The Research On Relationship Between HIF-1 Alpha Gene And Radiosensitivity In Esophageal Carcinoma Cells

Esophageal cancer (EC) is one of the most common malignant diseases. On a global basis, cancer of the esophagus is the sixth leading cause of cancer death worldwide. Due to cancer detection in advanced stages,it was over 50% of patients with unresectable or distant metastasis disease at diagnosis. Radiotherapy is one of the effective modalities for middle and advanced esophageal cancer. However, the 5-year overall survival rate remains poor by the high risk local or regeional recurrent after esophageal carcinoma chemoradiotherapy. Clearly, the effection strategies are needed to detect the early esophageal cancer and to improve systemic regeimen options to enhance radiosensitivity of esophageal carcinoma cells, and to increase local contral rate and long-time survival.It had been appreciated for more than 50 years since Thomlison and Gray that lowering the oxygenation of tissues made them more resistant to damage by ionizing radiation, and hypoxia in solid tumors is not only a major problem for radiation therapy but also leads to resistance to most anticancer drugs, importantly, appears to accelerate malignant progression and increase metastasis.With development of the molecular biology technology, there has been some definitive proof that hypoxia in human tumors contributes to chemoradiotherapy rasistance and a series of pathophysiological process.Until recently, some mechanism of radiated irresistance and tumor higher invasion induced by cell hypoxia in solid tumors had been proved: First, hypoxia causes cell cycle stop or progresse slower inhibits DNA replicate in S phase. As we known, radiotherapy and most anticancer drugs are more effective for rapidly those proliferating cells. The tumor hypoxia microenvironment leads to epigenetic and genetic adaptation of clones, such as genetic instability, aerobic glycolysis, loss of cell cycle control, and loss of normal apoptotic signals and increased invasiveness and metastasis. In turn, these hypoxic adaptations make the tumor cell more difficult with the treatment and confer increased resistance to current therapies. On the other hand, Hypoxia in solid tumor can cause the carcinoma cell chromosomal distortion and recombination to result in increasing tumor heterology. Second, the process of hypoxia-reoxygenation in tuomr generate free radical from oxygen, especially hydrogen-oxgen free radical increase genetic instability which make gene in cancer cell more invasion. Third, tumor hypoxia also stimulates tumor progression by promoting angiogenesis through the induction of proangiogenic proteins such as vascular endothelial growth factor (VEGF) and erythropoietin(EPO)and platelet-derived growth factorβ(PDGFβ) through hypoxia-responsive elements (HRE). Amplification of genes and the induction of various hypoxic stress proteins confer chemoradiotherapy resistance. Up to date, More than 70 hypoxia-regulated genes including vascular endothelial growth factor (VEGF), mediated by the hypoxia-inducible factor (HIF) complex, which is composed of a heterodimer pair of HIF-1 and HIF-1?. Given the overexpression of HIF-1 in so many tumor types and association of HIF-1 with tumor angiogenesis, many approaches have been taken to identify agents that reduce HIF-1 protein levels.RNA interference (RNAi) is a mechanism that is rapidly becoming exploited for both research and therapeutic applications. This process was first described by Fire et al. in nematodes, and has subsequently been elucidated as a multistep mechanism. Double-stranded RNAs are processed by the enzyme Dicer into short interfering RNAs (siRNA), 21 to 25 nucleotides in length. These siRNAs are bound to RNA-induced silencing complexes and mediate the degradation of their complementary RNA.In this study, firstly, expression of HIF-1αand VEGF were detected in esophageal carcinoma cells and tissus of esophageal carcinoma and its nomorl tisuss, relationship between protein expression of HIF-1αand VEGF, and development or prognosis after radiotherapy alone in esophageal carcinoma were observed. Secondly, protein expression of HIF-1αand VEGF, modified of cell cycle and radiosensitivity were detected in esophageal carcinoma cells in hypoxia culture in vitro. Finally, short hairpin RNA (shRNA)-plasmid vector of HIF-1αgene were transfected into esophageal carcinoma cells with lipofectamine reagent, effect on mRNA and protein expression of HIF-1αand VEGF, cell cycle and radiosensitivity after radiation were analysed.Part I The study on expression of HIF-1αand VEGF in cells and tissue of esophageal carcinoma and its clinical significaneObjective: To observed expression of HIF-1αand VEGF in esophageal carcinoma cells and tissues of esophageal carcinoma, and relationship between expression of HIF-1αand VEGF, and its progression or prognosis after rediotherapy was analysized.Methods: In human esophageal carcinoma cell lines of Eca109, expression of HIF-1αand VEGF mRNA were detected with reverse transcriptase-polymerase chain reaction(RT-PCR), expression of HIF-1αand VEGF protein were measured with western blotting. 50 specimens from patients with esophageal cancer were biopsied with gastroscopy and diagnosed as esophageal squamous cell carcinoma (ESCC). In adition to 10 cases of esophageal normal tissue were collected; protein of HIF-1αand VEGF in tissues of 50 cases of ESCC and 10 cases of nomorl tissue were detected with immunohistochemistry, prognostic factors were analysis after radiotherapy.Results:①Expression of HIF-1αand VEGF in Eca109 cells were observed in level of mRNA and protein in this study, expression of HIF-1αand VEGF protein were also observed in tissue of 50 cases ESCC while not in nomorl esophageal tissue. The positive expression rates of HIF-1αand VEGF were 68%and 74% in ESCC respectively. The expression rate of HIF-1αwas positively correlated with VEGF protein.②The expression rate of HIF-1αwere closely related to the clinical stage, radiotherapy effectiveness and survival rates, in adition to, the expression of HIF-1αwas closely related to distant matastasis. The effective rate to radiotherapy and mean survival periods of those patients with positive and negative expression of HIF-1αwere 8.8%, 10 months and 81.25 %, 25months, respectively, the total efficiency of clincial response was 84.0%(42/50)(16 cases CR and 26 cases of PR). The one,two,three years survival rate of all patients was 76% and 52% and 27%, madian survive was 13 months. Tumor length of X-ray and CT scan infiltration, clinical stage and lymph nodes and distant metastasis were correlated with prognosis of ESCCs after radiotherapy alone with one-way survival analysis. The expression of VEGF was only related to the distant matastasis. Only clinical stage and expression of HIF-1αis the independent prognostic factor of ESCC with Cox multivariate analysis. The one,two,three years survival rate of patients with positive and negative expression of HIF-1αwere 38.2%,6.0%,2.9%;81.3%,54.2%,15.8%, P=0.001, respectively.Conclusion: Expression of HIF-1αand VEGF were detected in esophageal carcinoma cells and tissues of esophageal squamous carcinoma, which was correlated with clinical stages. The expression of HIF-1αand clinical stages may serve as important parameter in evaluating response for radiotherapy and prognosis of ESCC.Part II CoCl2 induced radiation resistance in Eca109 cells and its mechanismObjective: To observe the cell cycle and radiosensitivity effect on expression of HIF-1αand VEGF mRNA and protein by CoCl2 induced hypoxia in Eca109 cells in vitro.Methods: The hypoxia culture Eca109 cell model was induced by CoCl2. Methyl thiazolyl tetrazolium (MTT) method was used to deteced proliferation activity of Eca109 cells in the presence of increasing concentrations of CoCl2(0,50,100,150,200μmol/L); cell cycle were measured with flow cytometry(FCM), and expression of HIF-1αand VEGF protein were detected with western blotting and the semi-quantitative reverse transcription PCR(RT-PCR) was applied to examine the expression of HIF-1αand VEGF mRNA at different hypoxia culture phase. The radiosensitivity was analysized with clonegentic assay after irradiation alone or combined with hypoxia in esophageal caicinoma cell line Eca109 in vitro.Result:①With the increasing concentrations of CoCl2, the proliferation activity of Eca109 cells was becoming slowly;②The expression of HIF-1αmRNA were observed in Eca109 cells at normol oxygen, the mRNA level of HIF-1αwas not changed significantly after the hypoxia. However, the protein expression of HIF-1αwas increased remarkably with correspondendence to the hypoxia time. Level of HIF-1αprotein expression were markedly increased in nucleoli but not in cytoplasm after hypoxia; VEGF mRNA and protein level were up-regulated under hypoxia.③CoCl2 induced hypoxia could increase cell cycle arrest in G0/G1 phase and decreasing aresst in S phase, with longer of hypoxiac time (0-24h), the other rate of cell cycle and apoptosis did not change obviously. The Eca109 esophageal cancer cell surviving fraction did not change obviously at 24h hypoxia or nomorl control, whereas, those surviving fraction which were radiated with hypoxia increased obviously compared with that not hypoxia. The G2/M phase block was arrested obviously in radiation alone, compared to the hypoxia plus irradiated group did not change remarkably. The apoptosis cells were increased in hypoxia plus radiation group.④The hypoxia decreased the radiosensitivity in espohageal cancer Eca109 cells with clonegenetic assay.Conclusions: Expression of HIF-1αprotein and VEGF mRNA and protein were up-regulated, increasing cell cycle arrest in G0/G1 phase and decreasing aresst in S phase, the radiosensitivity of Eca109 cell was decreased in hypoxia induced by CoCL2.Part III The radiosensitivity influenced by expression of HIF-1αand VEGF with RNA interference HIF-1αin esophageal carcinoma cell line Eca109Objective: To construct the small hairhip RNAs (shRNAs) eukaryotic expression vector targeting HIF-1αgene and under the hypoxia induced by CoCL2 to investigate the effect of the RNA silencing on the post transcription such as expression of HIF-1αand VEGF, and radiosensitivity and distribution fo cell cycle by irradiation in esophageal carcinoma cells treated with RNA interference HIF-1α.Methods:①Two small hairhip RNAs (shRNAs) eukaryotic expression vector targeting HIF-1αgene was constructed and connected with vector of pSilencer 2.1-U6 neo plasmid and were transformed into DH5αE coli, then selected and enlarged culture of E coli, extracted and identified with sequences of plasmid DNA, transfected into culture Eca109 cells via reagent of LiporfectamineTM 2000.Then the Eca109 cells were cultured under hypoxia conditions induced by CoCL2.②The inhibiting effects on HIF-1αgene were determined by RT-PCR and Western blotanalysis. Distribution of cell cycle was measured with folw cytomitry at 48h after interference with HIF-1αgenes after radiated by 5Gy. The radiosensitivity was analysized with clonegentic assay after irradiation alone or combined with RNA interference HIF-1αin esophageal caicinoma cell lin Eca109 in vitro.Result:①The successfully constructing of two shRNAs of HIF-1αplasmid was identified with sequencing.②After transfected with HIF-1αshRNA, the Eca109 cells mRNA and protein expression of HIF-1αand VEGF genes were inhibited obviously. Hypoxic induction of HIF-1-controlled target genes like vascular endothelial growth factor (VEGF) were markedly attenuated by HIF-1alpha-siRNA treatment.③Cycle cell and apoptosis did not changes obviously in Eca109 cells at 48h with interference of HIF-1αgenes after radiated by 5Gy. TheG0/G1 phase arreste was decreased obvioursly and G2/M phase arreste increased in shRNAi HIF-1αwith radiation while apoptosis increasing in radiation only.④The shRNAi HIF-1αcould enhance the radiosensitivity through increasing G2/M phase blocking in espohageal cancer Eca109 cells with clonegenetic assay.Conclusion: mRNA and protein expression of HIF-1αand VEGF genes were inhibited obviously, and the radiosensitivity of Eca109 cell was increased with shRNAi HIF-1αgene in Eca109 cells...