Expression Of ECRG4 In Nasopharyngeal Carcinoma And Its Role In The Malignant Biological Behavior Of Nasopharyngeal Carcinoma And Its Mechanism

Background and ObjectiveNasopharyngeal carcinoma is a malignant tumor originating from nasopharyngeal epithelial cells.The morbidity of nasopharyngeal carcinoma has obvious geographical distribution.It has a very high morbidity rate in Southeast Asia and South China.The annual incidence rate is 20 ~ 30 / 100,000.Reference to international authorities,global cancer statistics show that in 2008 more than nasopharyngeal cancer patients 84,000,of which 80% are in Asia and 5% are in Europe.Etiology study shows that the incidence of nasopharyngeal carcinoma and EB virus infection,ethnic background,eating habits,drinking and environmental factors.Now,with the imaging diagnosis,radiotherapy and chemotherapy combined with the progress of the application of localized nasopharyngeal lesions to get better control.However,the majority of nasopharyngeal carcinoma patients in the initial diagnosis of advanced,its treatment is not satisfactory,? and IV patients 5-year survival rates were 53% to 80%,28% to 61%.Therefore,it is very important to better understand the molecular mechanism of tumor development and find new specific therapeutic targets,which is very important in the early diagnosis,individual therapy and prognosis of nasopharyngeal carcinoma.Esophageal cancer related gene 4(ECRG4)is a novel gene that has the potentialof suppressing cancer.It is a differential expression sequence isolated from the cancerous family and normal esophageal tissue by m RNA differential display.It is expressed in almost all tissues,including the heart,brain,placenta,liver,lung,pancreas,bone and so on.Studies have shown that ECRG4 is involved in the development and loss of central nervous system response,articular chondrocyte differentiation and cartilage destruction and oligodendrocyte precursor cells and other physiological and pathological processes.Recent studies have shown that this gene is down-regulated in a variety of tumors,such as esophageal squamous cell carcinoma,colorectal cancer and breast cancer,suggesting that deletion of this gene may be one of the important factors involved in tumor development.The current study of ECRG4 is limited to several malignancies,and its regulatory mechanisms also need further study.Promoter hypermethylation is the main mechanism of low expression and transcription inactivation of ECRG4 in tumors.Gotze and other studies found that in colon cancer and glioma tissue,ECRG4 promoter methylation of high frequency appears,indicating that ECRG4 in cancer tissue down-regulated expression and its degree of methylation is closely related.At the same time,the expression of ECRG4 in tumors can be restored after treatment with demethylated drugs.Some studies showed that ECRG4 gene transfection into esophageal cancer cells can significantly inhibit the cell proliferation and clonogenic ability,ECRG4 overexpression of the cell cycle in the Gl phase.The overexpression of ECRG4 gene in head and neck tumor cell M2 can significantly reduce the proliferation and invasive ability of the cells.Matsuzaki et al.Also overexpressed ECRG4 in esophageal epithelial cells,and found that the gene and Ki-67 gene was negatively correlated,proved ECRG4 gene can inhibit cell proliferation.In the present study,we detected the mRNA and protein expression of ECRG4 in fresh nasopharyngeal carcinoma and nasopharyngeal carcinoma cell lines by RT-PCR and Western-blot respectively.Immunohistochemistry was used to detect the expression of ECRG4 in nasopharyngeal carcinoma ECRG4 si RNA in vitro and in vivo.Transwell assay and tail vein injection of nude mice were used to analyze the invasion and metastasis ability of ECRG4 in nasopharyngeal carcinoma cell lines5-8F and CNE2 in vitro and in vivo.Immunohistochemistry was used to detect theexpression of ECRG4.;4,Western blot analysis of ECRG4 on metastasis-related gene expression;6,respectively,by RT-PCR and gelatin zymography analysis of MMP2 mRNA expression and enzyme activity;7,upregulation,down-regulation of MMP2 expression,transwell The effect of MMP2 on the invasion and metastasis of nasopharyngeal carcinoma cells was observed by Western blotting.Western blot was used to analyze the phosphorylation of NF-kB p65.9.Cell proliferation assay,plate cloning assay and tumorigenesis in nude mice.The proliferation of nasopharyngeal carcinoma cells in vitro and in vivo;10,flow cytometry analysis of ECRG4 on the cell cycle of nasopharyngeal carcinoma;11,Western blo t analysis of ECRG4 on cell cycle regulation of protein-related effects.This study concludes three parts.Part 1: Expression of ECRG4 in nasopharyngeal carcinoma tissues and cells.Part 2: ECRG4 promotes the invasion and metastasis of nasopharyngeal carcinoma by down-regulating the expression of MMP2 by NF-?B.Part 3: ECRG4 promotes the proliferation of nasopharyngeal carcinoma cells.Part 1: Expression of ECRG4 in nasopharyngeal carcinoma tissues and cells Methods:1.Nasopharyngeal carcinoma cell lines(5-8F,6-10 B,CNE2)and an immortalized nasopharyngeal epithelial cell line(NP69)were cultured.2.The paraffin specimens from nasopharyngeal carcinoma and normal nasopharyngeal tissues were collected from People's hospital of henan province from 2006 to 2012.3.The expression of ECRG4 in nasopharyngeal carcinoma and normal nasopharyngeal tissues was detected by immunohistochemistry.4.7 cases of fine needle biopsy nasopharyngeal carcinoma fresh tissuecollection and 2 cases of normal nasopharyngeal tissue.5.The expression of ECRG4 in nasopharyngeal carcinoma tissues and normal nasopharyngeal tissues was detected by Western blot and RT-PCR.Results:1.ECRG4 expression in nasopharyngeal carcinoma was significantly lower than the adjacent tissue in 7 cases of fine needle aspiration biopsy of nasopharyngeal carcinoma and 2 cases of normal nasopharyngeal tissue from the protein level and mRNA expression levels of ECRG4,the results suggest that both at the mRNA level and in the protein.The expression of ECRG4 in nasopharyngeal carcinoma was significantly lower than that in normal nasopharyngeal tissues.2.ECRG4 in nasopharyngeal carcinoma cell lines.The results of Western-Blot and RT-PCR showed that ECRG4 was expressed in 6-10 B and CNE2 of nasopharyngeal carcinoma,and the expression of ECRG4 was higher than that of immortalized nasopharyngeal epithelial cell line Specific 5-8F cell line was significantly lower than the non-metastatic and low-metastatic CNE2 cell lines.3.ECRG4 expression in nasopharyngeal carcinoma tissues and clinicopathological significance.In order to further verify the expression of ECRG4 in nasopharyngeal carcinoma and normal nasopharyngeal tissues.We expanded the sample collected 153 cases of nasopharyngeal carcinoma and 30 cases of non-cancer nasopharyngeal paraffin tissue for immunohistochemical analysis,the results suggest that ECRG4 in nasopharyngeal carcinoma cells located mainly in the cytoplasm,a few in the nucleus.The expression of ECRG4 in non-nasopharyngeal carcinoma was high expression 93.3% and low expression 6.7% while in nasopharyngeal carcinoma high expression 52.9% and low expression 47.1%.The difference was significant(P<0.001)between nasopharyngeal carcinoma tissues and non-cancerous tissues.4.ECRG4 expression in nasopharyngeal carcinoma(NPC)and their clinicopathological significance.Further analysis of the expression of ECRG4 in nasopharyngeal carcinoma and the clinicopathological characteristics of nasopharyngeal carcinoma.Found that ECRG4 expression level and the patient'sgender,age,smoking,family history is not relevant.The expression intensity of ECRG4 was correlated with EBV infection,depth of invasion,differentiation degree,TNM stage,distant metastasis and lymph node metastasis of nasopharyngeal carcinoma.The positive expression rate of ECRG4 was lower in EBV-infected patients than in EBV-negative ones.The higher the tumor differentiation was,the higher the positive expression rate of ECRG4 was;the later the tumor stage,the lower the positive expression rate of ECRG4;the distant and lymph node metastasis Positive expression rate was significantly lower than the non-metastatic,the difference was statistically significant,p <0.01.Kaplan-Meier survival analysis showed that the degree of ECRG4 expression in nasopharyngeal carcinoma was closely related to the prognosis of patients with nasopharyngeal carcinoma.The overall survival and progression-free survival of patients with high expression of ECRG4 were higher than those with low ECRG4 expression.Part 2: ECRG4 inhibits the invasion and metastasis of nasopharyngeal carcinoma by down-regulating the expression of MMP2 by NF-KB Methods:1.Construction of pcDNA3.1 vector.2.Construction of ECRG4 and MMP2 overexpression vector.3.Construction of nasopharyngeal carcinoma cell line stably transfected with pcDNA3.1 vector.4.The migration and invasion of the recombinant lentivirus infection of 5-8F,CNE2 cells and control cells were detected by Transwell chamber assay.5.Data were analyzed using SPSS 13.0 and expressed as mean ± standard deviation.Paired-sample t-test was used to estimate the difference between the two groups.If p < 0.05,then the difference between the groups was considered statistically significant.Results:1.P3.1 / ECRG4-pcDNA3.1 1,p3.1 / ECRG4-pcDNA3.1 2 and p3.1 were transfected into 5-8F and CNE2 cell lines of nasopharyngeal carcinoma,and the positive clones were screened by G418 to obtain stable transfected 5-8F / pcDNA3.11,5-8F / pcDNA3.1 2,5-8F / control,respectively,CNE2 / pcDNA3.1 l,CNE2 /pcDNA3.12 and CNE2 / control.We successfully constructed a nasopharyngeal carcinoma cell line stably transfected with pcDNA3.1 vector.2.UP-regulation of ECRG4 inhibits the migration and invasion of nasopharyngeal carcinoma cells.ECRG4-pcDNA3.11 and ECRG4-pcDNA3.12 transfection reduced the number of migrating cells of 5-8F cells by 78.3% and 65.1%,respectively,and the number of invasive cells decreased by 70.4% and 59.8%,respectively,compared with those transfected with pSilencer3.1 empty vector cells /RTI & gt;The cell number of CNE2 cells decreased by 82.5% and 52.7%respectively,and the number of invasive cells decreased by 76.9% and 69.4%,respectively.In vitro migration and invasion experiments suggest that up-regulation of intracellular ECRG4 expression can inhibit the migration and invasion of nasopharyngeal carcinoma cells,suggesting that ECRG4 may have the ability to inhibit the invasion of nasopharyngeal carcinoma cells.3.ECRG4 can affect the expression of metastasis-associated genes in nasopharyngeal carcinoma.To investigate how ECRG4 regulates the migration and invasion of nasopharyngeal carcinoma cells.We used Western blot to detect the genes related to metastasis and to find the potential target gene of ECRG4.MMP2,MMP9,Snail,Vimentin,E-cadherin,H-cadherin and P-catenin,which have been reported to be the lytic substrates of ECRG4,have been selected in many genes related to tumor metastasis.The results showed that siRNA upregulated intracellular ECRG4.The expression of MMP-2,Snail and Vimentin in nasopharyngeal carcinoma cells was decreased and the expression of E-cadherin was increased,while the expression of MMP9,N-cadherin and P-catenin had no change.4.Up-regulated the expression of ECRG4 in nasopharyngeal carcinoma cells can decrease the expression and the activity of MMP-2.The degradation of extracellularmatrix is necessary for the invasion and metastasis of tumor cells to the surrounding cells.Much research has been reported on the relationship between matrix metalloproteinases(MMPs)and tumor metastasis,such as MMP2 plays an important role in the metastasis of many kinds of tumors.The role of MMP2 in particular is also involved in the transfer of nasopharyngeal carcinoma.A new study reported that ECRG4 can up-regulate the expression of MMP2 to promote intrahepatic cholangiocarcinoma metastasis.Therefore,in combination with the above Western blot results,we speculate that ECRG4 affects the metastasis of nasopharyngeal carcinoma cells is also regulated by MMP2.In this study,RT-PCR was used to confirm the up-regulation of ECRG4 expression in 5-8F and CNE2 cell lines,which resulted in the decrease of MMP2 expression.Finally,gelatin zymography was used to detect the MMP2 activity of the transfected cells.The results showed that upregulation of ECRG4 expression in both cell lines 5-8F and CNE2 resulted in a decrease in MMP2 activity.5.ECRG4 inhibits the migration and invasion of nasopharyngeal carcinoma cells by down-regulating the expression of MMP2.To determine whether ECRG4 regulates the migration and invasion of nasopharyngeal carcinoma cells by inducing the expression of MMP2,we first downregulated the expression of MMP2 in nasopharyngeal carcinoma cell lines using pcDNA3.1 overexpression technology.Cell migration and invasion ability,the experimental results show that:down-regulation of MMP2 expression can cause 5-8F,CNE2.Cell migration and invasion ability decreased,and the degree of decline similar to pcDNA3.1.ECRG4 cells,the difference was not statistically significant.Finally,we inhibited the expression of ECRG4 in nasopharyngeal carcinoma cells,overexpressed MMP2 expression,observed the cell migration and invasion ability,the results showed that:5-8F,CNE2 fine.The overexpression of MMP2 in the cell line could restore the cell migration and invasion induced by overexpressed ECRG4.These results suggest that ECRG4 may affect the migration and invasion of nasopharyngeal carcinoma cells by modulating the expression of MMP2.6.ECRG4 regulation of MMP2 expression through activation of NF-kB.It has been reported that NF-kB is a downstream effector of ECRG4 promoting cellmigration in hepatocellular carcinoma.In addition,NF-kB can up-regulate the expression of MMP2 in several studies to promote tumor cells.Therefore,we envision whether ECRG4 down-regulates the expression of MMP2 in nasopharyngeal carcinoma cell lines by activating NF-kB.Western blotting analysis showed siRNA knockdown ECRG4 significantly reduced the phosphorylation of NF-kB p65 in the cells;on the contrary,exogenous overexpression of ECRG4 Can increase the intracellular phosphorylation of NF-kB p65.Finally,we used NF-?B inhibitor helenalin to inhibit the activity of NF-kB and observe the changes of MMP2 expression in cells.Whether overexpression or knockdown of intracellular ECRG4,the expression of MMP2 in the cells was significantly lower than that of the control cells after the application of the helenalin inhibitor.The results showed that inhibition of NF-kB activity could inhibit the expression of MMP2 induced by ECRG4.These results suggest that up-regulation of MMP2 expression by ECRG4 may be mediated by phosphorylation of NF-kB p65.Part 3:ECRG4 promotes the proliferation of nasopharyngeal carcinoma cells Methods:1.Cell growth curve of the determination(CCK8 experiment).2.Cell plate colony formation experiment3.The cell cycle was analyzed by flow cytometry4.Nude mice in vivo tumor formation experiment Results:1.Up-regulation of ECRG4 inhibition of cell proliferation marker protein PCNA expression2.The up-regulation of ECRG4 significantly reduced the proliferation of nasopharyngeal carcinoma cells in vitro.3.Up-regulation of ECRG4 expression inhibited the cloning genic ability of nasopharyngeal carcinoma cells.4.Up-regulation of ECRG4 expression reduced the effect of nasopharyngeal carcinoma cells in vivo tumorigenic.5.ECRG4 could prevent nasopharyngeal carcinoma cells from G1 phase to S phase.6.ECRG4 play the role by reducing the expression of Cyclin,Cyclin D1 and D2 Cyclin E and up-regulated the expression of p27.7.Up-regulation of ECRG4 expression in nasopharyngeal carcinoma cells in vivo transfer capacity.To further study the effect of ECRG4 on the metastatic ability of nasopharyngeal carcinoma cells in vivo,we selected 5-8F / pcDNA3.11 cells and their control cells,respectively,injected into the nude mice via tail vein in vitro.The results showed that injection of 5-8F / pcDNA3.11 cells in nude mice formed less metastases,10 Nude mice had only one liver metastasis,two lung metastasis occurred,while the control group cells 5-8F / control 10 nude mice lung and liver metastasis were 7,the results suggest that ECRG4 have nasopharyngeal carcinoma cells inhibited in nude mice in the role of organ metastasis.Conclusions:1.The expression of ECRG4 in nasopharyngeal carcinoma was significantly lower than that in normal nasopharyngeal tissue for the first time.In nasopharyngeal carcinoma tissues the expression of ECGR4 is highly related to the expression of EB virus infection,tissue differentiation,TNM stage,tumor volume and metastasis;2.The up-regulation of ECRG4 expression could significantly inhibit the metastasis,invasion,proliferation and apoptosis of nasopharyngeal carcinoma cells,and tumorigenic ability;3.ECRG4 promotes the metastasis and invasion of nasopharyngeal carcinoma cells,possibly through the dependence of NF-kB signal pathway MMP2 expressionto achieve;4.Up-regulation of ECRG4 expression can prevent nasopharyngeal carcinoma cells from G 1 to S progress,the cells stagnated G0 / 1.The underlying mechanism may be partially down-regulated by Cyclin D1,Cyclin D 2 and Cyclin E,p27 to achieve.