| Gastric cancer is one of the most frequently occurring malignancies, and ranks as the second leading cause of cancer-related deaths worldwide. In China, mortality due to gastric cancer accounts for 25-30% of all cancer-related deaths, ranking first in all malignancies. Gastric cancer is thought to result from a combination of environmental factors, host factors such as infection by Helicobater pylori and accumulation of generalized and specific genetic alterations. Understanding the underlying molecular mechanism could provide insights leading to improved diagnosis and therapeutic approaches. Previous studies from clinical specimens have shown that activation of PI3K/Akt pathway is frequently found in human gastric cancer, as well as correlated with tumor progression, metastasis and prognosis. Studies in vitro have revealed that PI3K/Akt pathway plays important roles in gastric cell proliferation, apoptosis, migration and drug resistance. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) is a tumor suppressor that negatively regulates cell survival and proliferation by antagonizing Akt signaling. In our previous studies, a gastric caner mouse model was established with conditional deletion of PTEN in glandular epithelium. All of PTEN mutant mice showed accelerated cell proliferation and gastric tumorigenesis, which recapitulated the nature history and pathologic features of human gastric cancer.microRNAs are small, noncoding, single-stranded RNAs that suppress gene expression post-transcriptionally primarily through sequence-specific interaction with the 3'untranslated regions (UTRs) of cognate mRNA targets and inducing mRNA degradation or translational repression. Accumulating evidence has proved that miRNA deregulation was correlated with gastric tumorignenesis, histological subtype, progression, metastasis and prognosis, and involved in gastric cell proliferation, apoptosis, migration and drug resistance through regulating their downstream targets. However, the upstream regulator which modulates miRNA expression is still largely unknown.In this study, we screened miRNAs regulated by the Akt signaling pathway using the gastric cancer mouse model with PTEN deficiency in glandular epithelium. The function of these miRNAs on gastric cell proliferation, apoptosis and migration was evaluated. Their functional targets were also screened and confirmed. Moreover, we verified the correlation between the miRNA and the upstream Akt signaling and their downstream targets in mouse gastric cancer model and human gastric cancer tissues. Using this unique mouse model, we could identify Akt-regulating miRNAs in the early stage of tumor progression, which could be responsible for gastric tumorigenesis. This study will facilitate our understanding about the function and mechanism of Akt pathway and miRNA in gastric carcinogenesis, and benifit researches on early diagnostic biomarker and therapeutic target screening.First, we screened out Akt-regulating miRNAs which might be involved in initiation and development of gastric cancer. Using miRNA microarray analysis, 28 dysregulated miRNAs were identified in PTEN mutant gastric epithelium. Among them, 10 miRNAs were picked out and validated by Northern blot. The results were consistent with the findings from miRNA profiling. 4 miRNAs showed dramatically increased expression upon the repression of Akt signaling in gastric cancer cell line BGC-823, indicating that the 4 miRNAs could be regulated by Akt signaling. Among them, miR-365 stood out as an attractive candidate for the most significant inhibition of cell proliferation. So we subsequently focused on miR-365 for further testing whether it could be negatively regulated by Akt pathway. Upon the elevated Akt signaling by IGF-1/insulin treatment or overexpression of the constructively active mutant of Akt1, miR-365 expression was dramatically decreased. Moreover, the results from promoter luciferase assays suggested that Akt signaling might negatively regulate the transcription of miR-365. Evolutional conservation analysis and tissue expression profile indicated that miR-365 might play pivotal roles in development and diseases of gastrointestinal tissues.Due to the absent cellular function of miR-365 in previous studies, we next explored the biological effect of miR-365 on gastric cancer cells in vitro. To test the role of miR-365 in gastric cell proliferation, we transfected several gastric cell lines with miR-365 transiently or stably. Overexpression of miR-365 significantly suppressed cell proliferation and reduced clonal growth. Conversely, knockdown of endogenous miR-365 promoted the gastric cell proliferation and clonal growth. In addition, flow cytometric analysis revealed that miR-365 overexpression induced G1 arrest. Soft agar cloning assay and xenograft results revealed that miR-365 could inhibit tumorigenesis of gastric cancer cells. Taken together, the above data strongly demonstrated that miR-365 could inhibit gastric cell proliferation and carcinogenesis as a tumor suppressor. Bioinformatics analysis was performed to search for miR-365 targets which were responsible for regulating cell proliferation. CCND1 and E2F2, genes critical for transition through the G1-S checkpoint, were predicted as targets of miR-365. Western blot results proved that cyclin D1 and E2F2 expression was negatively regulated by miR-365. This was further confirmed by the upregulation of cyclin D1 and E2F2 in PTEN mutant gastric epithelium. The luciferase reporter assays suggested that cyclin D1 and E2F2 was direct targets of miR-365. Moreover,re-expression of cyclin D1 dramatically rescued miR-365-mediated growth inhibition, indicating that miR-365 inhibited cell proliferation by directly targeting cyclin D1.Finally, we examined whether Akt signaling might function in promoting cell proliferation by negatively regulating miR-365 expression. miR-365 overexpression dramatically abrogated the cell proliferation caused by activation of Akt signaling. Conversely, knockdown of miR-365 could restore cell proliferation inhibited by suppressing Akt signaling. FACS analysis also showed that miR-365 overexpression attenuated the effect of IGF-1 stimulation which showed the increased cells in S phase and decreased cells in G0-G1 phase. Moreover, the results from cyclin D1 and E2F2 expression were consistent with that from the proliferation and cell cycle assays. These data demonstrated that miR-365, as a downstream effector of Akt pathway, could regulate cell cycle and proliferation through its targets, cyclin D1 and E2F2. To genetically assess the possible contribution of p-Akt-miR-365-cyclin D1/E2F2 axis to tumorigenesis, the expression of the above molecules were tested in Akt1 and PTEN double mutant mice. The upregualtion of miR-365 and downregualtion of cyclin D1 and E2F2 strongly suggested that p-Akt-miR-365-cyclin D1/E2F2 axis might contribute to gastric cell proliferation and tumorigenesis in mouse. In addition, the correlation between dysregulation of p-Akt-miR-365-cyclin D1 axis and gastric tumorigenesis was also confirmed in human gastric cancer samples.In conclusion, we found that miR-365, which was dramatically downregulated in PTEN mutant mice, could inhibit cell proliferation, induce G1 arrest and suppress tumorigenesis in xenografts by direct targeting cyclin D1 and E2F2. Moreover, Akt pathway promoted cell proliferation by decreasing the abundance of miR-365 and consequently upregulating expression of cyclin D1 and E2F2. Remarkably, we also demonstrated that p-Akt-miR-365-cyclin D1 axis contributed to mouse and human gastric carcinomas. For the first time, our study revealed that the Akt signaling promotes gastric cancer cell proliferation and tumorigenesis by inhibiting tumor-suppressor miRNA, miR-365, which broadened our understanding about the epigentic mechanism underlying the regulation of Akt signaling pathway.
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