MicroRNA-34a Inhibits Stem-like Properties Through An MicroRNA-34a-c-Myc/Bmi-1 Negative Feedback Loop In Gastric Cancer

Part Ⅰ Bmi-1promotes stem-like properties andup-regulatesmicroRNA-34a in gastric cancerObjective:To explore whether Bmi-1 has an effect on stem cell-like propertiesin gastric cancer and try to explain how Bmi-1 up-regulates miR-34a.Methods:Serum-free culture was used to isolate gastric cancer stem-like cells(GCSC);Western-blotwas used to compare Bmi-1 protein levels between gastric cancer cells and microspheres; qRT-PCR was used to detectthe expression of miR-34aregulated by Bmi-1. In order to explore how Bmi-1 regulatesthe expression of miR-34a, we used Dual Luciferase assay to detect the transcriptional activity of NF-κB after Bmi-1’s up-regulation;then we used ChIP to detect whether NF-κB could combine with miR-34a’s promoter region; finally we detected the expression of miR-34a after using PDTC (aNF-κB inhibitor).Results:The gastric cancermicrospheres showed higher expression of Bmi-1; the microspheres formed more and grew lager after the up-regulation of Bmi-1. MiR-34a expression levels increased after Bmi-1 was regulated. Bmi-1 promoted thetranscription activityof NF-κB and NF-κB enhance the expression of miR-34a bye combining with its promoter region. PDTC could inhibitthe expression of miR-34a up-regulated by Bmi-1.CONCLUSION:Bmi-1 promotes stem cell-like properties in gastric cancer and up regulates the expression level of miR-34a through activating NF-κB transcriptional activity.Part Ⅱ miR-34a inhibits stem-like properties bydown-regulatesBmi-1 in gastric cancerObjective:To explore whether miR-34a has an effect on stem cell-like properties in gastric cancer and try to explain howmiR-34a regulatesBmi-1.Methods:Serum-free culture,Colony formation assay,CCK-8 cell proliferation assay,CCK-8 cell toxicity assay and Transwell migration assaywere used toassess the miR-34a’s role in stem cell-like properties of gastric cancer, such as self-renewal, resistance to chemotherapyand metastatic potential;Western-blot was used to detectprotein levels.Results:Bmi-linhibited theself-renewal, resistance to chemotherapyand metastatic potential. The expression level of Bmi-1 was suppressed after miR-34a’s up-regulation but restored after c-myc’s up-regulation. The compensation experiment of cell functiondemonstrated that up-regulating Bmi-1 could reverse the stem cell-like properties inhibited by miR-34a in gastric cancer cells.CONCLUSION:miR-34a down-regulatesBmi-land forms a negative feedback loop. miR-34ainhibits stem-like properties in gastric cancer through the feedback loop mentioned above.Part III the expression of miR-34a in gastric cancer and the relationship with clinical prognosisObjective:To detect the relative expression levels of Bmi-1 and miR-34a in several gastric cancer cell lines; To detect the relative expression levels of Bmi-1 and miR-34a in gastric carcinoma and adjacent tissues analysis their relationship with malignant phenotype and prognosis; To explore whether Bmi-1 and miR-34a’s expression levels in tissues are related.Methods:RT-PCR was used to compare the expression level of miR-34a amonghuman gastric mucosa GES-1 cells, gastric cancer cell lines AGS, HGC27, MKN28, MKN45,7901 and MGC803. Collected 42 cases of gastric cancer patients and detected theirthe relative expression levels of Bmi-1 and miR-34a in gastric carcinoma and adjacent tissues, then combined with clinical and pathological data and analysed miR-34a/Bmi-1’s relationship with the malignant phenotype and prognosis. Analysedwhether Bmi-1 and miR-34a’s expression levels was related in tissues.Results:Compared with human gastric mucosa cells GES-1, other gastric cancer cells had low expressions of miR-34a.42 casespatients’carcinoma showed higher relative expression levels of Bmi-1 than the adjacent tissues; perineural invasion-positive patients’carcinoma showed higher Bmi-1 expression levels thannegative patients; patients with lower Bmi-1 expression had a better prognosis; vascular invasion or perineural invasion patients’carcinoma showed lower expression level of miR-34a than negative patients.Conclusion:Compared with GES-1, miR-34a showed low levels in other gastric cancer cell lines; Bmi-1 expression lever has relationship with the malignant phenotype and the prognosis; the expression level of miR-34a and the malignant phenotype has some relevance.