Reactive oxygen species regulate ERBB2 and ERBB3 expression via miR‐199a/125b and DNA methylation

Overexpression of ERBB2 or ERBB3 is associated with cancer development and poor prognosis. In this study, we show that reactive oxygen species (ROS) induce both ERBB2 and ERBB3 expression in vitro and in vivo. We also identify that miR‐199a and miR‐125b target ERBB2 and/or ERBB3 in ovarian cancer cells, and demonstrate that ROS inhibit miR‐199a and miR‐125b expression through increasing the promoter methylation of the miR‐199a and miR‐125b genes by DNA methyltransferase 1. These findings reveal that ERBB2 and ERBB3 expression is regulated by ROS via miR‐199a and miR‐125b downregulation and DNA hypermethylation.     

MiR‐125b inhibits cell biological progression of Ewing's sarcoma by suppressing the PI3K/Akt signalling pathway

Objectives: Increasing evidence has suggested the close relationship between microRNAs (miRNAs) dysregulation and the carcinogenesis of Ewing's sarcoma (ES), among of which miR‐125b has been reported to be decreased in ES tissues recently. Strikingly, ectopic expression of miR‐125b could suppress cell proliferation of ES cell line A673, suggesting the tumor suppressor role of miR‐125b in ES. However, the other accurate mechanistic functions and relative molecule mechanisms are largely unknown. Materials and Methods: Herein, we completed a series of experiments to investigate the role of miR‐125b in Ewing's sarcoma. We restored the expression of miR‐125b in ES cell line A673 through transfection with miR‐125b mimics. To further understand the role of miR‐125b in ES, we detected the effects of miR‐125b on the cell proliferation, migration and invasion, cell cycle as well as cell apoptosis. Results: We found that restored expression of miR‐125b in ES cell line A673 inhibited cell proliferation, migration and invasion, arrested cell cycle progression, and induced cell apoptosis. Moreover, bioinformatic prediction suggested the oncogene, phosphoinositide‐3‐kinase catalytic subunit delta (PIK3CD), was a target gene of miR‐125b in ES cells. Further quantitative RT‐PCR and western blot assays identified over‐expression of miR‐125b suppressed the expression of PIK3CD mRNA and protein. PIK3CD participates in regulating the PI3K signaling pathway, which has been reported to play an important role in the development of ES. Suppression of PIK3CD down‐regulated the expression of phospho‐AKT and phospho‐mTOR proteins and inhibited the biologic progression of A673 cells. Conclusions: Collectively, these data suggest that miR‐125b functions as a tumor suppressor by targeting the PI3K/Akt/mTOR signaling pathway, and may provide potential therapy strategy for ES patients by targeting miRNA expression.     

MicroRNA signatures associated with immortalization of EBV-transformed lymphoblastoid cell lines and their clinical traits

Objective: MicroRNAs (miRNAs) are negative regulators of gene expression that play important roles in cell processes such as proliferation, development and differentiation. Recently, it has been reported that miRNAs are related to development of carcinogenesis. The aim of this study was to identify miRNAs associated with terminal immortalization of Epstein–Barr virus (EBV)‐transformed lymphoblastoid cell line (LCL) and associated clinical traits. Material and Methods: Hence, we performed miRNA microarray approach with early‐ (p6) and late‐passage (p161) LCLs. Results and Conclusion: Microarray data showed that nine miRNAs (miR‐20b*, miR‐28‐5p, miR‐99a, miR‐125b, miR‐151‐3p, miR‐151:9.1, miR‐216a, miR‐223* and miR‐1296) were differentially expressed in most LCLs during long‐term culture. In particular, miR‐125b was up‐regulated in all the tested late‐passage LCLs. miR‐99a, miR‐125b, miR‐216a and miR‐1296 were putative negative regulators of RASGRP3, GPR160, PRKCH and XAF1, respectively, which were found to be differentially expressed in LCLs during long‐term culture in a previous study. Linear regression analysis showed that miR‐200a and miR‐296‐3p correlated with triglyceride and HbA1C levels, respectively, suggesting that miRNA signatures of LCLs could provide information on the donor’s health. In conclusion, our study suggests that expression changes of specific miRNAs may be required for terminal immortalization of LCLs. Thus, differentially expressed miRNAs would be a potential marker for completion of cell immortalization during EBV‐mediated tumorigenesis.     

MicroRNA‐125b modulates inflammatory chemokine CCL4 expression in immune cells and its reduction causes CCL4 increase with age

Chemokines play a pivotal role in regulating the immune response through a tightly controlled expression. Elevated levels of inflammatory chemokines commonly occur with aging but the mechanism underlying this age‐associated change is not fully understood. Here, we report the role of microRNA‐125b (miR‐125b) in regulating inflammatory CC chemokine 4 (CCL4) expression in human immune cells and its altered expression with aging. We first analyzed the mRNA level of CCL4 in eight different types of immune cells including CD4 and CD8 T‐cell subsets (naïve, central and effector memory), B cells and monocytes in blood from both young (≤42 years) and old (≥70 years) adults. We observed that monocytes and naïve CD8 T cells expressed higher levels of CCL4 and exhibited an age‐related increase in CCL4. We then found the level of miR‐125b was inversely correlated with the level of CCL4 in these cells, and the level of miR‐125b was reduced in monocytes and naïve CD8 T cells of the old compared to the young adults. Knock‐down of miR‐125b by shRNA in monocytes and naïve CD8 T cells led to an increase of CCL4 protein, whereas enhanced miR‐125b expression by transfection in naïve CD8 T cells resulted in a reduction of the CCL4 mRNA and protein in response to stimulation. Finally, we demonstrated that miR‐125b action requires the ‘seed’ sequence in 3′UTR of CCL4. Together these findings demonstrated that miR‐125b is a negative regulator of CCL4 and its reduction is partially responsible for the age‐related increase of CCL4.     

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.     

MicroRNA‐181b negatively regulates the proliferation of human epidermal keratinocytes in psoriasis through targeting TLR4

Our study aims to explore the role of microRNA‐181b (miR‐181b) and TLR in the regulation of cell proliferation of human epidermal keratinocytes (HEKs) in psoriasis. Twenty‐eight patients diagnosed with psoriasis vulgaris were selected as a case group with their lesional and non‐lesional skin tissues collected. A control group consisted of 20 patients who underwent plastic surgery with their healthy skin tissues collected. Real‐time quantitative fluorescence polymerase chain reaction (RT‐qPCR), in situ hybridization and immunohistochemistry were used to detect the expressions of miR‐181b and TLR4 in HEKs of healthy skin, psoriatic lesional skin and non‐lesional skin respectively. The 3′ untranslated region (3′UTR) of TLR4 combined with miR‐181b was verified by a dual‐luciferase reporter assay. Western blotting and bromodeoxyuridine were applied for corresponding detection of TLR4 expression and cell mitosis. The expression of miR‐181b in HEKs of psoriatic lesional skin was less than healthy skin and psoriatic non‐lesional skin. In psoriatic lesional and non‐lesional skin, TLR4‐positive cell rates and the number of positive cells per square millimetre were higher than healthy skin. The dual‐luciferase reporter assay verified that miR‐181b targets TLR4. HEKs transfected with miR‐181b mimics had decreased expression of TLR4, along with the decrease of mitotic indexes and Brdu labelling indexes. However, HEKs transfected with miR‐181b inhibitors showed increased TLR4 expression, mitotic indexes and Brdu labelling indexes. HEKs transfected with both miR‐181b inhibitors and siTLR4 had decreased mitotic indexes and Brdu labelling indexes. These results indicate that miR‐181b can negatively regulate the proliferation of HEKs in psoriasis by targeting TLR4.     

MicroRNA‐125b induces tau hyperphosphorylation and cognitive deficits in Alzheimer's disease

Sporadic Alzheimer's disease (AD) is the most prevalent form of dementia, but no clear disease‐initiating mechanism is known. Aβ deposits and neuronal tangles composed of hyperphosphorylated tau are characteristic for AD. Here, we analyze the contribution of microRNA‐125b (miR‐125b), which is elevated in AD. In primary neurons, overexpression of miR‐125b causes tau hyperphosphorylation and an upregulation of p35, cdk5, and p44/42‐MAPK signaling. In parallel, the phosphatases DUSP6 and PPP1CA and the anti‐apoptotic factor Bcl‐W are downregulated as direct targets of miR‐125b. Knockdown of these phosphatases induces tau hyperphosphorylation, and overexpression of PPP1CA and Bcl‐W prevents miR‐125b‐induced tau phosphorylation, suggesting that they mediate the effects of miR‐125b on tau. Conversely, suppression of miR‐125b in neurons by tough decoys reduces tau phosphorylation and kinase expression/activity. Injecting miR‐125b into the hippocampus of mice impairs associative learning and is accompanied by downregulation of Bcl‐W, DUSP6, and PPP1CA, resulting in increased tau phosphorylation in vivo. Importantly, DUSP6 and PPP1CA are also reduced in AD brains. These data implicate miR‐125b in the pathogenesis of AD by promoting pathological tau phosphorylation.     

Arginase‐2, a miR‐1299 target,enhances pigmentation in melasma by reducing melanosome degradation via senescence‐induced autophagy inhibition

Expression profiles revealed miR‐1299 downregulation concomitant with arginase‐2 (ARG2) upregulation in hyperpigmented skin of melasma patients. Opposite regulation of tyrosinase and PMEL17 by miR‐1299 and inverse relationship between miR‐1299 and ARG2 expression denoted a role of miR‐1299 in pigmentation with ARG2 as a miR‐1299 target. ARG2 overexpression or knock‐down in keratinocytes, the main source of ARG2 in epidermis, positively regulated tyrosinase and PMEL17 protein levels, but not mRNA levels or melanosome transfer. ARG2 overexpression in keratinocytes reduced autophagy equivalent to 3‐MA, an autophagy inhibitor which also increased tyrosinase and PMEL17 protein levels, whereas ARG2 knock‐down induced opposite results. Autophagy inducer rapamycin reduced ARG2‐increased tyrosinase and PMEL17 protein levels. Also, autophagy was reduced in late passage‐induced senescent keratinocytes showing ARG2 upregulation. ARG2, but not 3‐MA, stimulated keratinocyte senescence. These results suggest that ARG2 reduces autophagy in keratinocytes by stimulating cellular senescence, resulting in skin pigmentation by reducing degradation of transferred melanosomes.     

The expression of miR‐125b in Nrf2‐silenced A549 cells exposed to hyperoxia and its relationship with apoptosis

Bronchopulmonary dysplasia (BPD) is a chronic lung disease that affects the quality of life of infants. At present, premature exposure to hyperoxia for extended periods of time is believed to affect the development of lung tissue and vascularity, resulting in BPD. The oxidative stress caused by hyperoxia exposure is an important risk factor for BPD in premature infants. Nuclear factor E2‐related factor 2 (Nrf2) is an important regulator of antioxidant mechanisms. As a microRNA, microRNA‐125b (miR‐125b) plays an important role in cell proliferation, differentiation and apoptosis. Although the Nrf2/ARE pathway has been extensively studied, little is known about the regulatory role of microRNAs in Nrf2 expression. In this study, the expression levels of Nrf2 and miR‐125b in the lung tissues of premature Sprague Dawley (SD) rats and A549 cells exposed to hyperoxia were detected by quantitative real‐time polymerase chain reaction (qRT‐PCR), and the apoptosis of A549 cells was detected by flow cytometry. The results showed that Nrf2 and miRNA‐125b in the lung tissues of premature rats increased significantly upon exposure to hyperoxia and played a protective role. Nrf2 was suppressed by small interfering RNA (siRNA) in A549 cells, miR‐125b was similarly inhibited, and apoptosis was significantly increased. These results suggest that miR‐125b helps protect against BPD as a downstream target of Nrf2.     

miR‐193b availability is antagonized by LncRNA‐SNHG7 for FAIM2‐induced tumour progression in non‐small cell lung cancer

Objectives

Long non‐coding RNAs have identified to involve into the tumour cell proliferation, apoptosis and metastasis. We previously found that up‐regulated LncRNA‐SNHG7 (SNHG7) positively correlated to the Fas apoptosis inhibitory molecule 2 (FAIM2) in lung cancer cells with unclear mechanism.

Methods

Non‐small cell lung cancer (NSCLC) and relative normal tissues (n = 25) were collected. The SNHG7 expression and function in NSCLC was determined. The SNHG7‐miR 193b‐FAIM2 network was analysed in vitro and vivo.

Results

We reported that oncogene SNHG7 predicted a poor clinical outcome and functioned as competitive endogenous RNA (ceRNA) antagonized microRNA‐193b (miR‐193b) to up‐regulate the FAIM2 level in NSCLC. Bioinformatic analysis predicted that SNHG7 harboured miR‐193b‐binding sites, and we found decreased miR‐193b levels in NSCLC tissues when compared to relative normal tissues. Luciferase assays indicated that overexpression of miR‐193b inhibited the Ruc expression of plasmid with miR‐193b‐binding sites of SNHG7 in a dose‐dependent manner. Ectopically expressed SNHG7 also as a molecular sponge sequestered endogenous miR‐193b. Besides, FAIM2 was found to be directly targeted by miR‐193b. The restoration of miR‐193b levels in NSCLC cell lines A549 and H125 suppressed the expression of FAIM2 and related tumour proliferation, metastasis and induced apoptosis. However, forced expression of SNHG7 could down‐regulate miR‐193b to elevate the FAIM2 level of tumour cells, leading to impaired miR‐193b/FAIM2‐induced tumour progression. Knockdown of SNHG7 in vivo significantly delayed the tumour growth with decreased tumour volume, which accompanied with enhanced miR‐193b expression and reduced FAIM2 levels.

Conclusion

The results indicated that miR‐193b is indispensible for the ceRNA role of SNHG7 in FAIM2‐supported tumourigenesis of lung cancer.     

MiR‐29b‐3p promotes chondrocyte apoptosis and facilitates the occurrence and development of osteoarthritis by targeting PGRN

This study was aimed to explore the role of miR‐29b‐3p and PGRN in chondrocyte apoptosis and the initiation and progress of osteoarthritis (OA). Both miR‐29b‐3p and PGRN were up‐regulated in cartilage tissue from patients with OA. Transfection of miR‐29b‐3p mimic into rat primary chondrocytes and SW1353 chondrosarcoma cells significantly suppressed PGRN expression and release, induced apoptosis, inhibited proliferation and scratch wound closure. By contrast, transfection of miR‐29b‐3p inhibitor exhibited the opposite effects. Moreover, the expression and secretion of cartilaginous degeneration‐related molecules were also altered by miR‐29b‐3p. Luciferase reporter gene assay showed rat GRN mRNA is directly targeted and repressed by miR‐29b‐3p. The fact that recombinant PGRN or shPGRN‐mediated PGRN interference abolished miR‐29b‐3p mimic‐induced cell apoptosis and growth inhibition suggested miR‐29b‐3p affect the cellular functions of chondrocyte through regulating PGRN expression. In vivo, joint cavity injection of miR‐29b‐3p antagomir prior to surgical induction of OA significantly suppressed the upregulation of miR‐29b‐3p, whereas further promoted the increased expression of PGRN. Articular chondrocytes apoptosis and cartilage loss in the knee joint of surgically induced OA rats were also ameliorated by the injection of miR‐29b‐3p antagomir, demonstrated by TUNEL and safranin O‐fast green staining. This work showed miR‐29b‐3p facilitates chondrocyte apoptosis and OA by targeting PGRN, and miR‐29b‐3p or PGRN may be the potential target for OA treatments.     

MiR‐130b promotes the progression of oesophageal squamous cell carcinoma by targeting SASH1

MiR‐130b and SAM and SH3 domain containing 1 (SASH1) play an important role in many types of human cancers. The aim of our research was to study their interactions in the process of the proliferation and aggressiveness of oesophageal squamous cell carcinoma (ESCC) cells. Microarray analysis was done to screen the differentially expressed genes in the ESCC tissues. miR‐130b and SASH1 mRNA levels in the ESCC tissues and cells were detected by qRT‐PCR. Dual luciferase reporter system was used to verify the target relationship between miR‐130b and SASH1. The effects of miR‐130b on SASH1 expression were explored by western blot in KYSE30 and TE1 cell lines. CCK‐8 assay, flow cytometry, Transwell, and wound healing assays were conducted to explore the effects of miR‐130b and SASH1 in vitro. In addition, in vivo experiments were conducted to study the roles of miR‐130b and SASH1. miR‐130b was highly expressed, while SASH1 was the opposite in both the ESCC tissues and cells. The expression of SASH1 was inhibited by the direct binding of miR‐130b. The inhibition of miR‐130b reduced the proliferation and aggressiveness of ESCC cells, while it also induced apoptosis and cell cycle arrest in the ESCC cells by suppressing SASH1. The in vivo assay suggested that the overexpression of miR‐130b promoted the growth of ESCC tumours. MiR‐130b was up‐regulated in the ESCC tumour tissues and cells, acting as a tumour promoter. A stimulating effect was demonstrated on ESCC cell growth and aggressiveness by suppressing SASH1, which is an anti‐oncogene.     

Epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer

Emerging evidence suggests that microRNAs (miRNAs) serve an important role in tumorigenesis and development. Although the low expression of miR‐125a‐5p in gastric cancer has been reported, the underlying mechanism remains unknown. In the current study, the low expression of miR‐125a‐5p in gastric cancer was verified in paired cancer tissues and adjacent non‐tumour tissues. Furthermore, the GC islands in the miR‐125a‐5p region were hypermethylated in the tumour tissues. And the hypermethylation was negatively correlated with the miR‐125a‐5p expression. Target gene screening showed that the histone methyltransferase Suv39H1 was one of the potential target genes. In vitro studies showed that miR‐125a‐5p could directly suppress the Suv39H1 expression and decrease the H3K9me3 levels. On the other hand, the Suv39H1 could induce demethylation of miR‐125a‐5p, resulting in re‐activation of miR‐125a‐5p. What is more, overexpessing miR‐125a‐5p could also self‐activate the silenced miR‐125a‐5p in gastric cancer cells, which suppressed cell migration, invasion and proliferation in vitro and inhibited cancer progression in vivo. Thus, we uncovered here that the epigenetic silenced miR‐125a‐5p could be self‐activated through targeting Suv39H1 in gastric cancer, suggesting that miR‐125a‐5p might be not only the potential prognostic value as a tumour biomarker but also potential therapeutic targets in gastric cancer.     

MicroRNAs mir‐184 and let‐7 alter Drosophila metabolism and longevity

MicroRNAs (miRNAs) are small RNA molecules that regulate gene expression associated with many complex biological processes. By comparing miRNA expression between long‐lived cohorts of Drosophila melanogaster that were fed a low‐nutrient diet with normal‐lived control animals fed a high‐nutrient diet, we identified miR‐184, let‐7, miR‐125, and miR‐100 as candidate miRNAs involved in modulating aging. We found that ubiquitous, adult‐specific overexpression of these individual miRNAs led to significant changes in fat metabolism and/or lifespan. Most impressively, adult‐specific overexpression of let‐7 in female nervous tissue increased median fly lifespan by ~22%. We provide evidence that this lifespan extension is not due to alterations in nutrient intake or to decreased insulin signaling.