Rapamycin-upregulated miR-29b promotes mTORC1-hyperactive cell growth in TSC2-deficient cells by downregulating tumor suppressor retinoic acid receptor β (RARβ)

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miR-29b has been identified as a rapamycin-induced microRNA (miRNA) in Tsc2-deficient, mTORC1-hyperactive cells. The biological significance of this induction of miR-29b is unknown. We have found that miR-29b acts as an oncogenic miRNA in Tsc2-deficient cells: inhibition of miR-29b suppressed cell proliferation, anchorage-independent cell growth, cell migration, invasion, and the growth of Tsc2-deficient tumors in vivo. Importantly, the combination of miR-29b inhibition with rapamycin treatment further inhibited these tumor-associated cellular processes. To gain insight into the molecular mechanisms by which miR-29b promotes tumorigenesis, we used RNA sequencing to identify the tumor suppressor retinoid receptor beta (RARβ) as a target gene of miR-29b. We found that miR-29b directly targeted the 3′UTR of RARβ. Forced expression of RARβ reversed the effects of miR-29b overexpression in proliferation, migration, and invasion, indicating that it is a critical target. miR-29b expression correlated with low RARβ expression in renal clear cell carcinomas and bladder urothelial carcinomas, tumors associated with TSC gene mutations. We further identified growth family member 4 (ING4) as a novel interacting partner of RARβ. Overexpression of ING4 inhibited the migration and invasion of Tsc2-deficient cells while silencing of ING4 reversed the RARβ-mediated suppression of cell migration and invasion. Taken together, our findings reveal a novel miR-29b/RARβ/ING4 pathway that regulates tumorigenic properties of Tsc2-deficient cells, and that may serve as a potential therapeutic target for TSC, lymphangioleiomyomatosis (LAM), and other mTORC1-hyperactive tumors.

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We thank Lorena Pantano of the Harvard Chan Bioinformatics Core for assistance with RNAseq analysis. This work was supported by the Engles Program in TSC and LAM research, Department of Defense grant W81XWH-15–1–0263 TS14003 (EPH) and a postdoctoral Fellowship from Tuberous Sclerosis Alliance (HJL).

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Correspondence to Elizabeth P. Henske.

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