MicroRNA-367 Inhibits Breast Cancer and Promotes Apoptosis by Targeting AT-Rich Interactive Domain-Containing Protein 1B

Introduction: Breast cancer (BC) developed in the glandular epithelial tissue of breast. microRNA (miR)-367 is an important player in cancer progression, but has never been studied in BC. This experiment tries to probe the mechanism of miR-367 in BC treatment with downstream target gene. Materials and Methods: Human BC cell lines and healthy breast epithelium cells were applied in this study. After the transfection of miR-367 inhibitor or mimic into BC cells, functional assays were conducted to measure cell growth. Afterwards, flow cytometry was employed in apoptosis verification. Then, target relation between miR-367 and ARID1B was certified. Furthermore, ARID1B level was also measured. Results: miR-367 was underexpressed in human BC cells (p < 0.05). Besides, overexpressed miR-367 inhibited BC cell proliferation and encouraged apoptosis, while underexpressed miR-367 led to an opposite outcome (p < 0.05). This experiment then implied that miR-367 dramatically suppressed the activity of cell transfected with ARID1B-wild type. miR-367 overexpression quenched ARID1B level in BC cells; while silencing miR-367 upregulated ARID1B expression (p < 0.05). Conclusion: Our experiment discovered that miR-367 quenched BC cell growth and promoted apoptosis by targeting ARID1B. This investigation may provide novel insights in BC treatment.

ALKBH5-mediated N6-methyladenosine modification of TRERNA1 promotes DLBCL proliferation via p21 downregulation

Long noncoding RNAs (lncRNAs) have crucial functions in the tumorigenesis and metastasis of cancers. N6-methyladenosine (m6A) modification of RNA is an important epigenetic regulatory mechanism in various malignancies. Nevertheless, the mechanism of m6A-modified lncRNA in diffuse large B cell lymphoma (DLBCL) has remained poorly defined. In the present study, we showed that lncRNA TRERNA1 was associated with the poor prognosis of DLBCL patients. TRERNA1 with internal m6A modification was highly correlated with the demethylase ALKBH5 expression. We further demonstrated that TRERNA1 was a potential downstream target of ALKBH5-mediated m6A modification by m6A-RNA sequencing and m6A-RIP assays. Decreased m6A methylation of TRERNA1 regulated by ALKBH5 was shown to regulate cell proliferation in vitro and in vivo. The results of mechanism analyses revealed that TRERNA1 recruited EZH2 to epigenetically silence the expression of the cyclin-dependent kinases inhibitor p21 by H3K27me3 modification of its promoter region. In addition, ALKBH5 further inhibited p21 expression. Taken together, our results elucidate the functional roles and epigenetic alterations of TRERNA1 through m6A modification in DLBCL. TRERNA1, the expression of which is upregulated by ALKBH5, acts as a scaffold that decreases p21 expression. The results of the present study provide novel targets for the diagnosis and treatment of DLBCL.

Phosphorylation-Induced Ubiquitination and Degradation of PXR through CDK2-TRIM21 Axis

Pregnane X receptor (PXR) is a member of the nuclear receptor superfamily that is activated by a variety of endogenous metabolites or xenobiotics. Its downstream target genes are involved in metabolism, inflammation and processes closely related to cancer. However, the stability regulation of PXR protein resulting from post-translational modification is still largely undefined. In the present study, primary mouse hepatocytes, hepatoma HepG2 cells and HEK 293T cells were used to investigate gene expression and protein interactions. The role of kinases was evaluated by RNA interference and overexpression constructs with or without PXR phosphorylation site mutations. The activity of CYP3A4 and P-gp was determined by enzymatic and substrate accumulation assays. It was found that E3 ubiquitin ligase TRIM21 mediates the ubiquitination and degradation of PXR and plays an important role in regulating the activity of PXR. On this basis, PXR phosphorylation-associated kinases were evaluated regarding regulation of the stability of PXR. We found cyclin dependent kinase 2 (CDK2) exclusively phosphorylates PXR at Ser350, promotes its disassociation with Hsp90/DNAJC7, and leads to subsequent TRIM21-mediated PXR ubiquitination and degradation. As well-known CDK inhibitors, dinaciclib and kenpaullone stabilize PXR and result in elevated expression and activity of PXR-targeted DMETs, including carboxylesterases, CYP3A4 and P-gp. The suppressed degradation of PXR by CDK2 inhibitors denotes dinaciclib-induced promotion of PXR-targeted genes. The findings of CDK2-mediated PXR degradation indicate a wide range of potential drug–drug interactions during clinical cancer therapy using CDK inhibitors and imply an alternative direction for the development of novel PXR antagonists.

MiR-135b-5p is an oncogene in pancreatic cancer to regulate GPRC5A expression by targeting transcription factor KLF4

KLF4 is implicated in tumor progression of pancreatic cancer, but the molecular regulatory mechanism of KLF4 needs to be further specified. We aimed to probe molecular regulatory mechanism of KLF4 in malignant progression of pancreatic cancer. qRT-PCR or western blot was completed to test levels of predicted genes. Dual-luciferase and chromatin immunoprecipitation (ChIP) assays were designed to validate binding between genes. Cell viability and oncogenicity detection were used for in vitro and vivo functional assessment. KLF4 was a downstream target of miR-135b-5p. KLF4 could regulate GPRC5A level. MiR-135b-5p was notably increased in cancer cells, and overexpressing KLF4 functioned a tumor repressive role, which could be restored by miR-135b-5p. Besides, cell malignant phenotypes could be inhibited through reducing miR-135b-5p level, but they were restored by GPRC5A. Our results stressed that KLF4, as a vital target of miR-135b-5p, could influence promoter region of GPRC5A, thus affecting the malignant progression of pancreatic cancer.

MiR-654-3p Constrains Proliferation, Invasion, and Migration of Sinonasal Squamous Cell Carcinoma via CREB1/PSEN1 Regulatory Axis

Background: MiR-654-3p can repress malignant progression of cancer cells, whereas no relative reports were about its modulatory mechanism in sinonasal squamous cell carcinoma (SNSCC). This research committed to approaching modulatory effect of miR-654-3p on SNSCC cells.Methods: Bioinformatics methods were utilized for analyzing interaction of miR-654-3p/cAMP-responsive element binding protein 1 (CREB1)/presenilin-1 (PSEN1). Expression levels of miR-654-3p, CREB1, and PSEN1 mRNA were assessed by quantitative real-time polymerase chain reaction. Western blot was completed for level assessment of CREB1, PSEN1, and epithelial–mesenchymal transition–related proteins. The targeted relationship between miR-654-3p and CREB1, or CREB1 and PSEN1 was authenticated via dual-luciferase assay and ChIP assay. A trail of experiments in vitro was used for detection of the effects of miR-654-3p/CREB1/PSEN1 axis on malignant progression of SNSCC cells.Results: CREB1 as the downstream target mRNA of miR-654-3p could activate transcription of its downstream target gene PSEN1. Besides, miR-654-3p could target CREB1 to repress PSEN1 expression, thus restraining proliferation, migration, invasion, epithelial–mesenchymal transition, and hastening apoptosis of SNSCC cells.Conclusion: MiR-654-3p as an antitumor gene targeted CREB1 to hamper malignant progression of SNSCC through miR-654-3p/CREB1/PSEN1 axis.

Mouse CHD4-NURD is Required for Neonatal Spermatogonia Survival and Normal Gonad Development.

Testis development and sustained germ cell production in adults rely on the establishment and maintenance of spermatogonia stem cells and their proper differentiation into spermatocytes. Chromatin remodeling complexes regulate critical processes during gamete development by restricting or promoting accessibility of DNA repair and gene expression machineries to the chromatin. Here, we investigated the role of CHD4 and CHD3 catalytic subunits of the NURD complex during spermatogenesis. Germ cell-specific deletion of Chd4 early in gametogenesis, but not Chd3, resulted in arrested early gamete development due to failed cell survival of neonate undifferentiated spermatogonia stem cell population. Candidate assessment revealed that CHD4 controls expression of Dmrt1 and its downstream target Plzf, both described as prominent regulators of spermatogonia stem cell maintenance. Our results show the requirement of CHD4 in mammalian gametogenesis pointing to functions in gene expression early in the process.

NEAT1 Enhances MPP+-Induced Pyroptosis In SH-SY5Y Cells Via Targeting Mir-5047/YAF2 Signaling

Parkinson’s disease (PD) is the second most frequent neurodegenerative disease. The aim of our study is to explore the role and regulatory mechanism of long non-coding RNA (lncRNA) NEAT1 in the MPP+-induced neuron pyroptosis. The levels of miR-5047 and YAF2 mRNA were determined through qRT-PCR. TUNEL staining was carried out to analyze neuronal apoptosis. Luciferase activity assay was accomplished to analyze the combination of miR-5047 and NEAT1 and YAF2 3’-UTR. Besides, the concentrations of IL-1β and IL-18 in supernatant were analyzed by ELISA assay. The levels of protein were examined through Western blot. NEAT1 and YAF2 expression were increased, while miR-5047 level was declined in the SH-SY5Y cells treated with MPP+. NEAT1 was a positively regulator for the SH-SY5Y cells pyroptosis induced by MPP+. In addition, YAF2 was a downstream target of miR-5047. NEAT1 promoted YAF2 expression via inhibiting miR-5047. Importantly, the promotion of NEAT1 to SH-SY5Y cells pyroptosis induced by MPP+ was recused by miR-5047 mimic transfection and YAF2 downregulation. In conclusion, NEAT1 was increased in the SH-SY5Y cells treated with MPP+, and it promoted the MPP+-induced pyroptosis through facilitating YAF2 expression by sponging miR-5047.

LRP6 modulates the phosphorylation of Cx43 via Gαs in ventricular tachycardia of myocardial infarction

Background Ventricular tachycardia (VT) and ventricular fibrillation are the most causes of early death in patients with myocardial infarction (MI). This study was aimed to explore whether LRP6 and its upstream genes circRNA1615 and miR-152-3p modulated the phosphorylation of Connexin-43 (Cx43) via Gαs in ventricular tachycardia of MI. Method we constructed the hypoxia cardiomyocyte model and AMI mice, and explored the modulation relationship of LRP6 and its upstream genes circRNA1615 and miR-152-3p. In addition, the immunoblot analysis with monoclonal and polyclonal antibodies were used to detect whether LRP6 and Cx43 were phosphorylated, further investigated that the LRP6 regulated the phosphorylation of its downstream target Cx43 via G-protein alpha subunit Gαs by using cell transfection, FISH assay, HE staining, RT-qPCR, and Western blot techniques. Result LRP6 mRNA expression was significantly reduced in AMI group compared with the control group. Hypoxia could inhibit the protein and phosphorylation levels of LRP6 and Cx43. The expression of circRNA1615 in AMI mice was significantly decreased, but overexpression of circRNA1615 significantly reversed it. Also overexpression of circRNA1615 could weaken the effect of miR-152-3p mimic, and the miR-152-3p mimic increased the hypoxia injury of LRP6 and Cx43, further LRP6 interference fragments could aggravate hypoxia injury of Cx43. The overexpression of LRP6 could significantly increase the protein level and phosphorylation level of Cx43, but the interference with LRP6 showed the opposite trend. Noticeably, the interference with Gαs weakened the protein and phosphorylation levels of Cx43, however, the interference with LRP6 further inhibited the protein and phosphorylation levels of Cx43. Finally, the transcriptions of circRNA1615 and LRP6 were inhibited in AMI, but the transcription of miR-152-3p was promoted, and the overexpression of circRNA1615 could weaken the damage effect and VT of AMI. Conclusion LRP6 and its upstream genes circRNA1615 and miR-152-3p modulated the phosphorylation of Cx43 via Gαs in ventricular tachycardia of myocardial infarction.

CircACC1 Promotes NSCLC Proliferation via miR-29c-3p/MCL-1 Signaling Pathway

Non-small cell lung cancer remains the leading cause of cancer-related deaths worldwide with high morbidity and mortality. There is an urgent need to reveal new molecular mechanisms that contribute to NSCLC progression to facilitate drug development and to improve overall survival. Much attention has been paid to the role of circRNAs in NSCLC development. However, the knowledge of circRNAs in NSCLC is still limited, and need to be further explored. The dysregulation of circACC1 was evaluated by qRT-PCR in NSCLC samples and cell lines. The oncogenic role of circACC1 in NSCLC progression was analyzed by CCK8 and colony formation assays. The interaction between the circACC1 and miR-29c-3p, as well as MCL-1, was verified by qRT-PCR, Western blot, luciferase reporter assay, and RIP experiment. Elevated levels of circACC1 were found in NSCLC patients and were negatively correlated with OS. Ectopic expression of circACC1 promoted the capacity of cell growth and clonogenicity, while the inhibition of circACC1 decreased the proliferation and clonogenicity potential. Mechanism studies elucidated that circACC1 contributes to cell growth via directly binding to miR-29c-3p. Transfection of miR-29c-3p mimic blocked circACC1 mediated NSCLC cell proliferation. MCL-1 is a downstream target of miR-29c-3p in NSCLC cells. The circACC1/miR-29c-3p/MCL-1 axis is important in NSCLS proliferation.

MiR-30c-5p loss-induced PELI1 accumulation regulates cell proliferation and migration via activating PI3K/AKT pathway in papillary thyroid carcinoma

Background The aberrant expression of E3 ubiquitin ligase Pellino-1 (PELI1) contributes to several human cancer development and progression. However, its expression patterns and functional importance in papillary thyroid cancer (PTC) remains unknown. Methods PELI1 expression profiles in PTC tissues were obtained and analyzed through the starBase v3.0 analysis. Real-time PCR, Immunohistochemical assays (IHC) and Western blot were used to investigate the mRNA and protein levels of PELI1 in PTC. The effects of PELI1 on PTC cell progression were evaluated through CCK-8, colony formation, Transwell, and Wound healing assay in vitro, and a PTC xenograft mouse model in vivo. The downstream target signal of PELI1 in PTC was analyzed by using Kyoto encyclopedia of genes and genomes (KEGG), and bioinformatics tools were used to identify potential miRNAs targeting PELI1. Human umbilical cord mesenchymal stem cells were modified by miR-30c-5p and the miR-30c-5p containing extracellular vesicles were collected (miR-30c-5p-EVs) by ultra-high-speed centrifugation method. Then, the effects of miR-30c-5p-EVs on PELI1 expression and PTC progression were evaluated both in vitro and in vivo. Results Both mRNA and protein expression of PELI1 were widely increased in PTC tissues, and overexpression of PELI1 was positively correlated with bigger tumor size and lymph node metastases. PELI1 promoted PTC cell proliferation and migration in vitro. While, PELI1 silencing significantly suppressed PTC growth in vivo accompanied with reduced expression of Ki-67 and matrix metallopeptidase 2 (MMP-2). Mechanistically, PI3K-AKT pathway was identified as the downstream target of PELI1, and mediated the functional influence of PELI1 in PTC cells. Moreover, we found that the expression of miR-30c-5p was inversely correlated with PELI1 in PTC samples and further confirmed that miR-30c-5p was a tumor-suppressive miRNA that directly targeted PELI1 to inhibit PTC cell proliferation and migration. Furthermore, we showed that miR-30c-5p-EVs could effectively downregulate PELI1 expression and suppress the PTC cell growth in vitro and in vivo. Conclusion This study not only supported the first evidence that miR-30c-5p loss-induced PELI1 accumulation facilitated cell proliferation and migration by activating the PI3K-AKT pathway in PTC but also provided novel insights into PTC therapy based on miR-carrying-hUCMSC-EVs.