Specific Suppression Of HIF-2α Oncoprotein By RNA Interference In Hela Cells Through Slow Virus Infection

Cervical cancer is one of common gynecological malignancies. The mortality rate is second in all women's cancer mortality rate. In recent years, the incidence of cervical cancer is increasing gradually. And the patients are younger and younger. The methods and the steps of cervical cancer's diagnosis has almost completed. However, the treatment of cervical cancer are constantly being improved. At present, the treatment of cervical cancer has maintained at the current situation of combined treatment,that include surgery, chemotherapy and radiotherapy. And treatment effectiveness did not be significantly improved. Particularly in patients with advanced cervical cancer, treatment is not satisfactory,and the quality of life is poor.The resistance to radiotherapy and chemotherapy have emerged in recent years.It makes the treatment of cervical cancer as "bottleneck". How to treat cervical cancer, effectively improve the efficacy and improve the cure rate and quality of life have become one of the most critical problems.With the development of science and technology in recent years, the treatment of cancer has been increased to the level of molecular biology. Parts of the target gene for the effective treatment method have been used in the clinical of cancer study. However, the molecular biology of cervical cancer treatment is still in the stage of research. The development of tumor is very hard to understand,because it include many genes. And the change of micro-environment can induce the process more difficult. Find the important genes, and study the gene's change in micro-environment is the first step of our study. Wether the gene can be interfered is the first step of whether it could be carried on treatment in clinic. We hope we can find a very important gene from the micro-environment of tumors,and the gene can be effect interfered.After all,we hope we can support the evident to the gene treatment on cervical cancer.This study is to find a valid target gene.And we can effectively interfere with the target gene. We hope that through interfering with the gene to improve the efficacy of cervical cancer. Or by methods of molecular biology,we can cure cervical cancer effectively. It contributes to find a new way to improve the efficacy and the treatment of cervical cancer.Cervical cancer is a solid tumor of the body. It found that the cervical cancer tissue of patients has lower PO2 than normal cervical tissues, and with the increaseing of cervical cancer, the level of PO2 is lower. That is to say there is hypoxia in cervical cancer. hypoxia plays an important role in the process of tumor formation and development. It participates in the metabolism of tumor cells to adapt to apoptosis resistance, angiogenesis and invasion, metastasis and so on. Hypoxic microenvironment of tumor cells achieve these roles by hypoxia inducible factor family (HIF). At present, the known hypoxia inducible factor includes HIF-1, HIF-2 and HIF-3. Each factor is made up with different functions and the same structural subunits.HIF-2a, that is the function subunit of HIF-2,has been found in 1997. It is the most important one of the found hypoxia inducible factors. The present study was less, but has attracted extensive attention from medical researchers at home and abroad, and the research on it has been constantly expanded. Now research has shown that HIF-2a gene was related with lung cancer, kidney cancer and other cancers, and was confirmed in these tumors HIF-2αgene plays the unique role by HIF,and can not be replaced. But there are also studies that HIF-2 gene expresses in tissue-specific, and affected by the surrounding environment. Ferthermore,the HIF-2a gene has be showed play an very important actions in avoid radiotherapy and chemotherapy, so we think it is very important in therapy of tumor. Whether HIF-2αgene expresses in cervical cancer, and is there any relationship between its expression and the environment? Whether the HIF-2αcan be interfered effectively? And whether HIF-2αcan effect on the active of cervical cance? The micro-environment can change the action of genes?How many genes are the aim gene?And how the micro-environment effect on it? There is no relevant research reported at home and abroad.In this study, the expression of HIF-2a gene incervical cancer Hela cell line is firstly studid. And then, we interfere with HIF-2a gene by RNAi technology, find the best target for interference,and interfere HIF-2a of Hela cells effectively to understand the expression of genes. So that we can explore the mechanism of the HIF-2a in cervical cancer Hela cells. Finally, after the effective interference with HIF-2αof Hela cells, we carried out cell cloning and cell invasion experiment to investigate the HIF-2a for the biological behavior of Hela cells. Through the experiment, we hope that it can provide new ideas and methods for the clinical treatment of cervical cancer. Part oneThe development of Hela cells and the expression level of HIF-2αin cervical cancer Hela cells in hypoxia and normoxia conditions.Purpose:In this study,we detect the development of Hela cells and the expression of HIF-2αin normal oxygen and hypoxic conditions. Comparison with normoxia, we investigate the trend of Hela cells and HIF-2α's expression in hypoxia conditions.Methods:we detect the development of Hela cells and drow the thread of Hela by MTT method and detect the expression levels of HIF-2αmRNA under normal oxygen and hypoxic by real-time PCR.Results:1. (1). Under normoxia conditions:OD value of Hela cell begins to grow from the 1st day, reaches the peak at the 5th day (p<0.05), and is slightly lower at the 7th and the 9th days. And, compared the 7th days and the 9th days, there was no significant difference (p> 0.05). (2). Under hypoxic conditions:OD value of Hela cell begins to grow from the 1st day,reaches the peak at 3rd day, and was significantly lower from the 5th day(p<0.05) 2.(1). Compared with the normal oxygen condition, the expression level of HIF-2αmRNA in Hela cells was significantly higher under hypoxia conditions(p<0.05); (2). As we extended the hypoxia time, the expression levels of HIF-2αmRNA in Hela cells was significantly higher, and reach the peak at the third day (p<0.05), but there was no significant difference between the 5th day and the 3rd day (p> 0.05)Conclusion:Compared with the normal oxygen condition, the expression level of HIF-2αmRNA in Hela cells was significantly higher under hypoxia conditions. As we extended the hypoxia time, the expression levels of HIF-2αmRNA in Hela cells was significantly higher, and reach the peak at the third day. It prompts the expressed abundance of HIF-2αmRNA has a certain time-dependent. The growth of Hela cells reached a plateau in 3-5 days.And in 7-9 days, most Hela cells have died. It prompts that HIF-2αcan induce the growth of Hela cells.But it can not make Hela cells be tolerant to hypoxia conditions for a long time. Part two The study of lentiviral vector for efficiency of HIF-2αgene silencing in Hela cellsPurpose:In this study, by screening the 4 siRNA interference sequence,which considers HIF-2αmRNA as target sequences,we find the most effective way to inhibite expression vector of HTF-2α, the basis for subsequent experiments. It lays the foundation for subsequent experiments.Method:We used RNAi design software, design role for HIF-2αmRNA in the four siRNA sequences, and inserted into the lentiviral vector respectively.Through the packaging of the viral vector, higher titer slow virus infecte 293T cells. We detecte 4 siRNA interference effect by using RT-PCR and werstern. Thus,we screened for interference sequence which has strong effect on HIF-2αgene silencing.Then use two dose lentivirus vector which have the most effective siRNA sequence to infect Hela cells,and test the expression of HIF-2αmRNA by real-time PCR method. To make sure the effect that the siRNA sequence silence the HIF-2αgene in Hela cells,and find the best dose of virus.Results: 1.In the four siRNA sequences, KD2 sequence has the strongest effect on the expression of HIF-2αinterference (p<0.05).2.The higher dose of virus has more effctive on silencing HIF-2αgene in Hela cells.Conclusion:1. The four siRNA sequences all has the effect on silencing HIF-2α, and KD2 sequence has the strongest effect on the expression of HIF-2αinterference (p<0.05). It prompts the KD2 can effectively inhibit the expression of HIF-2αgene.2.Higher dose virus vector can has more effective action on silencing HIF-2αgene. Part three HIF-2a gene silencing's effect on the biological characteristics of Hela cell.Purpose:In this study, before and after HIF-2αgene is silencing, in normoxia and hypoxia conditions, we detecte the expression of the biological characteristics of genes (cox-2, VEGF) in the Hela cells, cell cloning efficiency and invasiveness. We preliminarily study HIF-2a gene silencing's effect on the biological characteristics of Hela cells.Method:we cluture Hela cells in normoxia and hypoxia conditions,and then in different condition,Hela cell has divied three groups:ontrol group(CON), negative control group(NC) and knock down group(KD). We detect the expression of HIF-2a, cox-2 and VEGF in normoxic and hypoxic conditions by the use of real-time PCR. Before and after gene silencing we detect Hela cell cloning efficiency in normoxic and hypoxic conditions by the use of limit dilution cloning method. Before and after gene silencing, we detect the strength of Hela cell invasion in normoxic and hypoxic conditions by the use of Test of tumor cell invasion.Results: 1. In Hela cells the expression of HIF-2 gene in normoxic conditions and hypoxic conditions were significantly lower after being silence by siRNA (P<0.05).but there is no significantly difference between the gene expression under normoxic and hypoxic conditions (P> 0.05).2 (1). In normoxic and hypoxic conditions,,there is no significantly difference between expression levels of cox-2 mRNA in infected Hela cells and negative infected Hela cells. (2). I n the HIF-2a gene not silent Hela cells, in normoxia and hypoxia conditions, the expression levels of the cox-2 mRNA were not significantly different (p> 0.05); (3). In normoxic and hypoxic conditions, in HIF-2a gene silencing Hela cells, the expression of cox-2 mRNA was significantly lower than silent cells (P <0.05);But under normal oxygen conditions and hypoxic conditions, the expression of cox-2 mRNA levels were not significantly different (p> 0.05).3 (1). In normoxic and hypoxic conditions, compared the expression of VEGF mRNA not infected Hela cells with negative control virus-infected Hela cells,there was no significant difference (p> 0.05); (2). In normoxic conditions, compared the expression level of VEGF mRNA in HIF-2αgene silencing Hela cells with silence cell there is no significant change (p> 0.05). (3). Under hypoxic conditions, in HIF-2αgene silencing Hela cells, the expression of VEGF mRNA levels were significantly lower than silent cells (p<0.05); (4) In the HIF-2αgene silencing Hela cells, under hypoxic conditions, the expression of VEGF in Hela cells was significantly higher than in normal oxygen conditions (p <0.05); (5). In the HIF-2a gene silencing Hela cells, compared with the normal oxygen conditions, under hypoxic conditions, the expression of VEGF in Hela cells did not change significantly (p> 0.05).4 (1). In HIF-2αgene not silent Hela cells, in normoxia and hypoxia conditions, the cloning efficiency of Hela cells was not significantly different; (2). In normoxic and hypoxic conditions, in HIF-2a gene silencing Hela cell,colony formation were significantly lower than silent cells; (3). In the HIF-2a gene silencing Hela cells, compared with in the normoxic conditions, colony formation of Hela cells under hypoxia conditions was significantly reduced.5 (1). In HIF-2a gene not silencing in Hela cells, compared with the normal oxygen conditions, under hypoxic conditions, the invasion of Hela cells was significantly lower. (2). In hypoxia and normoxic conditions, HIF-2a gene silencing invasion of Hela cells'were significantly lower than silent cells', (3). In the HIF-2αgene silencing in Hela cells, compared with the normal oxygen conditions, under hypoxic conditions, invasion of Hela cells did not change significantly.Conclusion:1. HIF-2αin Hela cells can be effectively silent by KD2 sequence and will not be effected by hypoxia factors; 2 Down regulatation of HIF-2a gene expression can result in down regulatation of cox-2 gene.3 In the hypoxic conditions reduction of the HIF-2a's expression can result in reduction of VEGF's. (4). In HIF-2a not silence Hela cells, compared with the normal oxygen conditions, under hypoxic conditions, the expression of VEGF in Hela cells significantly increased.lt suggests that hypoxia can induce Hela cells to increase in the expression of VEGF mRNA; 4 After HIF-2a gene silencing, tumorigenicity and invasiveness in Hela cells was significantly decreased. Down regulation of HIF-2a gene in Hela cells can make Tumorigenicity of Hela cells be significantly lower than that in normoxic conditions, but cell invasion force isn't affected by the oxygen factors.