The Biobehaviour Of The Esophageal Carcinoma Cells Transfected With The Constructed SiRNA Expression System Targeting Against NS Gene

China is a country with the highest morbidity and mortality of esophageal carcinoma in the world.Each year,300 thousand people develop esophageal carcinoma,while more than half of them are Chinese people(167.2 thousand).Most patients suffered from esophageal carcinoma die of tumor infiltration and metastasis.So how to control tumors, especially esophageal carcinoma,to develop,infiltration,and metastasis has been the hotspot.Tumorigenesis arises from the acquisition of multi-genetic alterations,combined with multiple factors and develops in consecutive progression.Oncogenes and tumor suppressor genes are the two kinds of genes most involved in tumorigenesis,among which the alterations of multiple key genes lie the basis to carcinogenesis.The production of nucleostemin gene,nucleostemin(NS),a novel p53 binding protein,is found to play a role in controlling the cell cycle progression in stem cells and malignant cells,predominantly maintain the cell proliferation and lead to the cell cycle withdrawal before maturation.NS gene expression is positively associated with tumor invasion,which has been confirmed in gastric cancer,renal cancer,breast cancer,and bladder cancer,etc.NS regulates the stem cell and malignant cell proliferation by p53 gene in the late S stage and G2 stage during the cell cycle.That's why NS is supposed to be the pivotal regulator specifically controlling the cell cycle from G2 to M.Epidermal growth factor(EGF) is another vital factor in the cell proliferation and differentiation,which participates in transforming the normal cell to the neoplastic cell,and stimulates malignant cell to overgrowth,and which contributes to the neoplastic infiltration and metastasis.The excessive expression of EGF,together with the epidermal growth factor receptor(EGFR) has been indentified in many neoplasms.RNA interference(RNAi) is the process of mRNA degradation that is induced by double-stranded RNA in a sequence-specific manner.RNAi has been observed in all eukaryotes,from yeast to mammals.In the eukaryotes,the long dsRNAs get processed into 21-26 nucleotide small interfering RNAs(siRNAs) by an RNaseⅢ-like enzyme called Dicer.The power and utility of RNAi for specifically silencing the expression of any gene for which sequence is available has driven its incredibly rapid adoption as a tool for gene research.To demonstrate the application of RNAi on neoplasm,especially esophageal carcinoma,we designed the present research.We detected the NS,EGF,and EGFR in malignant esophagus tissues,constructed the siRNA expression system targeting NS, transfected the vector to esophageal carcinoma cell lines(EC9706),and finally,assessed the NS,EGF,and EGFR in the malignant cells as well as examined the neoplasm growth in vivo in the nude mice inoculated with tumor cells.The following was the abstract of the full article.Part One Expressions of NS,EGF and EGFR in esophageal squamous cell carcinoma and their associations with malignancyObjective To demonstrate the expressions of NS,EGF and EGFR in esophageal squamous cell carcinoma(ESCC) and their associations with neoplastic progression.Methods Sixty-two ESCC specimens,thirty-one adjacent dysplasia specimens and sixty-two normal mucosa specimens were used for determination.Histologically,fifteen neoplastic lesions were stageⅠ,twenty-five were stageⅡ,and twenty-two were stageⅢ. On metastasis,twenty had lymph node metastasis,and the other forty-two had not. According to invasion depth,seven cases were superficial,confined to the mucosa, submucosa,and superficial muscularis propria;while other fifty-five were deep to muscularis propria or adventitia.Immunohistochemi-stry was adopted to determine NS, EGF and EGFR proteins,and in situ hybridization was applied to measure NS,EGF and EGFR mRNA.RT-PCR and Real-time PCR were used to examine the expression levels of NS mRNA.The correlations of these proteins and mRNA with ESCC clinical data were statistically analyzed.Results1) The NS was expressed more(69.4%) in ESCC than in the adjacent hyperplasia and normal mucosa(41.9%and 17.7%,respectively) by immunohistochemisty(P＜0.05), and NS mRNA was more positively detected in ESCC(69.4%) than in the other two groups (25.8%and 21.0%,respectively,P＜0.05) by in situ hybridization.The following RT-PCR and Real-time PCR further confirmed that NS mRNA level in ESCC were higher than the other groups(P＜0.05).Correlation analysis revealed that NS and its mRNA were unrelated with age or gender(P＞0.05),but positively related with the histological evaluation, invasion depth and the lymph node metastasis.2) More EGF protein was expressed in ESCC(69.4%) than in adjacent hyperplasia and normal mucosa(45.2%and 27.4%respectively,P＜0.05),and the adjacent hyperplasia had more EGF than the normal mucosa(P＜0.05).In situ hybridization demonstrated the similar results:EGF mRNA in ESCC was expressed the most(77.4%) among the three groups,and the adjacent hyperplasia was less than the ESCC but more than the normal tissue(45.2%vs.27.4%,P＜0.05).It could not be confirmed that the EGF and its mRNA was associated with age or gender(P＞0.05),but what has been confirmed was that they were related to the tumor malignancy as well as the NS and NS mRNA(P＜0.05).3) EGFR and EGFR mRNA expression from the most to the least were ESCC(71.0% and 75.8%),adjacent hyperplasia(35.5%and 48.3%) and normal mucosa(14.5%and 40.3%)(P＜0.05).EGFR and EGFR mRNA were not relative to the age or gender but to the tumor stages,invasion depth,and lymph node metastasis.4) The NS in neoplastic tissues was positively correlated with the EGF and EGFR, with the correlation coefficients were 0.545 and 0.731 respectively.And so did their mRNA, with the coefficients were 0.394 and 0.604 respectively.Conclusion NS,EGF and EGFR are closely related with the carcinogenesis and development of ESCC. Part Two The biobehaviour of the EC9706 cells transfected with the constructed siRNA expression system targeting against NS geneObjective To construct and select an effective eukaryotic expression system of short interfering RNA(siRNA) suppressing NS gene,and evaluate the biobehaviour of EC9706 cells transfected with this expression system.Methods The sequences of siRNA for NS were taken from GenBank,and three 19 bp oligonucleotides were selected as targeting sequences(accession numbers:AY825265 126-144 nt,199-217 nt and 487-505 nt).The three pairs of siRNA duplex for NS were inserted with BamHI and XhoI sites and a pair of nonspecific siRNA duplex was as control. After annealing,the designed oligonucleotides were cloned into plasmid pRNAT-U6.1 as the expression vector.The plasmid was transformed into the host E.coli DH5αand extracted for identification by PCR and sequencing.Transfected the four siRNA expression system to EC9706 cell line and set the empty vector as control and cells without transfection as blank control.The most effective siRNA expression system was selected by RT-PCR and transfected into the tumor cell line EC9706.The NS mRNA,EGFR mRNA and their proteins in the transfected EC9706 cells were detected by Real-time PCR,in situ hybridization and immunocytochemistry.The cell migration was examined by Boyden chamber assay.The cell cycle and apoptosis were assayed by flow cytometry.Results1) The bands of designed siRNA duplex of siNS1,siNS2,siNS3 and siC were visualized at 50-60 bp by gel electrophoresis,identical in size to the designed.2) The recombinant plasmids were extracted from the proliferated host and amplified. The empty vector was 150 bp and the recombinant plasmids were 200 bp,suggesting the 50 bp hairpin DNA oligonucleotides were inserted.The sequences of inserted fragments in the recombinant plasmid pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2,pRNAT-U6.1-siNS3 and pRNAT-U6.1-siC were identical to the designed sequences.3) RT-PCR showed that the NS mRNA was down-regulated in the cells transfected with pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2 and pRNAT-U6.1-siNS3,compared with the three control groups.The NS mRNA in cells transfected with pRNAT-U6.1-siNS2 was almost suppressed completely,with the least NS produced in all the transfected cells. Contrary to the cells transfected with pRNAT-U6.1-siNS1,pRNAT-U6.1-siNS2,and pRNAT-U6.1-siNS3,the cells transfectd with nonspecific siRNA plasmid,empty plasmid and blank cells produced high levels of NS mRNA.4) NS mRNAs in siRNA silencing group,nonspecific group and blank group were 0.034±0.008,0.255±0.049,0.273±0.049,respectively,by Real-time PCR.The in situ hybridization demonstrated that(2.08±2.25)%cells in siRNA silencing group expressed NS mRNA,(13.86±1.38)%cells in nonspecific siRNA group expressed NS mRNA and (13.76±0.99)%cells expressed NS mRNA in blank group.Immunocytochemistry detected the NS protein in(6.08±3.35)%cells of siRNA silencing group,in(15.42±1.64)%cells of nonspecific siRNA group and(15.40±1.72)%cells of blank control.The NS gene was significantly suppressed by siRNA,compared with the nonspecific group and the blank control group(P＜0.01).5) EGFR mRNAs in siRNA silencing group,nonspecific group and blank group were 0.071±0.017,0.094±0.030 and 0.102±0.030 respectively,by Real-time PCR.The in situ hybridization demonstrated that(2.94±1.04)%cells in siRNA group expressed EGFR mRNA,(7.94±2.08)%cells in nonspecific siRNA group expressed EGFR mRNA and (6.46±1.49)%cells expressed EGFR mRNA in blank group,Immunocytochemistry detected the EGFR protein in(3.82±1.56)%cells of siRNA silencing group,in (9.26±2.92)%cells of nonspecific siRNA group and(9.48±1.94)%cells of blank control. The EGFR gene was significantly suppressed by siRNA,compared with the nonspecific group and the blank control group(P＜0.01).6) The invasion test showed that less EC9706 cells with NS knocked down migrated across the Matrigel compared with the nonspecific siRNA cells and blank cells(69.0±13.6 vs.126.6±10.8 and 69.0±13.6 vs.119.4±20.9,respectively,P＜0.05).7) The flow cytometry showed that NS suppression arrested the cell cycle in G0/G1 phase,and induced the cell apoptosis.Conclusion The NS specific siRNA expression system is established.The most effective expression system is selected and transfected into EC9706 cells.NS downregulation decreases the expression of EGFR and inhibites the aggression of EC9706. Furthermore,the NS suppression halts the cell cycle in G0/G1 phase and induces cell apoptosis.These data provide the foundation for the further gene therapy to esophageal cancer.Part Three The inhibition of siRNA-mediated NS gene silencing on the nude murine tumor xenograft modelObjective To assess the inhibition of siRNA induced NS gene suppression on growth of EC9706 cells in the nude murine tumor xenograft model.Methods Inoculated the EC9706 cells transfected with pRNAT-U6.1-siNS2 to five nude mice in each group subcutaneously,injected pRNAT-U6.1-siC as nonspecific siRNA control,and untransfected cells as blank control.The volume of the xenograft was measured.RT-PCR,in situ hybridization and Western-blot were applied for detecting NS, EGF and EGFR mRNAs and their products.Results The tumors were visible five to ten days after inoculation,and five weeks later,the tumor xenografts were formed in all the animals.The volumes of tumor xenografts were(1806.40±77.75) mm~3 in blank group,(1702.20±88.60) mm~3 in nonspecific siRNA group,and(847.00±82.25) mm~3 in NS silencing group.Further assays showed that the targeted NS gene had been effectively inhibited and NS mRNA and protein had been knocked down in the NS specific siRNA group.The EGF mRNA and EGFR mRNA and proteins were also downregulated(P＜0.05).Conclusion siRNA duplex targeting against NS delays the tumor xenograft growth, and reduces the expression of NS,EGF,and EGFR genes in the tumor xenograft models. The highly efficient siRNA mediated NS silence provides a novel gene analysis tool for therapeutic applications in malignant diseases.