The Occurrence And Mechanism Of Epithelial Mesenchymal Transformation In Malignant Tumors

Background and objectives:Malignant tumor because highly lethal become the most serious threat to human health.Approximately 90%of cancer-related death is caused by metastasis which has been proposed to the single most significant challenge to management of the disease.Therefore,there is an urgent need for a better understanding of the mechanisms of cancer spreading.EMT is an evolutionarily conserved development process during that cells lose epithelial characteristics and gain mesenchymal properties,resulting in the formation of migratory mesenchymal cells with invasive properties.Therefore,EMT is implicated in tumor progression and metastasis.The purpose of this study is to identifying new regulators and the underlying signaling mechanisms for EMT,from both own and external influence.1.Own influence:up-regulate miR-214 expression,inducing EMT and tumor metastasis.Studies reported that tumor cells inducing EMT and metastasis mediated by a series of gene expression and changing,including protein,miRNAs,et al.Researchers found that miRNAs function as oncogene and tumor suppressor gene,to control the EMT process in different levels.In lung cancer,previous reports have shown that miR-214 significantly correlates with advanced tumor stage,worse overall survival and higher recurrence rates.However,the underlying mechanisms about how miR-214 contribute to lung cancer progress and worse clinical outcomes remain largely unknown.Especially,there has no report about what's the role of miR-214 in tumor EMT progress.Depend on our previous studies showed that miR-214 up-regulated during EMT process,whether the miR-214 is the key factor in lung cancer EMT and metastasis?So in this part,we will unique in demonstrating the function of miR-214,and the mechanisms for inducing EMT and metastasis.2.External influence:CSCs in a paracrine manner to induce tumor cell gene changes,inducing EMT and tumor metastasis.For a long time,the mechanisms of tumorigenesis and metastasis mainly concentrated in the tumor cell own change in genes or miRNAs,finally inducing EMT and metastasis.Studies found that tumor cells can development or transfer to specific organs,the interaction of the tumor microenvironment also plays a key role.Tumor associated macrophage,fibroblasts,and mesenchymal stem cells played a key role in tumor metastasis by a paracrine manner.Previous studies have suggested that cancer stem cells,as a small subset of cancer cells that can generate tumors through their self-renewal and multi-differentiation capacities;are essential for metastatic colonization.But the CSCs control the occurrence and development of tumor by the other way was not have any reports.Around the tumor cells,MSCs comprise?6%of cells in human tumor ascites,however,the percentage of MSCs in solid tumors is much lower(?0.3%).CAFs,comprising?15%in cancer,are a major constituent of the tumor stroma.CSCs comprise 1%-5%of cells in human tumor ascites or solid tumors.Moreover,in tumors after chemotherapy CSCs are enriched,with some cases displaying a?9.5 fold increase in percentage such that CSCs constitute?20%of cells in the tumor.In addition,many studies have confirmed that CSCs can produce huge amount of key chemokine,which play a key role in tumor metastasis.Most importantly,tumor cells expressed the homologue receptors.So we hypothesized that CSCs in addition can control the tumor development by directly way,can also by paracrine way to control tumor development and metastasis in a new way.So in the second part,we will demonstrate and perfect the mechanisms for inducing EMT and metastasis.In a word,the project will elucidate the specific molecular mechanism of tumor EMT and metastasis by both own and external influence way,provides new scientific evidence for the prevention and treatment of metastasis in malignant tumor.Methods:Part I:The function and mechanisms of miR-214 in EMT and metastasis of lung cancer cells.1.The expression of miR-214 during EMT process.Identify the miR-214 expression in three kinds of EMT model(hypoxia,TGF-? and drug induced),CSCs,and the different invasion ability lung cancer cell lines by real-time PCR.2.The role of miR-214 in EMT and metastasis of lung cancer cells.Interference of miR-214 expression by over-expression or shRNA,then determine the EMT related protein changes by real-time PCR,western blot and immunofluorescence.Simultaneous detection of the influence of miR-214 up-regulation in EMT inducing models.And identify the role of miR-214 in CSCs characteristic of lung cancer cells by FACS,and real-time PCR.Identify the function of miR-214 on lung cancer cell metastasis by transwell migration or matrigel invasion assay in vitro,or tail vein injection mouse model in vivo.3.Identification and functional analysis of miR-214 target gene sufu.The online prediction method and the luciferase report experimental analysis of miR-214 and sufu gene 3'-UTR binding.Analysis the relationship between miR-214 and sufu by real-time PCR,immunofluorescence and western blot.Overexpression or suppression of sufu gene by lentivirals,observe the role of sufu in lung cancer EMT and metastasis by transwell or metrigel invison assay in vitro,or tail vein injection mouse model in vivo.And also detected the function of sufu in miR-214 mediated EMT and metastasis in lung cancer cells.Immunofluorescence and western blot were used to observe the location and expression of sufu downstream molecule Gli-1.4.Clinical validate the relationship between miR-214-sufu pathway and EMT in lung cancer.Analysis the relationship between miR-214 and sufu in para-cancerous,primary and metastatic lung cancer tissues by real-time PCR or immunofluorescence.To observe the relationship between sufu and miR-214 and EMT markers by immunofluorescence expression.5.Twist mediated the expression of miR-214 during the EMT process.Observe the expression of miR-214 and sufu by real-time PCR or western blot after twist gene blocked by shRNA.And analyze the correlation between the expression of twist,miR-214 and sufu.Part II:Effects of OCSLCs on NCSLCs EMT and metastasis.1.OCSLCs enhance the metastatic capability of NCSLCs through the secretion of soluble mediators.NCSLCs expressing RFP were co-cultured with CSLCs at different ratios,and the number of migrated RFP+ NCSLCs was quantified after co-culture for 24h.NCSLCs in the upper well with or without CSLCs in the lower chambers,or cultured in conditioned medium obtained from CSLCs,and the metastasis ability was detected by transwell migration or metrigel invasion assay.Observe the metastasis ability of NCSLCs in the presence of OCSLCs in vivo mouse model.The function of OCSLCs on the EMT process of NCSLCs were detected by real-time PCR,immunofluorescence and western blot.2.CSLC-produced CCL5 enhances the metastatic capacity of NCSLCs through its receptors CCR1/3/5 CSLCs were present in the culture system separated by chambers,we observed CCL5 co-localization with CCR1,CCR3,and CCR5 on the membranes of NCSLCs by immunofluorescence.Blocking CCL5 and its receptors by antibody in co-culture system,and the function of OCSLCs on EMT and metastasis of NCSLCs were observed by real-time PCR,immunofluorescence and western blot.To determine the effect of CCL5:CCR1/3/5 signaling on the enhancement of NCSLC metastasis by CSLCs in vitro and in vivo,transwell migration assay and tail vein injection mouse model were used.3.Clinical validate the relationship between CCL5:CCR1/CCR3/CCR5 pathway and the function of OCSLCs on EMT and metastasis of NCSLCs We collected cancer tissues,corresponding metastatic tissues,and paracancerous tissues from 20 patients with epithelial ovarian cancer and determined the expression levels of CCL5 and its receptors(CCR1,CCR3,and CCR5)by immunohistochemistry.4.Determine the mechanism involved in CCL5-enhancement of NCSLC migrationTo address whether CCL5 induces NF-?B activation in NCSLCs,the anti-CCL5 antibody(at 5 ng/ml)was added into the co-culture system,the nuclear translocation of NF-KB-p65 and NF-?B activity were determined by western blot,immunofluorescence and ELISA.We used pyrrolidine dithiocarbamate(PDTC,an inhibitor of NF-?B)to inhibit NF-?B activation in NCSLCs co-cultured with CSLCs or in NCSLCs treated with rhCCL5,the migration and invasion ability were determined by transwell migration and metrigel invasion assay.Results:Part I:The function and mechanisms of miR-214 in EMT and metastasis of lung cancer cells.1.MiR-214 is highly expressed in EMT induced lung cancer cells.We found that all of these three EMT inducing models showed up-regulated miR-214 expression during EMT in lung cancer cells.And low expression in predominant mesenchymal phenotype of E-cadherin that led to significant elevation of miR-214 expression,in established hypoxia induced EMT model.MiR-214 was definitely highly expressed in all these CSLCs,and began to decrease within 6 days and further over 14 days in differentiation condition.Furthermore,among five lung cancer cell lines with an descending order of metastatic potentials,the expression of miR-214 was corresponding decreased.Collectively,our findings suggested that miR-214 may play an important role for inducing EMT and participate lung cancer metastasis.2.MiR-214 induces EMTs and enhance the metastasis of lung cancer cells.Epithelial marker e-cadherin was significantly decreased in miR-214 over-expression cells,miR-214 over-expression contributes to enhance the EMT in lung cancer cells.In contrast,mesenchymal markers were increased.MiR-214 over-expression increased the percentage of CD133+CD326+ cells,a population presumably enriched for lung CLSCs,and also expressed high levels of CSC markers.At the meantime,over-expressed miR-214 also increased the ability of A549 cells to develop into sphere structures.Mir-214 strongly promotes lung cancer metastasis in vitro and in vivo.All the results suggesting that miR-214 not only triggers EMT but also further contribute to sternness transformation,and strongly promotes lung cancer metastasis.3.SUFU was a direct target of miR-214.MiR-214 mediated repression on Sufu was due to a direct interaction between miR-214 and the sufu gene at 3'UTR region.There was a striking inverse relationship between miR-214 and sufu expression in lung patients' cancer tissues.Sufu over-expressed group significantly enhanced the expression of epithelial maker E-cadherin,and decreased the expression of mesenchymal marker vimentin,and we also observed that ectopic sufu expression lead to a less metastasis of lung cancer cells.Our results suggested that sufu gene was indeed a functionally important target of miR-214.We found that sufu as a negative regulator of the Hh pathway,over-expression of Sufu was associated with a significant decrease in the level of GLi-1 in the nuclear extracts,consistent with this,the total level of GLi-1 in the cytoplasm increased,and these effect was rescued when over-expression miR-214 simultaneously.4.MiR-214 and Sufu expression in clinic lung cancer patients.There was a striking inverse relationship between miR-214 and sufu expression in lung patients' cancer tissues.Tumors with a high level of miR-214 tended to express high levels of vimentin,and low level of E-cadherin and sufu gene,whereas low level of miR-214 showed the reverse results using immunofluorescence staining.Furthermore,miR-214 expression levels were significantly increased and sufu levels were reduced in metastatic tumors compare to the primary.All these results demonstrated that miR-214 positively regulates to EMT and tumor metastasis in lung cancer patients by targeting sufu.5.Twist-1 promotes miR-214 expression in Lung cancer cells.MiR-214 expression was significantly decreased when blocked twist-1 expression by shRNA lentivirus.We also observed that forced miR-214 down-regulation increased the expression of sufu gene in both RNA and protein level,suggested that the effect of twist-1 on sufu expression was predominantly mediated by up-regulation of miR-214.Moreover,the significantly positively correlation between miR-214 and twist expression was observed in clinical samples.Taken together,these data demonstrate that miR-214 which regulated by twist was capable to inducing EMT and metastatic behavior through down-regulating sufu gene in lung cancer.Part II:Effects of OCSLCs on NCSLCs EMT and metastasis.1.OCSLCs enhance the metastatic capability of NCSLCs through the secretion of soluble mediators.The presence of OCSLCs markedly increased the migration and invasion capabilities of the NCSLCs compared with NCSLCs cultured alone in co-culture condition.Importantly,we still observed enhanced migration and invasion of NCSLCs when OCSLCs were plated in the lower chambers or treated directly by CM(from OCSLCs),compared to NSCLCs cultured alone.These results indicated that OCSLCs enhanced NCSLC migration and invasion,and this effect was dependent on OCSLC-secreted mediators rather than on direct cell-to-cell contact.We also found the same phenomenon utilized an established xenograft metastasis model in which GFP-labeled NCSLCs alone,or together with unlabeled CSLCs in vivo tumor model.Our results also indicate that CSLCs can drive NCSLCs to undergo EMT,NCSLCs co-transplanted with CSLCs expressed lower levels of E-cadherin but increased vimentin levels compared with NCSLCs transplanted alone.2.OCSLC-produced CCL5 induce EMT and enhances metastasis of NCSLCs through its receptors CCR1/3/5.We observed OCLSCs produce CCL5 can co-localization with CCR1,CCR3,and CCR5 on the membranes of NCSLCs.Our results also found that neither the CCL5 antibody nor antibodies to its receptors affected the EMT and invasion of NCSLCs in the absence of OCSLCs.And treated with rhCCL5 can significantly enhanced NCSLCs migration and invasion in a dose-dependent manner.Furthermore,the co-transfer of OCSLCs significantly increased the number of NCSLC-derived metastatic nodules in recipient mouse organs,but this effect was diminished when CCL5 was knocked-down in OCSLCs.Collectively,these data suggest that OCSLC-derived CCL5 is critical for the enhancement of NCSLCs metastasis by OCSLCs.3.Elevated expression of CCL5 and its receptors is associated with ovarian cancer metastasis.The results revealed that neither CCL5,nor CCR1,nor CCR3,nor CCR5 was expressed in the ovarian paracancerous tissues,whereas all these molecules were expressed in both primary cancer tissues and metastatic tissues.Compared with primary cancer tissues,the expression of both CCL5 and its receptors was two-fold higher in the metastatic cancer tissues.These clinical analyses suggest that the CCL5:CCR1/CCR3/CCR5 axis is involved in ovarian cancer metastasis.4.The NF-?B signaling pathway is involved in CCL5-enhancement of NCSLC migration.We found that NCSLCs co-cultured with CSLCs,or treated with rhCCL5,showed increased nuclear translocation of NF-?B-p65 compared with NCSLCs cultured alone,and neutralization of CCL5 significantly reduced NF-?B activity NCSLCs.We also found that the enhanced migration and invasion of NCSLCs induced by CSLC co-culture or rhCCL5 treatment was significantly reduced by the addition of an NF-?B inhibitor.Collectively,the NF-?B signaling pathway is involved in enhancement of NCSLC metastasis byCSLC-derived CCL5.Conclusions:Part I:The function and mechanisms of miR-214 in EMT and metastasis of lung cancer cells.Own influence:MiR-214 exerts its EMT inducing and metastatic potential through direct targeting the suppressor of fused homolog(Sufu),thereby affecting nuclear-cytoplasmic shuttling of GLi-1.1.MiR-214 is highly expressed in EMT induced lung cancer cells.2.MiR-214 induces EMTs and enhance the metastasis of lung cancer cells.3.SUFU was a direct target of miR-214.4.There was a striking inverse relationship between miR-214 and sufu expression in lung patients' cancer tissues.5.Twist-1 promotes miR-214 expression in Lung cancer cells.Part II:Effects of OCSLCs on NCSLCs EMT and metastasis.External influence:Paracrine CCL5 from OCSLCs activates the NF-?B signaling pathway in ovarian NCSLCs via binding CCR1/3/5,thereby inducing EMT and tumor invasion.1.OCSLCs enhance the metastatic capability of NCSLCs through the secretion of soluble mediators.2.OCSLC-produced CCL5 induce EMT and enhances metastasis of NCSLCs through its receptors CCR1/3/5.3.Elevated expression of CCL5 and its receptors is associated with ovarian cancer metastasis.4.The NF-?B signaling pathway is involved in CCL5-enhancement of NCSLC migration.