STAT3-induced lncRNA GNAS-AS1 accelerates keloid formation by mediating the miR-196a-5p/CXCL12/STAT3 axis in a feedback loop.

Keloids are pathological scar tissue resulting from skin trauma or spontaneous formation, often accompanied by itching and pain. Although GNAS antisense RNA 1 (GNAS-AS1) shows abnormal upregulation in keloids, the underlying molecular mechanism is unclear. The levels of genes and proteins in clinical tissues from patients with keloids and human keloid fibroblasts (HKFs) were measured using quantitative reverse transcription PCR, western blot and enzyme-linked immunosorbent assay. The features of HKFs, including proliferation and migration, were evaluated using cell counting kit 8 and a wound healing assay. The colocalization of GNAS-AS1 and miR-196a-5p in HKFs was measured using fluorescence in situ hybridization. The relationships among GNAS-AS1, miR-196a-5p and C-X-C motif chemokine ligand 12 (CXCL12) in samples from patients with keloids were analysed by Pearson correlation analysis. Gene interactions were validated by chromatin immunoprecipitation and luciferase reporter assays. GNAS-AS1 and CXCL12 expression were upregulated and miR-196a-5p expression was downregulated in clinical tissues from patients with keloids. GNAS-AS1 knockdown inhibited proliferation, migration, and extracellular matrix (ECM) accumulation of HKFs, all of which were reversed by miR-196a-5p downregulation. Signal transducer and activator of transcription 3 (STAT3) induced GNAS-AS1 transcription through GNAS-AS1 promoter interaction, and niclosamide, a STAT3 inhibitor, decreased GNAS-AS1 expression. GNAS-AS1 positively regulated CXCL12 by sponging miR-196-5p. Furthermore, CXCL12 knockdown restrained STAT3 phosphorylation in HKFs. Our findings revealed a feedback loop of STAT3/GNAS-AS1/miR-196a-5p/CXCL12/STAT3 that promoted HKF proliferation, migration and ECM accumulation and affected keloid progression.

MicroRNA-196a-5p facilitates the onset and progression via targeting ITM2B in esophageal squamous cell carcinoma.

Esophageal squamous cell carcinoma (ESCC) is a prevalent malignancy affecting the digestive tract, with an increasing incidence rate worldwide. Recently, numerous studies revealed that microRNAs were associated with gene expression regulation, particularly their involvement in the regulation of tumor cells, garnering widespread attention. Here, we discovered that miR-196a-5p was significantly upregulated in both ESCC tissues and cells, which was correlated with an unfavorable prognosis. Series functional in vitro investigations have confirmed that silencing miR-196a-5p obviously restrained the ESCC cells malignant phenotypes and promoted apoptosis. Bioinformatics analysis and rescue experiments revealed that miR-196a-5p directly targeted ITM2B, exerting influence on the development of ESCC cells through negative regulation of ITM2B expression. Xenograft mouse models were established for conducting in vivo experiments, providing further confirmation of the regulatory mechanism and biological significance of the miR-196a-5p/ITM2B axis in ESCC. Our research demonstrated miR-196a-5p promoted ESCC malignant progression by interacting with ITM2B, thereby providing novel clues and potential targets for the new diagnosis and thereby of ESCC.

Exosomal miR-196a-5p contributes to esophageal squamous cell carcinoma malignant progression by inhibiting ITM2B.

Exosomes from cancer cells function as carriers to spread or transport specific microRNAs (miRNAs) to distant sites to exert their effects, but the mechanism of exosomal miRNA action in esophageal squamous cell carcinoma (ESCC) has not been fully explained. Therefore, in this study, we were interested in the impact of exosomal miR-196a-5p in ESCC progression. We found that miR-196a-5p was expressed enriched in clinical tissues, ESCC cells, and exosomes. Functionally, depletion of miR-196a-5p impeded ESCC cell growth, migration, and invasion, whereas overexpression of miR-196a-5p produced the opposite results. Moreover, enhancement of exosomal miR-196a-5p in recipient ESCC cells triggered more intense proliferation and migration. Mechanistically, we identified integral membrane protein 2B (ITM2B) as a direct target of miR-196a-5p. Silencing of ITM2B partially counteracted the inhibitory effect of miR-196a-5p inhibitors on the malignant phenotype of ESCC. Furthermore, in vivo, lower miR-196a-5p levels triggered by the introduction of antagomiR-196a-5p resulted in the generation of smaller volume and weight xenograft tumors. Thus, our results demonstrated novel mechanisms of exosomal and intracellular miR-196a-5p-mediated ESCC growth and migration and identify the interaction of miR-196a-5p with ITM2B. These works might provide new targets and basis for the development of clinical treatment options for ESCC.

MicroRNA-152-3p and MicroRNA-196a-5p Are Downregulated When Müller Cells Are Promoted by Components of the Internal Limiting Membrane: Implications for Macular Hole Healing.

Müller cells play a critical role in the closure of macular holes, and their proliferation and migration are facilitated by the internal limiting membrane (ILM). Despite the importance of this process, the underlying molecular mechanism remains underexplored. This study investigated the effects of ILM components on the microRNA (miRNA) profile of Müller cells. Rat Müller cells (rMC-1) were cultured with a culture insert and varying concentrations of ILM component coatings, namely, collagen IV, laminin, and fibronectin, and cell migration was assessed by measuring cell-free areas in successive photographs following insert removal. MiRNAs were then extracted from these cells and analyzed. Mimics and inhibitors of miRNA candidates were transfected into Müller cells, and a cell migration assay and additional cell viability assays were performed. The results revealed that the ILM components promoted Müller cell migration (p < 0.01). Among the miRNA candidates, miR-194-3p was upregulated, whereas miR-125b-1-3p, miR-132-3p, miR-146b-5p, miR-152-3p, miR-196a-5p, miR-542-5p, miR-871-3p, miR-1839-5p, and miR-3573-3p were significantly downregulated (p < 0.05; fold change > 1.5). Moreover, miR-152-3p and miR-196a-5p reduced cell migration (p < 0.05) and proliferation (p < 0.001), and their suppressive effects were reversed by their respective inhibitors. In conclusion, miRNAs were regulated in ILM component-activated Müller cells, with miR-152-3p and miR-196a-5p regulating Müller cell migration and proliferation. These results serve as a basis for understanding the molecular healing process of macular holes and identifying potential new target genes in future research.

MicroRNA-196a-5p overexpression in Wharton's jelly umbilical cord stem cells promotes their osteogenic differentiation and new bone formation in bone defects in the rat calvarium.

The peri-tooth root alveolar loss often does not have sufficient space for repair material transplantation and plasticity. Mesenchymal stem cell (MSC) sheets have an advantage in providing more extracellular matrix (ECM) and may prove to be a new therapeutic consideration for this bone defect repair. The identification of key regulators that stimulate MSCs' osteogenic potential and sheet-derived ECM deposition is the key to promoting its application. In this study, we found that inhibition or overexpression of miR-196a-5p led to a decline or enhancement, respectively, in the alkaline phosphatase (ALP) activity, mineralization, and the levels of osteogenic markers, Osteocalcin (OCN), Dentin Matrix Protein 1 (DMP1), Bone Sialoprotein (BSP), and Dentin Sialophosphoprotein (DSPP) of Wharton's jelly of umbilical cord stem cells (WJCMSCs) in vitro. Moreover, the 5,6-Carboxyfluorescein Diacetate Succinimidyl Ester (CFSE) analysis revealed inhibition of the WJCMSCs' proliferative ability upon miR-196a-5p overexpression. Characterization of the sheet formation by picrosirius red and Masson staining indicated that miR-196a-5p overexpression significantly promoted the collagen content in whole WJCMSC sheet-derived ECM. Furthermore, micro-CT and histopathology results indicated that the miR-196a-5p-overexpressed WJCMSC sheets significantly promoted new bone regeneration and rat calvarial bone defect closure 12 weeks following transplantation. The mRNA microarray analysis of miR-196a-5p-overexpressed WJCMSCs revealed 959 differentially expressed genes (DEGs) (34 upregulated and 925 downregulated). Moreover, 241 genes targeted by miR-196a-5p were predicted by using miRNA function websites of which only 19 predicted genes were consistent with the microarray revealed DEGs. Hence, one unrevealed downregulated DEG Serpin Family B Member 2 (SERPINB2) was investigated. And the deletion of SERPINB2 enhanced the ALP activity and mineralization of WJCMSCs in vitro. In conclusion, our study found that miR-196a-5p, as a key regulator, could repress the proliferation tendency, while stimulating osteogenic ability and WJCMSC sheet-derived ECM deposition, thus promoting new bone formation and rat calvarial bone defect closure. Furthermore, SERPINB2 is a key downstream gene involved in the miR-196a-5p-promoted WJCMSC osteogenesis.

Upregulation of hsa-miR-196a-5p is associated with MIR196A2 methylation and affects the malignant biological behaviors of glioma.

Hsa-miR-196a-5p is involved in tumorigenesis and progression. However, the driving factors for hsa-miR-196a-5p overexpression and its correlation with the clinicopathological features and prognosis of patients remain unclear in glioma. Thus, this study aimed to investigate the prognostic value of hsa-miR-196a-5p and its correlation with MIR196A2 methylation in glioma. We observed that hsa-miR-196a-5p expression was upregulated in glioma. Next, 112 patients were divided into high (n = 56) and low (n = 56) hsa-miR-196a-5p expression groups. The chi-square test showed that hsa-miR-196a-5p expression was significantly related to age, WHO grade, histopathology, IDH mutation status, and 1p/19q codeletion. Univariate and multivariate Cox regression analyses showed that hsa-miR-196a-5p expression was an independent prognostic factor. GO and KEGG enrichment analyses showed that hsa-miR-196a-5p may be involved in the MAPK signaling, focal adhesion and cancer-related pathways. Compared with the normal astrocyte cell line, glioma cell lines had an unregulated MIR196A2 methylation level, which was confirmed by TCGA data. The hypermethylated CpG sites of MIR196A2 were mainly concentrated in the gene body region, which was significantly associated with hsa-miR-196a-5p overexpression. Kaplan-Meier curves revealed that MIR196A2 hypermethylation was a poor prognostic factor. These findings suggest that hsa-miR-196a-5p overexpression may be involved in malignant biological behaviors, and MIR196A2 hypermethylation of the gene body was significantly associated with hsa-miR-196a-5p overexpression, which was a poor prognostic factor of glioma. Therefore, MIR196A2 hypermethylation may act as an early marker of prognosis of patients with glioma.

A high level of lncFGD5-AS1 inhibits epithelial-to-Mesenchymal transition by regulating the miR-196a-5p/SMAD6/BMP axis in gastric Cancer.

BACKGROUND: Long non-coding RNA (lncRNA) was a vital factor in the progression and initiation of human cancers. This study found a new lncRNA, FGD5-AS1, which can inhibit EMT process, proliferation, and metastasis in vitro and in vivo. METHODS: qRT-PCR was employed to test the expression of lncFGD5-AS1 in 30 gastric cancer patients' cancer tissue and para-cancer tissue. Overexpressed lncFGD5-AS1 cells shown sharply decrease of proliferation, migration, and epithelial-mesenchymal transition (EMT). miR-196a-5p/SMAD6 was confirmed as downstream molecular mechanism of lncFGD5-AS1 by expression correlation analysis and mechanism experiments. In vivo study illustrated overexpression of lncFGD5-AS1 suppression tumor growth. RESULTS: LncFGD5-AS1 served as a ceRNA of miR-196a-5p to release its inhibition on SMAD6, a conventional inhibitor on the BMP pathway. Comparing with normal gastric cancer cells, FGD5-AS1 overexpressed group had fewer migration cells, lower cell viability, and lower EMT transformation rate. Meanwhile, xenografts nude mice injecting with overexpressed-FGD5-AS1 cells also shown smaller tumor weight and volume. CONCLUSION: In conclusion, this research supported the first evidence that FGD5-AS1 suppressed proliferation and metastasis in gastric cancer by regulating miR-196a-5p/SMAD6/BMP axis and suggested a potential therapeutic candidate for gastric cancer.

LncRNA LOXL1-AS1 controls osteogenic and adipocytic differentiation of bone marrow mesenchymal stem cells in postmenopausal osteoporosis through regulating the miR-196a-5p/Hmga2 axis.

INTRODUCTION: Exploring molecular mechanisms of human bone marrow mesenchymal stem cells (hBMMSCs) differentiation, a crucial step for bone formation, is a new direction for treating postmenopausal osteoporosis. LncRNAs are involved in lots of biological processes including hBMMSCs differentiation. The present study aimed to explore the effect of LOXL1-AS1 on hBMMSCs differentiation. MATERIALS AND METHODS: We examined the expression levels of LOXL1-AS1, miR-196a-5p and Hmga2 in peripheral blood from postmenopausal osteoporosis patients by RT-qPCR, and detected their changes during the osteogenic differentiation of hBMMSCs by RT-qPCR. RT-qPCR and western blot measured the level of biomarkers of bone formation and osteogenic differentiation (osteopontin, OPN; Alkaline phosphatase, ALP; Runt-related transcription factor-2, Runx-2). The effects of LOXL1-AS1 on the osteogenic and adipocytic differentiation of hBMMSCs were, respectively, determined by ALP, ARS staining assays and oil red O staining assay. RESULTS: The abnormal high expression of LOXL1-AS1 was found in patients. LOXL1-AS1 expression showed a gradual decrease during the osteogenic differentiation of hBMMSCs. However, LOXL1-AS1 overexpression inhibited the hBMMSCs osteogenic differentiation but promoted adipocytic differentiation. Furthermore, LOXL1-AS1 was identified to be a sponge of miR-196a-5p and Hmga2 as a target gene of miR-196a-5p. Besides, LOXL1-AS1 sponged miR-196a-5p to mediate Hmga2 expression, which played contrary effects on regulating osteogenic and adipocytic differentiation of hBMMSCs. Moreover, LOXL1-AS1/miR-196a-5p/Hmga2 axis regulated hBMMSCs differentiation through controlling C/EBPß-mediated PPARγ expression. CONCLUSION: These findings facilitate understanding the molecular mechanism of hBMMSCs differentiation and bring up a novel sight for postmenopausal osteoporosis therapy.

miR-196a-5p promotes metastasis of colorectal cancer via targeting IκBα.

BACKGROUND: MicroRNA-196a-5p (miR-196a-5p) has been reported to be involved in the metastatic process of several cancers. In present work, we aimed to investigate the effects of miR-196a-5p and its potential target IκBα on migration, invasion and epithelial-mesenchymal transition (EMT) of colorectal cancer (CRC) cells. METHODS: CCK-8 assay, wound healing assay and cell invasion assay were performed to evaluate the cell proliferation, migration and invasion. In vivo metastasis models were used to investigate the tumor metastasis ability. Real-time PCR, immunofluorescence staining or western blot were utilized to detect the expression of miR-196a-5p, IκBα, p-IκBα, nuclear p65 and EMT markers including E-cadherin, N-cadherin and fibronectin. Dual luciferase reporter assay was carried out to determine whether there is a direct interaction between miR-196a-5p and IκBα mRNA. RESULTS: Using SW480 cell with miR-196-5p over-expressed plus SW620 and HCT116 cells with miR-196a-5p knockdown, we found that miR-196a-5p promoted cell proliferation, migration and invasion in vitro and facilitated liver metastasis in vivo. We also observed that miR-196a-5p knockdown or NF-κB pathway inhibition up-regulated E-cadherin while down-regulated N-cadherin and fibronectin. By contrast, miR-196a-5p over-expression promoted EMT process of CRC. Data of dual luciferase reporter assay indicated that miR-196a-5p targeted the IκBα. Moreover, miR-196a-5p down-regulated IκBα expression while up-regulated nuclear p65 expression. Additionally, over-expression of IκBα in CRC cells attenuated the effects of miR-196a-5p on cell migration, invasion and EMT. CONCLUSIONS: miR-196a-5p may play a key role in EMT, invasion and metastasis of CRC cells via targeting the IκBα.

GAS5 suppresses malignancy of human glioma stem cells via a miR-196a-5p/FOXO1 feedback loop.

Glioma stem cells (GSCs) make up highly tumorigenic subpopulations within gliomas, and aberrant expression of GSC genes is a major underlying cause of glioma pathogenesis and treatment failure. The present study characterized the expression and function of long non-coding RNA growth arrest specific 5 (GAS5) in GSCs in order to elucidate the molecular mechanisms by which GAS5 contributes to glioma pathogenesis. We demonstrate that GAS5 suppresses GSC malignancy by binding to miR-196a-5p. miR-196a-5p, an onco-miRNA, stimulates GSC proliferation, migration, and invasion, in addition to reducing levels of apoptosis. miR-196a-5p specifically downregulates the expression of forkhead box protein O1 (FOXO1) by targeting its 3' untranslated region (3'-UTR). FOXO1 upregulates expression of phosphotyrosine interaction domain containing 1 (PID1), thereby inhibiting GSC tumorigenicity and growth. FOXO1 also upregulates migration and invasion inhibitory protein (MIIP), resulting in attenuation of migration and invasion activities. Interestingly, we also show that FOXO1 promotes GAS5 transcription, thus forminga positive feedback loop. These data provide insights into potential new pathways for GSC molecular therapy and suggest that GAS5 may be an efficacious target for glioma treatments.

Lowly-expressed lncRNA GAS5 facilitates progression of ovarian cancer through targeting miR-196-5p and thereby regulating HOXA5.

PURPOSE: This investigation was aimed at extrapolating whether and how lncRNA GAS5, miR-196a-5p and HOXA5 altered progression of ovarian cancer (OA). METHOD: Totally 195 pairs of OA tissues and adjacent normal tissues were collected. Also si-GAS5, pcDNA-GAS5, miR-196a-5p mimic, miR-196a-5p inhibitor and negative control (NC) were, respectively, transfected into OA cells. Besides, dual-luciferase reporter gene assay was performed to validate the targeted relationships between GAS5 and miR-196a-5p, as well as between miR-196a-5p and HOXA5. The impacts of GAS5, miR-196a-5p and HOXA5 on viability, proliferation and apoptosis of OA cells were appraised via conduction of colony formation assay, MTT assay and flow cytometry assay. RESULT: Lower GAS5 expression and higher miR-196a-5p expression were associated with larger tumor size (≥5 cm) and more advanced FIGO stage (III-IV) of OA patients (P < 0.05). Transfection of si-GAS5, miR-196a-5p mimic or si-HOXA5 conferred OA cells with stronger viability, faster proliferation and smaller percentage of apoptosis (P < 0.05). After injecting mice models with si-GAS5, miR-196a-5p mimic or si-HOXA5, a larger tumor size was also observed within the rats (P < 0.05). GAS5 was indicated to directly target miR-196a-5p and modify its expression, and the targeted relationship also seemed to exist between miR-196a-5p and HOXA5 (P < 0.05). The HOXA5 was found to reverse the effects imposed by miR-196a-5p on viability, proliferation and apoptosis of OA cells (P < 0.05). CONCLUSION: LncRNA GAS5 depressed OA development by targeting miR-196a-5p and thereby down-regulating HOXA5 expression, providing substance for developing lncRNA-based strategies to treat OA.

miR-196a Is Able to Restore the Aggressive Phenotype of Annexin A1 Knock-Out in Pancreatic Cancer Cells by CRISPR/Cas9 Genome Editing.

Annexin A1 (ANXA1) is a Ca2+-binding protein that is involved in pancreatic cancer (PC) progression. It is able to mediate cytoskeletal organization maintaining a malignant phenotype. Our previous studies showed that ANXA1 Knock-Out (KO) MIA PaCa-2 cells partially lost their migratory and invasive capabilities and also the metastatization process appeared affected in vivo. Here, we investigated the microRNA (miRNA) profile in ANXA1 KO cells finding that the modification in miRNA expression suggests the significant involvement of ANXA1 in PC development. In this study, we focused on miR-196a which appeared down modulated in absence of ANXA1. This miRNA is a well known oncogenic factor in several tumour models and it is able to trigger the agents of the epithelial to mesenchymal transition (EMT), like ANXA1. Our results show that the reintroduction in ANXA1 KO cells of miR-196a through the mimic sequence restored the early aggressive phenotype of MIA PaCa-2. Then, ANXA1 seems to support the expression of miR-196a and its role. On the other hand, this miRNA is able to mediate cytoskeletal dynamics and other protein functions promoting PC cell migration and invasion. This work describes the correlation between ANXA1 and specific miRNA sequences, particularly miR-196a. These results could lead to further information on ANXA1 intracellular role in PC, explaining other aspects that are apart from its tumorigenic behaviour.

MiR-196a-5p hinders vascular smooth muscle cell proliferation and vascular remodeling via repressing BACH1 expression.

Hyperproliferation of vascular smooth muscle cells (VSMCs) is a driver of hypertensive vascular remodeling. This study aimed to uncover the mechanism of BTB and CNC homology 1 (BACH1) and microRNAs (miRNAs) in VSMC growth and hypertensive vascular remodeling. With the help of TargetScan, miRWalk, miRDB, and miRTarBase online database, we identified that BACH1 might be targeted by miR-196a-5p, and overexpressed in VSMCs and aortic tissues from spontaneously hypertensive rats (SHRs). Gain- and loss-of-function experiments demonstrated that miR-196a-5p suppressed VSMC proliferation, oxidative stress and hypertensive vascular remodeling. Double luciferase reporter gene assay and functional verification showed that miR-196a-5p cracked down the transcription and translation of BACH1 in both Wistar Kyoto rats (WKYs) and SHRs. Silencing BACH1 mimicked the actions of miR-196a-5p overexpression on attenuating the proliferation and oxidative damage of VSMCs derived from SHRs. Importantly, miR-196a-5p overexpression and BACH1 knockdown cooperatively inhibited VSMC proliferation and oxidative stress in SHRs. Furthermore, miR-196a-5p, if knocked down in SHRs, aggravated hypertension, upregulated BACH1 and promoted VSMC proliferation, all contributing to vascular remodeling. Taken together, targeting miR-196a-5p to downregulate BACH1 may be a promising strategy for retarding VSMC proliferation and hypertensive vascular remodeling.

Exosomal miR-196a-5p enhances radioresistance in lung cancer cells by downregulating NFKBIA.

Radiation therapy is recognized as an effective modality in the treatment of lung cancer, but radioresistance resulting from prolonged treatment reduces the chances of recovery. MicroRNAs (miRNAs) play a pivotal role in radiotherapy immunity. In this study, we aimed to investigate the mechanism by which miR-196a-5p affects radioresistance in lung cancer. The radioresistant lung cancer cell line A549R26-1 was established by radiation treatment. Cancer-associated fibroblasts (CAFs) and normal fibroblasts (NFs) were observed by microscopy, and the expression levels of CAF-specific marker proteins were detected by immunofluorescence. The shape of the exosomes was observed by electron microscopy. A CCK-8 assay was used to detect cell viability, while clone formation assays were used to detect cell proliferative capacity. Flow cytometry was performed to investigate apoptosis. The binding of miR-196a-5p and NFKBIA was predicted and further verified by the dual luciferase reporter experiment. qRT-PCR and western blotting were used to detect gene mRNA and protein levels. We found that exosomes secreted by CAFs could enhance lung cancer cell radioresistance. Moreover, miR-196a-5p potentially bound to NFKBIA, promoting malignant phenotypes in radioresistant cells. Furthermore, exosomal miR-196a-5p derived from CAFs increased radiotherapy immunity in lung cancer. Exosomal miR-196a-5p derived from CAFs enhanced radioresistance in lung cancer cells by downregulating NFKBIA, providing a new potential target for the treatment of lung cancer.

miR-196a-5p Correlates with Chronic Atrophic Gastritis Progression to Gastric Cancer and Induces Malignant Biological Behaviors of Gastric Cancer Cells by Targeting ACER2.

BACKGROUND: As the prognosis of early gastric cancer (EGC) is significantly better than that of advanced gastric cancer (AGC), the development of biomarkers to monitor the progression of chronic atrophic gastritis (CAG) to gastric cancer (GC) is essential. METHODS: Stomach tissue miRNA and mRNA sequences from patients with chronic non-atrophic gastritis (CNAG), CAG, precancerous lesions of gastric cancer (PLGC), and GC were analyzed. A publicly available GC-related miRNA microarray dataset was obtained from the Gene Expression Omnibus database. Spearman's correlation and differential gene analyses, and clinical validation were used to identify novel miRNAs correlating with CAG progression to GC. miRNA targets were predicted using weighted gene co-expression analysis and databases. A dual-luciferase reporter assay was performed to check for direct interaction between miR-196a-5p and ACER2. The CCK-8 and wound healing assays, and flow cytometry were performed to evaluate cell proliferation, migration, and apoptosis. RESULTS: miR-196a-5p was correlated with CAG progression to GC. Overexpression of miR-196a-5p promoted GC cell proliferation and migration and inhibited apoptosis, whereas suppression of miR-196a-5p exerted the opposite effect. Based on the prediction and luciferase assays, ACER2 was identified as the target of miR-196a-5p. ACER2 was downregulated in GC cell lines. Knockdown of ACER2 increased GC cell proliferation rates and migration ability and inhibited apoptosis, while ACER2 overexpression led to the opposite effect. CONCLUSIONS: miR-196a-5p correlated with CAG progression to GC and induced malignant biological behaviors of GC cells by targeting ACER2, providing a novel monitoring biomarker and target for GC prevention.

LncRNA-IQCH-AS1 sensitizes thyroid cancer cells to doxorubicin via modulating the miR-196a-5p/PPP2R1B signalling pathway.

Thyroid cancer is a prevalent human endocrine tumour. Surgical resection is a primary approach for well-differentiated thyroid cancers. Currently, the combination of chemotherapy with subsequent irradiation is a therapeutic strategy for the late stage or metastatic thyroid cancer. Yet, drug resistance and side-effects greatly limit widely clinical applications of chemotherapeutic drugs. The long non-coding RNA IQCH antisense RNA 1 (IQCH-AS1) is correlated with survival and diagnosis of cancer patients. Currently, the precise roles of IQCH-AS1 in thyroid cancer and the chemosensitivity of doxorubicin remain unclear. Here, we report IQCH-AS1 was significantly down-regulated in thyroid cancer tissues and cell lines. Overexpression of IQCH-AS1 effectively sensitized thyroid cancer cells to doxorubicin. From the established doxorubicin-resistant thyroid cancer cell line, 8505 C Doxo R, we detected that IQCH-AS1 was remarkedly suppressed in doxorubicin-resistant cells. Bioinformatics analysis, RNA pull-down and luciferase assays illustrated that IQCH-AS1 functioned as a ceRNA of miR-196a-5p which showed an oncogenic role in thyroid cancer. Overexpression of miR-196a-5p, which was upregulated in 8505 C Doxo R cells, significantly de-sensitized thyroid cancer cells to doxorubicin. Furthermore, PPP2R1B, which encode the protein phosphatase 2 A regulatory subunit A, was directly targeted by miR-196a-5p in thyroid cancer cells. Rescue experiments validated that recovery of miR-196a-5p in IQCH-AS1-overexpressing 8508 C doxorubicin resistant cells successfully reversed the IQCH-AS1-promoted doxorubicin sensitization via targeting PPP2R1B. Summarily, our study revealed new functions and molecular targets of the lncRNA-IQCH-AS1-mediated chemosensitivity of thyroid cancer, contributing to the development of anti-chemoresistant strategies against thyroid cancer.

The Long Noncoding RNA-H19 Mediates the Progression of Fibrosis from Acute Kidney Injury to Chronic Kidney Disease by Regulating the miR-196a/Wnt/ß-Catenin Signaling.

INTRODUCTION: Long noncoding RNAs (lncRNAs) have been reported to be involved in the occurrence and development of various diseases. This study was to investigate the role of lncRNA-H19 in the transition from acute kidney injury (AKI) to chronic kidney disease (CKD) and its underlying mechanism. METHODS: Bilateral renal pedicle ischemia-reperfusion injury (IRI) was used to establish the IRI-AKI model in C57BL/6 mice. The expression levels of lncRNA-H19, miR-196a-5p, α-SMA, collagen I, Wnt1, and ß-catenin in mouse kidney tissues and fibroblasts were determined by quantitative real-time PCR and Western blotting. The degree of renal fibrosis was evaluated by hematoxylin and eosin staining. The interaction between lncRNA-H19 and miR-196a-5p was verified by bioinformatics analysis and luciferase reporter assay. Immunohistochemistry and immunofluorescence were used to evaluate the expression of α-SMA and collagen I in kidney tissues and fibroblasts of mice. RESULTS: lncRNA-H19 is upregulated, and miR-196a-5p is downregulated in kidney tissues of IRI mice. Moreover, miR-196a-5p is a direct target of lncRNA-H19. lncRNA-H19 overexpression promotes kidney fibrosis and activates fibroblasts during AKI-CKD development, while miR-196a-5p overexpression reversed these effects in vitro. Furthermore, lncRNA-H19 overexpression significantly upregulates Wnt1 and ß-catenin expression in kidney tissues and fibroblasts of IRI mice, while miR-196a-5p overexpression downregulates Wnt1 and ß-catenin expression in kidney tissues and fibroblasts of IRI mice. CONCLUSION: lncRNA-H19 induces kidney fibrosis during AKI-CKD by regulating the miR-196a-5p/Wnt/ß-catenin signaling pathway.

Tumor-associated macrophages secret exosomal miR-155 and miR-196a-5p to promote metastasis of non-small-cell lung cancer.

BACKGROUND: Understanding the molecular basis underlying metastasis of non-small cell lung cancer (NSCLC) may provide a new therapeutic modality for the treatment of NSCLC. However, the mechanisms by which tumor-associated macrophages (TAMs) affect NSCLC metastasis remain undefined. In this study, we aimed to discover a novel regulatory pathway involved in NSCLC metastasis. METHODS: Cell Counting Kit-8 (CCK-8), Transwell, western blot assays were used to assess cell viability, migration, invasion and epithelial-mesenchymal transition (EMT). Exosomes from macrophages medium were characterized, and in vitro cell coculture was further conducted to investigate M2 derived exosomes mediated crosstalk between TAMs and tumor cells. Besides, miRNA microarray was used to analyze miRNA expression profiles of M0 and M2 derived exosomes. Luciferase reporter assay was used to verify the potential binding between miRNA and mRNA. Moreover, 6-week-old male BALB/c nude mice were performed to establish transplantation tumor model using tail vein injection. Hematoxylin & eosin staining was used to detect the metastasis of tumor tissues. RESULTS: We found that M2 TAMs were the main TAMs in metastatic tissues of NSCLC patients and exosomes derived from M2 TAMs were able to promote cell viability, cell migration, cell invasion and EMT in NSCLC. We demonstrated that miR-155 and miR-196a-5p were abundant in M2 TAMs and exosomes secreted by M2 TAMs. Functional experiments demonstrated that the deletion of miR-155 and miR-196a-5p in M2 TAMs significantly prevented NSCLC metastasis in vitro and in vivo. To clarify the mechanism governing miR-155 and miR-196a-5p from M2 TAMs, we carried out bioinformatics analysis to predict potential target genes. Mechanistically, miR-155 and miR-196a-5p directly bound to the 3'-UTR of Ras association domain family member 4 (RASSF4), and negatively regulating RASSF4 expression. At last, rescue assays demonstrated that miR-155 and miR-196a-5p exerted its performance by RASSF4. CONCLUSIONS: Overall, we revealed a new regulatory pathway that was M2 TAMs secreted exosomal miR-155 and miR-196a-5p to promote NSCLC metastasis. This dynamic and reciprocal cross-talk between NSCLC and macrophages innovatively provided a potential opportunity for diagnosis and treatment of NSCLC.

Circular RNA circCRKL inhibits the proliferation of acute myeloid leukemia cells via the miR-196a-5p/miR-196b-5p/p27 axis.

As a new type of non-coding RNA, the role of circular RNA (circRNA) in various diseases and tumors has received considerable attention. Studies have shown that circRNAs play an important role in the progression of acute myeloid leukemia (AML) via different mechanisms. However, the specific underlying molecular mechanism of circRNAs in the proliferation of AML cells remians unclear. This study aimed to clarify the biological role and mechanism of circCRKL in AML. The results indicated low circCRKL expression in AML cell lines and samples. Moreover, the overexpression of circCRKL inhibited the proliferation and colony-forming ability of AML cells, while its silencing promoted them. In addition, bioinformatics tools and luciferase assays revealed that circCRKL could sponge miR-196a-5p and miR-196b-5p to promote the expression of p27. Furthermore, circCRKL inhibited AML cell proliferation via the miR-196a-5p/miR-196b-5p/p27 axis, suggesting a potential new target for AML therapy.

Psoralen Suppresses Cisplatin-Mediated Resistance and Induces Apoptosis of Gastric Adenocarcinoma by Disruption of the miR196a-HOXB7-HER2 Axis.

PURPOSE: The present study aimed to investigate the impact of psoralen on miR-196a-5p expression and function, and to reveal the mechanism underlying miR-196a-5p-mediated inhibition and the reversal of cisplatin (DDP) resistance. METHODS: Serum samples were collected from 50 patients with gastric cancer (GC), and the association between miR-196a-5p expression and the response to chemotherapy was assessed. A DDP-resistant GC cell line was also established to determine the effects of miR-196a-5p and psoralen on DDP resistance. MGC803 cells were transfected with miR-196a-5p mimic and inhibitor vectors for the overexpression and downregulation of miR-196a-5p, respectively. RESULTS: Clinical data analysis showed that the lower expression levels of miR-196a-5p were significantly associated with chemoresistance in patients with GC. Upregulation of miR-196a-5p significantly enhanced the anti-proliferative effect, apoptosis and sensitivity to DDP by regulating the protein expression levels of HOXB7, HER2, Bcl-2 and G1/S-specific cyclin-D1 (CCND1). Furthermore, psoralen reversed miR-196a-5p-induced DDP resistance and reduced the expression levels of HOXB7, HER2, Bcl-2 and CCND1. CONCLUSION: miR-196a-5p may be a novel biomarker of chemotherapeutic success in patients with GC and may also influence the sensitivity of GC cells to DDP. Moreover, psoralen can increase chemotherapeutic sensitivity by upregulating miR-196a-5p and then downregulating HOXB7-HER2 signaling axis.