Objective: Tuberous sclerosis complex (TSC) is a multisystem genetic disorder caused by mutations in the TSC1 and TSC2 genes, but the molecular events contributing to TSC are not well understood. However, little is known about the role of microRNAs in TSC. To explore the microRNA differential expression profile between tuberous sclerosis complex cell line TSC2-/- MEFs and normal type cell line TSC2+/+ MEFs, and to provide new clues to study the mechanism of microRNA function in tuberous sclerosis complex.
Methods: TSC2-/- MEFs and TSC2+/+ MEFs cell lines were cultured in vitro, each with three samples chosen as the experimental group and the control group respectively. Total RNA was isolated using TRizol and purified with RNeasy mini kit according to manufacturer's instructions. RNA quality and quantity were measured by using nanodrop spectrophotometer and RNA integrity was determined by gel electrophoresis. Total RNAs were extracted by TRizol, followed by RNA quantification and quality control. MicroRNA profiles were analyzed by microarray and the threshold value used to screen up-regulated more than 2-fold change or down-regulated less than 0.5-fold change compared with controls. Real-time PCR was used to validate the reliability of microarray. Cell counting kit-8 (CCK-8) assay was performed to evaluate the proliferation.
Results: Fourteen microRNAs, including miR-18a-5p, miR-376c-3p, miR-136-5p, miR-467c-5p, miR-467b-5p, miR-5104, miR-3098-3p, miR-30a-3p, miR-302b-3p, miR-18a-3p, miR-19b-1-5p, miR-19a-5p, miR-20a-5p, miR-155-5p, were up-regulated, while twenty-six microRNAs, including miR-200b-3p, miR-450a-1-3p, miR-542-5p, miR-199b-5p, miR-10a-5p, miR-466c-5p, miR-450a-5p, miR-450b-5p, miR-542-3p, miR-351-5p, miR-322-3p, miR-199a-3p, miR-335-5p, miR-10b-5p, miR-351-3p, miR-155-3p, miR-497a-5p, miR-503-5p, miR-148a-3p, miR-1843a-5p, miR-199a-5p, miR-490-5p, miR-450a-2-3p, miR-322-5p, miR-214-3p, miR-450b-3p, were down-regulated in tuberous sclerosis complex cell line TSC2-/- MEFs compared with normal type cell line TSC2+/+ MEFs (P<0.05). Real-time PCR confirmed the expressions of miR-136-5p, miR-30a-3p, miR-302b-3p, miR-10b-5p, miR-148a-3p, miR-199a-5p consistent with the microarray data (P<0.05). Furthermore, the overexpression of miR-199a-5p significantly inhibited cell proliferation (P<0.05).
Conclusion: There are differences in the expression of miRNA between the tuberous sclerosis complex cell line TSC2-/- MEFs and normal cell line TSC2+/+ MEFs. MiRNA-199a-5p plays an important role in tuberous sclerosis complex, which may be developed as an important molecular target for the treatment of tuberous sclerosis complex.