miR-200s Contribute to Interleukin-6 (IL-6)-induced Insulin Resistance in Hepatocytes

By influencing the activity of the PI3K/AKT pathway, IL-6 acts as an important regulator of hepatic insulin resistance. miR-200s have been shown to control growth by regulating PI3K, but the role of miR-200s in the development of hepatic insulin resistance remains unclear. The present study showed that elevated serum concentration of IL-6 is associated with decreased levels of miR-200s, impaired activation of the AKT/glycogen synthase kinase (GSK) pathway, and reduced glycogenesis that occurred in the livers of db/db mice. As shown in the murine NCTC 1469 hepatocytes and the primary hepatocytes treated with 10 ng/ml IL-6 for 24 h and in 12-week-old male C57BL/6J mice injected with 16 μg/ml IL-6 by pumps for 7 days, IL-6 administration induced insulin resistance through down-regulation of miR-200s. Moreover, IL-6 treatment inhibited the phosphorylation of AKT and GSK and decreased the glycogenesis. The effects of IL-6 could be diminished by suppression of FOG2 expression. We concluded that IL-6 treatment may impair the activities of the PI3K/AKT/GSK pathway and inhibit the synthesis of glycogen, perhaps via down-regulating miR-200s while augmenting FOG2 expression.     

miR‐20a‐5p regulates pulmonary surfactant gene expression in alveolar type II cells

MicroRNA (miRNA) critically controls gene expression in many biological processes, including lung growth and pulmonary surfactant biosynthesis. The present study was conducted to investigate whether miR‐20a‐5p had such regulatory functions on alveolar type II (AT‐II) cells. To accomplish this, miR‐20a‐5p–overexpressed and miR‐20a‐5p–inhibited adenoviral vectors were constructed and transfected into cultured AT‐II cells that were isolated from rat foetal lungs of 19 days' gestation. Transfection efficiency was confirmed by observing the fluorescence of green fluorescent protein (GFP) carried by the viral vector, whereas miR‐20a‐5p levels were verified by real‐time PCR. The CCK‐8 assay was used to compare the proliferation ability of AT‐II cells that had over‐ or underexpressed miR‐20a‐5p. The expression of surfactant‐associated proteins (SPs) and phosphatase and tensin homolog (PTEN) was measured by real‐time PCR and Western blotting. In AT‐II cells, transfection resulted in over‐ or under‐regulation of miR‐20a‐5p. While overexpression of miR‐20a‐5p promoted pulmonary surfactant gene expression, its underexpression inhibited it. Consistent with its role in negatively regulating the pulmonary surfactant gene, an opposite pattern was observed for miR‐20a‐5p regulation of PTEN. As a result, when miR‐20a‐5p was rendered overexpressed, PTEN was down‐regulated. By contrast, when miR‐20a‐5p was underexpressed, PTEN was up‐regulated. Neither overexpression nor underexpression of miR‐20a‐5p altered the cell proliferation. miR‐20a‐5p plays no role in proliferation of foetal AT‐II cells but is a critical regulator of surfactant gene expression. The latter appears to be achieved through a regulatory process that implicates expression of PTEN.     

The contrary functions of lncRNA HOTAIR/miR‐17‐5p/PTEN axis and Shenqifuzheng injection on chemosensitivity of gastric cancer cells

This study was implemented to figure out whether lncRNA HOTAIR/miR‐17‐5p/PTEN axis played a role that was opposite to Shenqifuzheng (SQFZ) injection in regulating the chemosensitivity of gastric cancer cells. The gastric cancer tissues were gathered and four gastric cancer cell lines were prepared, including BGC‐823, MGC‐803, SGC‐7901, and MKN28. Moreover, cisplatin, adriamycin, mitomycin, and 5‐fluoroura were managed as the chemo‐therapeutics, and SQFZ was prepared as a Chinese medicine. Striking distinctions of HOTAIR, miR‐17‐5p, and PTEN expressions were observed between gastric cancer tissues and para‐carcinoma normal tissues (P < 0.05). MKN28 was associated with the highest resistance to cisplatin, adriamycin, mitomycin, and 5‐fluoroura among all the cell types, and SQFZ significantly improved the MKN28 cells’ sensitivity to the drugs (P < 0.05). The over‐expressed HOTAIR and miR‐17‐5p, as well as under‐expressed PTEN tended to significantly facilitate the viability, EMT process and proliferation of MKN28 cells that were subject to treatment of chemo‐therapies (P < 0.05). SQFZ could amplify the effects of si‐HOTAIR, miR‐17‐5p inhibitor, and pcDNA‐PTEN on boosting the chemosensitivity of gastric cancer cells (P < 0.05). In addition, HOTAIR was also found to directly target miR‐17‐5p, and PTEN appeared to be subject to the modification of HOTAIR and miR‐17‐5p in its acting on the viability, proliferation, EMT process, and apoptosis of gastric cancer cells. The HOTAIR/miR‐17‐5p/PTEN axis could be regarded as the potential treatment targets for gastric cancer, and adjuvant therapy of SQFZ injection could assist in further improving the treatment efficacy of chemo‐therapies for gastric cancer.     

Circular RNA YAP1 acts as the sponge of microRNA‐21‐5p to secure HK‐2 cells from ischaemia/reperfusion‐induced injury

Circular RNA YAP1 (circYAP1) was reported to participate in progression of gastric cancer. However, the role of circYAP1 in acute kidney injury (AKI) remains obscure. We attempted to examine the effects of circYAP1 on ischaemia/reperfusion‐stimulated renal injury. AKI model was established by treating HK‐2 cells in ischaemia/reperfusion (I/R) environment. CircYAP1 expression in blood of AKI patients and I/R‐treated HK‐2 cells was evaluated via RT‐qPCR. CCK‐8, flow cytometry, ELISA and ROS assay were executed to test the impact of circYAP1 on cell viability, apoptosis, inflammatory cytokines and ROS generation. Bioinformatic analysis was executed to explore miRNA targets. The relativity between circYAP1 and miR‐21‐5p was verified by RT‐qPCR and luciferase assay. The functions of miR‐21‐5p in I/R‐triggered injury were reassessed. PI3K/AKT/mTOR pathway was detected by Western blot. Down‐regulated circYAP1 was observed in AKI blood samples and I/R‐treated HK‐2 cells. CircYAP1 overexpression expedited cell growth and weakened secretion of inflammatory factors and ROS generation in I/R‐disposed cells. Besides, we found circYAP1 could sponge to miR‐21‐5p. Interestingly, miR‐21‐5p overexpression overturned the repressive effects of circYAP1 on cell injury. Moreover, PI3K/AKT/mTOR pathway was activated by circYAP1 via inhibiting miR‐21‐5p. We demonstrated that circYAP1 activated PI3K/AKT/mTOR pathway and secured HK‐2 cells from I/R injury via sponging miR‐21‐5p.     

Endoplasmic reticulum stress‐induced exosomal miR‐27a‐3p promotes immune escape in breast cancer via regulating PD‐L1 expression in macrophages

Immune escape of breast cancer cells contributes to breast cancer pathogenesis. Tumour microenvironment stresses that disrupt protein homeostasis can produce endoplasmic reticulum (ER) stress. The miRNA‐mediated translational repression of mRNAs has been extensively studied in regulating immune escape and ER stress in human cancers. In this study, we identified a novel microRNA (miR)‐27a‐3p and investigated its mechanistic role in promoting immune evasion. The binding affinity between miR‐27a‐3p and MAGI2 was predicted using bioinformatic analysis and verified by dual‐luciferase reporter assay. Ectopic expression and inhibition of miR‐27a‐3p in breast cancer cells were achieved by transduction with mimics and inhibitors. Besides, artificial modulation of MAGI2 and PTEN was done to explore their function in ER stress and immune escape of cancer cells. Of note, exosomes were derived from cancer cells and co‐cultured with macrophages for mechanistic studies. The experimental data suggested that ER stress biomarkers including GRP78, PERK, ATF6, IRE1α and PD‐L1 were overexpressed in breast cancer tissues relative to paracancerous tissues. Endoplasmic reticulum stress promoted exosome secretion and elevated exosomal miR‐27a‐3p expression. Elevation of miR‐27a‐3p and PD‐L1 levels in macrophages was observed in response to exosomes‐overexpressing miR‐27a‐3p in vivo and in vitro. miR‐27a‐3p could target and negatively regulate MAGI2, while MAGI2 down‐regulated PD‐L1 by up‐regulating PTEN to inactivate PI3K/AKT signalling pathway. Less CD4⁺, CD8⁺ T cells and IL‐2, and T cells apoptosis were observed in response to co‐culture of macrophages and CD3⁺ T cells. Conjointly, exosomal miR‐27a‐3p promotes immune evasion by up‐regulating PD‐L1 via MAGI2/PTEN/PI3K axis in breast cancer.     

LncRNA SNHG1 functions as a ceRNA to antagonize the effect of miR‐145a‐5p on the down‐regulation of NUAK1 in nasopharyngeal carcinoma cell

How lncRNA SNHG1 influences the aggressiveness of nasopharyngeal carcinoma cells as well as the underlying mechanism was studied. The lncRNA differences were analysed by GSE12452 gene microarray. The expression of SNHG1, MiR‐145‐5p and NUAK1 was identified by qRT‐PCR and western blot. Transfection was conducted to construct nasopharyngeal carcinoma cells with different expressions of SNHG1, miR‐145‐5p and NUAK1. Dual‐luciferase reporter assay was performed to explore the relationship between SNHG1, miR‐145‐5p and NUAK1. Wound‐healing assay and transwell invasion experiments were employed to study changes in cell migration capacity and cell invasion, respectively. Tumour xenografts were performed to observe lung metastasis of nude mice inoculated with transfected CNE cells. SNHG1 is highly expressed in nasopharyngeal carcinoma tissues and in cell lines. Down‐regulation of SNHG1 facilitated the expression of miR‐145‐5p and further suppressed the level of NAUK1 in CNE and HNE‐1 cells. Silencing of SNHG1, up‐regulation of miR‐145‐5p and inhibition of NAUK1 by relative transfection all attenuated the aggressiveness of CNE and HNE‐1 cells both in vivo and in vitro. Moreover, the impaired cell migration and invasion by SNHG1 siRNA could be rescued by cotransfection of miR‐145‐5p in CNE and HNE‐1 cells. LncRNA SNHG1 promoted the expression of NUAK1 by down‐regulating miR‐145‐5p and thus promoted the aggressiveness of nasopharyngeal carcinoma cells through AKT signalling pathway and induced epithelial‐mesenchymal transition (EMT).     

Circ‐AKT3 inhibits the accumulation of extracellular matrix of mesangial cells in diabetic nephropathy via modulating miR‐296‐3p/E‐cadherin signals

Diabetic nephropathy is a leading cause of end‐stage renal disease globally. The vital role of circular RNAs (circRNAs) has been reported in diabetic nephropathy progression, but the molecular mechanism linking diabetic nephropathy to circRNAs remains elusive. In this study, we investigated the significant function of circ‐AKT3/miR‐296‐3p/E‐cadherin regulatory network on the extracellular matrix accumulation in mesangial cells in diabetic nephropathy. The expression of circ‐AKT3 and fibrosis‐associated proteins, including fibronectin, collagen type I and collagen type IV, was assessed via RT‐PCR and Western blot analysis in diabetic nephropathy animal model and mouse mesangial SV40‐MES13 cells. Luciferase reporter assays were used to investigate interactions among E‐cadherin, circ‐AKT3 and miR‐296‐3p in mouse mesangial SV40‐MES13 cells. Cell apoptosis was evaluated via flow cytometry. The level of circ‐AKT3 was significantly lower in diabetic nephropathy mice model group and mouse mesangial SV40‐MES13 cells treated with high‐concentration (25 mmol/L) glucose. In addition, circ‐AKT3 overexpression inhibited the level of fibrosis‐associated protein, such as fibronectin, collagen type I and collagen type IV. Circ‐AKT3 overexpression also inhibited the apoptosis of mouse mesangial SV40‐MES13 cells treated with high glucose. Luciferase reporter assay and bioinformatics tools identified that circ‐AKT3 could act as a sponge of miR‐296‐3p and E‐cadherin was the miR‐296‐3p direct target. Moreover, circ‐AKT3/miR‐296‐3p/E‐cadherin modulated the extracellular matrix of mouse mesangial cells in high‐concentration (25 mmol/L) glucose, inhibiting the synthesis of related extracellular matrix protein. In conclusion, circ‐AKT3 inhibited the extracellular matrix accumulation in diabetic nephropathy mesangial cells through modulating miR‐296‐3p/E‐cadherin signals, which might offer novel potential opportunities for clinical diagnosis targets and therapeutic biomarkers for diabetic nephropathy.     

Circular RNA ciRS‐7 promotes tube formation in microvascular endothelial cells through downregulation of miR‐26a‐5p

Atherosclerosis is one of the most common and crucial heart diseases involving the heart and brain. At present, atherosclerosis and its major complications comprise the leading causes of death worldwide. Our purpose was to identify the role of ciRS‐7 in atherosclerosis. Tubulogenesis of HMEC‐1 cell was evaluated utilizing tube formation assay. Cell Counting Kit‐8 assay and flow cytometry were utilized to test viability and apoptosis. Migration assay was utilized to determine the migration capacity of experimental cells. Western blot was applied to examine apoptosis and tube formation‐associated protein expression. In addition, the above experiments were repeated when silencing ciRS‐7, overexpressing ciRS‐7, and upregulating miR‐26a‐5p. HMEC‐1 cells formed tube‐like structures over time. Silencing ciRS‐7 suppressed viability, migration, and tube formation but promoted apoptosis. Oppositely, overexpressing ciRS‐7 reversed the effect in HMEC‐1 cells. miR‐26a‐5p expression was elevated by silencing ciRS‐7 and reduced by overexpressing ciRS‐7. Moreover, overexpressing ciRS‐7 facilitated viability, migration, and tube formation via upregulating miR‐26a‐5p. Conclusively, overexpressing ciRS‐7 mobilized phosphoinositide 3‐kinase/protein kinase B (PI3K/AKT) pathway and suppressed c‐Jun N‐terminal kinase (JNK)/p38 pathway. ciRS‐7 exerted influence on apoptosis, viability, migration, and tube formation through mediating PI3K/AKT and JNK/p38 pathways by miR‐26a‐5p downregulation in HMEC‐1 cells.     

Hypoxia‐induced miR‐3677‐3p promotes the proliferation,migration and invasion of hepatocellular carcinoma cells by suppressing SIRT5

Hepatocellular carcinoma (HCC), with life‐threatening malignant behaviours, often develops distant metastases and is the fourth most common primary cancer in the world, having taken millions of lives in Asian countries such as China. The novel miR‐3677‐3p is involved in a high‐expression‐related poor prognosis in HCC tissues and cell lines, indicating oncogenesis functions in vitro and in vivo. Initially, we confirmed the inhibition of proliferation, migration and invasion in miR‐3677‐3p knock‐down MHCC‐97H and SMMC‐7721 cell lines, which are well known for their high degree of invasiveness. Then, we reversed the functional experiments in the low‐miR‐3677‐3p‐expression Hep3B cell line via overexpressing miR‐3677‐3p. In nude mice xenograft and lung metastasis assays, we found suppressor behaviours, smaller nodules and low density of organ spread, after injection of cells transfected with shRNA‐miR‐3677‐3p. A combination of databases (Starbase, TargetScan and MiRgator) illustrated miR‐3677‐3p targets, and it was shown to suppress the expression of SIRT5 in a dual‐luciferase reporter system. To clarify the conclusions of previous ambiguous research, we up‐regulated SIRT5 in Hep3B cells, and rescue tests were established for confirmation that miR‐3677‐3p suppresses SIRT5 to enhance the migration and invasion of HCC. Interestingly, we discovered hypoxia‐induced miR‐3677‐3p up‐regulation benefited HCC malignancy and invasiveness. In conclusion, the overexpression of miR‐3677‐3p mediated SIRT5 inhibition, which could increase proliferation, migration and invasion of HCC in hypoxic microenvironments.     

MiR‐139‐5p,miR‐940 and miR‐193a‐5p inhibit the growth of hepatocellular carcinoma by targeting SPOCK1

The study was aimed to screen out miRNAs with differential expression in hepatocellular carcinoma (HCC), and to explore the influence of the expressions of these miRNAs and their target gene on HCC cell proliferation, invasion and apoptosis. MiRNAs with differential expression in HCC were screened out by microarray analysis. The common target gene of these miRNAs (miR‐139‐5p, miR‐940 and miR‐193a‐5p) was screened out by analysing the target genes profile (acquired from Targetscan) of the three miRNAs. Expression levels of miRNAs and SPOCK1 were determined by quantitative real time polymerase chain reaction (qRT‐PCR). The target relationships were verified by dual luciferase reporter gene assay and RNA pull‐down assay. Through 3‐(4,5‐dimethyl‐2‐thiazolyl)‐2,5‐diphenyl‐2‐H‐tetrazolium bromide,thiazolyl blue tetrazolium bromide (MTT) and transwell assays and flow cytometry, HCC cell viability, invasion and apoptosis were determined. In vivo experiment was conducted in nude mice to investigate the influence of three miRNAs on tumour growth. Down‐regulation of miR‐139‐5p, miR‐940 and miR‐193a‐5p was found in HCC. Overexpression of these miRNAs suppressed HCC cell viability and invasion, promoted apoptosis and inhibited tumour growth. SPOCK1, the common target gene of miR‐139‐5p, miR‐940 and miR‐193a‐5p, was overexpressed in HCC. SPOCK1 overexpression promoted proliferation and invasion, and restrained apoptosis of HCC cells. MiR‐139‐5p, miR‐940 and miR‐193a‐5p inhibited HCC development through targeting SPOCK1.     

MicroRNA‐876‐5p inhibits cell proliferation,migration and invasion by targeting c‐Met in osteosarcoma

Recently, aberrant expression of miR‐876‐5p has been reported to participate in the progression of several human cancers. However, the expression and function of miR‐876‐5p in osteosarcoma (OS) are still unknown. Here, we found that the expression of miR‐876‐5p was significantly down‐regulated in OS tissues compared to para‐cancerous tissues. Clinical association analysis indicated that underexpression of miR‐876‐5p was positively correlated with advanced clinical stage and poor differentiation. More importantly, OS patients with low miR‐876‐5p level had a significant shorter overall survival compared to miR‐876‐5p high‐expressing patients. In addition, gain‐ and loss‐of‐function experiments demonstrated that miR‐876‐5p restoration suppressed whereas miR‐876‐5p knockdown promoted cell proliferation, migration and invasion in both U2OS and MG63 cells. In vivo studies revealed that miR‐876‐5p overexpression inhibited tumour growth of OS in mice. Mechanistically, miR‐876‐5p reduced c‐Met abundance in OS cells and inversely correlated c‐Met expression in OS tissues. Herein, c‐Met was recognized as a direct target of miR‐876‐5p using luciferase reporter assay. Notably, c‐Met restoration rescued miR‐876‐5p attenuated the proliferation, migration and invasion of OS cells. In conclusion, these findings indicate that miR‐876‐5p may be used as a potential therapeutic target and promising biomarker for the diagnosis and prognosis of OS.     

Up‐regulation of miR‐10b‐3p promotes the progression of hepatocellular carcinoma cells via targeting CMTM5

In this study, we investigated how miR‐10b‐3p regulated the proliferation, migration, invasion in hepatocellular carcinoma (HCC) at both in vitro and in vivo levels. CMTM5 was among the differentially expressed genes (data from TCGA). The expression of miR‐10b‐3p and CMTM5 was detected by qRT‐PCR and Western blot (WB). TargetScan was used to acquire the binding sites. Dual‐luciferase reporter gene assay was used to verify the direct target relationship between miR‐10b‐3p and CMTM5. WB analysis proved that miR‐10b‐3p suppressed CMTM5 expression. Furthermore, proliferation, invasion and migration of HCC cells were measured by MTT assay, colony formation assay, transwell assay and wound‐healing assay, respectively. Kaplan‐Meier plotter valued the overall survival of CMTM5. Finally, xenograft assay was also conducted to verify the effects of miR‐10b‐3p/CMTM5 axis in vivo. Up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were detected in HCC tissues and cell lines. CMTM5 was verified as a target gene of miR‐10b‐3p. The overexpression of CMTM5 contributed to the suppression of the proliferative, migratory and invasive abilities of HCC cells. Moreover, the up‐regulation of miR‐10b‐3p and down‐regulation of CMTM5 were observed to be associated with worse overall survival. Lastly, we have confirmed the carcinogenesis‐related roles of miR‐10b‐3p and CMTM5 in vivo. We concluded that the up‐regulation of miR‐10b‐3p promoted the progression of HCC cells via targeting CMTM5.     

Increase in the nuclear localization of PTEN by the Toxoplasma GRA16 protein and subsequent induction of p53‐dependent apoptosis and anticancer effect

This study investigated the efficacy of Toxoplasma GRA16, which binds to herpes virus‐associated ubiquitin‐specific protease (HAUSP), in anticancer treatment, and whether the expression of GRA16 in genetically modified hepatocellular carcinoma (HCC) cells (GRA16‐p53‐wild HepG2 and GRA16‐p53‐null Hep3B) regulates PTEN because alterations in phosphatase and tensin homologue (PTEN) and p53 are vital in liver carcinogenesis and the abnormal p53 gene appears in HCC. For this purpose, we established the GRA16 cell lines using the pBABE retrovirus system, assessed the detailed mechanism of PTEN regulation in vitro and established the anticancer effect in xenograft mice. Our study showed that cell proliferation, antiapoptotic factors, p‐AKT/AKT ratio, cell migration and invasive activity were decreased in GRA16‐stable HepG2 cells. Conversely, the apoptotic factors PTEN and p53 and apoptotic cells were elevated in GRA16‐stable HepG2 cells but not in Hep3B cells. The change in MDM2 was inconspicuous in both HepG2 and Hep3B; however, the PTEN level was remarkably elevated in HepG2 but not in Hep3B. HAUSP‐bound GRA16 preferentially increased p53 stabilization by the nuclear localization of PTEN rather than MDM2‐dependent mechanisms. These molecular changes appeared to correlate with the decreased tumour mass in GRA16‐stable‐HepG2 cell‐xenograft nude mice. This study establishes that GRA16 is a HAUSP inhibitor that targets the nuclear localization of PTEN and induces the anticancer effect in a p53‐dependent manner. The efficacy of GRA16 could be newly highlighted in HCC treatment in a p53‐dependent manner.     

Down‐regulated lncRNA SLC25A5‐AS1 facilitates cell growth and inhibits apoptosis via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway in gastric cancer

Mounting evidence has illustrated the vital roles of long non‐coding RNAs (lncRNAs in gastric cancer (GC). Nevertheless, the majority of their roles and mechanisms in GC are still largely unknown. In this study, we investigate the roles of lncRNA SLC25A5‐AS1 on tumourigenesis and explore its potential mechanisms in GC. The results showed that the expressions of SLC25A5‐AS1 in GC were significantly lower than that of adjacent normal tissues, which were significantly associated with tumour size, TNM stage and lymph node metastasis. Moreover, SLC25A5‐AS1 could inhibit GC cell proliferation, induce G1/G1 cell cycle arrest and cell apoptosis in vitro, as well as GC growth in vivo. Dual‐luciferase reporter assay confirmed the direct interaction between SLC25A5‐AS1 and miR‐19a‐3p, rescue experiment showed that co‐transfection miR‐19a‐3p mimics and pcDNA‐SLC25A5‐AS1 could partially restore the ability of GC cell proliferation and the inhibition of cell apoptosis. The mechanism analyses further found that SLC25A5‐AS1 might act as a competing endogenous RNAs (ceRNA), which was involved in the derepression of PTEN expression, a target gene of miR‐19a‐3p, and regulate malignant phenotype via PI3K/AKT signalling pathway in GC. Taken together, this study indicated that SLC25A5‐AS1 was down‐regulated in GC and functioned as a suppressor in the progression of GC. Moreover, it could act as a ceRNA to regulate cellular behaviours via miR‐19a‐3p/PTEN/PI3K/AKT signalling pathway. Thus, SLC25A5‐AS1 might be served as a potential target for cancer therapeutics in GC.     

miR‐10b expression in breast cancer stem cells supports self‐renewal through negative PTEN regulation and sustained AKT activation

Cancer stem cells (CSCs) are linked to metastasis. Moreover, a discrete group of miRNAs (metastamiRs) has been shown to promote metastasis. Accordingly, we propose that miRNAs that function as metastatic promoters may influence the CSC phenotype. To study this issue, we compared the expression of 353 miRNAs in CSCs enriched from breast cancer cell lines using qRT–PCR analysis. One of the most altered miRNAs was miR‐10b, which is a reported promoter of metastasis and migration. Stable overexpression of miR‐10b in MCF‐7 cells (miR‐10b‐OE cells) promoted higher self‐renewal and expression of stemness and epithelial–mesenchymal transition (EMT) markers. In agreement with these results, inhibiting miR‐10b expression using synthetic antisense RNAs resulted in a decrease in CSCs self‐renewal. Bioinformatics analyses identified several potential miR‐10b mRNA targets, including phosphatase and tensin homolog (PTEN), a key regulator of the PI3K/AKT pathway involved in metastasis, cell survival, and self‐renewal. The targeting of PTEN by miR‐10b was confirmed using a luciferase reporter, qRT–PCR, and Western blot analyses. Lower PTEN levels were observed in CSCs, and miR‐10b depletion not only increased PTEN mRNA and protein expression but also decreased the activity of AKT, a downstream PTEN target kinase. Correspondingly, PTEN knockdown increased stem cell markers, whereas AKT inhibitors compromised the self‐renewal ability of CSCs and breast cancer cell lines overexpressing miR‐10b. In conclusion, miR‐10b regulates the self‐renewal of the breast CSC phenotype by inhibiting PTEN and maintaining AKT pathway activation.     

β‐arrestin2/miR‐155/GSK3β regulates transition of 5′‐azacytizine‐induced Sca‐1‐positive cells to cardiomyocytes

Stem‐cell antigen 1–positive (Sca‐1+) cardiac stem cells (CSCs), a vital kind of CSCs in humans, promote cardiac repair in vivo and can differentiate to cardiomyocytes with 5′‐azacytizine treatment in vitro. However, the underlying molecular mechanisms are unknown. β‐arrestin2 is an important scaffold protein and highly expressed in the heart. To explore the function of β‐arrestin2 in Sca‐1+ CSC differentiation, we used β‐arrestin2–knockout mice and overexpression strategies. Real‐time PCR revealed that β‐arrestin2 promoted 5′‐azacytizine‐induced Sca‐1+ CSC differentiation in vitro. Because the microRNA 155 (miR‐155) may regulate β‐arrestin2 expression, we detected its role and relationship with β‐arrestin2 and glycogen synthase kinase 3 (GSK3β), another probable target of miR‐155. Real‐time PCR revealed that miR‐155, inhibited by β‐arrestin2, impaired 5′‐azacytizine‐induced Sca‐1+ CSC differentiation. On luciferase report assay, miR‐155 could inhibit the activity of β‐arrestin2 and GSK3β, which suggests a loop pathway between miR‐155 and β‐arrestin2. Furthermore, β‐arrestin2‐knockout inhibited the activity of GSK3β. Akt, the upstream inhibitor of GSK3β, was inhibited in β‐arrestin2‐Knockout mice, so the activity of GSK3β was regulated by β‐arrestin2 not Akt. We transplanted Sca‐1+ CSCs from β‐arrestin2‐knockout mice to mice with myocardial infarction and found similar protective functions as in wild‐type mice but impaired arterial elastance. Furthermore, low level of β‐arrestin2 agreed with decreased phosphorylation of AKT and increased phophorylation of GSK3β, similar to in vitro findings. The β‐arrestin2/miR‐155/GSK3β pathway may be a new mechanism with implications for treatment of heart disease.     

Long non‐coding RNA cardiac hypertrophy‐associated regulator governs cardiac hypertrophy via regulating miR‐20b and the downstream PTEN/AKT pathway

Pathological cardiac hypertrophy (CH) is a key factor leading to heart failure and ultimately sudden death. Long non‐coding RNAs (lncRNAs) are emerging as a new player in gene regulation relevant to a wide spectrum of human disease including cardiac disorders. Here, we characterize the role of a specific lncRNA named cardiac hypertrophy‐associated regulator (CHAR) in CH and delineate the underlying signalling pathway. CHAR was found markedly down‐regulated in both in vivo mouse model of cardiac hypertrophy induced by pressure overload and in vitro cellular model of cardiomyocyte hypertrophy induced by angiotensin II (AngII) insult. CHAR down‐regulation alone was sufficient to induce hypertrophic phenotypes in healthy mice and neonatal rat ventricular cells (NRVCs). Overexpression of CHAR reduced the hypertrophic responses. CHAR was found to act as a competitive endogenous RNA (ceRNA) to down‐regulate miR‐20b that we established as a pro‐hypertrophic miRNA. We experimentally established phosphatase and tensin homolog (PTEN), an anti‐hypertrophic signalling molecule, as a target gene for miR‐20b. We found that miR‐20b induced CH by directly repressing PTEN expression and indirectly increasing AKT activity. Moreover, CHAR overexpression mitigated the repression of PTEN and activation of AKT by miR‐20b, and as such, it abrogated the deleterious effects of miR‐20b on CH. Collectively, this study characterized a new lncRNA CHAR and unravelled a new pro‐hypertrophic signalling pathway: lncRNA‐CHAR/miR‐20b/PTEN/AKT. The findings therefore should improve our understanding of the cellular functionality and pathophysiological role of lncRNAs in the heart.     

MicroRNA‐301b‐3p contributes to tumour growth of human hepatocellular carcinoma by repressing vestigial like family member 4

MicroRNAs (miRNAs) are key regulators in the tumour growth and metastasis of human hepatocellular carcinoma (HCC). Increasing evidence suggests that miR‐301b‐3p functions as a driver in various types of human cancer. However, the expression pattern of miR‐301b‐3p and its functional role as well as underlying molecular mechanism in HCC remain poorly known. Our study found that miR‐301b‐3p expression was significantly up‐regulated in HCC tissues compared to adjacent non‐tumour tissues. Clinical association analysis revealed that the high level of miR‐301b‐3p closely correlated with large tumour size and advanced tumour‐node‐metastasis stages. Importantly, the high miR‐301b‐3p level predicted a prominent poorer overall survival of HCC patients. Knockdown of miR‐301b‐3p suppressed cell proliferation, led to cell cycle arrest at G2/M phase and induced apoptosis of Huh7 and Hep3B cells. Furthermore, miR‐301b‐3p knockdown suppressed tumour growth of HCC in mice. Mechanistically, miR‐301b‐3p directly bond to 3′UTR of vestigial like family member 4 (VGLL4) and negatively regulated its expression. The expression of VGLL4 mRNA was down‐regulated and inversely correlated with miR‐301b‐3p level in HCC tissues. Notably, VGLL4 knockdown markedly repressed cell proliferation, resulted in G2/M phase arrest and promoted apoptosis of HCC cells. Accordingly, VGLL4 silencing rescued miR‐301b‐3p knockdown attenuated HCC cell proliferation, cell cycle progression and apoptosis resistance. Collectively, our results suggest that miR‐301b‐3p is highly expressed in HCC. miR‐301b‐3p facilitates cell proliferation, promotes cell cycle progression and inhibits apoptosis of HCC cells by repressing VGLL4.     

Bone marrow macrophage‐derived exosomal miR‐143‐5p contributes to insulin resistance in hepatocytes by repressing MKP5

ObjectiveIn this study, we aim to explore the role of bone marrow macrophage‐derived exosomes in hepatic insulin resistance, investigate the substance in exosomes that regulates hepatic insulin signalling pathways, reveal the specific molecular mechanisms involved in hepatic insulin resistance and further explore the role of exosomes in type 2 diabetes.Materials and methodsHigh‐fat diet (HFD)‐fed mice were used as obesity‐induced hepatic insulin resistance model, exosomes were isolated from BMMs which were extracted from HFD‐fed mice by ultracentrifugation. Exosomes were analysed the spectral changes of microRNA expression using a microRNA array. The activation of the insulin signalling pathway and the level of glycogenesis were examined in hepatocytes after transfected with miR‐143‐5p mimics. Luciferase assay and western blot were used to assess the target of miR‐143‐5p.ResultsBMMs from HFD‐fed mice were polarized towards M1, and miR‐143‐5p was significantly upregulated in exosomes of BMMs from HFD‐fed mice. Overexpression of miR‐143‐5p in Hep1‐6 cells led to decreased phosphorylation of AKT and GSK and glycogen synthesis. Dual‐luciferase reporter assay and western blot demonstrated that mitogen‐activated protein kinase phosphatase‐5 (Mkp5, also known as Dusp10) was the target gene of miR‐143‐5p. Moreover, the overexpression of MKP5 could rescue the insulin resistance induced by transfection miR‐143‐5p mimics in Hep1‐6.ConclusionBone marrow macrophage‐derived exosomal miR‐143‐5p induces insulin resistance in hepatocytes through repressing MKP5.