MALAT1: A Pivotal lncRNA in the Phenotypic Switch of Gastric Smooth Muscle Cells via the Targeting of the miR-449a/DLL1 Axis in Diabetic Gastroparesis

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  FIGURE 1. The expression of MALAT1 is upregulated in clinical samples and a murine model of DGP. (A) Contractility of circular gastric muscle strips from streptozotocin-induced diabetic and respective control mice after administration of 100 μmol/L CCh. (B) The hierarchical clustering and volcano analysis of differentially expressed lncRNAs. Here, 585 differentially expressed lncRNAs (210 downregulated and 375 upregulated) from hierarchical clustering were identified between DGP mice and the respective control group. The expression level of lncRNA is represented in red if it is above the average value of the gene in all samples. Conversely, green indicates that the expression level of lncRNA is below the average value. (C) qPCR was used to validate significantly differentially expressed lncRNAs, and lncRNA MALAT1 was significantly up-regulated in DGP mice. (D) qPCR analysis of the expression of MALAT1 in gastric tissues from a DGP mouse model (and control mice; n = 10 per group). (E) qPCR analysis of the expression of MALAT1 in (adjacent) normal stomach tissues from patients who underwent gastrectomy with or without DM and DGP symptoms (n = 10 per group). β-Actin was used as an internal…

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  FIGURE 2. MALAT1 knockdown attenuates the HG-induced promotion of cell viability, proliferation, migration, and phenotypic switching in HGSMCs. (A) qPCR analysis of the expression of MALAT1 in HGSMCs treated with different concentrations of glucose (5, 15, 25, and 35 mM) for 48 h. (B) qPCR analysis of the expression of MALAT1 in HGSMCs 48 h after transfection with shMALAT1s and shNC. (C–E) The CCK8, EdU, and Transwell assays were carried out to analyze the viability, proliferation, and migration of HGSMCs under different treatments. (F–G) qPCR and western blot analyses of SMC contractile markers (α-SMA, SM22α, and MHC) in HGSMCs under different treatments. For (C–G), four groups of cells were used: NG + shNC group, HG + shNC group, NG + shMALAT1 group, and HG + shMALAT1 group. β-Actin was used as an internal control. ***p < 0.001, **p < 0.01, *p < 0.05.

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  FIGURE 3. MALAT1 interacts with miR-449a in HGSMCs. (A) qPCR analysis was conducted to detect the endogenous microRNAs associated with MALAT1 in HGSMCs under HG conditions for about 48 h. (B) Predicted binding site of miR-449a in the MALAT1 sequence. (C) luciferase reporter assay analysis of the interaction between MALAT1 and miR-449a. (D) RIP assay analysis of the interaction between MALAT1 and miR-449a. U6 was used as an internal control. ***p < 0.001, **p < 0.01, *p < 0.05.

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  FIGURE 4. miR-449a targets DLL1. (A–B) HGSMCs were transfected with a miR-449a mimic, a miR-449a inhibitor, or the respective controls (NC), and 48 h later, the DLL1 mRNA and protein levels were detected by qPCR and western blotting. (C) Predicted binding site of miR-449a on the 3′ UTR of DLL1. (D) luciferase reporter assay analysis of the interaction between miR-449a and DLL1. (E) RIP assay analysis of the interaction between miR-449a and DLL1. U6 or β-Actin was used as an internal control. ***p < 0.001, **p < 0.01, *p < 0.05.

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  FIGURE 5. DLL1 is a target of MALAT1 via miR-449a. (A) qPCR analysis of DLL1 expressions in the adjacent normal tissues from the stomach of patients who underwent gastrectomy with or without DM and DGP symptoms (n = 10 per group) as well as in the gastric tissues from DGP and control mice (n = 10 per group). (B) Western blot analysis shows that the expression levels of DLL1 proteins were significantly upregulated in the gastric tissues of DGP clinical samples and DGP model mice. (C) qPCR and western blot analyses of the expression of DLL1 in HGSMCs treated with different concentrations of glucose (5, 15, 25, and 35 mM) for 48 h. (D) qPCR and western blot analyses of the expression of DLL1 in HGSMCs transfected with shDLL1s and shNC. (E–G) CCK8, EdU, and Transwell assays to analyze the viability, proliferation, and migration of HGSMCs under different treatments. (H–I) qPCR and western blot analyses of SMC contractile markers (α-SMA, SM22α, and MHC) in HGSMCs under different treatments. For (E–I) four groups were analyzed: NG + shNC group, HG + shNC group, NG + shDLL1 group, and HG + shDLL1 group. β-Actin was used as an internal control. ***p < 0.001, **p < 0.01, *p < 0.05.

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  FIGURE 6. The MALAT1/miR-499a/DLL1 pathway regulates the cellular processes, phenotypic switch and CCh-induced Ca2+ transient signals in HGSMCs under HG conditions. (A–C) CCK8, EdU, and Transwell assays for the analysis of the viability, proliferation, and migration of HGSMCs under different treatments. (D–E) qPCR and western blot analyses of SMC contractile markers (α-SMA, SM22α, and MHC) in HGSMCs under different treatments. (F) CCh (100 μmol/L)—induced [Ca2+]i changes in HGSMCs under different treatments. Changes in Ca2+ fluorescence intensity are expressed as F/F0, where F0 is the basal fluorescence intensity before stimulation. (G) Histogram analysis of the [Ca2+]i changes, where ΔF is the fluorescent intensity of the response minus F0. For (A–G), five groups were analyzed: NG + shNC group, HG + shNC group, HG + shMALAT1 group, HG + shMALAT1+miR-449a inhibitor, HG + shMALAT1+miR-449a inhibitor + shDLL1 group. β-Actin was used as an internal control. ***p < 0.001, **p < 0.01, *p < 0.05.

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  FIGURE 7. Proposed mechanism by which MALAT1 regulates the phenotypic switch of HGSMCs under HG condition.