The Preliminary Analysis Of Differential Expression Genes In The Development Of Esophageal Carcinoma

Objectives:1. To explore the differential expression genes preliminarily in ESCC by Oligomicroarray.2. To investigate the expression of CTHRC1 in human ESCC tissue and cell lines, and to discuss its clinicopathological features in development of ESCC.3. To observe the effects resulted from inhibition of CTHRC1 by RNAi on proliferation,invasion and migration of Eca-109 cells.Methods:1. The obviously differential expression genes among ESCC,PCT and NET were selected by Oligomicroarray,and was checked by Real-time Fluorescent Qantitation RT-PCR.2. Expression of CTHRC1 was observed in ESCC tissure by Immunohistochemistry and their relationship with clinicopathological features was evaluated.3. Expression of CTHRC1 in esophageal carcinoma cell lines Eca-109 and TE-13 was detected by Immunofluorescence and Western-blot.4. Eca-109 cells were transfected with siRNAs targeting CTHRC1.Then expression of CTHRC1 was detected by RT-PCR and Western-blot in order to assess the interference efficiency,accordingly the optimal siRNA was choosed.5. With RNA silencing of CTHRC1 in cell line Eca-109,the cellular proliferation was analyzed by MTT,the changes of invision and migration were assessed by Transwell and expression of MMP2 and MMP9 was detected by Western-blot.Results:1. Oligomicroarray:The total RNA,everse transcription product and fluorescence labeled cRNA were all of high quality;38 genes were up-regulated and 61 were down-regulated.2. Real-time Fluorescent Qantitation RT-PCR:The most differential gene was CTHRC1.3. Immunohistochemistry:The positive expression of CTHRC1 was existing in cellular membrane and cytoplasm of ESCC.The expression rate was 56.5%. There was significantly higher expression in the cases with metastasis than these with non-metastasis(P<0.05),and the expression of CTHRC1 was not related with age,sex,size of tumer and pathological type(P>0.05).4. Immunofluorescence and Western-blot:The expression of CTHRC1 was observed in esophageal carcinoma cell lines Eca-109 and TE-13.5. siRNA inhibited expression of CTHRC1 in Eca-109 cells dramatically:siRNA1 was selected for its excellent interference efficiency of CTHRC1 which was used in subsequent experiment.6. RNA silencing of CTHRC1 obviously inhibited proliferation and apoptosis of Eca-109 cells.Conclusions:1. The high throughput and effective Agilent oligomicroarray can screen novel therapy targeted genes by analyzing the differential expression genes in development and progression of ESCC.2. The development and progression of ESCC is a complicated process involving multigenes and multiprocedures.3. The expression of CTHRC1 was up-regulated in ESCC tissue and cell lines Eca-109,TE-13 and was associated with lymph-node metastasis.4. RNA interference can effectively silence CTHRC1 in cell line Eca-109.5. RNA silencing of CTHRC1 in cell line Eca-109 may lead to inhibition of cellular proliferation,migration and invision.It also leads to inhibition of MMP2 and MMP9 expression.