The Expression Of ALDH1A1 In Gastric Cancer And Role Of ALDH1A1 In Biological Function Of Gastric Cancer Cell

Objective:To detect the expression of ALDH1A1 in gastric cancer (GC) tissues,and to evaluate its clinical significance. Methods:ALDH1A1 expression in 1072 GC tissues and para-cacinoma(PC) tissues was detected by immunohistochemistry.Associations of ALDH1A1 with clinicopathological characteristics were subsequently assessed.8 frensh GC tissues and its para-cacinoma tissues were detected by Western blot. Six gastric cancer cell lines were detected by Western Blot. Results:Immunohistochemistry showed the expression of ALDH1A1 in gastric cancer tissue was higher than para-cacinoma tissues(P<0.05) .Immunohistochemistry showed the ALDH1A1 expression was associated with age of patients depth of gastic wall invasion,differentiation ,regional lymph, TNM stage and the expression of PCNA. Furthermore expression level of ALDH1A1 was correlated with poor prognosis.Western blot assay showed the positive expression level of ALDH1A1 were significantly higher than its para-cacinoma tissues in 4 of 8 gastric cancer fresh tisue.Western blot assay showed the expression level of ALDH1A1 of the poor differentiation gastric cancer cell line MKN-45 were higher than the moderately differentiated gastric cancer cell line NKN-28. Conclusions : ALDH1A1 was obviously up-regulated in GC tissues ,overexpression of ALDH1A1 might be the feature of GC and benefit us in predicting the prognosis.Chapter II Construction and identification of pEGFP-N1-ALDH1A1 and PGPU6/GFP/NEO-ALDH1A1Objective : To construct the PGPU6/GFP/NEO-ALDH1A1 expression vector targeting ALDH1A1 and evaluate its ability to down-regulate the expression of ALDH1A1 in human gastric cancer cell line MKN-45 and construct the pEGFP-N1-ALDH1A1 expression vector and evaluate its ability to up-regulate the expression of ALDH1A1 in human gastric cancer cell line MKN-28. Methods:The siRNA expression vector targeting ALDH1A1 were constructed using PGPU6/GFP/NEO.shNC was constructed as control,and it was identitied by Western blot analysis after recombinant plasmid PGPU6/GFP/NEO-ALDH1A1 transfected the gastric cancer cell line MKN-45. The protein expression of ALDH1A1 was detected by the Western blot assay. The activity of ALDH was detected with ultraviolet-visible light detector. The pEGFP-N1-ALDH1A1 was constructed by use of recombinant DNA technique and was demonstrated by restriction endonuclease mapping and sequencing from the company of Takara in Dalian.The pEGFP-N1-ALDH1A1 was transfected into human gastric cell line MKN-28 by using lipofectamine 2000.Results:Western blot showed the level of protein expression of ALDH1A1 in gastric cancer cell line MKN-45 transfected with PGPU6/GFP/NEO-ALDH1A1 were significantly decreased .And the assay of ALDH activity showed the ALDH activity of MKN-45 transfected with PGPU6/GFP/NEO-ALDH1A1 was significantly decreased. Western Blot showed the level of protein expression of ALDH1A1 in gastric cancer cell line MKN-28 transfected with pEGFP-N1-ALDH1A1 were significantly increased,and the assay of ALDH activity showed the activity of MKN-28 transfected with pEGFP-N1-ALDH1A1 were significantly increased. Conclusions : The recombinant plasmid of pEGFP-N1-ALDH1A1 was constructed corrected and the protein of ALDH1A1 could be expressed in human gastric cancer cell line MKN28. We successfully constructed the siRNA expression vetor PGPU6/GFP/NEO-ALDH1A1 down-regulating the expression of ALDH1A1 in gastric cancer cell line MKN-45.Chapter III The impact on the biological function of gastric cancer cell with overexpresion or downexpression of ALDH1A1objective:To evaluate the effect of ALDH1A1 silence or overexpression on the proliferation,clone formation and invasion of human gastric cancer cell lines.Materials and Methods:The cell cultures were measured for cell proliferation levels after transfection using MTT assay and cell cout;colony forming unit assay was used to evaluate the colony forming of gastric cancer cell.Transwell test was used to evaluate the invasion of gastric cancer cell lines MKN-45 and MKN-28 after transfection.Results:Cell count and MTT assay indicated that the proliferation level of MKN-45 cells were obviously decreased after transfection with PGPU6/GFP/NEO-ALDH1A1 as compared with control groups.Cell count and MTT assay indicated that the proliferation level of MKN-28 cells were obviously increased after transfection with pEGFP-N1-ALDH1A1 as compared with control groups.In the colony forming unit assay, the colony forming unit of MKN-45 cells transfected with PGPU6/GFP/NEO-ALDH1A1 obviously reduced as compared with control groups ,and the colony forming unit of MKN-28 cells transfected with pEGFP-N1-ALDH1A1 obviously increased as compared with control groups. In the transwell test,MKN-45 cells transfected with PGPU6/GFP/NEO-ALDH1A1 obviously reduced invasion, MKN-28 cells transfected with pEGFP-N1-ALDH1A1 obviously multiplied invasion. Conclusions:ALDH1A1 may affect the proliferation ,colony forming and invasion of human gastric cancer, is a potential target for treat gastric cancer .