| Background:Gastric cancer is one of the most common malignant tumors in the world,and the mortality rate is the second leading cause of death in malignant tumors.The progn-osis of gastric cancer is closely related to the clinical stage.The 5-year survival rate of patients with clinical ?-? can reach 90%,while the 5-year survival rate of patients with clinical ?-? is less than 10%.Therefore,early detection,early diagnosis and early treatment are helpful to reduce mortality.The study of the mechanism of gastric cancer can help to find an early intervention for gastric cancer.Helicobacter pylori(H.Pylori)is considered to be an important causative agent of gastric cancer.In 1994,WHO defined H.Pylori as a class I carcinogen.After infection of H.Pylori,it produce VacA,CagA protein and pathogenic factors such as urease.These pathogenic factors lead to loss of the cell cytoskeleton and polarity,reduced the adjacent cell adhesion ability and interfered the intracellular signal transduction and cell biological functions.Despite the deepening of the study,the molecular mechanisms underlying the maglig-ant transformation of gastric tissue induced by H.Pylori infection have not yet been elucidated.The mechanism of carcinogenesis of the agent needs further investigation.Studies have shown that H.Pylori infection can activate NF-?B singal pathway,which further regulate downstream oncogenes,involved of many non encoding RNA,protein and other bioactive molecules.MicroRNAs(miRNAs)is a small single stranded RNA that encodes no protein.It binds the 3' non encoding region of specific target gene,and downregulates the expression of genes at post transcriptional levels.A large number of studies have confirmed that miRNA is involved in the regulation of tumor related genes and important biological processes,including cell growth,proliferation,apoptosis,metastasis,angiogenesis and tumor immune response.Additonally it has been proven that H.Pylori infection afrects the expression of miRNA in gastric mucosal epithelial cells and is involved in the development of gastrie cancer.Biomformatics predict that miRNA-136 is regulated by the NF-?B and PDCD11 is the downstream regulator of miRNA-136.The recent study found that the expression of miRNA-136 is elevated in small cell lung cancer which plays a role in cancingenesis.There was no literature that miRNA-136 was involved in gastric cancer.Literature reported that PDCD11 can induce the transcription of FasL,thereby promoting the apoptosis mediated by Fas/FasL.PDCD11 also has been proven to promote the processing and mature of ribosomal RNA.Ribosome biogenesis disorder will increase the susceptibility of cancer.The abnormal expression of PDCD11 may be involved in the development of cancer.But the role of PDCD11 in the development and progression of gastric cancer has not been reported in the Iiterature.Through the review of literature combined with bioinformatics prediction,our papers assumed that the H.Pylori may activate the NF-k B and regulate the expression of miRNA-136 and PDCD11,which lead to gastric cancer.In this study,we explore the regulation of on the expression of miRNA-136 and PDCD11 and then provide a new approach for the diagnosis and treatment of gastric cancer.Purpose:To investigate the expression of miRNA-136 and PDCD11 in human gastric cancer and its correlation with clinical pathological features.To explore the molecular mechanism of H.pylori on miRNA-136 and PDCD11.To seek new diagnosis and treatment for gastric cancer through discussing the mechanism.Method:1.Relationship between miRNA-136 and PDCD11 and gastric cancer30 cases fresh gastric career tissue and adjacent normal tissues confirmed by pathology of surgical resection.The total RNA were extracted and expression of miRNA-136 and PDCD11 mRNA was detected by qRT.PCR,the expression of U6 and GAPDH as an internal control;The expression level of PDCD11 protein was detected by Western blot,GAPDH as an internal contrl.Statistical analyze the differential expression of miRNA-136 and PDCD11 in gastric cancer tissues and adjacent tissues and the correlation between miRNA-136 and PDCD11 and the clinicopathological factors,and H.Pylori infection,the survival analysis of Kaplan-Meier between miRNA-136 and PDCD11 and gastric cancer survival and prognosis.2.The effects of H.pylori on the expression of miRNA-136 and PDCD11H.Pylori(150:1?100:1?50:1)stimulate gastric cancer cells,At 12 hours,cells were collected.H.Pylori(100:1)stimulate gastric cancer cells,At 6,12,18,24 hours,cells were collected.The expression of miRNA-136 and PDCD11mRNA were detected by qRT-PCR.the expression of PDCD11 protein was detected by Western blot.Whether the expression of miRNA-136 and PDCD11 is present with the dose and time dependence on H.pylori was observed.3.Regulation of miRNA-136 with H.pylori by activating NF-?BTo verify whether the NF-?B signaling pathway is involved in H.Pylori on the expression of miRNA-136.Bioinforformation predict miRNA-136 promoter binding site.NF-?B binding site reporter vector and mutant miRNA-136 promoter reporter vector was transfected into gastric cancer cells.After transfection,H.pylori stimulated gastric cancer cells,the fluorescence intensity was detected.The fluorescence intensity change were analyzed.Through signaling pathway inhibition experiments,we further verify whether the NF-?B signaling pathway is involved in the regulation of H.Pylori on the expression of miRNA-136.4.miRNA-136 targeting regulation of PDCD11 influences the proliferation,apoptosis and invasion of gastric cancer cellsBioinformatics prediction and Luciferase experiment prove that PDCD11 3 'UTR was bind with miRNA-136.miRNA-136mimic and inhibitor was transfected into gastric cancer cells.PDCD11 protein and mRNA were detected by Western blot and qRT-PCR.After transfection,CCK8 assay and flow cytometry to detect the proliferation and apoptosis of gastric cancer cells,Transwell assay was used to observe the invasion of gastric cancer cells.After transfection of PDCD11 overexpre-ssion plasmid,the effects of overexpression of PDCD11 on proliferation,apoptosis and invasion of gastric cancer cells induced by miRNA-136 were observed.Result:1.Expression and clinical significance of miRNA-136 and PDCD11 in gastric carcinomaThe relative expression of miRNA-136 in gastric cancer tissues was significantly higher than that in normal tissues of.PDCD11mRNA and protein in gastric cancer tissues was significantly lower than that in adjacent normal tissues.PDCD1 lprotein was negatively related to miRNA-136 in gastric carcinoma and adjacent normal tissues.The expression of miRNA-136 in H.Pylori positive gastric cancer tissue was significantly higher than that in H.Pylori negative,and the expression of PDCD11 mRNA and protein was lower than that in H.Pylori negative.The expression of miRNA-136 in TNM ?-? stage and lymph node metastasis specimens of gastric cancer was significantly higher than that in stage ?-? and no lymph node metastasis,and the expression level of PDCD11mRNA and protein in ?-? phase and lymph node metastasis specimens of gastric cancer was significantly lower than that in stage?-? and no lymph node metastasis.The median survival time of gastric cancer patients with relative high expression of miRNA-136 and low expression of PDCD11 was significantly lower than that in patients with relative low expression of miRNA-136 and high expression of PDCD11.2.The effects of H.pylori on the expression of miRNA-136 and PDCD11The expression of miRNA-136 and PDCD11 is present with the dose and time dependence on H.pylori.3.H.Pylori regulate the expression of miRNA-136 by activating the NF-?BPromoter prediction results show that the binding sites of miRNA-136 promoter sequences containing NF-?B.Luciferase experiments verified binding of NF-?B to the promoter of miRNA-136,and signal transduction inhibitors furtherly showed that NF-?B signaling pathway was involved in the regulation of H.Pylori on the expression of miRNA-136.4.miRNA-136 targeting regulation of PDCD11 influences the proliferation,apoptosis and invasion of gastric cancer cellsAfter transfection with miRNA-136mimic and pGL-PDCD11-wt,luciferase activity decreased,but miRNA-136mimic and pGL-PDCD11-mut was transfected into gastric cancer cells,luciferase activity had no obvious change;After transfection with miRNA-136inhibitor and PGL-PDCD11-wt,luciferase activity increased,but transfection with miRNA-136inhibitor and pGL-PDCD11-mut,luciferase activity had no obvious change;After transfection with miRNA-136mimics,the expression of PDCD11 mRNA and protein was decreased.After transfection with miRNA-136 inhibitor,the expression level of PDCD11 mRNA and protein increased.After transfection with miRNA-136mimic,cell proliferation increased,cell apoptosis decreased,cell invasion increased.After transfection with miRNA-136inhibitor,cell proliferation decreased,cell apoptosis increased,cell invasion decreased.PDCD11 overexpression can affect proliferation,apoptosis and invasion of gastric cancer cells induced by miRNA-136Conclusion:1.The expression of miRNA-136 in gastric cancer increased and the expression of PDCD11 decreased,and the expression of them was negatively correlated.2.High expression of miRNA-136 and low expression of PDCD11 is associated with a poor prognosis of gastric cancer.3.High expression of miRNA-136 and low expression of PDCD11 are associated with H.Pylori infection and are involved in the development of H.Pylori associated gastric carcinoma.4.Through activation of NF-?B,H.pylori can upregulate the expression of miRNA-136,which inhibit expression of PDCD11,futher promotes proliferation,inhibitis apoptosis and invasion of gastric cancer cells,play a role in the development of gastric cancer. |
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