Under-expression of LATS1 promotes the differentiation, proliferation and migration of mesenchymal stem cells by inhibition the Hippo signaling pathwayin vitro / 中华危重病急救医学

ABSTRACT

Objective To explore the effects of under-expression of large tumor suppressor 1 (LATS1) on activation of Hippo signaling pathway and differentiation, proliferation, migration of bone marrow mesenchymal stem cells (mMSCs) of micein vitro.Methods mMSCs of C57BL/6 mice were divided into normal control (MSC) group, empty vector control (MSC-GFP) group, LATS1-over-expressing (MSC-LATS1) group, empty vector without LATS1 shRNA control (MSC-shControl) group and LATS1-under-expressing (MSC-shLATS1) group. Lentiviral vectors with activated,inactivated LATS1 (the key molecule of Hippo signaling pathway) modifications and empty vectors were constructed and were used to infect mMSCsin vitro. The transduction efficiencies mediated by the lentiviral vectors were evaluated by fluorescence microscopy and flow cytometry. The mRNA expression of LATS1 was quantified by quantitative real-time polymerase chain reaction (qRT-PCR), and the protein expressions of LATS1, YAP (p-YAP), 14-3-3 were quantified by Western Blot to evaluate the activation of Hippo signaling pathway. Osteogenic and adipogenic differentiation of mMSCs were evaluated through measurement of Runx2, OSX and C/EBPα, PPAR-γ mRNA by qRT-PCR, as well as Alizarin Red S and Oil red O staining. Proliferation of mMSCs was evaluated using methy thiazdyl tetrazolium (MTT) assay. The scratch test and Transwell chamber test were used to analyze the horizontal and vertical migration ability of mMSCs.Results The transduction efficiencies mediated by the lentiviral vectors were 94.74%-96.10%. Compared with MSC-GFP group, the activation of Hippo signaling pathway was promoted in MSC-LATS1 group [LATS1 mRNA (2-ΔΔCT) 4.37±0.21 vs. 1.20±0.04, LATS1 protein (gray value) 2.21±0.06 vs. 1.09±0.10, p-YAP/YAP protein (gray value) 1.51±0.13 vs. 0.98±0.05, 14-3-3 protein (gray value) 1.92±0.18 vs. 1.10±0.09, allP < 0.05], osteogenic and adipogenic differentiation of mMSCs were decreased in MSC-LATS1 group [mineralization (A value)0.13±0.02 vs. 0.40±0.03, Runx2 mRNA (2-ΔΔCT) 0.51±0.02 vs. 0.98±0.09, OSX mRNA (2-ΔΔCT) 0.41±0.04 vs. 1.04±0.09, lipid accumulation (A value) 0.10±0.02 vs. 0.25±0.03, C/EBPα mRNA (2-ΔΔCT) 0.33±0.03 vs. 1.11±0.09, PPAR-γ mRNA (2-ΔΔCT) 0.29±0.02 vs. 1.04±0.10, allP < 0.05], the proliferation rate of mMSCs at 4-7 days was decreased in MSC-LATS1 group and so were the horizontal and vertical migration of mMSCs [wound healing rate (18.65±3.53)% vs. (40.29±1.87)%, migrated cells (cells/MP) 35.99±6.18 vs. 103.67±17.77, bothP <0.05]. Compared with MSC-shControl group, the activation of Hippo signaling pathway was inhibited in MSC-shLATS1 group [LATS1 mRNA (2-ΔΔCT) 0.16±0.01 vs. 0.98±0.03, LATS1 protein (gray value) 0.38±0.03 vs. 1.04±0.07, p-YAP/YAP protein (gray value) 0.58±0.04 vs. 1.05±0.06, 14-3-3 protein (gray value) 0.14±0.02 vs. 1.02±0.09, allP < 0.05], osteogenic and adipogenic differentiation of mMSCs were increased in MSC-shLATS1 group [mineralization (A value) 0.93±0.13 vs. 0.44±0.05, Runx2 mRNA (2-ΔΔCT) 1.44±0.12 vs. 0.95±0.04, OSX mRNA (2-ΔΔCT)1.67±0.06 vs. 1.10±0.11, lipid accumulation (A value) 0.47±0.06 vs. 0.28±0.04, C/EBPα mRNA (2-ΔΔCT)3.98±0.61 vs. 0.99±0.10, PPAR-γ mRNA (2-ΔΔCT) 3.05±0.36 vs. 0.98±0.14, allP < 0.05], the proliferation rate of mMSCs at 3-7 days was increased in MSC-shLATS1 group and so were the horizontal and vertical migration of mMSCs [wound healing rate (80.18±6.98)% vs. (46.18±1.01)%, migrated cells (cells/MP) 212.69±41.21 vs. 115.87±35.15, bothP < 0.05].Conclusions Under-expression of LATS1 promotes the differentiation, proliferation, migration of mMSCs by inhibition of Hippo signaling pathwayin vitro.