| Objective: This study relies on the sequencing results of exons of Kazakh and Uighur patients with the esophageal squamous cell carcinoma.To investigate the difference of the expression of thrombin sensitive protein 1 type 7a(Thsd7a)gene in the esophageal squamous cell carcinoma in four ethnic groups.To investigate the relationship between the expression of Thsd7 a in Kazakh squamous cell carcinoma(ESCC)and the malignant phenotype of esophageal carcinoma.To further investigate the effects of Thsd7 a on biological behaviors such as proliferation,apoptosis and invasion of esophageal squamous cell carcinoma,as well as the pathway and downstream genes that play a role in esophageal squamous cell carcinoma.On the basis of suspected ownstream genes found on Thsd7 a microarray,through the screening of downstream driving genes and functional tests in vivo and in vitro,and a new driving gene downstream of Thsd7 a was identified.To analyze the clinical significance of Thsd7 a downstream drive genes in esophageal squamous cell carcinoma,and to explore the biological function of downstream genes in tumorigenesis and progression.It lays on the foundation for the study of individualized target therapy and further clinical application of esophageal carcinoma.Methods:(1)According to the current TNM staging criteria for esophageal cancer,esophageal tissue samples from four ethnic groups and tissue samples were collected.The expression of Thsd7 a in carcinomas and adjacent tissues was detected by immunohistochemistry.In accordance with the expression of target genes at the level of mRNA in RT-PCR,the ethnic group(Thsd7a)with significant differences in expression was used to determine whether there were ethnic differences and the characteristics of the expression.(2)The correlation between the expression changes of Thsd7 a and the clinicopathological features of Kazakh squamous cell carcinoma,and the prognosis of the patients were analyzed.(3)On the basis of clinical samples,we constructed Thsd7 a interference plasmid vector and lentiviral vector to examine the effects of Thsd7 a on cell proliferation,apoptosis,migration,invasion and other cell functions in vitro.(4)On the basis of vitro cytology experiments,the tumor growth and tumor inhibition rates were observed by constructing tumor suppression model in nude mice,and the effect of Thsd7 a gene on cell growth was further clarified.(5)Thsd7a functional related signaling pathway and downstream genes were screened by high throughput gene chip.(6)Cell were used to confirm the upstream and downstream relationship between suspected genes and Thsd7 a in RT-PCR.(7)Based on the mixtures of 3 interfering sequences of target gene,Celigo was used to monitor cell growth in real time,and the most significant downstream genes that affect cell growth were statistically analyzed.(8)Celigo was used for single target verification of significant differentially expressed genes.(9)Positive gene for single target validation,the cell function test(MTT,clone formation assay,cell cycle,apoptosis,wound healing assay and Transwell migration experiment,Western blot)gene functional verification.(10)Objective gene exon sequencing of esophageal squamous cell carcinoma cell was used to explore the root of gene function.Results: Part one:(1)The positive rates of Thsd7 a protein in normal esophageal tissues of Uygur,Han and Mongolian were not significantly different from those of ESCC.However,the positive rate of Thsd7 a protein in Kazakh normal tissues was lower than that in ESCC tissues.(2)Immunohistochemistry and RT-PCR showed that the expression of Thsd7 a in Kazakh squamous cell carcinoma was significantly higher than that in normal tissue adjacent to cancer.(3)The expression of Thsd7 a was significantly correlated with the clinical stage(P=0.04)and the differentiation(P<0.01).(4)The expression of Thsd7 a was not significantly correlated with the prognosis,but the clinical stage,T staging,N staging and differentiation were correlated with the prognosis of Kazakh squamous cell carcinoma patients.Part two:(1)After the knockdown of Thsd7 a,the MTT experiments showed that the proliferation of esophageal squamous cell carcinoma cell lines EC 9706 and Eca 109 were significantly inhibited;cell scratch assay and Transwell migration assay,the migration ability of EC 9706 and Eca 109 cells were significantly inhibited;invasion assay showed that EC 9706 and Eca 109 cell line was significantly inhibited in Thsd7 a knockdown after that gene can secrete metalloproteinases,with strong invasive ability;flowcytometry revealed that knockdown of Thsd7 a can cause cell cycle arrest in G0/G1 phase,and the cell apoptosis rate increased significantly.(2)Nude mouse tumor formation experiments showed that knockdown of Thsd7 a could effectively inhibit the tumorigenicity of Eca 109 cell line in nude mice.(3)Using microarray microarray analysis,the mRNA levels in Eca 109 cells expressed differentially expressed genes and signaling pathways after Thsd7 a knockdown.After further verification by RT-PCR,we found that Thsd7 a was significantly related to the five classical pathways: mTOR,Cell Cycle G1/S Checkpoint,Wnt,AMPK and ERK/MAPK.Part three:(1)The experimental results by using the method of bioinformatics analysis of gene chip screening: firstly,the corresponding gene in PUBMED has been published,inclusion criteria for the number of reported less than 100 papers,research in the tumor is relatively small,and in esophageal cancer have not been reported in gene.Through the above screening conditions,37 candidate genes were selected.(2)The Thsd7 a gene was transfected into Eca 109 cells to achieve a stable knockdown of the Thsd7 a gene,thereby observing the downstream genes affected by the cell level in vitro.It was found that 11 of these genes were down regulated(> 40%),so the 11 genes were screened for function.(3)3 interfering targets were designed and mixed into target gene one by one,and transfected into Eca 109 cells.By means of high throughput real-time detection with Celigo,2 genes(SCARA5 and SMCO4)could significantly affect the proliferation of esophageal squamous cell carcinoma.Furthermore,the interference sequences shSCARA5(PSC38272)and shSMCO4(PSC37528)were confirmed and identified by means of single target Celigo verification.(4)Background expression of SCARA5 in esophageal cancer cell lines showed that SCARA5 was highly expressed in Eca 109 and EC 9706 cells and low expression in TE-1 cells.(5)Knockdown of SCARA5,MTT test showed that the proliferation of esophageal cancer cell line Eca 109 in vitro significantly inhibited;cell clone formation assay showed that Eca cell clone formation ability was significantly inhibited by Eca 109;cell scratch assay and Transwell migration assay,Eca 109 cell migration ability was significantly inhibited;flow detection cytometry,knockdown of SCARA5 can cause cell cycle arrest in S phase and G2/M phase,and the cell apoptosis rate increased significantly.(6)Exon sequencing results showed that SCARA5 showed cancer promoting function is possible based on the gene exon 5 sequences of thirtieth C>T missense mutation(C/G).Conclusion:(1)The expression of Thsd7 a in Uygur,Kazakh,Han and Mongolian squamous cell carcinoma and adjacent tissues was detected by immunohistochemistry.The results showed that there was no significant difference in the expression of Thsd7 a between the carcinoma and the adjacent tissues of the Uigur,Han and Mongolian patients with esophageal cancer,and the difference was significant for the Kazakh squamous cell carcinoma and the adjacent tissues.(2)Thsd7a was highly expressed in Kazakh squamous cell carcinoma of the esophagus,and the expression of Thsd7 a was significantly correlated with the degree of differentiation and clinical stage.(3)Thsd7a could inhibit the apoptosis of esophageal squamous carcinoma cells,affect the cell cycle,and promote the proliferation and migration of cancer cells.In nude mice tumor bearing experiments,Thsd7 a could promote the growth of esophageal cancer cells in vivo and play the role of oncogenes.(4)Thsd7a plays the role of oncogene and needs to be accomplished through multiple classic tumor associated pathways.Moreover,Thsd7 a,a downstream driver gene SCARA5,also functions as a cancer promoter in esophageal cancer cells.(5)The esophageal cancer cell exome sequencing found that tumor suppressor gene SCARA5 expression of cancer promoting function in esophageal carcinoma cell lines,C>T may be mainly due to missense mutation in exon 5.This study provides new targets and ideas for targeted therapy of esophageal carcinoma. |
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