Study On The Regulating Site And The Expression Level Of α-1,4-N-acetylglucosaminyltransferase In Gastric Cancer Cell By The Induction Of Hp

Helicobacter pylori(Hp)is considered a leading cause of gastritis, gastric ulcer and gastric cancer. In spite of it's high infection rate about 50% in human population,most infected individuals remain asymptomatic, which suggests the presence of host defense mechanisms against Hp pathogenesis. Recent studies indicate thatα-1,4-N-acetylglucosaminyltransferase(A4GnT) exists in cardiac gland cells, pyloric gland cells, mucous neck cells of stomach and Bruuner's gland cells of duodenum. A4GnT is a glycosyltransferase that mediates transfer of GlcNAc toβGal of O-glycan to form the terminalα1,4-linked N-acetylglucosamine. The mucin with terminalα-1,4-GlcNAc in mucous cell exerts antimicrobial activity against Hp. This glycosyltransferase inhibits proliferation, motility and causes morphologic change of Hp due to the inhibition of the biosynthesis of cholesteryl-α-glucoside, a major cell wall component of the bacteria. So we suppose that A4GnT in gastric gland mucous cells prohibit Hp colonizing the deeper gastric mucosa by formingα-1,4-GlcNAc on mucous gland mucin, thus serves as a natural antibiotic protecting the host from Hp infection. In this study, after the co-culture of Hp and gastric cancer cells, we investigate the effcte of Hp on the expression level of A4GnT gene by RT-PCR and immunohistostaining and indentify the regulating site of the A4GnT gene expression by electorphoretic mobility shift assay.Objective: To study the effcte of Hp on the expression level of mRNA of the A4GnT gene by RT-PCR and the expression level of A4GnT protein by immunohistostaining. And to investigate the bingding of transcription factor and cisacting element in A4GnT gene under the induction of Hp by using electorphoretic mobility shift assay together for the further research on the regulatory mechanism of A4GnT gene expression.Methods:1 Cell culture: After resuscitation, gastric cancer cells MKN45 were cultured in RPMI 1640 media containing 10% FBS, 100 U/ml penicillin, 100μg/ml streptomycin at the incubator of 5%CO₂ atmosphere, temperature 37℃and a saturated hnmidity. After digested by trypsin, the cells were transfered in new culture flasks and new Petri dishes with the concentration of 2×10⁵/ml. The cells were used for study after 24 h.2 Bacterial culture: Hp strain J99 and SS1 were plated in Columbia agar containing 5% goat blood, 0.47μg/ml polymyxin B, 11μg/ml amphotericin B and 11μg/ml vancomycin. The culture plates were incubated at 37℃under microaerobic conditions and used for study after 3～5 d.3 Experimental group:Hp group: Hp strain J99 and SS1 with the concentration of 1×10⁶ CFU/ml were co-culture with nomal MKN45 cells respectively for 6 h,then changed the media and cultured to 24h and 48 h.Control group: MKN45 cells were cultured in the medium without Hp J99 and SS1 for 6 h, then changed the media and cultured to 24h and 48 h.4 After 24h, the total RNA of MKN45 cells were extracted, and RT-PCR was used to detect the expression level of A4GnT mRNA. The products and internal controlβ-actin were separated on 1.5% agarose gel and visualized under the ultraviolet light.5 After 48h, immunohistostaining was used to detect the expression level of A4GnT protein under the optical microscope. Each cover glass were counted 5000 cells and the positive rate were calculated for next assay.6 After 24h, the nuclear protein of MKN45 cells were extracted, and EMSA was used to detect the expression level of transcription factor binding to A4GnT cisacting element. The binding bands were exposed on X-ray for next assay.7 All the statistical analyses of tested results were performed using SPSS 17.0 statistical analysis software. The result of RT-PCR were expressed as Mean±S, using t test.Statistical significance was based on P<0.05.The result of immunohistostaining were expressed as the positive cells rate, usingχ² test.Results:1. RT-PCR analysis in Hp group and control group: the PCR product of A4GnT in Hp J99 group(0.6495±0.0129)is higher than that of control group(0.1241±0.0084), P<0.05; the PCR product of A4GnT in Hp SS1 group(0.3775±0.0293)is higher than that of control group(0.1241±0.0084), P<0.05;2. Immunohistochemical staining analysis in Hp group and control group: the rate of A4GnT positive cells in Hp J99 group (22.7%) had obviously increase than control group(4.5%), P<0.05; the rate of A4GnT positive cells in Hp SS1 group (14.2%) had obviously increase than control group(4.5%), P<0.05;3. EMSA analysis of the expression level of nuclear transcription factor binding to A4GnT cisacting element in Hp group and control group: with the induction of J99 and SS1, the binding complex of promoter region Sp1/Sp3 binding site and transcription factor Sp3 decreased; with the induction of J99, the binding complex of nuclear transcription factor and three potential NF-κB binding sites within A4GnT gene elevated.Conclusion:1 Hp J99 and SS1 can induce high expression level of A4GnT mRNA in MKN45 cells.2 Hp J99 and SS1 can induce high expression of the protein of A4GnT in MKN45 cells.3 With the induction of Hp, the high expression level of A4GnT gene is regulated in transcription process of A4GnT, and it may be regulated by the binding complex of transcription factor and cisacting element in A4GnT gene.