The microRNA miR-202 prevents precocious spermatogonial differentiation and meiotic initiation during mouse spermatogenesis.

Spermatogonial differentiation and meiotic initiation during spermatogenesis are tightly regulated by a number of genes, including those encoding enzymes for miRNA biogenesis. However, whether and how single miRNAs regulate these processes remain unclear. Here, we report that miR-202, a member of the let-7 family, prevents precocious spermatogonial differentiation and meiotic initiation in spermatogenesis by regulating the timely expression of many genes, including those for key regulators such as STRA8 and DMRT6. In miR-202 knockout (KO) mice, the undifferentiated spermatogonial pool is reduced, accompanied by age-dependent decline of fertility. In KO mice, SYCP3, STRA8 and DMRT6 are expressed earlier than in wild-type littermates, and Dmrt6 mRNA is a direct target of miR-202-5p. Moreover, the precocious spermatogonial differentiation and meiotic initiation were also observed in KO spermatogonial stem cells when cultured and induced in vitro, and could be partially rescued by the knockdown of Dmrt6. Therefore, we have not only shown that miR-202 is a regulator of meiotic initiation but also identified a previously unknown module in the underlying regulatory network.

Sperm-borne miR-202 targets SEPT7 and regulates first cleavage of bovine embryos via cytoskeletal remodeling.

In mammals, sperm-borne regulators can be transferred to oocytes during fertilization and have different effects on the formation of pronuclei, the first cleavage of zygotes, the development of preimplantation embryos and even the metabolism of individuals after birth. The regulatory role of sperm microRNAs (miRNAs) in the development of bovine preimplantation embryos has not been reported in detail. By constructing and screening miRNA expression libraries, we found that miR-202 was highly enriched in bovine sperm. As a target gene of miR-202, co-injection of SEPT7 siRNA can partially reverse the accelerated first cleavage of bovine embryos caused by miR-202 inhibitor. In addition, both a miR-202 mimic and SEPT7 siRNA delayed the first cleavage of somatic cell nuclear transfer (SCNT) embryos, suggesting that miR-202-SEPT7 mediates the delay of first cleavage of bovine embryos. By further exploring the relationship between miR-202/SEPT7, HDAC6 and acetylated α-tubulin during embryonic development, we investigated how sperm-borne miR-202 regulates the first cleavage process of bovine embryos by SEPT7 and demonstrate the potential of sperm-borne miRNAs to improve the efficiency of SCNT.

MicroRNA-202 safeguards meiotic progression by preventing premature SEPARASE-mediated REC8 cleavage.

MicroRNAs (miRNAs) are believed to play important roles in mammalian spermatogenesis but the in vivo functions of single miRNAs in this highly complex developmental process remain unclear. Here, we report that miR-202, a member of the let-7 family, plays an important role in spermatogenesis by phenotypic evaluation of miR-202 knockout (KO) mice. Loss of miR-202 results in spermatocyte apoptosis and perturbation of the zygonema-to-pachynema transition. Multiple processes during meiosis prophase I including synapsis and crossover formation are disrupted, and inter-sister chromatid synapses are detected. Moreover, we demonstrate that Separase mRNA is a miR-202 direct target and provides evidence that miR-202 upregulates REC8 by repressing Separase expression. Therefore, we have identified miR-202 as a new regulating noncoding gene that acts on the established SEPARASE-REC8 axis in meiosis.

A novel indel within the bovine SEPT7 gene is associated with ovary length.

The ovary can generate oocytes and secrete female hormones and thus is of great significance to animal fertility. In turn, the functioning of this organ has an effect on the profit margins of the livestock breeding industry. As the development-regulating gene and target gene of miR-202, SEPT7 might play an important role in ovarian growth. Therefore, we hypothesized that SEPT7 is related to ovarian traits owing to the regulation of gonad-specific miR-202. To further investigate the connection between bovine SEPT7 and ovarian development, we analyzed data from 408 samples. After genotyping and analyzing three selected loci, we found that two out of the three loci (L1 and L5) were polymorphic, of which the minimum allelic frequencies were 0.417 (L1) and 0.094 (L5). Moreover, one novel indel L1 of SEPT7 was associated with ovarian length (p < 0.05). More specifically, individuals with II and ID genotypes have longer ovaries than those with the DD genotype. Our work shows that SEPT7 can be selected as a testing marker gene for animal fertility. Our findings contribute to improving the prospects of the cattle industry and the wider use of genetic techniques in breeding.

MiR-202-5p Regulates Geese Follicular Selection by Targeting BTBD10 to Regulate Granulosa Cell Proliferation and Apoptosis.

The regulation of granulosa cells (GCs) proliferation and apoptosis is the key step in follicular selection which determines the egg production performance of poultry. miR-202-5p has been reported to be involved in regulating the proliferation and apoptosis of mammalian ovarian GCs. However, its role in regulating the proliferation and apoptosis of goose GCs is still unknown. In the present study, the GCs of pre-hierarchical follicles (phGCs, 8-10 mm) and those of hierarchical follicles (hGCs, F2-F4) were used to investigate the role of miR-202-5p in cell proliferation and apoptosis during follicle selection. In phGCs and hGCs cultured in vitro, miR-202-5p was found to negatively regulate cell proliferation and positively regulate cell apoptosis. The results of RNA-seq showed that BTB Domain Containing 10 (BTBD10) is predicted to be a key target gene for miR-202-5p to regulate the proliferation and apoptosis of GCs. Furthermore, it is confirmed that miR-202-5p can inhibit BTBD10 expression by targeting its 3'UTR region, and BTBD10 was revealed to promote the proliferation and inhibit the apoptosis of phGCs and hGCs. Additionally, co-transfection with BTBD10 effectively prevented miR-202-5p mimic-induced cell apoptosis and the inhibition of cell proliferation. Meanwhile, miR-202-5p also remarkably inhibited the expression of Phosphatidylinositol-4,5-Bisphosphate 3-Kinase Catalytic Subunit Beta (PIK3CB) and AKT Serine/Threonine Kinase 1 (AKT1), while it was significantly restored by BTBD10. Overall, miR-202-5p suppresses the proliferation and promotes the apoptosis of GCs through the downregulation of PIK3CB/AKT1 signaling by targeting BTBD10 during follicular selection. Our study provides a theoretical reference for understanding the molecular mechanism of goose follicular selection, as well as a candidate gene for molecular marker-assisted breeding to improve the geese' egg production performance.

Localization and in silico-based functional analysis of miR-202 in bull testis.

Bull fertility is pivotal to the prosperity of the cattle industry worldwide. miR-202 has been shown to be gonad specific and to have key roles in gonad function in different species. To further understand the involvement of miR-202 in bull reproduction, this study aimed to establish its localization in bovine testicular tissue and to identify putative biological functions using bioinformatics approaches. We assessed the miR-202 expression in paraffin-embedded tissue samples collected form an abattoir using in situ hybridization. miR-202 was present in Sertoli cells and in germ cells at different stages of development. Using available databases, a total of 466 predicted gene targets of miR-202 were identified. Functional annotation revealed that miR-202 target genes were mainly associated with protein modification and phosphorylation processes as well as longevity regulating pathway. Moreover, genes in the longevity regulating pathway mapped to PI3K/Akt/mTOR pathway which is involved in promoting proliferation of testicular cells and spermatogenesis. These findings suggest that miR-202 plays important roles in regulating proliferation and viability of testicular cells including somatic and germ cells.

Circulating miRNA repertoire as a biomarker of metabolic and reproductive states in rainbow trout.

BACKGROUND: Circulating miRNAs (c-miRNAs) are found in most, if not all, biological fluids and are becoming well-established non-invasive biomarkers of many human pathologies. However, their features in non-pathological contexts and whether their expression profiles reflect normal life history events have received little attention, especially in non-mammalian species. The aim of the present study was to investigate the potential of c-miRNAs to serve as biomarkers of reproductive and metabolic states in fish. RESULTS: The blood plasma was sampled throughout the reproductive cycle of female rainbow trout subjected to two different feeding regimes that triggered contrasting metabolic states. In addition, ovarian fluid was sampled at ovulation, and all samples were subjected to small RNA-seq analysis, leading to the establishment of a comprehensive miRNA repertoire (i.e., miRNAome) and enabling subsequent comparative analyses to a panel of RNA-seq libraries from a wide variety of tissues and organs. We showed that biological fluid miRNAomes are complex and encompass a high proportion of the overall rainbow trout miRNAome. While sharing a high proportion of common miRNAs, the blood plasma and ovarian fluid miRNAomes exhibited strong fluid-specific signatures. We further revealed that the blood plasma miRNAome significantly changed depending on metabolic and reproductive states. We subsequently identified three evolutionarily conserved muscle-specific miRNAs or myomiRs (miR-1-1/2-3p, miR-133a-1/2-3p, and miR-206-3p) that accumulated in the blood plasma in response to high feeding rates, making these myomiRs strong candidate biomarkers of active myogenesis. We also identified miR-202-5p as a candidate biomarker for reproductive success that could be used to predict ovulation and/or egg quality. CONCLUSIONS: Together, these promising results reveal the high potential of c-miRNAs, including evolutionarily conserved myomiRs, as physiologically relevant biomarker candidates and pave the way for the use of c-miRNAs for non-invasive phenotyping in various fish species.

miR-450-5p and miR-202-5p Synergistically Regulate Follicle Development in Black Goat.

Follicle maturation is a complex biological process governed by numerous factors, and researchers have observed follicle development by studying the proliferation and apoptosis of follicular granulosa cells (GCs). However, the regulatory mechanisms of GCs proliferation and death during follicle development are largely unknown. To investigate the regulatory mechanisms of lncRNAs, mRNAs, and microRNAs, RNA sequencing (RNA-seq) and small RNA-seq were performed on large (>10 mm) and small follicles (<3 mm) of Leizhou black goat during estrus. We discovered two microRNAs, miR-450-5p and miR-202-5p, which can target GCs in goats and may be involved in follicle maturation, and the effects of miR-450-5p and miR-202-5p on ovarian granulosa cell lines were investigated (KGN). Using cell counting kit-8 (CCK-8) assays, 5-Ethynyl-2'-deoxyuridine (EdU) assay and flow cytometry, miR-202-5p overexpression could suppress the proliferation and induce apoptosis of GCs, whereas miR-450-5p overexpression induced the opposite effects. The dual-luciferase reporter assay confirmed that miR-450-5p could directly target the BMF gene (a BCL2 modifying factor), and miR-202-5p targeted the BCL2 gene. A considerable rise in phosphorylated Akt (p-AKT) protein was observed following the downregulation of BMF by miR-450-5p mimics. After BMF gene RNAi therapy, a notable elevation in p-AKT was detected. Mimics of miR-202-5p inhibited BCL2 protein expression, significantly decreasing p-AMPK protein expression. These results imply that during the follicular development in black goats, the miR-450-5p-BMF axis favored GC proliferation on a wide scale, while the miR-202-5p-BCL2 axis triggered GC apoptosis.

Effects of miR-202-5p silencing PIK3CA gene expression on proliferation, invasion, and epithelial-mesenchymal transition of cervical cancer SiHa cells through inhibiting PI3K/Akt/mTOR signaling pathway activation.

To explore the mechanism of miR-202-5p targeting the expression of PIK3CA and mediating the activation of PI3K/Akt/mTOR signaling pathway on the proliferation, invasion, and epithelial-mesenchymal transition (EMT) of cervical cancer. The objects of study were 105 cases of cervical cancer and their corresponding normal tissues. qRT-PCR was used to detect the expression of miR-202-5p and PIK3CA in adjacent normal tissue and cervical cancer tissue. Dual luciferase reporter assay was used to verify the targeting relationship between miR-202-5p and PIK3CA gene. Human cervical cancer cell lines HPV-16E6, SiHa, HeLa, and CaSki were purchased for our cell experiments. The expression levels of PIK3CA in the cells were detected by qRT-PCR. The cell line with higher expression levels was selected to complete the follow-up experiment. The cultured cells were transfected and divided into the miR-202-5p mimic NC group, miR-202-5p mimic group, miR-202-5p inhibitor NC group, miR-202-5p inhibitor group, siRNA-PIK3CA NC group, siRNA-PIK3CA group, miR-202-5p inhibitor NC + siRNA-PIK3CA NC group, miR-202-5p inhibitor + siRNA-PIK3CA NC group, and miR-202-5p inhibitor + siRNA-PIK3CA group. QRT-PCR was used to detect the expression of miR-202-5p. Western blot and qRT-PCR were applied to detect the mRNA and protein expression levels of related pathway proteins (PIK3CA, PI3K, PTEN, p-Akt1, and p-mTOR) and epithelial-mesenchymal transition-related factors (N-cadherin, E-cadherin, and vimentin). Cell proliferation was detected by plate colony formation assay. Transwell assay was used to detect the invasion ability of each group. When compared with the adjacent tissues, PIK3CA mRNA expression level was significantly increased and miR-202-5p expression level was significantly decreased in cervical cancer tissues (all P < 0.05). PIK3CA was a target gene of miR-202-5p. The mRNA expression level of PIK3CA in SiHa cervical cancer cells was significantly higher than that in CaSki, HeLa, and HPV-16E6 cells (all P < 0.05), and SiHa cervical cancer cells were selected to complete the follow-up experiments. When compared with the corresponding NC group, the expression of miR-202-5p in miR-202-5p mimic group was increased. In addition, the mRNA and protein expression levels of E-cadherin and PTEN in miR-202-5p mimic and siRNA-PIK3CA groups were increased, and the protein expression of p-Akt1 and p-mTOR was decreased, and also, the mRNA and protein expression levels of PIK3CA, PI3K, N-cadherin, and vimentin were decreased (all P < 0.05); in miR-202-5p inhibitor group, the expression levels of miR-202-5p, E-cadherin, and PTEN decreased, the protein expression of p-Akt1 and p-mTOR increased, and the mRNA and protein expression of PIK3CA, PI3K, N-cadherin, and vimentin increased in miR-202-5p inhibitor group (all P < 0.05); in miR-202-5p inhibitor + siRNA-PIK3CA group, the expression of miR-202-5p decreased (P < 0.05), but the mRNA and protein expression of PIK3CA, PI3K, p-Akt1, p-mTOR, N-cadherin, E-cadherin, and vimentin had no significant changes (all P > 0.05). When compared with the corresponding NC group, the number of cell clones in miR-202-5p mimic group and siRNA-PIK3CA group was decreased, and the invasion ability of miR-202-5p inhibitor group was increased, and the invasion ability was enhanced (all P < 0.05); miR-202-5p inhibitor + siRNA-PIK3CA group showed no significant change in the number of cell clones and the rate of invasion (P > 0.05). In conclusion, the overexpression of miR-202-5p can suppress PIK3CA gene expression and the activation of PI3K/Akt/mTOR signaling pathway to suppress the proliferation, invasion, and EMT of cervical cancer.

Proper Balance of Small GTPase rab10 Is Critical for PGC Migration in Zebrafish.

MicroRNAs (miRNAs) play important roles in post-transcriptional repression in nearly every biological process including germ cell development. Previously, we have identified a zebrafish germ plasm-specific miRNA miR-202-5p, which regulates PGC migration through targeting cdc42se1 to protect cdc42 expression. However, knockdown of cdc42se1 could not significantly rescue PGC migration in maternal miR-202 mutant (MmiR-202) embryos, indicating that there are other target genes of miR-202-5p required for the regulation of PGC migration. Herein, we revealed the transcriptional profiles of wild type and MmiR-202 PGCs and obtained 129 differentially expressed genes (DEGs), of which 42 DEGs were enriched cell migration-related signaling pathways. From these DEGs, we identified two novel miR-202-5p target genes prdm12b and rab10. Furthermore, we found that disruption of rab10 expression led to significantly migratory defects of PGC by overexpression of rab10 siRNA, or WT, inactive as well as active forms of rab10 mRNA, and WT rab10 overexpression mediated migratory defects could be partially but significantly rescued by overexpression of miR-202-5p, demonstrating that rab10 is an important factor involved miR-202-5p mediated regulation of PGC migration. Taken together, the present results provide significant information for understanding the molecular mechanism by which miR-202-5p regulates PGC migration in zebrafish.

Retinal pigment epithelial cells secrete miR-202-5p-containing exosomes to protect against proliferative diabetic retinopathy.

Previous studies have reported that endothelial-to-mesenchymal transition (EndoMT) contributes to pathological fibrosis in proliferative diabetic retinopathy (PDR). The hypothesis of our study was that exosomes from high glucose (HG)-treated ARPE19 cells reprogram endothelial cell behavior in HG conditions by transferring their genetic contents. Our study showed that ARPE19-derived exosomes were internalized by human umbilical vein endothelial cells (HUVECs). Additionally, miR-202-5p, a miRNA known to target TGFßR2, was enriched in ARPE19-derived exosomes. A dual luciferase reporter assay, qPCR, and western blotting were used to characterize the expression of miR-202-5p and phosphorylation of the TGF/Smad pathway proteins. We showed that miR-202-5p-containing exosomes suppressed HUVEC cell growth, migration, and tube formation. Furthermore, TGFßR2 was confirmed as the target of miR-202-5p. A dual luciferase reporter assay showed that TGFßR2 expression was negatively regulated by miR-202-5p. We also showed that miR-202-5p-containing exosomes suppressed HG-induced EndoMT. These collective results suggested that ARPE-derived exosomes may serve as significant mediators of cell-to-cell crosstalk to suppress EndoMT by transferring miR-202-5p through the TGF/Smad pathway, and may be a potential treatment for PDR patients.

MiR-202-5p attenuates neurological deficits and neuronal injury in MCAO model rats and OGD-induced injury in Neuro-2a cells by targeting eIF4E-mediated induction of autophagy and inhibition of Akt/GSK-3ß pathway.

Ischemic stroke is a common cerebrovascular disease caused by insufficient blood supply to the brain. In recent years, studies have demonstrated that microRNAs (miRNAs) are involved in a variety of biological processes in the nervous system. However, the effects of miR-202-5p on cerebral ischemic stroke injury have not been completely elucidated. In our study, N2a cells were subjected to oxygen-glucose deprivation/reoxygenation (OGD/R) treatment, and middle cerebral artery occlusion (MCAO) rat models were constructed. Our results indicated that decreased miR-202-5p expression was connected to N2a cells after OGD/R-induced injury and rats after MCAO. In addition, high miR-202-5p expression increased proliferation and prevented apoptosis and autophagy of OGD/R-treated N2a cells, while also effectively decreasing the infarct volume in MCAO model rats. We validated the interplay between miR-202-5p and eukaryotic translation initiation factor 4E (eIF4E), and found that miR-202-5p downregulated eIF4E by targeted combination. Moreover, we demonstrated that miR-202-5p accelerated proliferation and suppressed autophagy of OGD/R-induced N2a cells by targeting eIF4E. Meanwhile, our other results suggest that upregulation of miR-202-5p may activate the Akt/GSK-3ß pathway in ischemic brain injury. Our findings suggest that miR-202-5p may serve as a protective agent for ischemia-reperfusion injury in stroke via eIF4E.

Diagnostic Value of Circulating miR-202 in Early-Stage Breast Cancer in South Korea.

Background and objectives: Breast cancer is the most common cancer among women worldwide. Early stage diagnosis is important for predicting increases in treatment success rates and decreases in patient mortality. Recently, circulating biomarkers such as circulating tumor cells, circulating tumor DNA, exosomes, and circulating microRNAs have been examined as blood-based markers for the diagnosis of breast cancer. Although miR-202 has been studied for its function or expression in breast cancer, its potential diagnostic value in a clinical setting remains elusive and miR-202 has not been investigated in South Korea. In this study, we aimed to evaluate the diagnostic utility of miR-202 in plasma samples of breast cancer patients in South Korea. Materials and Methods: We investigated miR-202 expression in the plasma of 30 breast cancer patients during diagnosis along with 30 healthy controls in South Korea by quantitative reverse transcription PCR. Results: The results showed that circulating miR-202 levels were significantly elevated in the breast cancer patients compared with those in healthy controls (p < 0.001). The sensitivity and specificity of circulating miR-202 were 90.0% and 93.0%, respectively. Additionally, circulating miR-202 showed high positivity at early stage. The positive rate of miR-202 was as follows: 100% (10/10) for stage I, 90% (9/10) for stage II, and 80% (8/10) for stage III. miR-202 was also a predictor of a 9.6-fold high risk for breast cancer (p < 0.001). Conclusions: Additional alternative molecular biomarkers for diagnosis and management of pre-cancer patients are needed. Circulating miR-202 might be potential diagnostic tool for detecting early stage breast cancer.

From incomplete penetrance with normal telomere length to severe disease and telomere shortening in a family with monoallelic and biallelic PARN pathogenic variants.

PARN encodes poly(A)-specific ribonuclease. Biallelic and monoallelic PARN variants are associated with Hoyeraal-Hreidarsson syndrome/dyskeratosis congenita and idiopathic pulmonary fibrosis (IPF), respectively. The molecular features associated with incomplete penetrance of PARN-associated IPF have not been described. We report a family with a rare missense, p.Y91C, and a novel insertion, p.(I274*), PARN variant. We found PARN p.Y91C had reduced deadenylase activity and the p.(I274*) transcript was depleted. Detailed analysis of the consequences of these variants revealed that, while PARN protein was lowest in the severely affected biallelic child who had the shortest telomeres, it was also reduced in his mother with the p.(I274*) variant but telomeres at the 50th percentile. Increased adenylation of telomerase RNA, human telomerase RNA, and certain small nucleolar RNAs, and impaired ribosomal RNA maturation were observed in cells derived from the severely affected biallelic carrier, but not in the other, less affected biallelic carrier, who had less severely shortened telomeres, nor in the monoallelic carriers who were unaffected and had telomeres ranging from the 1st to the 50th percentiles. We identified hsa-miR-202-5p as a potential negative regulator of PARN. We propose one or more genetic modifiers influence the impact of PARN variants on its targets and this underlies incomplete penetrance of PARN-associated disease.

The long noncoding RNA NORAD enhances the TGF-ß pathway to promote hepatocellular carcinoma progression by targeting miR-202-5p.

Hepatocellular carcinoma (HCC) is one of the most fatal cancers with common features of invasion and metastasis. Recent evidence indicate that the long noncoding RNA NORAD is a potential oncogene and is significantly upregulated in several cancers. However, the general biological role and clinical value of NORAD in HCC remains unknown. Here, NORAD expression was measured in 29 paired tumor and paratumor tissues via quantitative real-time polymerase chain reaction (qPCR). The effects of NORAD on HCC cell malignant potential were investigated via NORAD overexpression and knockdown both in vitro and in vivo. The mechanism of competitive endogenous RNAs (ceRNAs) was acquired and identified by bioinformatics analyses and luciferase assays. Moreover, the impact of NORAD level on the transforming growth factor ß (TGF-ß) pathway was further determined by qPCR. We found that HCC tissues had a high level of NORAD compared with the paratumor tissues, and NORAD upregulation was associated with the shorter overall survival of patients with HCC. Furthermore, NORAD overexpression was demonstrated to promote HCC cell migration and invasion. Mechanically, NORAD might function as a ceRNA to regulate miR-202-5p, which served as a tumor-suppressing microRNA via the TGF-ß pathway. We address that NORAD has a tumor-promoting effect in HCC and describes a novel mechanism whereby NORAD regulates the TGF-ß pathway as a ceRNA of Homo sapiens (hsa)-miR-202-5p.

MiR-202 inhibits cell proliferation, invasion, and migration in breast cancer by targeting ROCK1 gene.

PURPOSE: The aim of this study was to research the effect of miR-202 on breast cancer cells proliferation, invasion, and migration. METHODS: TCGA analysis was used to research miR-202 expression and overall survival in patients with breast cancer. Transfection with miR-202 mimics or cotransfection with miR-202 mimics and ROCK1 expression vector was performed on breast cancer cells. Reverse transcription polymerase chain reaction was used to detect the miR-202 expression of breast cancer tissues and cells. Western blot was conducted to research the expression of ROCK1, E-cadherin, Twist, N-cadherin, and MMP2. Dual luciferase reporter assay was performed to detect the targeted relationship between ROCK1 and miR-202. MTT and transwell assay were used to detect breast cancer cells proliferation, invasion, and migration. RESULTS: Downregulation of miR-202 was positively correlated with poor prognosis of patients with breast cancer. miR-202 in breast cancer tissues and breast cancer cells was significantly downregulated. Upregulation of miR-202 inhibited the proliferation, migration, and invasion of breast cancer cells. miR-202 promoted E-cadherin expression and inhibited the expression of N-cadherin, Twist, and MMP2. ROCK1 was the target gene of miR-202. miR-202 regulated proliferation, migration, invasion, and expression of related proteins in breast cancer cells by targeting ROCK1 expression. CONCLUSIONS: miR-202 inhibited breast cancer cells proliferation, invasion, and migration by targeting the ROCK1 gene.

miR-202 modulates the progression of neuropathic pain through targeting RAP1A.

Neuropathic pain is a somatosensory disorder which is caused by disease or nerve injury that affects the nervous system. microRNAs (miRNAs) are proved to play crucial roles in the development of neuropathic pain. However, the role of miR-202 in neuropathic pain is still unknown. Sprague-Dawley rats were used for constructing the neuropathic pain model. The expression of miR-202 was determined by quantitative real-time polymerase chain reaction. Potential target gene for miR-202 was measured using bioinformatics methods and Western blot analysis. In this study, we used rats to establish a neuropathic pain model and measured the effect of miR-202 in neuropathic pain. We demonstrated that miR-202 expression was downregulated in the spinal dorsal horn of bilateral sciatic nerve chronic constriction injury (bCCI) rat. However, miR-202 expression was not changed in the dorsal root ganglion, hippocampus, and anterior cingulated cortex of bCCI rat. We identified that RAP1A was a direct target gene of miR-202 in the PC12 cell. RAP1A expression was upregulated in the spinal dorsal horn of bCCI rat. Overexpression of miR-202 could improve the pain threshold for bCCI rats in both hindpaws, indicating that miR-202 overexpression could lighten the pain threshold for model rats. Moreover, RAP1A overexpression increased the pain threshold effect of miR-202 overexpression treated bCCI rats, indicating that miR-202 could lighten the pain threshold through inhibiting RAP1A expression. These data suggested that miR-202 acted pivotal roles in the development of neuropathic pain partly through targeting RAP1A gene.

Network of three specific microRNAs influence type 2 diabetes through inducing insulin resistance in muscle cell lines.

Insulin resistance has been implicated as one of the best predictors for type 2 diabetes. Growing evidence propose the involvement of microRNAs (miRNAs) as short regulatory molecules in modulating and inducing resistance. In this regard, we have investigated the role of three selected miRNAs in insulin resistance development (miR-135, miR-202, and miR-214), via assessing glucose uptake levels in C2C12 and L6 muscle cell lines. Interestingly, miRNA-transfected cells demonstrated a significantly different glucose uptake compared to the positive control cells. In addition, we evaluated the expression levels of three putative miRNA target genes (Rho-associated coiled-coil containing protein kinase 1, serine/threonine kinase 2, and vesicle-associated membrane protein 2) in transfected cells, recruiting luciferase assay. Our results indicated the targeting and downregulation of Rho-associated coiled-coil containing protein kinase 1 and serine/threonine kinase 2 genes in all miR-transfected cell lines ( P ≤ 0.05), but not for vesicle-associated membrane protein 2. MiRNA upregulation led to the poor stimulation of glucose uptake through insulin and developed insulin-resistant phenotype in both muscle cell lines. Our study showed the role of three miRNAs in the induction of insulin resistance in cell lines and making them prone to type 2 diabetes development.

miR-202-5p protects rat against myocardial ischemia reperfusion injury by downregulating the expression of Trpv2 to attenuate the Ca 2+ overload in cardiomyocytes.

BACKGROUND: This study was aimed to unveil micro RNA (miRNA) expression profiles in myocardial ischemia-reperfusion (MI/R) rats and explore whether and how dysregulated miRNAs were involved in the initiation and progression of MI/R in a calcium-dependent manner. METHOD AND RESULTS: Rat model of MI/R was established and cardiomyocytes isolated from neonatal rats cardiomyocytes were induced. Both miRNA and messenger RNA expression profiles were analyzed by Microarray. Quantitative reverse-transcription polymerase chain reaction, immunoblotting, bioinformatics analysis, dual-luciferase reporter gene assay, hematoxylin and eosin, Evans blue, and triphenyl tetrazolium chloride were also used in this study. Serum concentrations of myocardial enzymes (phosphocreatine kinase [CK], creatine kinase [CK-MB], lactate dehydrogenase [LDH]), cardiomyocytes loadage of Ca2+ , as well as the expression level of inositol 1,4,5-trisphosphate receptors (IP3R) and sarcoplasmic reticulum Ca2+ -ATPase 2a (SERCA2a) were measured, respectively. Effects of upregulation or downregulation of miR-202-5p or Trpv2 on these indicators were investigated in vivo and in vitro. In MI/R rats and hypoxia/reoxygenation-induced NCMs, miR-202-5p was downregulated, while Trpv2 was upregulated. Trpv2 was a promising target of miR-202-5p and negatively regulated by miR-202-5p. Upregulation of miR-202-5p or downregulation of Trpv2 significantly reduced the serum concentration of myocardial enzymes, as well as cardiomyocyte-produced reactive oxygen species, but inhibition of miR-202-5p or overexpression of Trpv2 brought the worsening situation for these indicators. Besides, upregulation of miR-202-5p upregulation or downregulation of Trpv2 also inhibited Ca2+ overload in cardiomyocytes, accompanied with the increase of SERCA2a and suppression of IP3R. The reduced damage degree and infarct size in myocardial tissue were contrarily worsened by miR-202-5p inhibitor. CONCLUSION: Overexpression of miR-202-5p or downregulation of its downstream Trpv2 presented the cardioprotective effects to MI/R rats.

MiR-202-5p Promotes M2 Polarization in Allergic Rhinitis by Targeting MATN2.

BACKGROUND: The aim of this study was to investigate the effect of MiR-202-5p on macrophages involved in allergic rhinitis. METHODS: Thirty allergic rhinitis patients and 10 healthy controls were recruited. Nasal lavage was utilized to collect mucosal tissues and mucus-derived macrophages. Flow cytometry was used to detect and sort type 2 macrophages, and qRT-PCR and Western blot were adopted to determine the expression of miR-202-5p and MATN2. Luciferase reporter assay and biotin-RNA pulldown were performed to testify the direct binding of miR-202-5p with MATN2. MATN2 was further overexpressed to test its effect on M2 polarization. RESULTS: miR-202-5p, which can promote M2 polarization, was highly expressed in allergic rhinitis mucosal tissues and macrophages compared with the healthy controls. MATN2 could be directly targeted by miR-202-5p and can reverse miR-202-5p-mediated M2 phenotype polarization in allergic rhinitis. CONCLUSIONS: miR-202-5p could target MATN2 to induce M2 polarization involved in allergic rhinitis.