Total Flavonoids of Aurantii Fructus Immaturus Regulate miR-5100 to Improve Constipation by Targeting Fzd2 to Alleviate Calcium Balance and Autophagy in Interstitial Cells of Cajal.

Aurantii Fructus Immaturus total flavonoids (AFIF) is the main effective fraction extracted from AFI, which has a good effect on promoting gastrointestinal motility. This study aimed to investigate AFIF which regulates miR-5100 to improve constipation symptoms in mice by targeting Frizzled-2 (Fzd2) to alleviate interstitial cells of Cajal (ICCs) calcium ion balance and autophagy apoptosis. The constipated mouse model was induced by an antibiotic suspension, and then treated with AFIF. RNA-seq sequencing, luciferase assay, immunofluorescence staining, transmission electron microscopy, ELISA, flow cytometry, quantitative polymerase chain reaction (PCR), and Western blot were applied in this study. The results showed that AFIF improved constipation symptoms in antibiotic-induced constipated mice, and decreased the autophagy-related protein Beclin1 levels and the LC3-II/I ratio in ICCs. miR-5100 and its target gene Fzd2 were screened as key miRNAs and regulator associated with autophagy. Downregulation of miR-5100 caused increased expression of Fzd2, decreased proliferation activity of ICCs, increased apoptotic cells, and enhanced calcium ion release and autophagy signals. After AFIF treatment, miR-5100 expression was upregulated and Fzd2 was downregulated, while autophagy-related protein levels and calcium ion concentration decreased. Furthermore, AFIF increased the levels of SP, 5-HT, and VIP, and increased the expression of PGP9.5, Sy, and Cx43, which alleviated constipation by improving the integrity of the enteric nervous system network. In conclusion, AFIF could attenuate constipation symptoms by upregulating the expression of miR-5100 and targeting inhibition of Fzd2, alleviating calcium overload and autophagic death of ICCs, regulating the content of neurotransmitters, and enhancing the integrity of the enteric nervous system network.

The Peptide AWRK6 Alleviates Lipid Accumulation in Hepatocytes by Inhibiting miR-5100 Targeting G6PC.

Non-alcoholic fatty liver disease (NAFLD) is the leading chronic liver disease, with a worldwide prevalence of more than 25%, and there is no approved drug for NAFLD specifically. In our previous study, the synthetic peptide AWRK6 was found to ameliorate NAFLD in mice. However, the mechanisms involved are still largely unknown. Here, AWRK6 treatment presented an alleviative effect on lipid accumulation induced by oleic acid in hepatocytes. Meanwhile, miR-5100 and miR-505 were found to be elevated by oleic acid induction and reversed by AWRK6 incubation. Further, the miR-5100 inhibitor inhibited oleic acid-induced lipid accumulation, and the alleviation effect of AWRK6 was partially counteracted by miR-5100 mimics. The screening of potential target genes revealed that a catalytic subunit of G6Pase G6PC was significantly inhibited by miR-5100 mimics transfection in both mRNA and protein levels. The direct targeting of miR-5100 on G6PC was verified by a Dual-Luciferase Reporter Assay. Moreover, the mRNA and protein levels of G6PC were found to be significantly increased by AWRK6 treatment. These results suggested that the peptide AWRK6 could alleviate lipid accumulation in hepatocytes, partly through reducing miR-5100 to restore one of its targets: G6PC. Thus, AWRK6 has the potential to treat NAFLD. Additionally, miR-5100 is a mediator of lipid accumulation in hepatocytes, which could be targeted by AWRK6.

Metformin inhibits melanoma cell metastasis by suppressing the miR-5100/SPINK5/STAT3 axis.

Melanoma is the most lethal skin cancer characterized by its high metastatic potential. It is urgent to find novel therapy strategies to overcome this feature. Metformin has been confirmed to suppress invasion and migration of various types of cancer. However, additional mechanisms underlying the antimetastatic effect of metformin on melanoma require further investigation. Here, we performed microarray analysis and uncovered an altered mRNA and miRNA expression profile between melanoma and nevus. Luciferase reporter assay confirmed that miR-5100 targets SPINK5 to activate STAT3 phosphorylation. Migration and wound healing assays showed that the miR-5100/SPINK5/STAT3 axis promotes melanoma cell metastasis; the mechanism was proven by initiation of epithelial-mesenchymal transition. Co-immunoprecipitation (Co-IP) further confirmed an indirect interaction between SPINK5 and STAT3. Furthermore, metformin dramatically inhibited miR-5100/SPINK5/STAT3 pathway, and decreased B16-F10 cell metastasis to lung in C57 mouse module. Intriguingly, pretreatment of metformin before melanoma cell injection improved this effect further. These findings exposed the underlying mechanisms of action of metformin and update the use of this drug to prevent metastasis in melanoma.

Long Non-Coding LEF1-AS1 Sponge miR-5100 Regulates Apoptosis and Autophagy in Gastric Cancer Cells via the miR-5100/DEK/AMPK-mTOR Axis.

DEK and miR-5100 play critical roles in many steps of cancer initiation and progression and are directly or indirectly regulated by most promoters and repressors. LEF1-AS1 as a long non-coding RNA can regulate tumor development through sponge miRNA. The effect and regulatory mechanism of DEK on autophagy and apoptosis in gastric cancer (GC), and the role between miR-5100 and DEK or miR-5100 and LEF1-AS1 are still unclear. Our study found that DEK was highly expressed in gastric cancer tissues and cell lines, and knockdown of DEK inhibited the autophagy of cells, promoted apoptosis, and suppressed the malignant phenotype of gastric cancer. DEK regulates autophagy and apoptosis through the AMPK/mTOR signaling pathway. In addition, miR-5100 inhibits autophagy and promotes apoptosis in GC cells while LEF1-AS1 had the opposite effect. Studies have shown that miR-5100 acts by targeting the 3'UTR of DEK, and LEF1-AS1 regulates the expression of miR-5100 by sponging with mIR-5100. In conclusion, our results found that LEF1-AS1 and miR-5100 sponge function, and the miR-5100/DEK/AMPK/mTOR axis regulates autophagy and apoptosis in gastric cancer cells.

The miR151 and miR5100 Transfected Bone Marrow Stromal Cells Increase Myoblast Fusion in IGFBP2 Dependent Manner.

BACKGROUND: Bone marrow stromal cells (BMSCs) form a perivascular cell population in the bone marrow. These cells do not present naïve myogenic potential. However, their myogenic identity could be induced experimentally in vitro or in vivo. In vivo, after transplantation into injured muscle, BMSCs rarely fused with myofibers. However, BMSC participation in myofiber reconstruction increased if they were modified by NICD or PAX3 overexpression. Nevertheless, BMSCs paracrine function could play a positive role in skeletal muscle regeneration. Previously, we showed that SDF-1 treatment and coculture with myofibers increased BMSC ability to reconstruct myofibers. We also noticed that SDF-1 treatment changed selected miRNAs expression, including miR151 and miR5100. METHODS: Mouse BMSCs were transfected with miR151 and miR5100 mimics and their proliferation, myogenic differentiation, and fusion with myoblasts were analyzed. RESULTS: We showed that miR151 and miR5100 played an important role in the regulation of BMSC proliferation and migration. Moreover, the presence of miR151 and miR5100 transfected BMSCs in co-cultures with human myoblasts increased their fusion. This effect was achieved in an IGFBP2 dependent manner. CONCLUSIONS: Mouse BMSCs did not present naïve myogenic potential but secreted proteins could impact myogenic cell differentiation. miR151 and miR5100 transfection changed BMSC migration and IGFBP2 and MMP12 expression in BMSCs. miR151 and miR5100 transfected BMSCs increased myoblast fusion in vitro.

Two Novel Ceramide-Like Molecules and miR-5100 Levels as Biomarkers Improve Prediction of Prostate Cancer in Gray-Zone PSA.

Reliable liquid biopsy-based tools able to accurately discriminate prostate cancer (PCa) from benign prostatic hyperplasia (BPH), when PSA is within the "gray zone" (PSA 4-10), are still urgent. We analyzed plasma samples from a cohort of 102 consecutively recruited patients with PSA levels between 4 and 16 ng/ml, using the SANIST-Cloud Ion Mobility Metabolomic Mass Spectrometry platform, combined with the analysis of a panel of circulating microRNAs (miR). By coupling CIMS ion mobility technology with SANIST, we were able to reveal three new structures among the most differentially expressed metabolites in PCa vs. BPH. In particular, two were classified as polyunsaturated ceramide ester-like and one as polysaturated glycerol ester-like. Penalized logistic regression was applied to build a model to predict PCa, using six circulating miR, seven circulating metabolites, and demographic/clinical variables, as covariates. Four circulating metabolites, miR-5100, and age were selected by the model, and the corresponding prediction score gave an AUC of 0.76 (C.I. = 0.66-0.85). At a specified cut-off, no high-risk tumor was misclassified, and 22 out of 53 BPH were correctly identified, reducing by 40% the false positives of PSA. We developed and applied a novel, minimally invasive, liquid biopsy-based powerful tool to characterize novel metabolites and identified new potential non-invasive biomarkers to better predict PCa, when PSA is uninformative as a tool for precision medicine in genitourinary cancers.

Mir-5100 Mediates Proliferation, Migration and Invasion of Oral Squamous Cell Carcinoma Cells Via Targeting SCAI.

PURPOSE: We aimed to investigate the role of microRNA-5100 (miRNA-5100) in oral squamous cell carcinoma (OSCC) and its underlying mechanisms.Material/Methods: The expression of miR-5100 and suppressor of cancer cell invasion (SCAI) in OSCC cell lines were examined. A luciferase reporter assay was applied to confirm the combination between miR-5100 and SCAI. Then, miR-5100 inhibitor or small hairpin RNA (shRNA)-SCAI were transfected into cells. Cell Counting Kit-8 assay was executed for testing cell proliferation ability. Flow cytometry assay was exploited for measuring cell cycle. Invasion and migration of OSCC cells were assessed using Transwell assay and wound healing assay. The expression of proteins were detected using western blotting. RESULTS: The results demonstrated that the level of miR-5100 was upregulated while SCAI was downregulated in OSCC cells. SCAI was verified as a direct target of miR-5100. MiR-5100 silencing suppressed proliferation of OSCC cells, increased cells in the G1 and G2 phases, and reduced those in the S phase, which was reversed after transfection with shRNA-SCAI. Moreover, miR-5100 inhibitor downregulated the expression of cyclin-dependent kinase-2 (CDK-2) and cyclinD1, accompanied by upregulation in p27 expression, whereas SCAI silencing had the opposite results. The invasion and migration abilities of OSCC cells were reduced after treatment with miR-5100 inhibitor, whereas SCAI silencing suppressed the effects of miR-5100 inhibitor on OSCC cell behaviors. CONCLUSION: These findings suggested that miR-5100 silencing inhibit proliferation, invasion and migration of OSCC cells via upregulating the expression of SCAI, which provides theoretical basis and treatment strategies for the treatment of OSCC.

MKL1/miR-5100/CAAP1 loop regulates autophagy and apoptosis in gastric cancer cells.

PURPOSE: miR-5100 participates in the proliferation of lung cancer and pancreatic cancer cells, and participates in the differentiation of osteoblasts. However, the regulation of gastric cancer cells in gastric cancer cells remains unclear. EXPERIMENTAL DESIGN: The blood of patients was collected to detect the expression level of miR-5100, and the apoptosis and autophagy levels of cells were detected using western blot, flow cytometry, and confocal. At the same time, in vitro tumor formation experiments in nude mice were used to verify the results of in vitro experiments. RESULTS: The expression of miR-5100 is related to the prognosis of gastric cancer, miR-5100 can enhance the apoptosis level of gastric cancer cells and inhibit the occurrence of autophagy by targeting CAAP1. MKL1 can inhibit the apoptosis of gastric cancer cells and promote the occurrence of autophagy by targeting CAAP1. At the same time, MKL1 can also increase the expression of miR-5100. CONCLUSIONS: Our research reveals the mechanism by which the MKL1/miR-5100/CAAP1 loop regulates apoptosis and autophagy levels in gastric cancer cells, and miR-5100 is expected to become a new potential target for gastric cancer treatment.

MiR-5100 increases the cisplatin resistance of the lung cancer stem cells by inhibiting the Rab6.

Cisplatin-based chemotherapy is the most commonly used treatment regimen for lung cancer. Cancer stem cells (CSCs) are postulated to be important promoters of drug resistance. We previously found that miR-5100 is overexpressed in lung cancer, but it is unknown whether and how miR-5100 regulates cisplatin resistance. Here, we demonstrated that miR-5100 was significantly up-regulated in CD44+ CD133+ lung cancer stem cells (LCSCs) compared with non-CSCs. Additionally, over-expression of miR-5100 increased CSC properties, cell growth, and tumor sphere formation in lung cancer cell line A549 or H1299, and that miR-5100 inhibitor significantly increased sensitivity of LCSCs to cisplatin in vitro. Surprisingly, the combination with miR-5100 inhibitor significantly decreased the IC50 of LCSCs to cisplatin. Furthermore, miR-5100 increased CSC properties and cisplatin resistance by inhibiting Rab6, a direct target gene of miR-5100. We demonstrated that miR-5100 overexpression increases the cisplatin resistance of the LCSCs through the mitochondrial apoptosis pathway. In conclusion, our results suggest that miR-5100 increases the cisplatin resistance of the LCSCs by inhibiting the Rab6. This study provides novel insight into the regulation of LCSCs by miRNA.

MiR-5100 targets TOB2 to drive epithelial-mesenchymal transition associated with activating smad2/3 in lung epithelial cells.

Idiopathic pulmonary fibrosis (IPF) is a devastating disease and the pathogenesis of IPF remains unclear. Our previous study indicated that miR-5100 promotes the proliferation and metastasis of lung epithelial cells. In this study, we investigated the effect and mechanism of miR-5100 on bleomycin (BLM)-induced mouse lung fibrosis and transforming growth factor ß (TGF-ß1) or epidermal growth factor (EGF) induced EMT-model in A549 and Beas-2B cells. The elevated level of miR-5100 was observed in both the mouse lung fibrosis tissues and EMT cell model. Furthermore, the exogenous expression of miR-5100 promoted the EMT-related changes, enhanced TGF-ß1 or EGF-induced EMT and activated the smad2/3 in lung epithelial cells, while silencing miR-5100 had the converse effects. In addition, transwell assay showed that miR-5100 can enhance cell migration. Using target prediction software and luciferase reporter assays, we identified TOB2 as a specific target of miR-5100 and miR-5100 can decrease the accumulation of endogenous TOB2 in A549 and Beas-2B cells. Moreover, the exogenous expression of TOB2 relieves the promotion of miR-5100 on EMT process and migration ability. Taken together, our results indicate that miR-5100 promotes the EMT process by targeting TOB2 associated with activating smad2/3 in lung epithlium cells. Our findings may provide novel insights into the pathogenesis of IPF.

MiR-5100 promotes osteogenic differentiation by targeting Tob2.

MicroRNAs have emerged as pivotal regulators in various physiological and pathological processes, including osteogenesis. Here we discuss the contribution of miR-5100 to osteoblast differentiation and mineralization. We found that miR-5100 was upregulated during osteoblast differentiation in ST2 and MC3T3-E1 cells. Next, we verified that miR-5100 can promote osteogenic differentiation with gain-of-function and loss-of-function experiments. Target prediction analysis and experimental validation demonstrated that Tob2, which acts as a negative regulator of osteogenesis, was negatively regulated by miR-5100. Furthermore, we confirmed that the important bone-related transcription factor osterix, which can be degraded by binding to Tob2, was influenced by miR-5100 during osteoblast differentiation. Collectively, our results revealed a new molecular mechanism that fine-tunes osteoblast differentiation through miR-5100/Tob2/osterix networks.

miR-5100 promotes tumor growth in lung cancer by targeting Rab6.

Our previous study demonstrated that microRNA 5100 (miR-5100) is overexpressed in lung cancer tissues; however, the function of miR-5100 remained elusive. In this study, we demonstrate that miR-5100 is highly expressed in a wide variety of lung cancer tissues and lung cancer cell lines. Exogenous expression of miR-5100 in A549 and H1299 lung cancer cells enhanced proliferation and colony formation, and conversely, suppression of miR-5100 exhibited inhibitory effects. Furthermore, we demonstrate that miR-5100 promotes tumor growth in nude mice. These effects may result from the ability of miR-5100 to promote G1/S transition and downregulate cyclin D1 and cyclin-dependent kinases 2 (CDK2) expressions in lung cancer stable cells. Using a bioinformatics target prediction tool, we identified Rab6 as a potential target of miR-5100. Consistently, overexpression of miR-5100 specifically reduced the expression of a luciferase reporter containing the predicted binding site from the 3'untranslated region (3'UTR) of Rab6 and decreased the accumulation of endogenous Rab6 in A549 and H1299 cells. Moreover, exogenous expression of Rab6 compromised the effects of miR-5100 on cell proliferation and colony formation. Our data suggest that miR-5100 promotes tumor growth by facilitating the G1/S transition and targeting Rab6.