A Hyperglycemic Microenvironment Inhibits Tendon-to-Bone Healing through the let-7b-5p/CFTR Pathway

Abstract

Background: Tendon-to-bone healing is a difficult process in treatment of rotator cuff tear (RCT). In addition, diabetes is an important risk factor for poor tendon-to-bone healing. Therefore, we investigated the specific mechanisms through which diabetes affects tendon-to-bone healing by regulating the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR).

Methods: Tendon-derived stem cells (TDSCs) were extracted from rats after which their proliferative capacities were evaluated by the MTT assay. The expression levels of CFTR and tendon-related markers were determined by qRT-PCR. Then, bioinformatics analyses and dual luciferase reporter gene assays were used to identify miRNAs with the ability to bind CFTR mRNA. Finally, CFTR was overexpressed in TDSCs to validate the specific mechanisms through which the high glucose microenvironment inhibits tendon-to-bone healing.

Results: The high glucose microenvironment downregulated mRNA expression levels of tendon-related markers and CFTR in TDSCs cultured with different glucose concentrations. Additionally, bioinformatics analyses revealed that let-7b-5p may be regulated by the high glucose microenvironment and can regulate CFTR levels. Moreover, a dual luciferase reporter gene assay was used to confirm that let-7b-5p targets and binds CFTR mRNA. Additional experiments also confirmed that overexpressed CFTR effectively reversed the negative effects of the hyperglycaemic microenvironment and upregulation of let-7b-5p on TDSC proliferation and differentiation. These findings imply that the hyperglycemic microenvironment inhibits CFTR transcription and, consequently, proliferation and differentiation of TDSCs in vitro by upregulating let-7b-5p.

Conclusions: A hyperglycemic microenvironment inhibits TDSC proliferation in vitro via the let-7b-5p/CFTR pathway, and this is a potential mechanism in diabetes-induced poor tendon-to-bone healing.

Figure 1

Hyperglycemia inhibited cell proliferation and suppressed the expression of key tendon-related markers of TDSCs. (a) TDSCs were cultured in different glucose concentrations (5.5 mM, 15 mM, and 25 mM), and cell activities were measured by the MTT assay. (b) qRT-PCR assays showed that expressions of CFTR, Tnmd, Col-l, and Scx mRNAs were significantly downregulated in 15 mM and 25 mM glucose-treated cells, relative to the normoglycemic group (p < 0.05). (c) A high glucose microenvironment downregulated the mRNA expressions of tendon-related markers and CFTR in TDSCs. (d) Expressions of the CFTR protein were downregulated in the high glucose environment (15 mM and 25 mM).

Figure 2

Let-7b-5p targets and regulates CFTR in a hyperglycemic microenvironment. (a) Intersections of CFTR-related miRNAs, T2DM-related miRNAs, and TDSCs-related miRNAs were shown by the VEEN plot. (b) A volcanic map of miRNAs related to TDSCs. (c) Relative expression levels of let-7b-5p in the diabetic and normal groups. (d) Expressions of let-7b-5p were significantly upregulated after high glucose (15 mM and 25 mM) treatment of TDSCs, compared to the control (5.5 mM), p < 0.05. (e). Let-7b-5p targeting regulated CFTR transcription, as predicted by RNAhybrid (minimum free energy = −28.9 kcal/mol). (f). TDSCs were transfected with the 3′UTR CFTR luciferase reporter gene along with a let-7b-5p mimic or let-7b-5p inhibitor, respectively. (g) Western blot assays demonstrated that the let-7b-5p mimic significantly downregulated CFTR protein levels in TDSCs.

Figure 3

Histogram of Let-7c-5p target gene enrichment analysis.

Figure 4

A hyperglycemic microenvironment downregulated CFTR by upregulating let-7b-5p. (a). TDSCs were cultured in a high glucose (25 mM) medium after which cell activities in relation to let-7b-5p were evaluated by the MTT assay. (b) Treatment of TDSCs with DMEM (high glucose), overexpressed CFTR and the let-7b-5p mimic, reduced cell proliferations on days 1, 3, and 5. (c) Western blot analysis showed that upregulations of CFTR protein expressions, caused by overexpressed CFTR, were suppressed by the let-7b-5p mimic and DMEM (high glucose). (d) qRT-PCR assay showed that overexpressed CFTR upregulated CFTR, Tnmd, Col-l, and Scx mRNA levels. In addition, the let-7b-5p mimic and DMEM (high glucose) suppressed the upregulation of CFTR, Tnmd, Col-l, and Scx, caused by the overexpressed CFTR; ^∗p < 0.05.