MicroRNA-146a modulates TGF-beta1-induced hepatic stellate cell proliferation by targeting SMAD4

Activation of hepatic stellate cells (HSC) plays a pivotal role in the development of hepatic fibrosis. Transforming growth factor-β1 (TGF-β1) is considered to be the main stimuli factor responsible for the activation of HSC. MicroRNAs (miRNAs) have recently been shown to regulate cell proliferation, differentiation, and apoptosis. The involvement of miRNAs and their roles in TGF-β1-induced HSC activation remains largely unknown. Our study found that the expression of miR-146a was downregulated in HSC in response to TGF-β1 stimulation in dose-dependent manner by one-step real-time quantitative PCR. Moreover, we sought to examine whether miR-146a became dysregulated in CCl(4)-induced hepatic fibrosis in rats. Our study revealed that miR-146a was downregulated in liver fibrotic tissues. In addition, The HSC transfected with miR-146a mimics exhibited attendated TGF-β1-induced α-smooth muscle actin (α-SMA) expression compared with the control. Furthermore, overexpression of miR-146a suppressed TGF-β-induced HSC proliferation, and increased HSC apoptosis. Bioinformatics analyses predict that SMAD4 is the potential target of miR-146a. MiR-146a overexpression in TGF-β1-treated HSC did not decrease target mRNA levels, but significantly reduced target protein expression. These results suggested that miR-146a may function as a novel regulator to modulate HSC activation during TGF-β1 induction by targeting SMAD4.     

Silencing PRSS1 suppresses the growth and proliferation of gastric carcinoma cells via the ERK pathway

Background: Gastric carcinoma (GC) is one of the most common malignant tumors and seriously threatens human life and health.Methods: In the present study, 243 differentially expressed proteins in GC were identified using laser capture microdissection (LCM) combined with isotopically labeled quantitative proteomics technology. The expression of serine protease 1 (PRSS1) protein was analyzed by immunohistochemistry and Western blot. MTT and colony formation assays were employed to determine the effect of PRSS1 expression on the growth and proliferation of GC cells. Then, we observed the expression of miR-146a-5p in GC by qRT-PCR. A dual luciferase assay was performed to determine whether PRSS1 is a target gene of miR-146a-5p. We also explored the influence of miR-146a-5p expression on PRSS1 expression and on the growth and proliferation of GC cells. Finally, Western blotting was used to analyze the effect of PRSS1 expression on the activation of the ERK signaling pathway.Results: We confirmed that PRSS1 expression was significantly increased and was positively correlated with the differentiation, tumor size and lymph node metastasis of GC. Subsequently, we found that overexpression of PRSS1 promoted the growth and proliferation of cells, whereas silencing PRSS1 expression inhibited the growth and proliferation of MGC803 cells by inhibiting activation of the ERK signaling pathway via reductions in PAR-2 activation. MiR-146a-5p targets PRSS1 and suppresses the growth and proliferation of MGC803 cells.Conclusions: miR-146a-5p targets PRSS1 and suppresses the growth and proliferation of MGC803 cells. Silencing PRSS1 expression inhibits the ERK signaling pathway by reducing PAR-2 activation, resulting in suppressed growth and proliferation of MGC803 GC cells.     

Exosomal miR-146a-5p and miR-155-5p promote CXCL12/CXCR7-induced metastasis of colorectal cancer by crosstalk with cancer-associated fibroblasts

C-X-C motif chemokine receptor 7 (CXCR7) is a newly discovered atypical chemokine receptor that binds to C-X-C motif chemokine ligand 12 (CXCL12) with higher affinity than CXCR4 and is associated with the metastasis of colorectal cancer (CRC). Cancer-associated fibroblasts (CAFs) have been known to promote tumor progression. However, whether CAFs are involved in CXCR7-mediated metastasis of CRC remains elusive. We found a significant positive correlation between CXCR7 expression and CAF activation markers in colonic tissues from clinical specimens and in villin-CXCR7 transgenic mice. RNA sequencing revealed a coordinated increase in the levels of miR-146a-5p and miR-155-5p in CXCR7-overexpressing CRC cells and their exosomes. Importantly, these CRC cell-derived miR-146a-5p and miR-155-5p could be uptaken by CAFs via exosomes and promote the activation of CAFs through JAK2–STAT3/NF-κB signaling by targeting suppressor of cytokine signaling 1 (SOCS1) and zinc finger and BTB domain containing 2 (ZBTB2). Reciprocally, activated CAFs further potently enhanced the invasive capacity of CRC cells. Mechanistically, CAFs transfected with miR-146a-5p and miR-155-5p exhibited a robust increase in the levels of inflammatory cytokines interleukin-6, tumor necrosis factor-α, transforming growth factor-β, and CXCL12, which trigger the epithelial–mesenchymal transition and pro-metastatic switch of CRC cells. More importantly, the activation of CAFs by miR-146a-5p and miR-155-5p facilitated tumor formation and lung metastasis of CRC in vivo using tumor xenograft models. Our work provides novel insights into CXCR7-mediated CRC metastasis from tumor–stroma interaction and serum exosomal miR-146a-5p and miR-155-5p could serve as potential biomarkers and therapeutic targets for inhibiting CRC metastasis.Subject terms: Cancer microenvironment, Colon cancer     

METTL3 inhibition ameliorates liver damage in mouse with hepatitis B virus-associated acute-on-chronic liver failure by regulating miR-146a-5p maturation

Hepatitis B virus (HBV)-associated acute-on-chronic liver failure (ACLF) is a clinical syndrome of severe liver damage. HBV infection is affected by N6-methyladenosine (m⁶A) RNA modification. Here, we investigated whether methyltransferase-like 3 (METTL3)-mediated m⁶A methylation can affect ACLF. Human hepatic cells (THLE-2) were treated with lipopolysaccharide (LPS) to induce cell damage. Proliferation, apoptosis and m⁶A modification were measured by MTT assay, flow cytometry and Dot blot assay. Our results showed that HBV infection significantly enhanced the levels of m⁶A modification and elevated the expression of METTL3 and mature-miR-146a-5p in THLE-2 cells, which was repressed by cycloleucine (m⁶A inhibitor). METTL3 overexpression enhanced m⁶A modification and promoted mature-miR-146a-5p expression. METTL3 overexpression promoted HBV replication and apoptosis, enhanced the levels of pro-inflammatory cytokines, hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg), and repressed cell proliferation in THLE-2 cells, which attributed to repress miR-146a-5p maturation. Moreover, a severe liver failure mouse model was established by HBV infection to verify the impact of METTL3 knockdown on liver damage in vivo. HBV-infection led to a severe liver damage and increase of apoptosis in hepatic tissues of mice, which was abolished by METTL3 knockdown. METTL3 knockdown reduced METTL3 expression and impeded miR-146a-5p maturation in HBV-infected mice. In conclusion, this work demonstrates that METTL3 inhibition ameliorates liver damage in mouse with HBV-associated ACLF, which contributes to repress miR-146a-5p maturation. Thus, this article suggests a novel therapeutic avenue to prevent and treat HBV-associated ACLF.     

miR-146a-5p circuitry uncouples cell proliferation and migration,but not differentiation,in human mesenchymal stem cells

Administration of mesenchymal stem cells (MSCs) has the potential to ameliorate degenerative disorders and to repair damaged tissues. The homing of transplanted MSCs to injured sites is a critical property of engraftment. Our aim was to identify microRNAs involved in controlling MSC proliferation and migration. MSCs can be isolated from bone marrow and umbilical cord Wharton’s jelly (BM-MSCs and WJ-MSCs, respectively), and WJ-MSCs show poorer motility yet have a better amplification rate compared with BM-MSCs. Small RNA sequencing revealed that miR-146a-5p is significantly overexpressed and has high abundance in WJ-MSCs. Knockdown of miR-146a-5p in WJ-MSCs inhibited their proliferation yet enhanced their migration, whereas overexpression of miR-146a-5p in BM-MSCs did not influence their osteogenic and adipogenic potentials. Chemokine (C-X-C motif) ligand 12 (CXCL12), together with SIKE1, which is an I-kappa-B kinase epsilon (IKKε) suppressor, is a direct target of miR-146a-5p in MSCs. Knockdown of miR-146a-5p resulted in the down-regulation of nuclear factor kappa-B (NF-κB) activity, which is highly activated in WJ-MSCs and is known to activate miR-146a-5p promoter. miR-146a-5p is also downstream of CXCL12, and a negative feedback loop is therefore formed in MSCs. These findings suggest that miR-146a-5p is critical to the uncoupling of motility and proliferation of MSCs. Our miRNome data also provide a roadmap for further understanding MSC biology.     

miR-34a-5p对三阴性乳腺癌细胞的影响及相关机制研究*

目的: 探讨miR-34a-5p在三阴性乳腺癌(triple negative breast cancer,TNBC)中的表达,分析miR-34a-5p对TNBC细胞增殖、凋亡、迁移的作用,对TNBC荷瘤小鼠肿瘤生长的影响以及在TNBC中对B7-H1表达的影响。方法: 利用RT-qPCR、Western blot分析TNBC细胞中miR-34a-5p、B7-H1的表达,并利用Kaplan-Meier分析二者的表达与TNBC患者的生存关系;将miR-34a-5p转染TNBC细胞,通过CCK-8、流式细胞术及划痕实验检测miR-34a-5p对TNBC细胞增殖、凋亡、迁移的影响;利用RT-qPCR、Western blot检测miR-34a-5p、B7-H1表达水平的变化,双荧光素酶基因报告验证miR-34a-5p与B7-H1的相互作用;利用RT-qPCR、Western blot、IHC检测miR-34a-5p对MDA-MB-231荷瘤小鼠miR-34a、B7-H1表达的影响。结果: TNBC细胞中miR-34a-5p呈低表达,B7-H1呈高表达,二者均与TNBC患者的不良预后有关,差距具有统计学意义(P<0.01);miR-34a-5p抑制TNBC细胞增殖、侵袭,促进细胞凋亡,并且在TNBC细胞中靶向抑制B7-H1;miR-34a-5p agomir在体内抑制MDA-MB-231成瘤裸鼠的肿瘤生长和B7-H1表达。结论: miR-34a-5p在TNBC发生、发展中发挥着重要作用,靶向miR-34a-5p/B7-H1可能成为TNBC患者新的分子治疗策略。     

LncRNA NEAT1 promotes docetaxel resistance in prostate cancer by regulating ACSL4 via sponging miR-34a-5p and miR-204-5p

Docetaxel resistance remains one of the main problems in clinical treatment of metastatic prostate cancer (PCa). Previous studies identified differently expressed lncRNAs in docetaxel-resistant PCa cell lines, while the potential mechanisms were still unknown. In the present study, we found NEAT1 was expressed at high levels in docetaxel-resistant PCa clinical samples and related cell lines. When knockdown NEAT1, cell proliferation and invasion in docetaxel-resistant PCa cells in vitro and in vivo were downregulated. Our further researches explained that NEAT1 exerts oncogenic function in PCa by competitively ‘sponging’ both miR-34a-5p and miR-204-5p. Inhibition of miR-34a-5p or miR-204-5p expression mimics the docetaxel-resistant activity of NEAT1, whereas ectopic expression of miR-34a-5p or miR-204-5p attenuates the anti-drug function of NEAT1 in PCa cells. Besides, we also found ACSL4 is a target of both miR-34a-5p and miR-204-5p, and ACSL4 was also inhibited by miR-34a-5p and miR-204-5p. Moreover, suppression of miR-34a-5p or/and miR-204-5p greatly restrained the expression of ACSL4 upon NEAT1 overexpression. Joint expression of miR-34a-5p and miR-204a-5p synergistically decreased the expression of ASCL4, indicating miR-34a-5p and miR-204a-5p collaboratively inhibit the expression of ACSL4. Innovatively, we concluded that NEAT1 contributes to the docetaxel resistance by increasing ACSL4 via sponging miR-34a-5p and miR-204-5p in PCa cells.     

The deoxycholic acid targets miRNA-dependent CAC1 gene expression in multidrug resistance of human colorectal cancer

There is evidence indicating that bile acid is a promoter of colorectal cancer. Deoxycholic acid modifies apoptosis and proliferation by affecting intracellular signaling and gene expression. We are interested in revealing the relationship between deregulated miRNAs and deoxycholic acid in colorectal cancer development. We found that miR-199a-5p was expressed at a low level in human primary colonic epithelial cells treated with deoxycholic acid compared with control, and miR-199a-5p was significantly down-regulated in colorectal cancer tissues. The miR-199a-5p expression in colorectal cancer cells led to the suppression of tumor cell growth, migration and invasion. We further identified CAC1, a cell cycle-related protein expressed in colorectal cancer, as a miR-199a-5p target. We demonstrated that CAC1 is over-expressed in malignant tumors, and cellular CAC1 depletion resulted in cancer growth suppression. HCT-8 cells transfected with a miR-199a-5p mimic or inhibitor had a decrease or increase in CAC1 protein levels, respectively. The results of the luciferase reporter gene analysis demonstrated that CAC1 was a direct miR-199a-5p target. The high miR-199a-5p expression and low CAC1 protein expression reverse the tumor cell drug resistance. We conclude that miR-199a-5p can regulate CAC1 and function as a tumor suppressor in colorectal cancer. Therefore, the potential roles of deoxycholic acid in carcinogenesis are to decrease miR-199a-5p expression and/or increase the expression of CAC1, which contributes to tumorigenesis in patients with CRC. These findings suggest that miR-199a-5p is a useful therapeutic target for colorectal cancer.     

Downregulation of lysyl oxidase-like 4 LOXL4 by miR-135a-5p promotes lung cancer progression in vitro and in vivo

Aberrant microRNAs are widely identified in multiple cancers, including lung cancer. miR-135a-5p can function as a significant tumor regulator in diverse cancers via impacting multiple genes in oncogenic pathways. Nevertheless, the biological role of miR-135a-5p in lung cancer is poorly known. Here, we investigated its function in lung cancer. As exhibited, miR-135a-5p was elevated in lung cancer cells in contrast to BEAS-2B cells. Then, we inhibited miR-135a-5p expression by transfecting LV-anti-miR-135a-5p into lung cancer cells. As displayed, miR-135a-5p was obviously reduced in A549 and H1299 cells. Knockdown of miR-135a-5p repressed lung cancer cell growth and cell proliferation. Meanwhile, cell colony formation capacity was depressed, cell apoptosis was enhanced and cell cycle progression was blocked in G1 phase by inhibition of miR-135a-5p in vitro. Additionally, the migration and invasion of A549 and H1299 cells was strongly depressed by LV-anti-miR-135a-5p. For another, by using informatics analysis, lysyl oxidase-like 4 (LOXL4) was speculated as the downstream target of miR-135a-5p. We validated their direct correlation and moreover, overexpression of miR-135a-5p restrained LOXL4 levels in lung cancer cells. Subsequently, we proved that miR-135a-5p promoted lung cancer development via targeting LOXL4 by carrying out the in vivo assays. Taken these together, our study revealed miR-135a-5p might be indicated as a perspective for lung cancer via targeting LOXL4.     

PGE2 induces apoptosis of hepatic stellate cells and attenuates liver fibrosis in mice by downregulating miR-23a-5p and miR-28a-5p

MicroRNAs (miRNAs), small noncoding RNAs modulating messenger RNA (mRNA) and protein expression, have emerged as key regulatory molecules in chronic liver diseases, whose end stage is hepatic fibrosis, a major global health burden. Pharmacological strategies for prevention or treatment of hepatic fibrosis are still limited, what makes it necessary to establish a better understanding of the molecular mechanisms underlying its pathogenesis. In this context, we have recently shown that cyclooxygenase-2 (COX-2) expression in hepatocytes restricts activation of hepatic stellate cells (HSCs), a pivotal event in the initiation and progression of hepatic fibrosis. Here, we evaluated the role of COX-2 in the regulation of a specific set of miRNAs on a mouse model of CCl₄ and bile duct ligation (BDL)-induced liver fibrosis. Our results provide evidence that COX-2 represses miR-23a-5p and miR-28-5p expression in HSC. The decrease of miR-23a-5p and miR-28-5p expression promotes protection against fibrosis by decreasing the levels of pro-fibrogenic markers α-SMA and COL1A1 and increasing apoptosis of HSC. Moreover, we demonstrate that serum levels of miR-28-5p are decreased in patients with chronic liver disease. These results suggest a protective effect exerted by COX-2-derived prostanoids in the process of hepatofibrogenesis.     

Differentiation Induces Dramatic Changes in miRNA Profile,Where Loss of Dicer Diverts Differentiating SH-SY5Y Cells Toward Senescence

MicroRNAs (miRNAs) are generated by endonuclease activity of Dicer, which also helps in loading of miRNAs to their target sequences. SH-SY5Y, a human neuroblastoma and a cellular model of neurodevelopment, consistently expresses genes related to neurodegenerative disorders at different biological levels (DNA, RNA, and proteins). Using SH-SY5Y cells, we have studied the role of Dicer and miRNAs in neuronal differentiation and explored involvement of P53, a master regulator of gene expression in differentiation-induced induction of miRNAs. Knocking down Dicer gene induced senescence in differentiating SH-SY5Y cells, which indicate the essential role of Dicer in brain development. Differentiation of SH-SY5Y cells by retinoic acid (RA) or RA + brain-derived neurotrophic factor (BDNF) induced dramatic changes in global miRNA expression. Fully differentiated SH-SY5Y cells (5-day RA followed by 3-day BDNF) significantly (p < 0.05 and atleast >3-fold change) upregulated and downregulated the expression of 77 and 17 miRNAs, respectively. Maximum increase was observed in the expression of miR-193-5p, miR-199a-5p, miR-192, miR-145, miR-28-5p, miR-29b, and miR-222 after RA exposure and miR-193-5p, miR-146a, miR-21, miR-199a-5p, miR-153, miR-29b, and miR-222 after RA + BDNF exposure in SH-SY5Y cells. Exploring the role of P53 in differentiating SH-SY5Y cells, we have observed that induction of miR-222, miR-192, and miR-145 is P53 dependent and expression of miR-193a-5p, miR-199a-5p, miR-146a, miR-21, miR-153, and miR-29b is P53 independent. In conclusion, decreased Dicer level enforces differentiating cells to senescence, and differentiating SH-SY5Y cells needs increased expression of P53 to cope up with changes in protein levels of mature neurons.     

MiR-181a-5p is involved in the cardiomyocytes apoptosis induced by hypoxia–reoxygenation through regulating SIRT1

ABSTRACT

MiR-181a-5p’s mechanism in hypoxia–reoxygenation (H/R)-induced cardiomyocytes apoptosis has not been clarified. This study verified that SIRT1 was the target of miR-181a-5p. MiR-181a-5p expression was up-regulated or down-regulated in H/R-induced cardiomyocytes, and SIRT1 was transfected into cells alone or in combination with miR-181a-5p. Cell viability, apoptosis, levels of released lactate dehydrogenase (LDH), malondialdehyde (MDA), and superoxide dismutase (SOD), as well as the Bcl-2, Bax, and Caspase 3 levels in treated cells were tested. On the one hand, down-regulated miR-181a-5p promoted cell viability, reduced released LDH and MDA, and increased SOD level in H/R-induced cardiomyocytes. On the other hand, miR-181a-5p inhibited apoptosis and elevated Bcl-2 expression while decreasing the expressions of Bax and Caspase 3 in treated cells, but the effects of miR-181a-5p could be rescued by SIRT1. In conclusion, miR-181a-5p involved in H/R-induced cardiomyocytes apoptosis through regulating SIRT1, which might become a novel direction for related diseases.     

miR-125a-5p通过靶向调控GAB2抑制乳腺癌细胞的迁移能力

miR-125a-5p可负性调节GAB2表达,抑制胶质瘤细胞的侵袭和转移。本研究旨在证明miR-125a-5p抑癌作用的普遍性,即miR-125a-5p是否可通过靶向抑制GAB2抑制乳腺癌细胞的迁移。荧光素酶实验结果显示,miR-125a-5p可特异识别GAB2的3′-UTR,抑制报告酶的表达。荧光定量PCR结果揭示,与正常乳腺上皮细胞MCF-10A比较,miR-125a-5p在乳腺癌细胞MDA231和MCF-7中的表达明显降低;与迁移能力相对较低的MCF-7细胞比较,miR-125a-5p在迁移能力较高的MDA231细胞中的表达量更低。Western 印迹结果证明,与空载体（对照）和anti-miR125a 5p转染细胞比较,转染miR-125a-5p明显抑制GAB2蛋白在乳腺癌细胞中的表达。Transwell结果显示,与空载体转染的对照细胞比较,转染miR-125a-5p的乳腺癌细胞穿过基质胶的细胞数明显减少;相反,转染anti-miR125a-5p的细胞穿过基质胶的细胞数却明显增多。上述结果提示,miR-125a-5p在正常的乳腺细胞中高表达,而在乳腺癌细胞中低表达,其表达水平与癌细胞的迁移能力和GAB2表达呈反向关系。本研究结果还提示,miR-125a-5p通过靶向负调控GAB2抑制乳腺癌细胞的迁移能力。总之,本研究证明,miR-125a-5p在肿瘤中发挥抑癌作用。     

MiR-378a-5p直接靶ZEB1向上调鼻咽癌CNE-1细胞E-cadherin表达*

目的： Mir-378a-5p是一种被发现多年的微小RNA,其在包括肺癌、结肠癌和乳腺癌等多种肿瘤中都被认为具有抑制肿瘤生 长的作用。Mir-378a-5p与细胞增殖的关系在多篇文章中已经有较为详细的阐述,然而,目前没有报道提及miR-378a-5p是否通过 作用于细胞迁移和细胞粘附途径从而达到抑制肿瘤生长的作用。方法与结果：在本研究中,我们通过wound healing 和trans-well 的方法发现在鼻咽癌细胞CNE-1 中过表达miR-378a-5p显著的抑制了细胞迁移以及细胞浸润的过程。通过免疫印迹方法,我们 揭示了细胞粘附因子E-cadherin在过表达miR-378a-5p后显著上调。通过生物信息学的方法,我们预测了miR-378a-5p的可能作 用靶点,并通过双荧光报告载体的方法证实了ZEB1是miR-378a-5p的直接靶点。结论：我们的研究提示了miR-378a-5p造成的E-cadherin 的上调是通过直接抑制E-cadherin的负调控因子ZEB1造成的。E-cadherin的上调不但影响了细胞的迁移和粘附,而且 通过间接阻断Wnt通路抑制了下游控制细胞增殖的基因表达。本研究为理解miR-378a-5p的肿瘤抑制作用提供了一个新的作用 机理。     

miR-103a-2-5p/miR-30c-1-3p inhibits the progression of prostate cancer resistance to androgen ablation therapy via targeting androgen receptor variant 7

Androgens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly used to treat advanced prostate cancer. However, the acquisition of androgen ablation therapy resistance remains a challenge. Recently, androgen receptor splicing variants lacking the ligand-binding domain have been reported to play a critical role in the acquisition of androgen ablation therapy resistance. In the present study, we revealed that the messenger RNA expression and the protein levels of an androgen receptor variant 7 (AR-V7) were higher in prostate cancer tissue samples and in the AR-positive prostate cancer cell line, VCaP. In contrast, microRNA (miR)-30c-1-3p/miR-103a-2-5p expression was significantly downregulated in tumor tissues and cells. miR-30c-1-3p/miR-103a-2-5p overexpression could inhibit AR-V7 expression, suppress VCaP cell growth, and inhibit AR-V7 downstream factor expression by directly targeting the 3′-untranslated region of AR-V7. Under enzalutamide (Enza) treatment, the effects of AR-V7 overexpression were the opposite of those of miR-103a-2-5p/miR-30c-1-3p overexpression; more importantly, the effects of miR-103a-2-5p/miR-30c-1-3p overexpression could be significantly reversed by AR-V7 overexpression under Enza. In summary, we demonstrated a novel mechanism of the miR-30c-1-3p/miR-103a-2-5p/AR-V7 axis modulating the cell proliferation of AR-positive prostate cancer cells via AR downstream targets. The clinical application of miR-30c-1-3p/miR-103a-2-5p needs further in vivo validation.     

Family of microRNA-146 Regulates RARβ in Papillary Thyroid Carcinoma

Retinoic acid is a promising tool in adjuvant cancer therapies, including refractory thyroid cancer, and its biological role is mediated by the retinoic acid receptor beta (RARβ). However, expression of RARβ is lowered in papillary thyroid carcinoma (PTC), contributing to promotion of tumor growth and inefficiency of retinoic acid and radioactive iodine treatment. The causes of aberrant RARB expression are largely unknown. We hypothesized that the culpable mechanisms include the action of microRNAs from the miR-146 family, previously identified as significantly upregulated in PTC tumors. To test this hypothesis, we assessed the expression of RARB as well as miR-146a-5p and miR-146b-5p in 48 PTC tumor/normal tissue pairs by Taqman assay to reveal that the expression of RARB was 3.28-fold decreased, and miR-146b-5p was 28.9-fold increased in PTC tumors. Direct interaction between miRs and RARB was determined in the luciferase assay and further confirmed in cell lines, where overexpression of miR-146a-5p and miR-146b-5p caused a 31% and 33% decrease in endogenous RARB mRNA levels. Inhibition of miR-146a and miR-146b resulted in 62.5% and 45.4% increase of RARB, respectively, and a concomitant decrease in proliferation rates of thyroid cancer cell lines, analyzed in xCELLigence system.We showed that two microRNAs of the miR-146 family directly regulate RARB. Inhibition of miRs resulted in restoration of RARB expression and decreased rates of proliferation of thyroid cancer cells. By restoring RARB levels, microRNA inhibitors may become part of an adjuvant therapy in thyroid cancer patients.     

MiR-216a-5p protects 16HBE cells from H2O2-induced oxidative stress through targeting HMGB1/NF-kB pathway

Asthma is a complex, chronic inflammatory disorder of the bronchial tree, and can affect patients of all ages including children. High mobility group box 1 (HMGB1) has been proved as a therapeutic target in children with asthma, and was predicted to be the target gene of microRNA-216a-5p (miR-216a-5p). The present study aimed to investigate the function of miR-216a-5p in asthma by creating a human bronchial epithelial cell (16HBE) injury model using H?O?. A significantly elevation of HMGB1 protein expression and a reduction of miR-216a-5p expression were observed in children with asthma as well as in H?O? stimulated 16HBE cells. Dual luciferase reporter assays confirmed the target reaction between HMGB1 and miR-216a-5p. MiR-216a-5p repressed HMGB1 protein expression in H?O? induced 16HBE cells. Moreover, miR-216a-5p inhibited H?O? induced cell injury by elevating cell proliferation and decreasing cell apoptosis in 16HBE cells. Furthermore, miR-216a-5p repressed NF-kB pathway activation in H?O? induced 16HBE cells. In conclusion, these results suggested that miR-216a-5p functions as a negative regulator of H?O? induced 16HBE cell injury through targeting HMGB1/NF-kB pathway, provided a potential therapeutic target for asthma.     

Troxerutin attenuates myocardial cell apoptosis following myocardial ischemia-reperfusion injury through inhibition of miR-146a-5p expression

The aim of the current study was to investigate the effects and the underlying mechanisms of troxerutin on myocardial cell apoptosis during ischemia-reperfusion (I/R) injury. Hypoxia/reoxygenation (H/R) model in neonatal rat cardiomyocytes, and I/R model in rats, were established following troxerutin preconditioning. The quantitative real-time polymerase chain reaction analysis was performed to examine the messenger RNA miR-146a-5p expression in cardiomyocytes and myocardial tissues. Hemodynamic parameters and serum creatine kinase, lactate dehydrogenase, tumor necrosis factor-α, and interleukin-10 were evaluated. Infarct size was examined by 2,3,5-triphenyltetrazolium chloride staining. Besides, myocardial apoptosis was detected by terminal deoxynucleotidyl transferase (dUTP) nick end labeling (TUNEL) assay. Western blot analysis was performed to determine the protein levels of caspase-3, Bax, and Bcl-2. The results showed that, troxerutin decreased rat cardiomyocyte apoptosis during H/R injury. Furthermore, the antiapoptotic effect of troxerutin against I/R injury was mediated by miR-146a-5p downregulation. In vivo experiments suggested that troxerutin alleviated myocardial I/R injury in rats via inhibition of miR-146a-5p. In conclusion, troxerutin exerted cardioprotective effects during I/R injury by downregulating miR-146a-5p.