Identification of novel homozygous asthenoteratospermia-causing ARMC2 mutations associated with multiple morphological abnormalities of the sperm flagella.

PURPOSE: To identify the genetic causes of multiple morphological abnormalities in sperm flagella (MMAF) and male infertility in patients from two unrelated Han Chinese families. METHODS: Whole-exome sequencing was conducted using blood samples from the two individuals with MMAF and male infertility. Hematoxylin and eosin staining and scanning electron microscopy were performed to evaluate sperm morphology. Ultrastructural and immunostaining analyses of the spermatozoa were performed. The HEK293T cells were used to confirm the pathogenicity of the variants. RESULTS: We identified two novel homozygous missense ARMC2 variants: c.314C > T: p.P105L and c.2227A > G: p.N743D. Both variants are absent or rare in the human population genome data and are predicted to be deleterious. In vitro experiments indicated that both ARMC2 variants caused a slightly increased protein expression. ARMC2-mutant spermatozoa showed multiple morphological abnormalities (bent, short, coiled, absent, and irregular) in the flagella. In addition, the spermatozoa of the patients revealed a frequent absence of the central pair complex and disrupted axonemal ultrastructure. CONCLUSION: We identified two novel ARMC2 variants that caused male infertility and MMAF in Han Chinese patients. These findings expand the mutational spectrum of ARMC2 and provide insights into the complex causes and pathogenesis of MMAF.

Comprehensive analysis of the biological functions of endoplasmic reticulum stress in prostate cancer.

Introduction: Endoplasmic reticulum stress (ERS) has sizeable affect on cancer proliferation, metastasis, immunotherapy and chemoradiotherapy resistance. However, the effect of ERS on the biochemical recurrence (BCR) of prostate cancer patients remains elusive. Here, we generated an ERS-related genes risk signature to evaluate the physiological function of ERS in PCa with BCR. Methods: We collected the ERS-related genes from the GeneCards. The edgeR package was used to screen the differential ERS-related genes in PCa from TCGA datasets. ERS-related gene risk signature was then established using LASSO and multivariate Cox regression models and validated by GEO data sets. Nomogram was developed to assess BCR-free survival possibility. Meanwhile, the correlations between ERS-related signature, gene mutations, drug sensitivity and tumor microenvironment were also investigated. Results: We obtained an ERS risk signature consisting of five genes (AFP, COL10A1, DNAJB1, EGF and PTGS2). Kaplan Meier survival analysis and ROC Curve analysis indicated that the high risk score of ERS-related gene signature was associated with poor BCR-free prognosis in PCa patients. Besides, immune cell infiltration and immune checkpoint expression levels differed between high- and low-risk scoring subgroups. Moreover, drug sensitivity analyzed indicated that high-risk score group may be involved in apoptosis pathway. Discussion: This study comprehensively analyzed the characteristics of ERS related genes in PCa, and created a five-gene signature, which could effectively predict the BCR time of PCa patients. Targeting ERS related genes and pathways may provide potential guidance for the treatment of PCa.

The mechanosensitive lncRNA Neat1 promotes osteoblast function through paraspeckle-dependent Smurf1 mRNA retention.

Mechanical stimulation plays an important role in bone remodeling. Exercise-induced mechanical loading enhances bone strength, whereas mechanical unloading leads to bone loss. Increasing evidence has demonstrated that long noncoding RNAs (lncRNAs) play key roles in diverse biological, physiological and pathological contexts. However, the roles of lncRNAs in mechanotransduction and their relationships with bone formation remain unknown. In this study, we screened mechanosensing lncRNAs in osteoblasts and identified Neat1, the most clearly decreased lncRNA under simulated microgravity. Of note, not only Neat1 expression but also the specific paraspeckle structure formed by Neat1 was sensitive to different mechanical stimulations, which were closely associated with osteoblast function. Paraspeckles exhibited small punctate aggregates under simulated microgravity and elongated prolate or larger irregular structures under mechanical loading. Neat1 knockout mice displayed disrupted bone formation, impaired bone structure and strength, and reduced bone mass. Neat1 deficiency in osteoblasts reduced the response of osteoblasts to mechanical stimulation. In vivo, Neat1 knockout in mice weakened the bone phenotypes in response to mechanical loading and hindlimb unloading stimulation. Mechanistically, paraspeckles promoted nuclear retention of E3 ubiquitin ligase Smurf1 mRNA and downregulation of their translation, thus inhibiting ubiquitination-mediated degradation of the osteoblast master transcription factor Runx2, a Smurf1 target. Our study revealed that Neat1 plays an essential role in osteoblast function under mechanical stimulation, which provides a paradigm for the function of the lncRNA-assembled structure in response to mechanical stimulation and offers a therapeutic strategy for long-term spaceflight- or bedrest-induced bone loss and age-related osteoporosis.

Vascular smooth muscle cell-specific miRNA-214 knockout inhibits angiotensin II-induced hypertension through upregulation of Smad7.

Vascular remodeling is a prominent trait during the development of hypertension, attributable to the phenotypic transition of vascular smooth muscle cells (VSMCs). Increasing studies demonstrate that microRNA plays an important role in this process. Here, we surprisingly found that smooth muscle cell-specific miR-214 knockout (miR-214 cKO) significantly alleviates angiotensin II (Ang II)-induced hypertension, which has the same effect as that of miR-214 global knockout mice in response to Ang II stimulation. Under the treatment of Ang II, miR-214 cKO mice exhibit substantially reduced systolic blood pressure. The vascular medial thickness and area in miR-214 cKO blood vessels were obviously reduced, the expression of collagen I and proinflammatory factors were also inhibited. VSMC-specific deletion of miR-214 blunts the response of blood vessels to the stimulation of endothelium-dependent and -independent vasorelaxation and phenylephrine and 5-HT induced vasocontraction. In vitro, Ang II-induced VSMC proliferation, migration, contraction, hypertrophy, and stiffness were all repressed with miR-214 KO in VSMC. To further explore the mechanism of miR-214 in the regulation of the VSMC function, it is very interesting to find that the TGF-ß signaling pathway is mostly enriched in miR-214 KO VSMC. Smad7, the potent negative regulator of the TGF-ß/Smad pathway, is identified to be the target of miR-214 in VSMC. By which, miR-214 KO sharply enhances Smad7 levels and decreases the phosphorylation of Smad3, and accordingly alleviates the downstream gene expression. Further, Ang II-induced hypertension and vascular dysfunction were reversed by antagomir-214. These results indicate that miR-214 in VSMC established a crosstalk between Ang II-induced AT1R signaling and TGF-ß induced TßRI /Smad signaling, by which it exerts a pivotal role in vascular remodeling and hypertension and imply that miR-214 has the potential as a therapeutic target for the treatment of hypertension.

Lnc-STYK1-2 regulates bladder cancer cell proliferation, migration, and invasion by targeting miR-146b-5p expression and AKT/STAT3/NF-kB signaling.

BACKGROUND: Epigenetic modulation by noncoding RNAs substantially contributes to human cancer development, but noncoding RNAs involvement in bladder cancer remains poorly understood. This study investigated the role of long noncoding RNA (lncRNA) lnc-STYK1-2 in tumorigenesis in cancerous bladder cells. METHODS: Differential lncRNA and mRNA profiles were characterized by high-throughput RNA sequencing combined with validation via quantitative PCR. Bladder cancer cell proliferation was assessed through MTS, and bladder cancer cell migration and invasion were assessed through a Transwell system. The in vivo tumorigenesis of bladder cancer cells was evaluated using the cancer cell line-based xenograft model. The dual-luciferase reporter assay verified the association of miR-146b-5p with lnc-STYK1-2 and the target gene. Protein abundances and phosphorylation were detected by Western blotting. RESULTS: Alterations in lncRNA profiles, including decreased lnc-STYK1-2 expression, were detected in bladder cancer tissues compared with adjacent noncancerous tissues. lnc-STYK1-2 silencing effectively promoted proliferation, migration, and invasion in two bladder cancer cell lines, 5637 and T24, and their tumorigenesis in nude mice. lnc-STYK1-2 siRNA promoted miR-146b-5p and reduced ITGA2 expression in bladder cancer cells. Moreover, miR-146b-5p suppressed ITGA2 expression in bladder cancer cells through direct association. Also, lnc-STYK1-2 directly associated with miR-146b-5p. Finally, miR-146b-5p inhibitors abrogated the alterations in bladder cell functions, ITGA2 expression, and phosphorylation of AKT, STAT3, and P65 proteins in 5637 and T24 cells induced by lnc-STYK1-2 silencing. CONCLUSION: lnc-STYK1-2 inhibited bladder cancer cell proliferation, migration, and tumorigenesis by targeting miR-146b-5p to regulate ITGA2 expression and AKT/STAT3/NF-kB signaling.

3' untranslated region of Ckip-1 inhibits cardiac hypertrophy independently of its cognate protein.

AIMS: 3' untranslated region (3' UTR) of mRNA is more conserved than other non-coding sequences in vertebrate genomes, and its sequence space has substantially expanded during the evolution of higher organisms, which substantiates their significance in biological regulation. However, the independent role of 3' UTR in cardiovascular disease was largely unknown. METHODS AND RESULTS: Using bioinformatics, RNA fluorescent in situ hybridization and quantitative real-time polymerase chain reaction, we found that 3' UTR and coding sequence regions of Ckip-1 mRNA exhibited diverse expression and localization in cardiomyocytes. We generated cardiac-specific Ckip-1 3' UTR overexpression mice under wild type and casein kinase 2 interacting protein-1 (CKIP-1) knockout background. Cardiac remodelling was assessed by histological, echocardiography, and molecular analyses at 4 weeks after transverse aortic constriction (TAC) surgery. The results showed that cardiac Ckip-1 3' UTR significantly inhibited TAC-induced cardiac hypertrophy independent of CKIP-1 protein. To determine the mechanism of Ckip-1 3' UTR in cardiac hypertrophy, we performed transcriptome and metabolomics analyses, RNA immunoprecipitation, biotin-based RNA pull-down, and reporter gene assays. We found that Ckip-1 3' UTR promoted fatty acid metabolism through AMPK-PPARα-CPT1b axis, leading to its protection against pathological cardiac hypertrophy. Moreover, Ckip-1 3' UTR RNA therapy using adeno-associated virus obviously alleviates cardiac hypertrophy and improves heart function. CONCLUSIONS: These findings disclose that Ckip-1 3' UTR inhibits cardiac hypertrophy independently of its cognate protein. Ckip-1 3' UTR is an effective RNA-based therapy tool for treating cardiac hypertrophy and heart failure.

Targeting E3 Ubiquitin Ligase WWP1 Prevents Cardiac Hypertrophy Through Destabilizing DVL2 via Inhibition of K27-Linked Ubiquitination.

BACKGROUND: Without adequate treatment, pathological cardiac hypertrophy induced by sustained pressure overload eventually leads to heart failure. WWP1 (WW domain-containing E3 ubiquitin protein ligase 1) is an important regulator of aging-related pathologies, including cancer and cardiovascular diseases. However, the role of WWP1 in pressure overload-induced cardiac remodeling and heart failure is yet to be determined. METHODS: To examine the correlation of WWP1 with hypertrophy, we analyzed WWP1 expression in patients with heart failure and mice subjected to transverse aortic constriction (TAC) by Western blotting and immunohistochemical staining. TAC surgery was performed on WWP1 knockout mice to assess the role of WWP1 in cardiac hypertrophy, heart function was examined by echocardiography, and related cellular and molecular markers were examined. Mass spectrometry and coimmunoprecipitation assays were conducted to identify the proteins that interacted with WWP1. Pulse-chase assay, ubiquitination assay, reporter gene assay, and an in vivo mouse model via AAV9 (adeno-associated virus serotype 9) were used to explore the mechanisms by which WWP1 regulates cardiac remodeling. AAV9 carrying cardiac troponin T (cTnT) promoter-driven small hairpin RNA targeting WWP1 (AAV9-cTnT-shWWP1) was administered to investigate its rescue role in TAC-induced cardiac dysfunction. RESULTS: The WWP1 level was significantly increased in the hypertrophic hearts from patients with heart failure and mice subjected to TAC. The results of echocardiography and histology demonstrated that WWP1 knockout protected the heart from TAC-induced hypertrophy. There was a direct interaction between WWP1 and DVL2 (disheveled segment polarity protein 2). DVL2 was stabilized by WWP1-mediated K27-linked polyubiquitination. The role of WWP1 in pressure overload-induced cardiac hypertrophy was mediated by the DVL2/CaMKII/HDAC4/MEF2C signaling pathway. Therapeutic targeting WWP1 almost abolished TAC induced heart dysfunction, suggesting WWP1 as a potential target for treating cardiac hypertrophy and failure. CONCLUSIONS: We identified WWP1 as a key therapeutic target for pressure overload induced cardiac remodeling. We also found a novel mechanism regulated by WWP1. WWP1 promotes atypical K27-linked ubiquitin multichain assembly on DVL2 and exacerbates cardiac hypertrophy by the DVL2/CaMKII/HDAC4/MEF2C pathway.

Breast cancer exosomes contribute to pre-metastatic niche formation and promote bone metastasis of tumor cells.

Rationale: Breast cancer preferentially develops osteolytic bone metastasis, which makes patients suffer from pain, fractures and spinal cord compression. Accumulating evidences have shown that exosomes play an irreplaceable role in pre-metastatic niche formation as a communication messenger. However, the function of exosomes secreted by breast cancer cells remains incompletely understood in bone metastasis of breast cancer. Methods: Mouse xenograft models and intravenous injection of exosomes were applied for analyzing the role of breast cancer cell-derived exosomes in vivo. Effects of exosomes secreted by the mildly metastatic MDA231 and its subline SCP28 with highly metastatic ability on osteoclasts formation were confirmed by TRAP staining, ELISA, microcomputed tomography, histomorphometric analyses, and pit formation assay. The candidate exosomal miRNAs for promoting osteoclastogenesis were globally screened by RNA-seq. qRT-PCR, western blot, confocal microscopy, and RNA interfering were performed to validate the function of exosomal miRNA. Results: Implantation of SCP28 tumor cells in situ leads to increased osteoclast activity and reduced bone density, which contributes to the formation of pre-metastatic niche for tumor cells. We found SCP28 cells-secreted exosomes are critical factors in promoting osteoclast differentiation and activation, which consequently accelerates bone lesion to reconstruct microenvironment for bone metastasis. Mechanistically, exosomal miR-21 derived from SCP28 cells facilitates osteoclastogenesis through regulating PDCD4 protein levels. Moreover, miR-21 level in serum exosomes of breast cancer patients with bone metastasis is significantly higher than that in other subpopulations. Conclusion: Our results indicate that breast cancer cell-derived exosomes play an important role in promoting breast cancer bone metastasis, which is associated with the formation of pre-metastatic niche via transferring miR-21 to osteoclasts. The data from patient samples further reflect the significance of miR-21 as a potential target for clinical diagnosis and treatment of breast cancer bone metastasis.

Ginsenoside Re Treatment Attenuates Myocardial Hypoxia/Reoxygenation Injury by Inhibiting HIF-1α Ubiquitination.

Previous studies have shown an attenuating effect of ginsenoside Re on myocardial injury induced by hypoxia/reoxygenation (H/R). However, the underlying mechanism remains unclear. This study was designed to determine the underlying mechanism by which ginsenoside Re protects from myocardial injury induced by H/R. HL-1 cells derived from AT-1 mouse atrial cardiomyocyte tumor line were divided into control, H/R, and H/R + ginsenoside Re groups. Cell viability was measured by CCK-8 assay. ATP levels were quantified by enzymatic assays. Signaling pathway was predicted by network pharmacology analyses and verified by luciferase assay and gene-silencing experiment. The relationship between ginsenoside Re and its target genes and proteins was analyzed by docking experiments, allosteric site analysis, real-time PCR, and ubiquitination and immunoprecipitation assays. Our results showed that ginsenoside Re treatment consistently increased HL-1 cell viability and significantly up-regulated ATP levels after H/R-induced injury. Network pharmacology analysis suggested that the effect of ginsenoside Re was associated with the regulation of the Hypoxia-inducing factor 1 (HIF-1) signaling pathway. Silencing of HIF-1α abrogated the effect of ginsenoside Re on HL-1 cell viability, which was restored by transfection with an HIF-1α-expressing plasmid. Results of the bioinformatics analysis suggested that ginsenoside Re docked at the binding interface between HIF-1α and the von Hippel-Lindau (VHL) E3 ubiquitin ligase, preventing VHL from binding HIF-1α, thereby inhibiting the ubiquitination of HIF-1α. To validate the results of the bioinformatics analysis, real-time PCR, ubiquitination and immunoprecipitation assays were performed. Compared with the mRNA expression levels of the H/R group, ginsenoside Re did not change expression of HIF-1α mRNA, while protein level of HIF-1α increased and that of HIF-1α[Ub]n decreased following ginsenoside Re treatment. Immunoprecipitation results showed that the amount of HIF-1α bound to VHL substantially decreased following ginsenoside Re treatment. In addition, ginsenoside Re treatment increased the expression of GLUT1 (glucose transporter 1) and REDD1 (regulated in development and DNA damage response 1), which are targets of HIF-1α and are critical for cell metabolism and viability. These results suggested that Ginsenoside Re treatment attenuated the myocardial injury induced by H/R, and the possible mechanism was associated with the inhibition of HIF-1α ubiquitination.

MiR-200a Regulates Nasopharyngeal Carcinoma Cell Migration and Invasion by Targeting MYH10.

Nasopharyngeal carcinoma (NPC), is one of the most common malignant tumor in southern China and southeast Asia. MYH10 is a coding gene of the NMMHC-IIB protein. Previous studies have shown that MYH10 expression was up-regulated in breast cancer, glioma and meningioma. Moreover, it was targeted by miR200 family. However, no relevant studies have been found in NPC. In present study, we found in 48 NPC specimens, MYH10 level was lower in most cancer areas than that in the adjacent normal tissue. Moreover, the depletion of MYH10 can promote the migration and invasion of NPC. In addition, we demonstrated that miR-200a has the strongest regulation to MYH10 among miR-200 family. miR-200a mimics could decrease MYH10 expression, while miR-200a inhibitor increase MYH10 expression. Next, we found that miR-200a bound directly to MYH10 using Dual-luciferase reporter. Finally, it was demonstrated that siMYH10 could reverse the effect of miR-200a inhibitor on NPC cell migration and invasion. Taken together, it can be concluded that MYH10 is lowly expressed in NPC compared with adjacent tissues, and the loss of MYH10 can promote the migration and invasion of NPC cells; Among the miR-200 family, miR-200a has the strongest regulatory effect on MYH10; MYH10 is a direct target gene of miR200a, and miR200a targets MYH10 to regulate the migration and invasion of NPC cells.

A multicenter randomized trial of personalized acupuncture, fixed acupuncture, letrozole, and placebo letrozole on live birth in infertile women with polycystic ovary syndrome.

BACKGROUND: Traditional Chinese medicine (TCM) usually involves syndrome differentiation and treatment. Acupuncture, one form of TCM, requires the selection of appropriate acupoints and needling techniques, but many clinical trials on acupuncture have used fixed acupuncture protocols without accounting for individual patient differences. We have designed a multicenter randomized controlled trial (RCT) to evaluate whether personalized or fixed acupuncture increases the likelihood of live births in infertile women with polycystic ovary syndrome (PCOS) compared with letrozole or placebo letrozole. We hypothesize that letrozole is more effective than personalized acupuncture, which in turn is more effective than fixed acupuncture, and that placebo letrozole is the least effective intervention. Moreover, we hypothesize that personalized acupuncture is more likely to reduce the miscarriage rate and the risk of pregnancy complications compared with letrozole. METHODS/DESIGN: The study is designed as an assessor-blinded RCT. A total of 1100 infertile women with PCOS will be recruited from 28 hospitals and randomly allocated to 4 groups: personalized acupuncture, fixed acupuncture, letrozole, or placebo letrozole. They will receive treatment for 16 weeks, and the primary outcome is live birth. Secondary outcomes include ovulation rate, conception rate, pregnancy rate, pregnancy loss rate, changes in hormonal and metabolic parameters, and changes in quality of life scores. Adverse events will be recorded throughout the trial. All statistical analyses will be performed using IBM SPSS Statistics version 21.0 software (IBM Corp., Armonk, NY, USA), and a P value < 0.05 will be considered statistically significant. DISCUSSION: This study will be the first multicenter RCT to compare the effect of personalized or fixed acupuncture with letrozole or placebo letrozole on live birth in infertile women with PCOS. The findings will inform whether personalized acupuncture therapy can be considered an alternative treatment to improve the live birth rate in infertile women with PCOS. TRIAL REGISTRATION: ClinicalTrials.gov, NCT03625531. Registered on July 13, 2018. Chinese Clinical Trial Registry, ChiCTR1800017304. Registered on July 23, 2018.

Alteration of calcium signalling in cardiomyocyte induced by simulated microgravity and hypergravity.

OBJECTIVES: Cardiac Ca2+ signalling plays an essential role in regulating excitation-contraction coupling and cardiac remodelling. However, the response of cardiomyocytes to simulated microgravity and hypergravity and the effects on Ca2+ signalling remain unknown. Here, we elucidate the mechanisms underlying the proliferation and remodelling of HL-1 cardiomyocytes subjected to rotation-simulated microgravity and 4G hypergravity. MATERIALS AND METHODS: The cardiomyocyte cell line HL-1 was used in this study. A clinostat and centrifuge were used to study the effects of microgravity and hypergravity, respectively, on cells. Calcium signalling was detected with laser scanning confocal microscopy. Protein and mRNA levels were detected by Western blotting and real-time PCR, respectively. Wheat germ agglutinin (WGA) staining was used to analyse cell size. RESULTS: Our data showed that spontaneous calcium oscillations and cytosolic calcium concentration are both increased in HL-1 cells after simulated microgravity and 4G hypergravity. Increased cytosolic calcium leads to activation of calmodulin-dependent protein kinase II/histone deacetylase 4 (CaMKII/HDAC4) signalling and upregulation of the foetal genes ANP and BNP, indicating cardiac remodelling. WGA staining indicated that cell size was decreased following rotation-simulated microgravity and increased following 4G hypergravity. Moreover, HL-1 cell proliferation was increased significantly under hypergravity but not rotation-simulated microgravity. CONCLUSIONS: Our study demonstrates for the first time that Ca2+ /CaMKII/HDAC4 signalling plays a pivotal role in myocardial remodelling under rotation-simulated microgravity and hypergravity.

Lnc-MUC20-9 binds to ROCK1 and functions as a tumor suppressor in bladder cancer.

The study aimed to investigate the expression and function of bladder cancer (BC) long noncoding RNAs (lncRNAs) using a high-throughput platform. High-throughput sequencing was used to compare the expression profiles of lncRNA in BC and adjacent normal tissues. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR), in situ hybridization, gene ontology, and Kyoto Encyclopedia of Genes and Genomes analysis were performed to verify differential expression of lncRNA. The effect of lncRNA overexpression on cellular proliferation, apoptosis, migration, and invasion was analyzed following the transfection of lncRNA overexpressing lentivirus into 5637 and T24 cell lines. The overexpressing cells were subcutaneously injected into nude mice to evaluate their effects on tumor growth. Tumor-associated RNA-binding proteins of lncRNA were analyzed by RNA pull-down combined with mass spectrometry. A total of 93 lncRNA genes were upregulated and 352 lncRNA genes were downregulated in the tissues of patients with BC. Of which, we investigated the function of downregulated lnc-MUC20-9. Overexpression of lnc-MUC20-9 in 5637 and T24 cells resulted in decreased tumor cell viability and cell clones, decreased migration and invasion, and increased apoptosis. Similarly, nude mice bearing lnc-MUC20-9 overexpressing tumor cells exhibited smaller tumor size and volume than that of mice bearing control cells. Mass spectrometry analysis showed that lnc-MUC20-9 binds to ROCK1, an oncogene whose expression was decreased in lnc-MUC20-9 overexpressing cells. The study revealed that lnc-MUC20-9 has the function of inhibiting tumor growth, migration, and invasion. In BC cells, lnc-MUC20-9 binds to ROCK1 and may be involved in lnc-MUC20-9-mediated tumor suppressor function, which might be potential therapeutic targets for BC.

The analysis of a ceRNA network and the correlation between lncRNA, miRNA, and mRNA in bladder cancer.

BACKGROUND: To explore the correlation between the lncRNA-miRNA-mRNA and ceRNA network through the differential expression analysis of lncRNAs, miRNAs and mRNAs in bladder cancer based on The Cancer Genome Atlas (TCGA) database combined with Gene Ontology (GO) and Kyoto Encyclopedia of Genes Genomes (KEGG) enrichment analysis. METHODS: Firstly, the expression profile data and corresponding clinical data of RNAs in bladder cancer were searched and downloaded from TCGA database, and aberrantly expressed long non-coding RNA (lncRNA), microRNA (miRNA), and messenger RNA (mRNA) were screened and found by using TCGA database. The relationship between lncRNA-miRNA-mRNA was established by comparing these lncRNAs, miRNAs, and mRNAs, while the ceRNA network was constructed. Combined with the analysis of the GO annotation and KEGG pathway, the effects of lncRNA-miRNA-mRNA interaction on the development of bladder cancer were explored. RESULTS: A total of 1,742 differentially expressed lncRNA, 511 differentially expressed miRNAs, and 4,373 differentially expressed mRNAs were identified, and 328 lncRNAs, 73 miRNAs, and 677 mRNAs were screened by survival analysis. With the lncRNA-miRNA-mRNA correlation analysis, a ceRNA network consisting of 45 lncRNAs, 14 miRNAs, and 29 mRNAs was successfully constructed. The GO annotation and functional enrichment of target gene mRNAs in the network are mainly concentrated in the signal pathways and include fatty acid biosynthesis, gap junction, insulin signaling pathway, and the MAPK signaling pathway biological processes such as positive regulation of cellular process and system development. CONCLUSIONS: We successfully identified the target gene correlating lncRNA, miRNA, and mRNA, and constructed a ceRNA network. Our findings can provide a potential target for the study of the occurrence, development, diagnosis, treatment, and prognosis of bladder cancer.

The mechanosensitive Piezo1 channel is required for bone formation.

Mechanical load of the skeleton system is essential for the development, growth, and maintenance of bone. However, the molecular mechanism by which mechanical stimuli are converted into osteogenesis and bone formation remains unclear. Here we report that Piezo1, a bona fide mechanotransducer that is critical for various biological processes, plays a critical role in bone formation. Knockout of Piezo1 in osteoblast lineage cells disrupts the osteogenesis of osteoblasts and severely impairs bone structure and strength. Bone loss that is induced by mechanical unloading is blunted in knockout mice. Intriguingly, simulated microgravity treatment reduced the function of osteoblasts by suppressing the expression of Piezo1. Furthermore, osteoporosis patients show reduced expression of Piezo1, which is closely correlated with osteoblast dysfunction. These data collectively suggest that Piezo1 functions as a key mechanotransducer for conferring mechanosensitivity to osteoblasts and determining mechanical-load-dependent bone formation, and represents a novel therapeutic target for treating osteoporosis or mechanical unloading-induced severe bone loss.

Panax quinquefolium saponin attenuates cardiac remodeling induced by simulated microgravity.

BACKGROUND: Cardiac atrophy and reduced cardiac distensibility have been reported following space flight. Cardiac function is correspondingly regulated in response to changes in loading conditions. Panax quinquefolium saponin (PQS) improves ventricular remodeling after acute myocardial infarction by alleviating endoplasmic reticulum stress and Ca2+overload. However, whether PQS can ameliorate cardiac atrophy following exposure to simulated microgravity remains unknown. PURPOSE: To explore the protective role of PQS in cardiac remodeling under unloading conditions and its underlying mechanisms. METHODS: Hindlimb unloading (HU) model was used to simulate unloading induced cardiac remodeling. Forty-eight male rats were randomly assigned to four groups, including control, PQS, HU and HU + PQS. At 8 weeks after the experiment, cardiac structure and function, serum levels of Creatine Kinase-MB (CK-MB), Cardiactroponin T (cTnT), ischemia modified albumin (IMA), and cardiomyocyte apoptosis were measured. Network pharmacology analysis was used to predict the targets of the six major constituents of PQS, and the signaling pathways they involved in were analyzed by bioinformatics methods. Changes in the key proteins involved in the protective effects of PQS were further confirmed by Western Blot. RESULTS: Simulated microgravity led to increases in serum levels of CK-MB, cTnT and IMA, remodeling of cardiac structure, impairment of cardiac function, and increased cardiomyocyte apoptosis as compared with control. PQS treatment significantly reduced serum levels of CK-MB, cTnT and IMA, improved the impaired cardiac structure and function, and decreased cardiomyocyte apoptosis induced by unloading. The activation of AMPK and inhibition of Erk1/2 and CaMKII/HDAC4 were demonstrated in the cardiocytes of HU rats after PQS treatment. CONCLUSION: PQS provides protection against cardiac remodeling induced by simulated microgravity, partly resulting from changes in the signaling pathways related to energy metabolism reduction, calcium overloading and cell apoptosis.

Long Noncoding RNA PRRG4-4 Promotes Viability, Cell Cycle, Migration, and Invasion in Lung Cancer Cells.

There is a perception that long noncoding RNA (lncRNA) has relationship with carcinogenesis. Many studies have previously identified and validated that the section of chromosome 11p13 is associated with high incidence of tumor. In this study, we investigated a new lncRNA, named lncPRRG4-4, mapped to 11p13 and suspected that lncPRRG4-4 was a potential lung cancer-related gene. To explore its role in carcinogenesis, we first demonstrated that lncPRRG4-4 was upregulated in lung cancer tissues compared with adjacent nontumor tissues and functioned as an oncogene in lung cancer cells. The lncPRRG4-4 was significantly upregulated in lung cancer tissues compared with adjacent normal counterparts (mean ± standard deviation: 0.12 ± 0.84 vs. 0.05 ± 0.22; p < 0.001). Patients with metastasis exhibited high levels of lncPRRG4-4 expression than those without metastasis in both the southern samples (p = 0.045) and eastern samples (p = 0.030), total samples (p = 0.004). In addition, downregulation of lncPRRG4-4 expression inhibited lung cancer proliferation, viability, migration, and invasion ability, arrested cell cycle, facilitated apoptosis, and vice versa. Taken together, these observations suggested that the lncPRRG4-4 functions as an oncogene in lung cancer cells.

Upregulation of LncRNA FEZF-AS1 is associated with advanced clinical stages and family history of cancer in patients with NSCLC.

Antisense RNA (AS) is a type of long non-coding RNAs that functions as a post-transcriptional regulatory element on regulating parental coding gene expression via directly binding to complementary mRNA sequences. We aimed to investigate the effect of the AS to FEZF1 gene on non-small cell lung cancer (NSCLC) development. The expression level of lncRNA FEZF-AS1 and FEZF1 was determined by the quantitative Real-time PCR in 160 cases of NSCLC tissues and their adjacent non-tumour tissues. We found that lncRNA FEZF-AS1 was significantly up-regulated in tumour tissues when compared to the adjacent non-cancerous tissues (P = 0.001), and it's high expression correlated with advanced stages (P = 0.002) and Tumour Family History (P = 0.029). Meanwhile, In 58 cases of NSCLC tissues the expression of lncRNA FEZF-AS1 was positively associated with that of FEZF1expression (r = 0.8810, p = 1.6575E-20). By GEPIA database analysis, we also found that the expression of lncRNA FEZF-AS1 and FEZF1 were significantly higher in tumour tissues than those of the adjacent non-cancerous tissues in 969 NSCLC patients (P < 0.05), and lncRNA FEZF-AS1 was positively correlated with FEZF1 (r = 0.90, P < 0.001). These results suggest that lncRNA FEZF-AS1 relate to the progression of lung cancer patients and it may be a potential target for cancer therapy.

A Peptide Encoded by a Putative lncRNA HOXB-AS3 Suppresses Colon Cancer Growth.

A substantial fraction of eukaryotic transcripts are considered long non-coding RNAs (lncRNAs), which regulate various hallmarks of cancer. Here, we discovered that the lncRNA HOXB-AS3 encodes a conserved 53-aa peptide. The HOXB-AS3 peptide, not lncRNA, suppresses colon cancer (CRC) growth. Mechanistically, the HOXB-AS3 peptide competitively binds to the ariginine residues in RGG motif of hnRNP A1 and antagonizes the hnRNP A1-mediated regulation of pyruvate kinase M (PKM) splicing by blocking the binding of the ariginine residues in RGG motif of hnRNP A1 to the sequences flanking PKM exon 9, ensuring the formation of lower PKM2 and suppressing glucose metabolism reprogramming. CRC patients with low levels of HOXB-AS3 peptide have poorer prognoses. Our study indicates that the loss of HOXB-AS3 peptide is a critical oncogenic event in CRC metabolic reprogramming. Our findings uncover a complex regulatory mechanism of cancer metabolism reprogramming orchestrated by a peptide encoded by an lncRNA.