| BackgroundCervical cancer is a great threaten to women’s health and is the second factor contributed to women death. Currently, the treatment of cervical cancer still depends on surgery, radiotherapy and chemotherapy or comprehensive therapies. Athough the treatments are effective, the cancer reccurrence remains the most tough problem encountered in the treatments.Previous researches indicated that although the standardization of treatments are conducted,the recurrence of cervical cancer is nearly 35%, and nearly 90% were occurred in 3 years after the initial treatments.The metastasis followed the relapse of cervical cancer are still the most tough problem encountered in its treatments, which is also the major cause of death and treatments failure in cervical cancer. Also, the current treatments for the advanced cervical cancer suffers with metastasis are still very limited. It is widely accepted that persistent infection of high-risk human papillomavirus(Hr-HPV) is the main cause of cervical cancer. Recently, due to the development of screening strategies and the application of HPV preventive vaccines,the occurrence and mortality of cervical cancer in developed countries is decreased significantly. However,these screening methods and HPV vaccines could not be widely used in developing country,due to their high cost. Therefore, the occurrence of cervical cancer in china is still increased. So to further elucidate process and mechanism of cervical cancer progression is the cornerstone of developing new therapeutic strategies.Epithelialmesenchymal transition(EMT) is the main reason leading to tumor metastasis and plays an important role in cervical cancer progression.In this process,the polarized epithelial cells loose the epithelial surface markers and transforms into mesenchymal cells with the ability of invasion and metastasis. The most important characteristics of EMT is the downregulation of epithelial cell markers,like E-cadherin and the up-regulation of mesenchymal cell surface markers such as vimenti,along with the increased cell mobility and decreased adhesion.Previous studies indicated that EMT also involved in the process of embryonic development and organogenesis, wound healing and tissue remodeling, organ fibrosis, chronic inflammation, tumor invasion and formation.In addition, EMT plays an important role in cervical cancer metastasis and progression. A variety of factors in vivo or vitro can lead to the occurrence of EMT in cervical cancer, including hypoxia, HPV infection, activation of oncogenes and the inbalance of cervical microenvironment. These factoers could swith on the programme of EMT and promote migration in cervical cancer through activating EMT-related transcription factors.Previous studies demonstreated that activation of EMT signaling pathways were appeared in a variety of epithelial tumors. Interestingly, the activation of EMT is a dynamic process: EMT can promote local tumor invasion, however,the reverse EMT to MET was an important step essential for distant organ metastasis,which suggested EMT is not regulated by genetic or epigenetic,instead by a variety of molecular from the tumor microenvironment(TME).Tumor microenvironment(TME) is an external environment surrounded the tumor cells, which is composed by fibroblasts, immune cells, extracellular matrix and a variety of cytokines secreted by these cells. Studies showed TME played an important role in tumor torlerance and growth,drug resistance and tumor metastasis,which provided a sutable environment for tumor growth and metastasis and inducing drug resistance and tumor relapse.The cytokines in the TME such as tumor necrosis factor(TNF-α), interleukin(IL-1,6,8), transforming growth factor(TGFβ), angiogenic factors(VEGF & PLGF) and some non-tumor cells, including macrophages, lymphocytes, all play an important role in promoting tumor progression. HPV infection is the major cause of cervical cancer and is an important factor caused the changes of cervical microenvironment.Persistent infection of HPV could result in persistent inflammation status in cervix, inducing and activating many inflammatory cells secreted a variety of cytokines and chemical materials to form a complex cervical vaginal microenvironment,which play a vitol role in the development and progression of cervical cancer.The transformation of normal epithelial cells to malignant epithelial cells caused by HPV infection is a gradual process, where EMT is an important factor leading to the normal epithelial cells to invasive cancer cells.It is clinicopathologically confirmed that cervical cancer is gradually transformed from cervical intraepithelial neoplasia(CIN).Some CIN lesions may be reversed to normal epithelium. These finds at least confirmed that the differentiate direction of the transformed epithelium is not regulated by genic or epigenic way, instead by cervical microenvironment. The inbalance of the cervical microenvironment caused by HPV infection may play an important role in regulating EMT and progression of cervical cancer. Previous studies found that PLGF is overexpressed in various tumor tissues and actvating its receptoer Flt-1 could promote EMT in breast cancer and pancreatic cancer cells. PLGF promote tumor invasion and progression through activating tumor angiogenesis. Also it is confirmed that HPV infection can cause the inbalance of cervical microenvironment and induce the secretion of various proinflammatory cytokines, including PLGF, which will work together to promote the development from CIN to invasive carcinoma. Meanwhile,The overexpression of PLGF mRNA could be detected the cervix epithelium infected with HPV,and the expression of PLGF and its mRNA in cervican cancer tissues is significantly higher than the matched adjacent normal tissues. Thus, PLGF, as an inportant components of cervical microenvironment caused by HPV infection,is closely correlated with cervical cancer invasion and metastasis.Placenta growth factor(PLGF) is a member of vascular endothelial growth factor(VEGF) family. It is different from other VEGFs, the expression of PLGF in normal physiological condition is very low. However, its expression is significantly up-regulated in pathological conditions, such as ischemia, inflammation and cancer, suggesting that PLGF may serve as an important prognosis biomarker. Studies found that PLGF could promote tumor invasion and metastasis through activating tumor angiogenesis. Numerous studies indicated that PLGF might serve as a valuable prognostic markers for various tumors. Previous studies demonstrated that PLGF exert its biological function through activating its specific receptor Flt-1. The overexpression of PLGF and Flt-1 could be obversed in ischemic and inflammatory diseases and many cancers including non-small cell lung cancer(NSCLC), prostate cancer, breast cancer and colon cancer, lung cancer, hepatocellular carcinoma, glioblastoma, multiple myeloma and Wilms’ tumors. Numerous studies reported that PLGF could promote tumor invasion and metastasis through activating Flt-1 signaling pathway. Recent studies show that PLGF can also promote tumor invasion and metastasis by activating the EMT signaling pathway.However,whether PLGF could regulated EMT and its mechanism in cervical cancer is not fully unstood.ObjectiveIn this study,we detected the expression of PLGF,Flt-1 and EMT-related proteins named E-cadherin and viemntin in cervical cancer tissues and the matched adjacent normal tissues and analyze the correlation between them. Also, we explored the mechanism and function of PLGF on the cervical cancer cells and its association with EMT-related proteins in the cultured cervical cancer cell line named siha.Methods1.HE staining was applied to identify the the collected tissue samples and the confirmed accurate specimen were accepeted in further researches.2.Immunohistochemical stains were used to detect the expression of PLGF, receptor Flt-1 and EMT-associated protein named E-cadherin and vimentin in the cervical cancer tissues and the matched normal tissues, and the chi-quear test and spearman analysis were used to nanlyze the a the correlation between them.3.The western blot analysis was applied to further analyze the expression of PLGF, receptor Flt-1 and EMT-associated protein named E-cadherin and vimentin in the cervical cancer tissues and the matched normal tissues.4.The expression levels of PLGF in serum and vaginal lavage from the cervical cancer suffers and normal control groups were detected by enzyme-linked immunosorbent assay(ELISA) and analyzed its correlations with the HPV status and tumor stage.5.The PLGF on the migration of cervical cancer cells was detected by transwell assay in the cultured cervical cancer cells line in vitro.6.The expression of EMT-related proteins in siha cells treated with PLGF were detected by western blot.7.The puromycin was used to screen the stably expressing shRNA-Flt-1 siha cells after infected with with shRNA-Flt-1 lentiviral vectors.8.We applied the transwell assay to analysze the migration and the western blot to detected the expressions of EMT-related proteins in the siha and Flt-1 silenced siha cells and compared their differences before and after treated with PLGF.9.The cultured siha cells were pretreated with a specific ERK/MAPK inhibitor named PD98059, the transwell assay and western blot were applied to analyze the PLGF on the migration and EMT-related proteins in siha cells after treated with PD98059.Results1.the expression of PLGF and Flt-1 in cervical cancer tissues are significantly higher than the matched adjacent normal tissues.Their positive expression in cervical cancer tissues are 61.8% and 60.9% respectively;while,the E-cadherin was decreased and vimentin was increased in cancer tissues.Correlation analysis indicated that the high expression of PLGF and Flt-1 is closly associated with the low expression of E-cadherin and high expression of vimentin in cervical cancer tissues(r=-0.618,P<0.01;r=0.450,P<0.01).2.It was further demonstrated by western blot that the expression of PLGF,Flt-1 and vimentin in cervical cancer tissues were significantly higher than the adjacent normal tissues,and the expression of E-cadherin was decreased in cervical cancer tissues.3.The serum PLGF level in cervical cancer suffers was significantly higher than normal control groups(22.96±0.90ng/mlVS9.926±1.049ng/ml,P<0.001) and was up-regulated as the cancer progession(P<0.01);The lavage PLGF level in cervical cancer patients was significantly higher than the normal women(24.80±0.73ng/mlvs 11.29±0.90ng/ml,P<0.001).However,whatever in serum or in lavage,the PLGF levels have no significant difference in stage I and II patients(P>0.05).4.The mean lavage PLGF level in HPV positive suffers is significantly higher than that of HPV negative group(25.74 ± 0.6258ng/ml VS 11.40 ± 0.7439ng/ml, P<0.05, respectively).5.Experiments in vitro demonstated that the migration of siha cells were significantly elevated after treated with PLGF,which could be inhibited by silencing the expression of Flt-1(P<0.05); Meanwhile, the E-cadherin was up-regulated and vimentin was down-reguated in siha cells treated with PLGF,which could be reversed by inhibiting the expression of Flt-1.6.The migration promotion and regulation of EMT-related proteins of PLGF in siha cells could only be inhibited by the ERK/MAPK specific inhibitor PD98059.Conclusions1.The expression of PLGF and its receptor Flt-1 in cervical cancer tissues is higher than the adjacent normal tissues,which is closely correlated with the stage and lymph node status of cervical cancer,indicating that PLGF plays an important role in the progression of cervical cancer.In addition,the low expression of E-cadherin and high expression of vimentin in cervical cancer tissues are also associated with the stage and lymph node status of cervical cancer,which is the same as their expression in EMT,indicating that EMT also plays an important role in the progression of cervical csncer.2.Correlation analysis indicated that The high expression of PLGF is significantly associated with low expression of E-cadherin and high expression of vimentin,which suggested that PLGF may regulate the expression of EMT-renlated proteins.,and may be an important biomarker for predicting the metastasis of cervical cancer.3.The expression level of PLGF in serum and lavage of the cervical cancer patients is significantly higher than that of the normal control group, which is also associated with the stage of the cervical cancer and the mean lavage PLGF level in HPV positive suffers is significantly higher than that of HPV negative group,which suggested that HPV may be an important factor that promote PLGF expression and PLGF may be an important biomarker for screening and predicting the severity and prognosis of cervican cancer.4.PLGF promotes the migration of cervical cancer cells and regulates the expression of EMT-related proteins through activating the Flt-1 signaling pathway which is also depends on the ERK/MAPK signaling pathway activation,indicating that PLGF/Flt-1 /ERK/MAPKsignailing pathway plays an important role in the progression of cervical cancer,which will provide the therapeutic targets for inhibiting the metastasis of cervical cancer. |
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