The Effect Of Transcription Factor Sox2 On The Phenotype Shift Of Gastric Intestinal Metaplasia

Objective:Gastric intestinal metaplasia (IM),an intermediate step in Correa's cascade ofthe intestinal type of gastric carcinogenesis,is generally regarded as a premalignantlesion although the question remains controversial.The mechanism of itsdevelopment hasn't been clarified in detail.Studies on the factors governing thisprogression are necessary,if we are to prevent cancer developing in such lesions.Theprogress during which stem cells and their descendants are diverted from gastricphenotype to intestine phenotype,is usually triggered by transcription factors.There have been some studies on the expression and potential function ofintestinal transcription factors,Cdxl and Cdx2,in gastric IM,but little is knownabout factors controlling the gastric phenotype.One candidate is encoded by the Sox2gene,a member of the transcription factor family containing a Sry-like high mobilitygroup (HMG)box.We designed following experiments to elucidate whether and howthe transcription factors Cdx2 and Sox2 regulate the phenotype shift during gastricintestinal metaplasia.Methods:1.The expression of SOX2 and CDX2 protein in 80 gastric mucosa specimensembedded in paraffin and pathologically diagnosed as gastritis,mild IM,moderateIM and severe IM were investigated by immunohistochemistry.Then the mRNAlevels of Sox2 and Cdx2 in 40 endoscopic biopsy tissues pathologically diagnosedgastritis,mild IM,moderate and severe IM were quantified by real time Q-PCR.2.The promoter methylation status of gastric IM tissues in 40 endoscopic biopsytissues pathologically diagnosed gastritis,mild IM,moderate and severe IM wasdetected by methylation-specific PCR (MSP).3.pcDNA3.1/Cdx2 plasmid and Sox2siRNAs were transfected into gastricepithelial cell line GES-1 to up-regulate Cdx2 and down-regulate Sox2 respectively. Muc2,Sox2 and Cdx2 mRNA were detected by RT-PCR.The appearance of MUC2protein,an intestinal goblet cell marker,was determined as formation of intestinalphenotype.Results:1.SOX2 and CDX2 proteins were located in the nuclei of normal gastric andnormal intestinal epithelial cells respectively.The positive rates of SOX2 and CDX2proteins in gastritis,mild IM,moderate IM and severe IM were 94.4 %vs5.6 %,75%vs50%,23.5%vs85.7%,9.5%vs90.5%,respectively (P＜0.05).There weredecreased expression of SOX2 and ectopic emergence of CDX2 protein in IM glands.2.Sox2 and Cdx2 mRNA in gastritis,mild IM,moderate and severe IM were0.5778±0.0778 vs 0.0517±0.0218,0.1496±0.0384 vs 0.1402±0.0300,0.1131±0.0384 vs 0.3453±0.0537,respectively (P＜0.05);Sox2 transcripts decreased with theprogression of IM,while Cdx2 transcripts were up-regulated,showing inversecorrelation (r＜0).3.The methylated alleles of Sox2 and Cdx2 were found in 33.3%vs83 % ofgastritis,56.25%vs62.5 % of mild IM,and 83.33%vs25% of moderate and severe IM(P＜0.05).Sox2 and Cdx2 mRNA expression was significantly inversely correlatedwith their methylation status of promoter region (r＜0).4.pcDNA3.1/Cdx2 plasmid and Sox2siRNAs significantly up-regulated of Cdx2and down-regulated of Sox2 in GES-1 cells,respectively.Down-regulation of Sox2could up-regulate Muc2 and Cdx2.Up-regulation of Cdx2 could up-regulate Muc2and down-regulate Sox2.There was synergy between down-regulation of Sox2 andup-regulation of Cdx2.It suggested that both intestinal transcription factor Cdx2 andgastric transcription factor Sox2 are the sufficient and essential factors in gastric IMformation.Conclusions:1.There were down-regulation of Sox2 and the ectopic expression of Cdx2 inthe development of gastric IM.2.Epigenetic mechanisms may play an important role in the transcriptionregulation of Sox2 and Cdx2,especially DNA methylation.3.Both of intestinal transcription factor Cdx2 and gastric transcription factor Sox2 may initiate the shift from the gastric phenotype to the intestinal phenotype ingastric IM development. The Expression of IGF-â… R in Human Gastric Cancer Cells Inhibited by Special Small Interfering RNAObjective:Gastric cancer is one of the most common cancers in the world. Although a declinedincidence has been observed in recent years in our country, it is still the leading causeof cancer mortality. To study the molecular mechanism of gastric cancer from genelevel contributes to clinical treatment and prevention, which has become the hot spotsof cancer reseaches. In this study, we examined the expression of IGF-â… R in humangastric cancer MGC803 cells inhibited by special siRNAs, and investigate thefollowing effect of the cell proliferation and apoptosis after down-regulation ofIGF-â… R.Methods:Two different siRNAs targeted to IGF-â… R were designed,synthesized and transfectedinto MGC803 cells. The changes of IGF-â… R protein level were detected by westernblot 48h after transfection, then the cell proliferation was examined by MTT and thecells were counted and the growth curve was obtained. The early-stage apoptosis wasdetected by flow cytometry.Results:Sequence-specific siRNAs reduced IGF-â… R protein level effectively in MGC803cells at 48h after transfection (siRNA₂-L:64.41%±4.11%, siRNA₂-H:74.14%±6.15%, siRNA₁:89.8%±4.10%) ; the cell proliferation gradually decreased by 49.9%±1.2%, 45.9%±4.4%, 39.1%±5.1%, and 29%±4.0% 2～5 day after transfection,the cell number decreased by 65.58%±4.89%, 55.59%±0.82%, 44.18%±3.17%,and 21.15%+1.1% at the same time; but the early-stage apoptosis percentage of eachgroup detected by FCM was less than 5%. No significant difference was found inearly-stage apoptosis among groups.Conclusions:siRNAs targeted to IGF-â… R can effectively silence the IGF-â… R gene of MGC803 cells and inhibit the cell proliferation, without increasing the cell apoptosis.