in HIF-1αprotein staining.The microvessel density(MVD)and apoptotic index were 19.83±4.09/0.155mm~2,2.78±0.26 in A549 group, 11.61±3.04/0.155mm~2,2.65±0.23 in A549/HIF-1α(-)group.(3)Radiotherapy could inhibit tumors growth apparently.The tumor growth delay was prolonged with radiation-dose increasing.It was longer in HIF-1αsiliencing group than in A549 group irradiated by the same-dose X-ray.The sensitivity enhancement ratio were 1.40,1.34 and 1.31.Conclusions The construction and production of lentiviral vectors was successful.The selected ceils demonstrated significant disruption of HIF-1αexpression at both mRNA and protein levels.A549 and A549/HIF-1α(-)cell lines had good tumorgenic ability in nude mice.The tumor xenografts of A549/HIF-1α(-) grew more slowly than those of A549.The immunochemistry and TUNEL analysis showed that tumors of A549/HIF-1α(-)had decreasing HIF-1αexpression,reduced vascularity and similar apoptotic rate.Both A549 and A549/HIF-1α(-)tumor growth could be inhibited by radiotherapy.The A549/HIF-1α(-)xenografts were more sensitive to radiotherapy than A549 xenografts.