| Objective:Malignant lesions of gastric mucosa is a gradual process. It often experiences intestinalmetaplasia→dysplasia→early gastric cancer→advanced gastric cancer. If we canintervene effectively in the early stage,the processing of carcergenesis may be block. As akind of early malignant lesions of gastric mucosa, we pay more and more attention tointestinal metaplasia. Telomerase is a kind of mRNA enzyme, which has the function ofreverse transcriptase activity and this is necessary for cell immortalization and infiniteproliferation. As the limit step of Telomerase, the expression of hTERT is as the same asTelomerase. Therefore, hTERT will be defined as the indicator of measuring the activity oftelomerase. The study shown that in the process of the malignant lesions of gastric mucosa,with the severity of cancergenesis the expression of hTERT will be increased, the sameresult was found in the intestinal epithelium. So, we suggest that hTERT may play animportant role in the process, but the mechanism is not clearly and We will do someresearch on it.Methods：⑴qPCR to test the expression of hTERT and CDX2in normal and gastric intestinalmetaplasia；⑵Use immunohistochemical staining to detect the expression of hTERT,CDX2and MUC2；⑶To detect the expression change of related gene CDX1, CDX2andCDX4after transfected hTERT, then Western Blot to test the change of protein level；⑷UseqPCR to test the change of CDX2after transfected hTERT si-RNA, then to the proteinlevel；⑸Co-IP to detect the interaction between hTERT and p65；⑹qPCR, Western Blotand the dual-luciferase to test the function of NF-κB inhibitor (BAY78-1105) to hTERTup-regulates CDX2；⑺qPCR and the dual-luciferase to test the expression change ofKLF4after transfected p65；⑻qPCR, Western Blot and the dual-luciferase to test the expression change of KLF4after transfected hTERT and immunohistochemical staining todetect the expression of KLF4in the normal and gastric intestinal metaplasia；⑼Dual-luciferase to test the function of interfering KLF4in hTERT up-regulate CDX2expression;⑽Western Blot to test the function of interfering p65in hTERT increasing theexpression of CDX2;⑾qPCR and dual-luciferase to test the expression of CDX2aftertransfected p65and p50；⑿Co-IP to test the interaction of hTERT and c-Rel and p50.Results.1. hTERT is closely with malignant lesions of gastric mucosa(1) The qPCR of intestinal metaplasis tissue results show that hTERT and CDX2areincreased in mRNA level;(2) Immunohistochemical of the tissue resulted hTERT andCDX2are up-regulated；(3) Over-expression and interference of hTERT, the expression ofCDX2is increased and decreased in mRNA and protein levels which show that hTERTjoins the process of intestinal metaplasis by regulates the expression of CDX2. All theresults indicate that hTERT is closely with malignant lesions of gastric mucosa and may bea part of the process of intestinal metaplasis.2. hTERT up-regulates the expression of CDX2in gastric cancer by p65-KLF4pathway(1) Co-IP verified hTERT and p65are interaction with each other in protein level andpromote the expression of p-p65which shows hTERT has some function with p65in orderto promote the transcription of downstream gene;(2) Immunohistochemical shows that the expression of KLF4is sharply increased inintestinal metaplasis tissue which expressed hTERT highly, over-expression of hTERT canpromote the expression of KLF4in mRNA/protein and promoter levels;(3) The function of hTERT up-regulates CDX2and KLF4will be inhibited by NF-κBinhibitor and interference of p65RNA.(4) Interference KLF4can inhibit the function of hTERT increased the expression ofCDX2.These results suggest that p65-KLF4-CDX2pathway is involved in the occurrence ofgastric intestinal metaplasia.3. hTERT up-regulates CDX2by c-Rel/p50(1) Interference p50can inhibit the function of hTERT increased the expression of CDX2(2) The activation of CDX2in mRNA, protein and promoter will be increased byover-expression of p50.(3) Co-IP results show that hTERT is not only has interaction with c-Rel but also p50in protein level which indicate that hTERT, c-Rel and p50will be a trimer and regulate theexpression of downstream gene.The above results suggest that hTERT can form a trimer with c-Rel/p50CDX2expression regulation, and promote the occurrence of gastric intestinal metaplasiaConclusionIn the process of gastric intestinal metaplasia，hTERT up-regulates CDX2throughp65-KLF4-CDX2(indirectly) and hTERT/c-Rel/p50-CDX2(directly) two pathways,promoting gastric intestinal metaplasia occurs. |
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