The Expression And Significance Of HIF-1α In Cervical Carcinoma And Cervical Intraepithelial Neoplasia

IntroductionCervical carcinoma is onlae of the most frequently founded malignant tumors in women which severely threats the health and life of women patients. Its incidence rate is in the second place and inferior to breast cancer. Recently its incidence rate increases significantly and has the tendency of juvenescence. With the popularity of the screening methods, such as vaginoscope, it is easier to diagnose and treat as possible as quickly. Because of the improvement and enhancement in the therapeutics, such as operation, radiotherapy and chemotherapy, the mortality of cervical carcinoma has dropped enormously. In spite of this, the overall survival rate of cervical cancer is only 40%. Recent research indicates that hypoxia is the initiating agent in the malignant transformation and metastasis of tumor. Hypoxia-inducible factor la widely resides in the mammalian and homo-sapiens whose activity contributes to the energy metabolism and angiogenesis and proliferation. The genesis of cervical carcinoma needs a long process. Generally it takes more than ten years to develop into the cancer from the cervical intraepithelial neoplasia. So we investigate the genesis and development of cervical cancer from the molecular level who hope to provide new orientation for the therapy and intercepting its malignant transformation. Although the expression of HIF-1αhas been reported recently, there are less reports about its expression in cervical liquid-based cells and their correlations. We use the methods of immunohistochemistry,western blot and RT-PCR to detect the expression of HIF-1αin cervical tissue and cervical liquid-based cells. We judge its expressional characters and provide help for revealing the genesis mechanism and understanding the biological behaviour and judging the prognosis and choosing the reasonable therapeutic regimen.Materials and Methods一,SamplesThe tissue paraffin blocks of 78 cervical carcinoma and 40 CINⅠand 12 normal cervical tissues were obtained from the pathological department of the First Affiliated Hospital of China Medical University between 1996 and 2004. The other 41 liquid-based cells samples and fresh cervical tissues were obtained from the department of gynecology in the First Affiliated Hospital of China Medical University between September, 2005 and December, 2006.二,Main Reagents:Mouse anti-human monoclonal antibody and bovine anti-mouse IgG HRP were bought from Santa Cruz company of USA. Ultra-sensitive S-P kit and DAB agent kit were from MaiXin biological technology company in Fuzhou. Rabbit anti-human polyclonalβ-actin antibody and Goat anti-rabbit antibody labelled with horse radish peroxidase were obtained from ZhongShanJinQiao biological technological limited company in Beijing. RT-PCR kit and PVDF membrane were provided by Dalianbaoshengwu biological company.三,Methods1. ImmunohistochemistryAdopt immunohistochemistry S-P method. Result determination: The positive signal of HIF-1αis in the nucleus or cytoplasm stained by buff or yellow brown. The expression of HIF-1αis assessed by the plus between density and positive rate of staining. Select 5 fields of vision of high power at random to carry out the criterion. The staining intensity was scored as negative (0), weak (1), moderate (2) and strong (3); The number of cells stained was graded as follows:≤5%(0),5%～25%(1),25%～ 50%(2),＞50%(3). Above-mentioned two items were added. 0 score is negative(-), 1-2 score are 1+, 3-4 scores are 2+ and 5-6 scores are 3+.2. Western blotGet fresh sample and 5 volumes of schizolysis buffering. Clip to pieces and homogenate the tissues. Centrifugate it with high speed and low temperature to collect supernatant protein. Undergo the procedures such as electrophoresis, transferring protein from gel to PVDF membrane, blocking, incubation with primary antibody and second antibody, staining and so on. Result determination is that there is brown strap in membrane. Measure the gray scale of protein strap.3. RT-PCRThe main procedures are follows: extracting RNA, measuring OD value, reverse-transcriptional and PCR reaction, electrophoresis. The light strap in agarose gel in proper position is the positive result. Determine the gray scale.4. Statistical analysisAll the datas were analyzed with SPSS 11.0 software. We used x~2 to test the expression and correlation with the clinical pathological factors and significance in different cervical leisions.Results一,Immunohistochemistry result of HIF-1αin cervical tissueHIF-1αis mainly localized in the nucleus and(or) cytoplasm. The positive rate of HIF-1αprotein in normal, CINⅠ, CINⅡ-Ⅲand cervical carcinoma respectively is 0,6.67%,48% and 58.9%. The expression of HIF-1αisn't correlated to age of the patient but to the size,FIGO stage,differentiation and metastasis of lymphonode.二,Variation of protein and mRNA of HIF-1αin cervical tissueThe result of Western shows: There isn't expression of protein in normal and para cancerous tissues. It increases with the aggravation of cervical lesions. There is distinct discrepancy between cervical carcinoma and CINⅡ-Ⅲor between normal group and CINⅠgroup. There is not statistical significance of protein between CINⅠand normal as well as between para cancerous and normal tissue or between CINⅡ-Ⅲand carcinoma.The result of RT shows: There are specific objective gene straps of HIF-1αandβ-actin in 315 and 485 kd. The expression of HIF-1αmRNA increases with the aggravation of cervical lesions. There is significant discrepancy between carcinoma or CINⅡ-Ⅲand normal or CINⅠ. There is no statistical significance of mRNA between CINⅠand normal as well as between para cancerous and normal tissue or between CINⅡ-Ⅲand carcinoma.三,The variation of protein and mRNA of HIF-1αin cervical liquid-based cellsThe result of Western shows: There isn't protein expression of HIF-1αin normal group. It increases gradually with the aggravation of lesions and achieves the maximum. There is significant discrepancy between carcinoma or HSIL and normal or LSIL by comparison. There isn't statistic significance between LSIL and normal or between HSIL and carcinoma.The result of RT shows: There isn't expression of mRNA in normal exfoliate cell. It is low in LSIL and high in HSIL and carcinoma. There is distinct discrepancy between HSIL or normal and LSIL or normal. There is no statistic significance between LSIL and normal or between LSIL and normal or between HSIL and carcinoma.DiscussionCervical carcinoma is one of the most founded malignant tumors in female reproductive system. There are anoxic cells in it like other tumors. Hypoxia is not only involved in the neovascularization but also in all kinds of physiological functions by many complicate mechanisms. It is the main driving force of growth, apoptosis, invasion, metastasis and tolerance to radiotherapy and chemotherapy of tumor cells. HIF-1αis in the core in a serious of effe'cts.We adopt immunohistochemistry method to observe the expression of protein level of HIF-1αin cervical tumor at first. Result shows that there are 58.9% and 48.0% of HIF-1αover-expressed in cervical carcinoma and CINⅡ-Ⅲrespectively. The positive site is in the nucleus or cytoplasm. There is statistic significance of above slides compared to the normal tissues which shows that HIF-1αmay be expressed in cervical carcinoma and precancerosis lesions. It is also the early event of genesis of uterine cervix cancer and promote the malignant transformation of cellular phenotype. Zhong suggested that HIF-1αwas over-expressed in some lesions precancerous and early cancerations. All of these suggest that there is up-regulation of HIF-1αexpression in prophase and nonage of carcinogenesis. At the same time, we also find that the degree of expression of HIF-1αis correlated with the size, metastasis of lymph node, clinical stage and pathological grading. There are more expression of HIF-1αwith the more late of clinical stage and the rise of pathological grading. It is significantly higher in the group with the metastasis of lymph node than that in the non-metastasis of lymph node which suggests that HIF-1αis involved in the invasion and metastasis of tumor in the advanced stage. It takes an important role in the carcinogenesis and development of uterine cervix cancer and is an important prognostic indicator. Its mechanism may be that there is deficiency in blood supply with the growth and invasion of tumor which induces the expression of HIF-1α. It promotes the transcription of many target genes, angiogenesis, energy metabolism, continuous growth, invasion to periphery tissue and distant metastasis.We further adopt Western blot method to detect the expression of protein in different cervical tissues and liquid-based cells which shows that it is higher in cervical carcinoma and precancerous change than that in normal cervical tissue and para-cancer normal tissue. We consider that there are more oncogene activation and anti-oncogene inactivation in poorly differentiated tumor which delay the degradation and increase its endocellular level. We detect the level of mRNA in cervical tissue and liquid-based cell which shows that it is higher in cervical carcinoma and CINⅡ-Ⅲthan that in normal cervical tissue. The expression of mRNA in HSIL and carcinoma is higher than that in normal cervical cell and ASCUS. On the whole there is similar result between transcriptional level and translational level. Volm found that it was high in transcriptional level and apoptosis rate in lung cancer cell. Liquid-based cytology test is the great revolution in the collection of cellular specimen and making procedure of slide. It is a test facility of the oecumenical, high performance, micro-trauma, shortcut and convenience which suits to the crowd census and elevates the detection rate of uterine cervix cancer in early stage. We first utilize the specimen to detect the expression of HIF-1αand get the similar result and expand its application realm.Therefore, there is important effect of HIF-1αin occurrence and development of tumor. Monitoring and studying to the protein and mRNA of HIF-1αwill contribute to the early diagnosis of uterine cervical cancer. It can predict the malignant degree, metastasis potential and prognosis. It is most likely that it be a new kind of clinical diagnosis criteria and tumor marker. It is detected in precancerous change tissue which may be the early event in the process of tumor and has the value of forecasting tumor. With the further investigation of HIF-1αtargeting it may be an important means to tumor therapy.Conclusions1. HIF-1αprotein is over-expressed in cervical carcinoma and CINⅡ-Ⅲ.2. HIF-1αmRNA increases with the aggravation of the cervical lesions and coincides with the level of protein. There are similar results between tissue specimen and cell specimen.