Study On Functioning Of Astrocyte Elevated Gene-1and E-cadherin In Cervical Cancer And Effects Of AEG-1gene Silencing On Epithelial To Mesenchymal Transition Of HeLa Cells

BackgroundCervical cancer is the most common female genital tract malignancy around the world, particularly in Africa, Asia and South America. The morbidity and mortality of the cervical cancer are in the second place only after breast cancer in women. There is about13million newly diagnosis cases in China. Those new cases in china accounting for1/3of the world’s total new cases. There are around53,000deaths annually in China, ranking second in the world. The high risk of cervical cancer related to many factors. Age is one of them, cervical cancer patients between20-50years of age growth rapidly then slow down thereafter. In situ cervical carcinoma peak among the age of40-44, the peak age of invasive cervical cancer is between45and54. In recent years the incidence of cervical cancer appears a trend of advancing in age. Secondly, ethnicity, region, occupation, socioeconomic status also have an impact on the prevalence of cervical cancer. There is a lot of research reports about cervical cancer risk factors, such as HPV infection, sexual behavior, immune function, socioeconomic status, maternal history of herpes simplex virus type II, food, smoking and genetic susceptibility etc. These factors are affecting the incidence of cervical cancer alone and also synergistically. Compared with other malignancies, cervical cancer is relatively with good prognosis if detected early. Many patients without been screening arrive at very late stage once discovered. Cervical lesions occur in the cervical squamous columnar junction cells. The cancer cells will break through the basement membrane to the deeper tissues and even spread to nearby organs.Epithelial-mesenchymal transition (EMT) is a multi-step morphogenetic process during which epithelial cells downregulate their epithelial properties and upregulate mesenchymal characteristics. Namely, static epithelial cells lose cell to cell junctions and as a consequence they lose apico-basal polarity to become migratory mesenchymal-like cells. As a feature of aggressive tumors, epithelial to mesenchymal transition is characterized by reduced E-cadherin and increased N-cadherin and vimentin expression. Meanwhile, transcription factors such as Snail increased their expression. There are tightly relationship between EMT and tumor invasion and metastasis. Presently, little is known about the regulation of EMT in cervical cancer cells and tissues and how the cervical cancer cells moved to far regions have been studied.Astrocyte elevated gene-1(AEG-1) was initially identified in primary human fetal astrocytes as a novel gene induced by human immunodeficiency virus (Su et al.,2002). Human AEG-1gene is located in Chromosome8q22having12exons/11introns. The mRNA encodes a single pass transmembrane protein of predicted molecular mass of64kDa and pl9.3that predominantly localizes in the endoplasmic reticulum and perinuclear region. In cancer, AEG-1is markedly overexpressed in all cancer indications studied so far, including prostate, breast, neuroblastoma, esophageal squamous cell carcinoma (ESCC), gastric, colorectal, non-small cell lung cancer (NSCLC), et al. and are associated with disease progression and poor clinical outcomes. Moreover, AEG-1has been found to promote cancer chemoresistance, metastasis, invasion, and angiogenesis. Based on the previous studies, we hypothesize that AEG-1might be associated with EMT in cervical cancer.RNA interference (RNAi) is a phenomenon, in which double strand RNA (dsRNA) silences the homologous gene expression. RNA interference has been a powerful tool in the research of specific gene function and it has also been used in gene therapy function research of cancers. It is extensive that lentiviral vectors are used in mediating RNAi for two features:more efficient transduction of non-dividing cells and stable RNAi by integrating shRNA expression cassette into genome of host cells.In this study, we addressed this hypothesis by RNAi knockdown of AEG-1in cervical cancer HeLa cell line and found that AEG-1knockdown not only decreased EMT in cervical cancer cells but also attenuated their aggressive behavior as shown by reduced colony formation and increased sensitivity to cancer drugs.Chapter one:The expression of astrocyte elevated gene-1and E-cadherin in cervical cancer1. Aim(1) To detect the expression of AEG-1/MTDH and E-cadherin in cervical cancer tissue samples.(2) To study the relationship between AEG-1/MTDH and clinical characteristics, the relationship between AEG-1/MTDH and E-cadherin.2. Materials and Methods(1) This study was conducted on a total of52paraffin-embedded cervical cancer samples, which were clinically diagnosed at Department of Pathology, the First Hospital of Lanzhou University from January2009to July2011. The fresh surgical cervical cancer samples were selected from Department of Obstetrics and Gynecology, Gansu Provincial People’s Hospital and Hysteroscopic Center, Maternal and Child Health Care Hospital of Lanzhou City.(2) Immunohistochemistry and Western blot for AEG-1protein expression using a rabbit polyclonal anti-AEG-1antibody3. Results(1) AEG-1/MTDH was strongly positive in37of52cervical cancers (71.2%)(2) We analyzed the clinical data of52cervical cancer samples. AEG-1expression was65.1%(28/43) among early stage (I+11) samples, and100%(9/9) among advanced stage (Ⅲ+Ⅳ) samples. Compared to the group with positive AEG-1expression, AEG-1expression was significantly associated with clinical staging (P=0.046); AEG-1expression was55.6%(15/27) among high-differentiated samples, and88%(22/25) among low-differentiated samples, Compared to the group with positive AEG-1expression, AEG-1expression was significantly associated with histology classification (P=0.014). E-cadherin expression was88.4%(38/43) among early stage (Ⅰ+Ⅱ) samples, and33.3%(3/9) among advanced stage (Ⅲ+Ⅳ) samples. Compared to the group with positive E-cadherin expression, E-cadherin expression was significantly associated with clinical staging (P=0.001); E-cadherin expression was92.6%(25/27) among high-differentiated samples, and64%(16/25) among low-differentiated samples, Compared to the group with positive E-cadherin expression, E-cadherin expression was significantly associated with histology classification (P=0.017).(3) The expression of AEG-1was increased with clinical stage and histology classification. The expression of E-cadherin was decreased with clinical stage and histology classification. Positive expression of AEG-1was associated with expression of E-cadherin (r=-0.707,P=0.009) by spearman correlation analysis in advanced stage.4. Conclusions(1) AEG-1was strongly positive in37of52cervical cancers (71.2%)(2) AEG-1and E-cadherin expression was significantly associated with clinical staging and histology classification.(3) Positive expression of AEG-1was associated with expression of E-cadherin (r=-0.707, spearman correlation analysis in advanced stage. Chapter Two:Effects of AEG-1gene silence on biological behavior of cervical cancer HeLa cells1. Aim(1) To constructe AEG-lgene specific silenced HeLa cells and the non-specific controls.(2) To determine AEG-1mRNA level and analyze AEG-1protein.(3) To analyze the EMT markers protein(4) To test the function of AEG-1in migration of cervical cancer HeLa cells.(5) To determine the function of AEG-1in invasion.(6) To illustrate the function of AEG-1in proliferation(7) To elucidate the function of AEG-1in drug sensitivity(8) To analyse the NF-κB p65protein2. Materials and Methods(1) AEG-1/MTDH gene specific silenced HeLa cells and the non-specific controls were constructed by virus transduction and puromincin screening.(2) AEG-1/MTDH mRNA level was determined by RT-PCR.(3) AEG-1/MTDH protein and EMT markers protein were analyzed by Western Blot.(4) Migration of cervical cancer cells were tested by scrasy assay.(5) Invasion of cells were researched by transwell method.(6) Proliferation of cells were tested by clonogenic forming assay.(7) Drug sensitivity of HeLa cells were tested by MTT and soft agar assay3. Results(1) AEG-1gene specific silenced HeLa cells were established constructed and named HeLa-AEG-1shRNAl, HeLa-AEG-1shRNA2(2) Transduction of cells with AEG-1RNAi vector resulted in sequence specific silencing with82%decreases of AEG-1mRNA transcription and91%of protein expressions respectively.(8) Cells transfected with AEG-1shRNAl had significantly decreased levels of AEG-1compared with the control cells. Knockdown of AEG-1downregulated the mesemchymal vimentin and EMT transcription factor Snail. N-cadherin was downregulated while the level of E-cadherin was not significantly altered. Since AEG-1shRNA2knockdown did not work as well as shRNA1construct, we only used AEG-1shRNA1construct as AEG-1knockdown for subsequent assays.(9) Migration distance of AEG-1shRNA cells was greatly reduced compared with the vector control cells by24h (P<0.05). Moreover, AEG-1shRNA cells (284.25±39.9) showed significantly decreased invasion ability in the transwell assay in comparison with the shRNA vector cells (525.4±83.7; P<0.01)(10) AEG-1shRNA cells (45±11.1) showed significantly decreased CFU in soft agar assay in comparison with the shRNA vector cells (102±17.2P=0.05)(11) Paclitaxel (2.5nM) and cisplatin (5μM) inhibited colony formation ability of AEG-1shRNA cells to about3-8colonies/5000cells, but the same treatment produced about30-170colonies/5000cells for the vector cells (P<0.01).(12) NF-κB p65activity was decreased in AEG-1shRNA cells compared with shRNA vector cells4. Conclusion(1) Knockdown the AEG-1decreased EMT.(2) AEG-1serves in regulating cell migration, invasion and cell proliferation.(3) Knockdown the AEG-1increased the sensitivity of chemotherapy in HeLa cells.(4) Knockdown the AEG-1decreased the expression of NF-κB p65.