Activation of the HNRNPA2B1/miR-93-5p/FRMD6 axis facilitates prostate cancer progression in an m6A-dependent manner.

It is becoming increasingly clear that N6-methyladenosine (m6A) plays a key role in post-transcriptional modification of eukaryotic RNAs in cancer. The regulatory mechanism of m6A modifications in prostate cancer is still not completely elucidated. Heterogeneous nuclear ribonucleoprotein A2/B1 (HNRNPA2B1), an m6A reader, has been revealed to function as an oncogenic RNA-binding protein. However, its contribution to prostate cancer progression remains poorly understood. Here, we found that HNRNPA2B1 was highly overexpressed and correlated with a poor prognosis in prostate cancer. In vitro and in vivo functional experiments demonstrated that HNRNPA2B1 knockout impaired proliferation and metastasis of prostate cancer. Mechanistic studies indicated that HNRNPA2B1 interacted with primary miRNA-93 and promoted its processing by recruiting DiGeorge syndrome critical region gene 8 (DGCR8), a key subunit of the Microprocessor complex, in an METTL3-dependent mechanism, while HNRNPA2B1 knockout significantly restored miR-93-5p levels. HNRNPA2B1/miR-93-5p downregulated FERM domain-containing protein 6 (FRMD6), a cancer suppressor, and enhanced proliferation and metastasis in prostate cancer. In conclusion, our findings identified a novel oncogenic axis, HNRNPA2B1/miR-93-5p/FRMD6, that stimulates prostate cancer progression via an m6A-dependent manner.

LncFALEC recruits ART5/PARP1 and promotes castration-resistant prostate cancer through enhancing PARP1-meditated self PARylation.

Accumulating evidence indicates that long noncoding RNAs (lncRNAs) are abnormal expression in various malignant tumors. Our previous research demonstrated that focally amplified long non-coding RNA (lncRNA) on chromosome 1 (FALEC) is an oncogenic lncRNA in prostate cancer (PCa). However, the role of FALEC in castration-resistant prostate cancer (CRPC) is poorly understood. In this study, we showed FALEC was upregulated in post-castration tissues and CRPC cells, and increased FALEC expression was associated with poor survival in post-castration PCa patients. RNA FISH demonstrated FALEC was translocated into nucleus in CRPC cells. RNA pulldown and followed Mass Spectrometry (MS) assay demonstrated FALEC directly interacted with PARP1 and loss of function assay showed FALEC depletion sensitized CRPC cells to castration treatment and restored NAD+. Specific PARP1 inhibitor AG14361 and NAD+ endogenous competitor NADP+ sensitized FALEC-deleted CRPC cells to castration treatment. FALEC increasing PARP1 meditated self PARylation through recruiting ART5 and down regulation of ART5 decreased CRPC cell viability and restored NAD+ through inhibiting PARP1meditated self PARylation in vitro. Furthermore, ART5 was indispensable for FALEC directly interaction and regulation of PARP1, loss of ART5 impaired FALEC and PARP1 associated self PARylation. In vivo, FALEC depleted combined with PARP1 inhibitor decreased CRPC cell derived tumor growth and metastasis in a model of castration treatment NOD/SCID mice. Together, these results established that FALEC may be a novel diagnostic marker for PCa progression and provides a potential new therapeutic strategy to target the FALEC/ART5/PARP1 complex in CRPC patients.

Genome-Wide Identification and Expression Analysis of Senescence-Associated Genes in Grapevine (Vitis vinifera L.) Reveal Their Potential Functions in Leaf Senescence Order.

Natural leaf senescence is an acclimation strategy that enables plants to reallocate nutrients. In the present study, interestingly, we found that the basal mature leaves of grapevine primary shoots (P) exhibited the earliest senescence, followed by the apical young leaves of secondary shoots (ST), and then the basal mature leaves of secondary shoots (S). The Chl level decreased with the extent of leaf senescence. According to the genome-wide identification and expression analysis, sixteen senescence-associated genes (SAGs) involved in Chl breakdown were identified in the grapevine genome. Their expression patterns showed that the transcript changes in VvSGR, VvPPH2, and VvFtsH6-2 corresponded to the changes in Chl content among P, S, and ST. The changes in the transcription of VvNYC1, VvSGR, VvPAO1, VvPAO2, VvPAO4, VvPPH1, VvPPH3, and VvFtsH6-1 only contributed to low Chl levels in P. The cis-element analysis indicated that these SAGs possessed several light- and hormone-responsive elements in their promoters. Among them, ABA-responsive elements were found in twelve of the sixteen promoters of SAGs. Correspondingly, ABA-signaling components presented various changes in transcription among P, S, and ST. The transcription changes in VvbZIP45 and VvSnRK2.1 were similar to those in VvSGR, VvPPH2, and VvFtsH6-2. The other nine ABA-signaling components, which included VvRCAR2, VvRCAR4, VvRCAR6, VvRCAR7, VvRCAR2, VvPP2C4, VvPP2C9, VvbZIP25, and VvSnRK2.3, were highly expressed in P but there was no difference between S and ST, with similar expression patterns for VvNYC1, VvSGR, VvPAO1, VvPAO2, VvPAO4, VvPPH1, VvPPH3, and VvFtsH6-1. These results suggested that the senescence of P and ST could be regulated by different members of Chl breakdown-related SAGs and ABA-signaling components. These findings provide us with important candidate genes to further study the regulation mechanism of leaf senescence order in grapevine.

Tumor-associated macrophages in direct contact with prostate cancer cells promote malignant proliferation and metastasis through NOTCH1 pathway.

Background: M2 macrophages are well accepted to promote cancer progression in the prostate cancer (PCa). Paracrine is the principally studied mode of communication between M2 macrophages and tumor cells. In addition to this, we present here a novel model to demonstrate these cellular communications. Methods: PCa cells were co-cultured with THP-1/ human peripheral blood mononuclear cells derived M2 macrophages in direct contact manner. Cancer cell proliferation and invasion were examined to explain how direct contact communicates. Cell-based findings were validated in two xenograft models and patients samples. Results: M2 macrophage direct contact induced a higher proliferation and invasion in PCa cells when compared with noncontact coculture manner. In direct contact manner, NOTCH1 pathway was greatly activated in PCa cells, induced by elevated γ-secretase activity and higher coactivator MAML2 expression. Additionally, blocking γ-secretase activity and depletion of MAML2 completely abolished M2 macrophage direct contact-mediated PCa cell proliferation and invasion. In vivo, inhibiting NOTCH1 signalling impaired M2 macrophage-mediated PCa tumor growth and lung metastasis. Notably, M2 macrophage infiltration as well as high NOTCH1 signaling in cancer cells indicated more aggressive features and worse survival in PCa patients. Conclusion: Our results demonstrated the cell-cell direct contact pattern is an important way in PCa microenvironment cell communication. In this manner, elevated γ-secretase activity and MAML2 expression induced higher NOTCH1 signalling in PCa cells, which increased tumor cells proliferation and invasion. This potentially provided a therapeutic target for PCa.

Cepharanthine Ameliorates Chondrocytic Inflammation and Osteoarthritis via Regulating the MAPK/NF-κB-Autophagy Pathway.

Osteoarthritis is a worldwide joint disease caused by abnormal chondrocytic metabolism. However, traditional therapeutic methods aimed at anti-inflammation for early-stage disease are palliative. In the present study, we demonstrated that cepharanthine (CEP), extracted from the plant Stephania cepharantha, exerted protective medicinal efficacy on osteoarthritis for the first time. In our in vitro study, CEP suppressed the elevated expression of matrix metalloproteinases (MMPs), a disintegrin and metalloproteinase with thrombospondin motifs 5 (ADAMTS5) and inducible nitric oxide synthase (iNOS) stimulated by IL-1ß or TNF-α by inhibiting the activation of MAPK and NF-κB signaling pathways, and upregulated the protein expression of aggrecan, collagen II, and Sox9. Also, CEP could reverse the reduced level of cellular autophagy in IL-1ß or TNF-α-induced chondrocytes, indicating that the protective effect of CEP on osteoarthritis was achieved by restoring MAPK/NF-κB-mediated autophagy. Furthermore, in a murine OA model, CEP mitigated cartilage degradation and prevented osteoarthritis in the CEP-treated groups versus the OA group. Hence, our results revealed the therapeutic prospect of CEP for anti-osteoarthritic treatment.

Therapeutic Potential of POU3F3, a Novel Long Non-coding RNA, Alleviates the Pathogenesis of Osteoarthritis by Regulating the miR-29a- 3p/FOXO3 Axis.

BACKGROUND: Osteoarthritis (OA) is the predominant threat to the health of the elderly, and it is crucial to understand the molecular pathogenetic mechanisms involved in it. This study aims to investigate the role of a well-studied cancer-related long non-coding RNA (lncRNA)-POU3F3 in OA and its implicated molecular mechanisms. METHODS: The expression of POU3F3 and miR-29a-3p was examined in osteoarthritis patients, as well as destabilization of the medial meniscus (DMM) mouse OA model and IL- 1ß induced chondrocytes cell OA model, by quantitative real-time PCR. The interaction between POU3F3, miR-29a-3p and transcription factor forkhead box O3 (FOXO3) was verified via dual-luciferase reporter analysis and RNA immunoprecipitation analyses. Cell proliferation and apoptosis were evaluated by cell viability assay and flow cytometry, respectively. Cartilage extracellular matrix (ECM) degradation was investigated with ELISA and western blotting. In addition, the in vivo regulation of POU3F3 in OA was verified by intra-articular injection of lentivirus overexpression POU3F31 in mice models. RESULTS: The expression level of POU3F3 was decreased in OA patients/animal cartilage tissues and IL-1ß-stimulated in vitro chondrocyte model. POU3F3 overexpression inhibited IL-1ß-induced injury of chondrocytes, enhancing cell viability, suppressing apoptosis and inflammatory cytokine secretion, rescuing metabolic dysfunction, and restraining autophagy in vitro. Mechanistically, Luciferase reporter and RNA immunoprecipitation (RIP) assays indicated that miR-29a-3p could directly bind to POU3F3, and FOXO3 was a target gene of miR-29a-3p. Functional rescue assays confirmed this POU3F3/miR-29a-3p/FOXO3 axis in chondrocytes during OA occurrence. Furthermore, intraarticularly delivery of lentivirus containing POU3F3 alleviates the damage in mouse OA model in vivo. CONCLUSION: In conclusion, this work highlights the role of the POU3F3/miR-29a-3p/FOXO3 axis in the OA pathogenesis, suggesting this axis as a potential therapeutic target for OA.

Transcription Profiles Reveal Age-Dependent Variations of Photosynthetic Properties and Sugar Metabolism in Grape Leaves (Vitis vinifera L.).

Leaves, considered as the 'source' organs, depend on the development stages because of the age-dependent photosynthesis and assimilation of leaves. However, the molecular mechanisms of age-dependent limitations on the function of leaves are seldom reported. In the present study, the photosynthesis-related characteristics and photoassimilates were investigated in grape leaves at six different age groups (Ll to L6) at micro-morphological, biochemical, and molecular levels. These results showed lower expression levels of genes associated with stomatal development, and chl biosynthesis resulted in fewer stomata and lowered chlorophyll a/b contents in L1 when compared to L3 and L5. The DEGs between L5 and L3/L1 were largely distributed at stomatal movement, carbon fixation, and sucrose and starch metabolism pathways, such as STOMATAL ANION CHANNEL PROTEIN 1 (SLAC1), FRUCTOSE-1,6-BISPHOSPHATE ALDOLASE (FBA1), SUCROSE-PHOSPHATE SYNTHASE (SPP1), and SUCROSE-PHOSPHATE PHOSPHATASE (SPS2, 4). These genes could be major candidate genes leading to increased photosynthesis capacity and sugar content in L5. The accumulation of starch grains in the chloroplast and palisade tissue of L5 and higher transcription levels of genes related to starch biosynthesis in L5 further supported the high ability of L5 to produce photoassimilates. Hence, our results provide insights for understanding different photosynthetic functions in age-dependent leaves in grape plants at the molecular level.

Long Noncoding RNA PVT1 Promotes Prostate Cancer Metastasis by Increasing NOP2 Expression via Targeting Tumor Suppressor MicroRNAs.

BACKGROUND: Metastatic disease caused by prostate cancer (PCa) is the principal cause of PCa-related mortality. Long non-protein-coding RNAs may possess significant cellular functions. Plasmacytoma variant translocation 1 (PVT1), a long non-coding RNA encoded by the human PVT1 gene, is an oncogene, which can regulate several tumor-related genes. In PCa, the function and mechanism of PVT1 are unclear. NOP2 is being pursued as a prognostic marker for cancer aggressiveness, which promotes mouse fibroblast growth and tumor formation. Essentially, nothing is known about the specific interactions between the PVT1 and NOP2. METHODS:  190 pairs of PCa tissues and adjacent normal tissues were collected and RNA sequencing was used to identify the differential lncRNAs. Real-time quantitative real-time PCR (RT-qPCR) confirmed these results and gene regulatory relationship. Lentiviral vectors were used to alter PVT1 and genes to analyze their effects on PCa progression. Transwell migration and invasion assays were performed to test the metastasis ability. Biofunction of PVT1 and NOP2 were confirmed in vitro and in vivo. RESULTS: In this study, we reported that the long noncoding RNA-PVT1 was upregulated in PCa metastasis tissues and promoted migration of PCa cells in vitro and their metastasis in vivo. High levels of PVT1 significantly downregulated tumor suppressor microRNAs (miRNAs), such as miR-15b-5p, miR-27a-3p, miR-143-3p, and miR-627-5p, whose levels in metastasis tissues were low compared to those in non-metastasis tissues. In vitro and in vivo, PVT1 promotes PCa metastasis via targeting miRNAs. Furthermore, the expression level of PVT1 was positively associated with the expression of NOP2, a cancer metastasis-related protein. We demonstrated that NOP2 promoted invasion and migration of PCa. For specific mechanism, correlation analysis showed that PVT1 promoted metastasis by up-regulating NOP2. CONCLUSION: Taken together, our results show that PVT1 acts as an inducer of PCa metastasis via targeting miRNAs, thereby promoting NOP2. This axis may have diagnostic and therapeutic potential for advanced PCa.

Long noncoding RNA LINC00963 induces NOP2 expression by sponging tumor suppressor miR-542-3p to promote metastasis in prostate cancer.

Metastatic disease caused by castration-resistant prostate cancer (CRPC) is the principal cause of prostate cancer (PCa)-related mortality. CRPC occurs within 2-3 years of initiation of androgen deprivation therapy (ADT), which is an important factor of influencing PCa metastasis. Recent studies have revealed that non-coding RNAs in PCa can enhance metastasis and progression, while the mechanisms are still unclear. In this study, we reported that the long noncoding RNA-LINC00963 was increased in CRPC tissues and promoted migration of PCa cells in vitro and their metastasis in vivo. High levels of LINC00963 significantly decreased tumor suppressor miR-542-3p, whose levels in metastasis tissues were low compared to those in non-metastasis tissues. LINC00963 promotes and miR-542-3p inhibits metastasis. Furthermore, the expression levels of LINC00963 and miR-542-3p were positively and negatively associated with the expression of NOP2. We demonstrated that NOP2 promoted PCa by activating the epithelial-mesenchymal transition (EMT) pathway. For specific mechanism, dual luciferase reporter assays showed that miR-542-3p directly binds to both 3'-untranslated region (UTR) of LINC00963 and NOP2 mRNA. Taken together, our results show that LINC00963 acts as an inducer of PCa metastasis by binding miR-542-3p, thereby promoting NOP2. This axis may have diagnostic and therapeutic potential for advanced PCa.

CAFs-derived MFAP5 promotes bladder cancer malignant behavior through NOTCH2/HEY1 signaling.

Cancer-associated fibroblasts (CAFs) are an important component of the tumor microenvironment and contribute to tumor cell proliferation and metastasis. Microfibrillar-associated protein 5 (MFAP5), a component of elastic microfibers and an oncogenic protein in several types of tumors, is secreted by CAFs. However, the role of MFAP5 in the bladder cancer remains unclear. Here, we report that MFAP5 is upregulated in bladder cancer and is associated with poor patient survival. Downregulation of MFAP5 in CAFs led to an impairment in proliferation and invasion of bladder cancer cells. Luciferase reporter assays and electrophoretic mobility shift assays (EMSA) showed QKI directly downregulates MFAP5 in CAFs. In addition, CAFs-derived MFAP5 led to an activation of the NOTCH2/HEY1 signaling pathway through direct interaction with the NOTCH2 receptor, thereby stimulating the N2ICD release. RNA-sequencing revealed that MFAP5-mediated PI3K-AKT signaling activated the DLL4/NOTCH2 pathway axis in bladder cancer. Moreover, downregulation of NOTCH2 by short hairpin RNA or the inactivating anti-body NRR2Mab was able to reverse the adverse effects of MFAP5 stimulation in vitro and in vivo. Together, these results demonstrate CAFs-derived MFAP5 promotes the bladder cancer proliferation and metastasis and provides new insight for targeting CAFs as novel diagnostic and therapeutic strategy.

Rebamipide-loaded chitosan nanoparticles accelerate prostatic wound healing by inhibiting M1 macrophage-mediated inflammation via the NF-κB signaling pathway.

A large proportion of benign prostatic hyperplasia (BPH) patients suffer from lower urinary tract symptoms after surgery due to the presence of prostatic urothelium wounds. Rebamipide (RBM) exerts wound healing promotion and anti-inflammatory effects on various tissues, including the urothelium. However, intravesical administration of RBM is hindered due to its low solubility and resulting unsustainable drug concentrations in the bladder. In this study, RBM-loaded chitosan nanoparticles (RBM/CTS NPs) were prepared using the ionic cross-linking method. Physicochemical characteristics and the wound healing promotion effect, as well as in vitro influence on macrophages were evaluated. The results show that RBM/CTS NPs are spherical with uniform size distribution, while slower and sustained in vitro release of RBM is presented. In vivo, faster wound healing and improved re-epithelialization progress were observed after treatment with RBM/CTS NPs in a model of thulium laser resection of the prostate (TmLRP). The degree of local inflammatory response decreased, as confirmed by decreasing numbers of pro-inflammatory M1 phenotype macrophages and levels of IL-1ß, IL-6, IL-12 and TNF-α in the urine of canines. We also found that RBM/CTS NPs suppress macrophage M1 polarization induced by lipopolysaccharide and interferon-γ and inhibit the activation of the NF-κB signaling pathway. Therefore, as a novel therapeutic strategy, intravesical administration of RBM/CTS NPs can effectively avoid drug intolerance and drug wastage, accelerating the postoperative wound repairing of the prostatic urethra by suppressing macrophage M1 phenotype polarization.

Heat injured stromal cells-derived exosomal EGFR enhances prostatic wound healing after thulium laser resection through EMT and NF-κB signaling.

BACKGROUND: This study investigated shallow heat injury to prostate stromal fibroblasts and epithelial cells and their interaction to regulate the wound healing and the underlying molecular events. METHODS: Prostate stromal fibroblasts and epithelial cells were cultured individually or cocultured and subjected to shallow heat injury for assessments of cell proliferation, migration, apoptosis, cell cycle distribution, and gene expression. The supernatant of heat-injured WPMY-1 cells was collected for exosome extraction and assessments. Furthermore, beagle dogs received thulium laser resection of the prostate (TmLRP) and randomly divided into Gefitinib, GW4869, and control treatment for the histological analysis, tissue re-epithelialization, and epidermal growth factor receptor (EGFR) expression on the prostatic wound surface. Immunofluorescence was to evaluate p63-positive basal progenitor cell trans-differentiation and macrophage polarization and ELISA was to detect cytokine levels in beagles' urine. RESULTS: Shallow heat injury caused these cells to enter a stressed state and enhanced their crosstalk. The prostate stromal fibroblasts produced and secreted more exosomal-EGFR and other cytokines and chemokines after shallow heat injury, resulting in increased proliferation and migration of prostate epithelial cells during wound healing. The wound healing of the canine prostatic urethra following the TmLRP procedure was slower in the Gefitinib and GW4869 treatment group than in the control group of animals. Immunofluorescence and ELISA showed that reduced EGFR expression interrupted macrophage polarization but increased the inflammatory response. CONCLUSIONS: Shallow heat injury was able to promote the interaction of prostate stromal cells with prostate epithelial cells to enhance wound healing. Stromal-derived exosomal-EGFR plays a crucial role in the balance of the macrophage polarization and prostatic wound healing.

M1 macrophage mediated increased reactive oxygen species (ROS) influence wound healing via the MAPK signaling in vitro and in vivo.

Thulium laser resection of the prostate (TmLRP), a major treatment for benign prostatic hyperplasia (BPH), has several postoperative complications that affect the patients' quality of life. The aim of this study was to investigate the effect of the M1 macrophage-secreted reactive oxygen species (ROS) on prostatic wound healing, and the role of MAPK signaling in this process. A co-culture model in vitro was established using macrophages and prostate epithelial or stromal cells. Cell proliferation, migration, apoptosis, MAPK pathway-related gene expression levels were evaluated by standard assays. In addition, an in vivo model of prostatectomy was established in beagles by subjecting them to TmLRP, and were either treated with N-acetyl-L-cysteine (NAC) and or placebo. Wound healing and re-epithelialization were analyzed histopathologically in both groups, in addition to macrophage polarization, oxidative stress levels and MAPK pathway-related proteins expressions. Intracellular ROS levels were significantly increased in the prostate epithelial and stromal cells following co-culture with M1-like macrophages and H2O2 exposure via MAPK activation, which affected their proliferation, migration and apoptosis, and delayed the wound healing process. The cellular functions and wound healing capacity of the prostate cells were restored by blocking or clearing the macrophage-secreted ROS. In the beagle model, increased ROS levels impaired cellular functions, and appropriate removing ROS accelerated the wound healing process.

Thulium laser VapoResection of the prostate versus traditional transurethral resection of the prostate or transurethral plasmakinetic resection of prostate for benign prostatic obstruction: a systematic review and meta-analysis.

PURPOSE: To compare the efficacy and safety of thulium laser VapoResection of the prostate (ThuVaRP) versus standard traditional transurethral resection of the prostate (TURP) or plasmakinetic resection of prostate (PKRP) for benign prostatic obstruction. METHODS: Systematic searches were performed in the Medline, EMBASE, the Cochrane Library, Web of Science, and CNKI in December 2017. The outcomes of demographic and clinical characteristics, perioperative variables, complications, and postoperative efficacy including International Prostate Symptom Score (IPSS), quality of life (QoL), maximum flow rate (Qmax), and postvoid residual (PVR) were assessed. RESULTS: 16 studies were selected in the meta-analysis including nine randomized controlled trials (RCTs) and seven non-RCTs. Among of them, nine studies compared ThuVaRP with PKRP, while seven studies compared ThuVaRP with TURP. It seemed that ThuVaRP needed longer operation time than TURP (WMD = 6.41, 95% CI 1.38-11.44, p = 0.01) and PKRP (WMD = 10.15, 95% CI 5.20-15.10, p < 0.0001). ThuVaRP was associated with less serum hemoglobin decreased, catheterization time, and the length of hospital stay compared with TURP (WMD = - 0.58, 95% CI - 0.77 to 0.38, p < 0.00001; WMD = - 1.89, 95% CI - 2.67 to 1.11, p < 0.00001; WMD = - 2.25, 95% CI - 2.91 to 1.60, p < 0.00001) and PKRP (WMD = - 0.28, 95% CI - 0.46 to 0.10, p = 0.002; WMD = - 1.88, 95% CI - 2.87 to 0.89, p = 0.0002; WMD = - 2.08, 95% CI - 2.63 to 1.54, p<0.00001). According to our assessment, there was no significantly difference in postoperative efficacy. CONCLUSIONS: The pooled data indicated that ThuVaRP had a nearly efficacy to TURP and PKRP based on IPSS, QoL, Qmax, and PVR. Although ThuVaRP was associated with longer operation time, it got distinct superiority on serum hemoglobin decreased, catheterization time, and hospital stay.