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BackgroundLong-term diabetes-associated complications are the major causes of morbidity and mortality in individuals with diabetes. These diabetic complications are closely linked to immune system activation along with chronic, non-resolving inflammation, but therapies to directly reverse these complications are still not available. Our previous study demonstrated that mesenchymal stem cells (MSCs) attenuated chronic inflammation in type 2 diabetes mellitus (T2DM), resulting in improved insulin sensitivity and islet function. Therefore, we speculated that MSCs might exert anti-inflammatory effects and promote the reversal of diabetes-induced kidney, liver, lung, heart, and lens diseases in T2DM rats.MethodsWe induced a long-term T2DM complication rat model by using a combination of a low dose of streptozotocin (STZ) with a high-fat diet (HFD) for 32 weeks. Adipose-derived mesenchymal stem cells (ADSCs) were systemically administered once a week for 24 weeks. Then, we investigated the role of ADSCs in modulating the progress of long-term diabetic complications.ResultsMultiple infusions of ADSCs attenuated chronic kidney disease (CKD), nonalcoholic steatohepatitis (NASH), lung fibrosis, and cataracts; improved cardiac function; and lowered serum lipid levels in T2DM rats. Moreover, the levels of inflammatory cytokines in the serum of each animal group revealed that ADSC infusions were able to not only inhibit pro-inflammatory cytokines IL-6, IL-1β, and TNF-α expression but also increase anti-inflammatory cytokine IL-10 systematically. Additionally, MSCs reduced the number of iNOS(+) M1 macrophages and restored the number of CD163(+) M2 macrophages.ConclusionsMultiple intravenous infusions of ADSCs produced significant protective effects against long-term T2DM complications by alleviating inflammation and promoting tissue repair. The present study suggests ADSCs may be a novel, alternative cell therapy for long-term diabetic complications.

MicroRNA-9 (miR-9) dysregulation is implicated in a variety of human malignancies including hepatocellular carcinoma (HCC), but its role remains contradictory. In this study, we explored the expression and methylation status of miR-9 in HCC samples, as well as the tumor-related functions of miR-9 in vitro. Bioinformatics analysis, array-based RNA expression profile, and literature retrieval were used to identify miR-9 targets in HCC. The potential downstream candidates were then validated by luciferase reporter assay, real-time quantitative PCR, and western blot or enzyme linked immunosorbent assay (ELISA). The expression status and clinicopathologic significances of miR-9 target genes in clinical samples were further explored. The results showed that miR-9 was frequently downregulated in primary HCC. Its silencing was largely contributed by a high frequency (42.5%) of mir-9-1 hypermethylation, which was correlated with bigger tumor size (P = 0.0234). In vitro functional studies revealed that miR-9 restoration retarded HCC cell proliferation and migration. IL-6, AP3B1, TC10, ONECUT2, IGF2BP1, MYO1D, and ANXA2 were confirmed to be miR-9 targets in HCC. Among them, ONECUT2, IGF2BP1, and ANXA2 were confirmed to be aberrantly upregulated in HCC. Moreover, upregulation of ONECUT2, IGF2BP1, and IL-6 were significantly associated with poor post-surgery prognosis (P = 0.0458, P = 0.0037 and P = 0.0461, respectively). Mechanically, miR-9 plays a tumor suppressive role partially through a functional miR-9/IGF2BP1/AKT&ERK axis. Our study suggests that miR-9 functions as a tumor suppressor in HCC progression by inhibiting a series of target genes, including the newly validated miR-9/IGF2BP1/AKT&ERK axis, thus providing potential therapeutic targets and novel prognostic biomarkers for HCC patients.

Retinoic acid (RA) contributes to cleft palate; however, the cellular and molecular mechanisms responsible for the deleterious effects on the developing palate are unclear. Wnt signaling is a candidate pathway in the cleft palate and is associated with RA in organ development; thus, we aim to investigate whether RA-induced cleft palate also results from altered Wnt signaling. Administration of RA to mice altered cell proliferation and apoptosis in craniofacial tissues by regulating molecules controlling cell cycle and p38 MAPK signaling, respectively. This altered cell fate by RA is a crucial mechanism contributing to 100% incidence of cleft palate. Moreover, Wnt/β-catenin signaling was completely inhibited by RA in the early developing palate via its binding and activation with RA receptor (RAR) and is responsible for RA-induced cleft palate. Furthermore, PI3K/Akt signaling was also involved in actions of RA. Our findings help in elucidating the mechanisms of RA-induced cleft palate.

Progressive pancreatic β-cell dysfunction is recognized as a fundamental pathology of type 2 diabetes (T2D). Recently, mesenchymal stem cells (MSCs) have been identified in protection of islets function in T2D individuals. However, the underlying mechanisms remain elusive. It is widely accepted that β-cell dysfunction is closely related to improper accumulation of macrophages in the islets, and a series of reports suggest that MSCs possess great immunomodulatory properties by which they could elicit macrophages into an anti-inflammatory M2 state. In this study, we induced a T2D mouse model with a combination of high-fat diet (HFD) and low-dose streptozotocin (STZ), and then performed human umbilical cord-derived MSCs (hUC-MSCs) infusion to investigate whether the effect of MSCs on islets protection was related to regulation on macrophages in pancreatic islets. hUC-MSCs infusion exerted anti-diabetic effects and significantly promoted islets recovery in T2D mice. Interestingly, pancreatic inflammation was remarkably suppressed, and local M1 macrophages were directed toward an anti-inflammatory M2-like state after hUC-MSC infusion. In vitro study also proved that hUC-MSCs inhibited the activation of the M1 phenotype and induced the generation of the M2 phenotype in isolated mouse bone marrow-derived macrophages (BMDMs), peritoneal macrophages (PMs) and in THP-1 cells. Further analysis showed that M1-stimulated hUC-MSCs increased the secretion of interleukin (IL)-6, blocking which by small interfering RNA (siRNA) largely abrogated the hUC-MSCs effects on macrophages both in vitro and in vivo, resulting in dampened restoration of β-cell function and glucose homeostasis in T2D mice. In addition, MCP-1 was found to work in accordance with IL-6 in directing macrophage polarization from M1 to M2 state. These data may provide new clues for searching for the target of β-cell protection. Furthermore, hUC-MSCs may be a superior alternative in treating T2D for their macrophage polarization effects.

ANO1, a calcium-activated chloride channel, has been reported to be amplified or overexpressed in tissues of several cancers. However, reports on its roles in tumor progression obtained from cancer cell lines are inconsistent, suggesting that the role of ANO1 in tumorigenesis is likely dependent on either its expression level or cell-type expressing ANO1. To investigate the biological roles of ANO1 in different tumor cells, we, in this study, selected several cancer cell lines and a normal HaCaT cell line with high expression levels of ANO1, and examined the function of ANO1 in these cells using approaches of lentiviral knockdown and pharmacological inhibition. We found that ANO1 knockdown significantly inhibited cell proliferation and induced cell apoptosis in either tumor cell lines or normal HaCaT cell line. Moreover, silencing ANO1 arrested cancer cells at G1 phase of cell cycle. Treatment with ANO1 inhibitor CaCCinh-A01 reduced cell viability in a dose-dependent manner. Furthermore, both ANO1 inhibitors CaCCinh-A01 and T16Ainh-A01 significantly suppressed cell migration. Our findings show that ANO1 overexpression promotes cancer cell proliferation and migration; and genetic or pharmacological inhibition of ANO1 induces apoptosis and cell cycle arrest at G1 phase in different types of epithelium-originated cancer cells.

Overexpression of the Ca2+-activated chloride channel ANO1/TMEM16A is implicated in tumorigenesis, and inhibition of ANO1 overexpression suppresses xenograft tumor growth and invasiveness. However, the underlying molecular mechanism for ANO1 inhibition in suppression of tumorigenesis remains unknown. Here, we show that silencing or inhibition of endogenous ANO1 inhibits cell growth, induces apoptosis and upregulates TNF-α expression in prostate cancer PC-3 cells. Enhancement of TNF-α signaling by ANO1 knockdown leads to upregulation of phosphorylated Fas-associated protein with death domain and caspase activation. Furthermore, silencing of ANO1 inhibits growth of PC-3 xenograft tumors in nude mice and induces apoptosis in tumors via upregulation of TNF-α signaling. Taken together, our findings provide mechanistic insight into promoting apoptosis in prostate cancer cells by ANO1 inhibition through upregulation of TNF-α signaling.

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