Construction Of HIF-1α Silencing Vector And The Effect On PDT Of Esophageal Squamous Cells

BackgroundEsophageal carcinoma is known as one of the most common malignant tumors and ranks as the fourth mortality in China following that of Hepatic carcinoma, Lung carcinoma and Gastric carcinoma. One third of the patients suffered from this type of cancer died every year. It is believed that early detection, diagnosis and treatment of patients can effectively improve the survival. Surgery is the common clinical therapy for Esophageal cancer, but the survival rate can be decreased due to local recurrence after surgery and the distant metastasis. Photodynamic therapy (PDT) is a treatment modalities using photosensitizer and an appropriate wave lenth that has been applied in the treatment of curative therapies of early esophageal cancer or premalignancies and palliative treatment of invasive esophageal carcinoma over the last 20 years, but the local recurrence after treatment and individual low sensitivity of response to PDT still remian big problems. A better understanding of the determinants in the sensitivity of individuals to PDT can further promote the responses and improve the survival after clinical treatment by rationalizing therapies in different cases.Previous studies has shown that several determinants, including the light, the photosensitizers and the oxygen, play their important roles in PDT. But the biologic characters of tumor cells, the immunity of individuals and some anti- or pro-apoptosis proteins are also involved in these processes. Oxygen play important roles for an effective PDT. but as a feature of solid tumors, hypoxia can reduce responses of tumor cells to PDT. HIF-1αis an essential transcription factor of Hypoxia signal transduction pathway that accumulates in cells due to the low protein degradation by the Ubiquitin-Proteasome which can stimulate the expression of various genes including erythropoietin (EPO). vascular endothelial growth factor (VEGF). multidrug resistance gene (MDR1) and enzymes that relate to energy metabolism. All these processes have been related to cell survival, cell proliferation. cell invasion, angiogenesis and drug resistance. There are also evidences that suggest a potential role of HIF-1αin modulating cell apoptosis. Because inducing apoptosis in tumor cells is one of the essential mechanisms, the expression level of HIF-la may have something to do with the phototoxicity of PDT. This study focuses on the relationship between interference of HIF-1αexpression and the effect of PDT on esophageal cancer cells.In this study, chemical induction was used to establish the mimic cell hypoxia. Protein HIF-1αsilencing vector was constructed for the study of the effect of HIF-1αon PDT of Esophageal squamous cells, which hopefully establish the theoriotical and experimental bases for bettering PDT efficacy through genetic manipulation.The studies are going to be performed as follows:Part one, construction and identification of HIF-1αshRNA recombinant plasmids. Part two, interference of protein HIF-la expression and the effect on PDT of Esophageal squamous cells.Objectives1. To construct HIF-la shRNA vector and to establish the long-term HIF-la inhibition cell line by stable transfection esophageal cancer cells EC-9706 with HIF-1αshRNA vector for further studies on the functions of HIF-1α.2. To test protein HIF-la cellular expression of esophageal cancer cells after chemical induction and the effects on cell proliferation.3. To evaluate the survival rate and apoptosis induction after PDT treatment of esophageal cancer cells following HIF-1αchemical induction and inhibition for iditification the relationships between HIF-la expression and the effect of PDT on esophageal cancer cells.Methods1. According to the principle of siRNA interference, siRNA interference sequence was designed with WHITEHOUSE, siDirect and Rational siRNA Design Online software.2. After changing the siRNA sequnce to shRNA according to the uninsert site of the vector. shRNA was cloned into pENTRTM/Hl/TO and pEZsiRNA6.1 vector with molecular techniques. 3. HIF-1α-shRNA-pEZsiRNA6.1 vector was transfected into Het-1a/EC-97076 cells with liposome complex method. Fluo-rescence photographs was used to test transfection and Western blot method was used to test the HIF-1αprotein expression. Positive transfected EC-9706 cell clones were screened with G418.4. Cell proliferation after protein HIF-la chemical induction and cell survival rate after PDT treatment following chemical protein induction and inhibition were measured by MTT assay.5. Apoptosis was investigated with flow cytometry before and after interference of HIF-1αexpression.Results1. Instant transfection and Western blot showed that protein HIF-la induction was inhibited by designed siRNA. After changing the siRNA into shRNA according to the insert site of the vector, recombinant pEZsiRNA6.1-hif-105 vector was constructed and identified by PCR and sequencing. Fluorescence observation and western blot showed that stable cell transfect was obtained after transfect with the vector. Western blot also showed that protein HIF-la induction was inhibited in this cell line compared to the untransfected Ec-9706/Het-la cells.2. Mimic cell hypoxia was established with chemical method. No obvious differences were obtained in cell growth rate and growth characteristics between cells with and without HIF-1αinterference (p> 0.05).3. The cell survival rate after PDT treatment following cobalt chloride induction in HIF-la protein interference group (EC-9706-pEZsiRNA6.1-hif-105) and non-interference group (EC-9706-pEZsiRNA6.1-hif-neg) were 69.27%±5.21 and 88.36%±2.81 respectively (p<0.01), showing that HIF-1αprotein expression attenuated PDT effect..4. Flow cytometry results indicated that the apoptosis rate in HIF-la protein interference group (EC-9706-pEZsiRNA6.1-hif-105) and non-interference group (EC-9706-pEZsiRNA6.1-hif-neg) were 29.31±2.75% and 13.99±0.89%. respectively (p<0.05). suggesting that the attenuated PDT effect was achieved partially by apoptosis inhibition. Conclusion1. EC-9706-pEZsiRNA6.1-hif-105 cell line with stable interference of HIF-1αexpression was successfully2. High expression of protein HIF-la in esophageal cancer cells attenuated PDT phototoxity. Manipulation of HIF-1αprotein expression restores PDT efficacy.3. Attenuated PDT effect rendered by HIF-1αwas achieved partially by apoptosis inhibition.