In this study, we demonstrated that miR-221 and miR-222 regulate gastric cancer cell viability, apoptosis, cell cycle progression and invasive ability. Our data suggests that downregulation of PTEN expression and enhanced Akt phosphorylation (p-Akt) are important mediators of these cellular processes. As pAkt impacts cell proliferation, cell transit from the G0/G1 to the S phase, apoptosis, cell invasive ability, and cell radiosensitivity, downregulation of miR-221 and miR-222 expression have important biologic effects on the malignant phenotype of SGC7901 cells. These results identify AS-miR-221/222 as a potential therapeutic approach for gastric cancer via upregulation of PTEN.
PTEN functions as a tumor suppressor gene, specifically by negatively regulating the Akt/PKB signaling pathway. Genetic inactivation of PTEN is a hallmark of many cancers, including glioblastoma, endometrial and prostate cancers, and reduced expression occurs in many other tumor types. Deficiency of PTEN in the intestine has been reported to induce precancerous polyps, via the induction of formation and fission of crypts, structures located at the base of the intestine containing a rapidly dividing pool of intestinal stem cells [40
]. Guo JM et al studied the microRNAs expression in primary gastric cancer tissues via microRNA microarray assay and were the first to demonstrate that PTEN was the target of miR-21 [41
]; however, little is known regarding the impact of miR-221 and miR-222 on PTEN expression in gastric cancer.
miR-221 and miR-222 expression is abnormally increased in gastric cancer [42
], however the mechanism by which miR-221 and miR-222 modulates tumor progression within the gut remains unknown. Here, we observed miR-221 and miR-222 upregulation in the human gastric cancer cell line SGC7901 compared with HEK293 epithelial cells, corroborating the findings of Young-kook et al [23
]. miR-221 and miR-222 modulate a variety of biological functions in the SGC7901 cell, including cell proliferation, apoptosis, invasion, and radioresistance. We identified binding sites for miR-221 and miR-222 in the PTEN 3'-UTR by bioinformatics analysis, suggesting that increased expression of the miR-221/222 cluster might impact on PTEN expression. Indeed, we demonstrated that PTEN is a target gene of miR-221 and miR-222 by luciferase reporter assay. As PTEN can antagonize PI3K activity by dephosphorylating PIP3 and thereby negatively regulates the activity of Akt pathway [24
]. Several studies suggest that the loss of the PTEN function might be the underlying factor in Akt pathway activation [43
]; thus, our findings are consistent with an emerging body of literature.
Akt represents a subfamily of the serine/threonine kinase family [46
]. It modulates the function of numerous substrates related to the cell proliferation, apoptosis and invasion and is putatively involved in the development of some cancers, such as in colon [47
], prostate [48
], lung [49
] and thyroid cancer [50
]. It has been shown that Akt activation in cancer cells can increase their invasive ability and resistance to radiotherapy [51
]. In our study, we found that knockdown of miR-221 and miR-222 in SGC7901 cells resulted in downregulation of pAkt expression, affecting the expression of several Akt-regulated proteins including cyclin D1, Bcl-2, and MMP2/9. The malignant phenotype of the SGC7901 cells was reversed by knockdown miR-221 and miR-222, and cells were sensitized to radiation, corroborating the results of Garofalo et al [54
]. As PTEN is a target of miR-221 and miR-222, and has been described previously as an important regulator of radiation sensitivity [24
], these results suggest that increasing PTEN expression by silencing miR-221/222 could enhance the radiosensitivity of SGC7901 cells. Whether PTEN/Akt signaling is the sole target for miRNA-221/222 regulation of radiosensitivity remains unknown.
While our work identifies a role for PTEN in miRNA-221/222-induced biology, it remains possible other factors might be at least partially involved. Negative regulation of p27 and p57 by miRNA221/222 might also contribute to radioresistance [56
], however they are more likely to contribute to cell proliferation and viability [57
]. Moreover, TIMP3, as a target of miR-221 and miR-222, might also affect cell invasion[54
]. In sum, our results suggest that inhibition of the miR221/222 cluster represents a molecular therapeutic approach that impacts on multiple genes involved in anti-tumor growth and radiosensitization, as summarized in Figure .
Figure 6 miR-221/222-mediated regulation of signal transduction via PTEN. Hypothetical representation of the role of miR-221 and miR-222 in regulation of Akt phosphorylation and downstream signaling of proliferation, apoptosis, invasion, and radiosensitivity via (more ...)