Hsa-miRNA-765 as a key mediator for inhibiting growth, migration and invasion in fulvestrant-treated prostate cancer

Fulvestrant (ICI-182,780) has recently been shown to effectively suppress prostate cancer cell growth in vitro and in vivo. But it is unclear whether microRNAs play a role in regulating oncogene expression in fulvestrant-treated prostate cancer. Here, this study reports hsa-miR-765 as the first fulvestrant-driven, ERβ-regulated miRNA exhibiting significant tumor suppressor activities like fulvestrant, against prostate cancer cell growth via blockage of cell-cycle progression at the G2/M transition, and cell migration and invasion possibly via reduction of filopodia/intense stress-fiber formation. Fulvestrant was shown to upregulate hsa-miR-765 expression through recruitment of ERβ to the 5′-regulatory-region of hsa-miR-765. HMGA1, an oncogenic protein in prostate cancer, was identified as a downstream target of hsa-miR-765 and fulvestrant in cell-based experiments and a clinical study. Both the antiestrogen and the hsa-miR-765 mimic suppressed HMGA1 protein expression. In a neo-adjuvant study, levels of hsa-miR-765 were increased and HMGA1 expression was almost completely lost in prostate cancer specimens from patients treated with a single dose (250 mg) of fulvestrant 28 days before prostatectomy. These findings reveal a novel fulvestrant signaling cascade involving ERβ-mediated transcriptional upregulation of hsa-miR-765 that suppresses HMGA1 protein expression as part of the mechanism underlying the tumor suppressor action of fulvestrant in prostate cancer. © 2014 Leung et al

Atrasentan and renal events in patients with type 2 diabetes and chronic kidney disease (SONAR): a double-blind, randomised, placebo-controlled trial

Background: Short-term treatment for people with type 2 diabetes using a low dose of the selective endothelin A receptor antagonist atrasentan reduces albuminuria without causing significant sodium retention. We report the long-term effects of treatment with atrasentan on major renal outcomes. Methods: We did this double-blind, randomised, placebo-controlled trial at 689 sites in 41 countries. We enrolled adults aged 18–85 years with type 2 diabetes, estimated glomerular filtration rate (eGFR)25–75 mL/min per 1·73 m 2 of body surface area, and a urine albumin-to-creatinine ratio (UACR)of 300–5000 mg/g who had received maximum labelled or tolerated renin–angiotensin system inhibition for at least 4 weeks. Participants were given atrasentan 0·75 mg orally daily during an enrichment period before random group assignment. Those with a UACR decrease of at least 30% with no substantial fluid retention during the enrichment period (responders)were included in the double-blind treatment period. Responders were randomly assigned to receive either atrasentan 0·75 mg orally daily or placebo. All patients and investigators were masked to treatment assignment. The primary endpoint was a composite of doubling of serum creatinine (sustained for ≥30 days)or end-stage kidney disease (eGFR <15 mL/min per 1·73 m 2 sustained for ≥90 days, chronic dialysis for ≥90 days, kidney transplantation, or death from kidney failure)in the intention-to-treat population of all responders. Safety was assessed in all patients who received at least one dose of their assigned study treatment. The study is registered with ClinicalTrials.gov, number NCT01858532. Findings: Between May 17, 2013, and July 13, 2017, 11 087 patients were screened; 5117 entered the enrichment period, and 4711 completed the enrichment period. Of these, 2648 patients were responders and were randomly assigned to the atrasentan group (n=1325)or placebo group (n=1323). Median follow-up was 2·2 years (IQR 1·4–2·9). 79 (6·0%)of 1325 patients in the atrasentan group and 105 (7·9%)of 1323 in the placebo group had a primary composite renal endpoint event (hazard ratio [HR]0·65 [95% CI 0·49–0·88]; p=0·0047). Fluid retention and anaemia adverse events, which have been previously attributed to endothelin receptor antagonists, were more frequent in the atrasentan group than in the placebo group. Hospital admission for heart failure occurred in 47 (3·5%)of 1325 patients in the atrasentan group and 34 (2·6%)of 1323 patients in the placebo group (HR 1·33 [95% CI 0·85–2·07]; p=0·208). 58 (4·4%)patients in the atrasentan group and 52 (3·9%)in the placebo group died (HR 1·09 [95% CI 0·75–1·59]; p=0·65). Interpretation: Atrasentan reduced the risk of renal events in patients with diabetes and chronic kidney disease who were selected to optimise efficacy and safety. These data support a potential role for selective endothelin receptor antagonists in protecting renal function in patients with type 2 diabetes at high risk of developing end-stage kidney disease. Funding: AbbVie

Processing of speech in Chinese students with different reading abilities – an FMRI study

While imaging studies with English-speaking children showed that reading achievement of children is associated with their brain profiles during speech perception tasks, little is known about the neurocognitive processing of normal\ud Chinese students with different reading proficiencies: To study the neurocognitive processing on Chinese and the input of reading level on the process, seventeen six year old students with Chinese as their first language were recruited for the research study. These students were further assessed by a reading test to distinguish their Chinese reading ability. Those scored 2/3 deviation\ud below the cultural mean score were classified as low-proficiency readers. Those scored between 2/3 below or above the mean score were classified as intermediate-proficiency readers. Those scored 2/3 standard deviation above the mean score were regarded as high-proficiency readers. During the experiment, 12 pairs of Chinese syllables evenly distributed in three stimuli blocks were presented to the students acoustically and they had to judge whether each pair of syllables was the same in rhyme\ud or not. Throughout the whole process the students' brain activation profiles were assessed by FMRl examination using the BOW (blood oxygen level dependent) contrast method in a 1. 5 T MRI system. Result analysis was focussed on the possible neurocognitive discrepancies between students of varying reading proficiency on phonological sensitivity

Morphometry of the corpus callosum in Chinese children: relationship with gender and academic performance

Background: the corpus callosum has been widely studied,\ud but no study has demonstrated whether its size and shape have any relationship with language and calculation\ud performance.\ud \ud Objective: to examine the morphometry of the corpus callosum of normal Chinese children and its relationship with gender and academic performance.\ud \ud Materials and methods: One hundred primary school children (63 boys, 37 girls; age 6.5–10 years) were randomly\ud selected and the standardized academic performance for each was ascertained. On the mid-sagittal section of a brain MRI, the length, height and total area of the corpus\ud callosum and its thickness at different sites were measured. These were correlated with sex and academic\ud performance. \ud \ud Results: apart from the normal average dimension of the\ud different parts of the corpus callosum, thickness at the body-splenium junction in the average-to-good performance group was significantly greater than the below-average performance group in Chinese language (P=0.005), English language (P=0.02) and mathematics (P=0.01). The remainder of the callosal thickness showed no significant relationship with academic performance. There was no significant sex difference in the thickness of any part of the corpus callosum.\ud \ud Conclusions: these findings raise the suggestion that language and mathematics proficiency may be related to\ud the morphometry of the fibre connections in the posterior parietal lobes

Prevalence, Patterns, and Clinical Severity of Long COVID among Chinese Medicine Telemedicine Service Users: Preliminary Results from a Cross-Sectional Study

Introduction: The emergence and persistence of symptoms after acute COVID-19 is expected to become a major burden on healthcare systems. We assessed the features of the post-COVID-19 Syndrome (Long COVID) burden in a cohort of COVID-19 patients during the fifth major wave in Hong Kong. Methods: A cross-sectional study of 135 patients with confirmed COVID-19 from Feb to Apr 2022 who utilized traditional Chinese medicine telemedicine services was conducted. The COVID-19 Yorkshire Rehabilitation Scale was administered using an online survey 12 weeks after the COVID-19 infection. Prevalence of symptom severity and functional impairments were assessed to identify burdens and patterns. The correlation between symptom severity, functional impairments, patient characteristics, and overall health was evaluated. Results: The mean age was 46.8 years, with 46 (34.1%) males. Symptoms, functional impairments, and overall health worsened significantly when compared to the status prior to the infection. More than 50% reported the following sequelae 12 weeks after the acute infection: breathlessness, laryngeal or airway complications, fatigue, weakness, sleep, cognition, and anxiety. The presence of a single symptom or functional impairment significantly correlated with at least seven other problems positively, except for pain. Severity tended to be higher among vulnerable groups, including those who were chronic disease patients, older, less well educated, female, or had incomplete COVID-19 vaccinations. Conclusions: Long COVID is a significant healthcare burden among telemedicine users in Hong Kong, with complex needs for symptom and functional impairment management. Designing relevant health and rehabilitation services tailored to the needs of these patients is warranted

<i>Hsa-miR-765</i> suppresses DU145 cell growth, migration, and invasion.

<p>(A) <i>Hsa-miR-765</i> mimic effectively recognizes reporter with complementary sequence of <i>hsa-miR-765</i> in DU145 cells. Fold changes of luciferase activities of the <i>hsa-miR-765</i> mimic treated cells relative to the cells treated with the negative-control mimic are presented (n = 3). Transfection reagents were used as control. (B) <i>Hsa-miR-765</i> mimic reduces DU145 cell growth. MTS assay was performed on the cells treated with <i>hsa-miR-765</i> mimic or negative-control mimic or transfection control for 4 days (n = 8). (C) <i>Hsa-miR-765</i> mimic significant reduces G0/G1 to G2/M ratio in DU145. Representative DNA histograms (n = 3) are presented. (D) <i>Hsa-miR-765</i> mimic treatment causes up-regulation of cyclin A, cyclin B, and phosphorylated-cdc2 expression in DU145 cells. Protein expression levels of cell cycle regulator proteins were determined by Western blot analyses. Two independent experiments were performed and one representative set of data was presented. (E) <i>Hsa-miR-765</i> mimic suppresses DU145 cell migration and invasion as shown in transwell migration assay (top left) and invasion assay (top right), respectively. Representative micrographs of the cells after transwell migration (top left) or invasion assay (top right) are presented. Fold changes of migration (bottom left) and invasion (bottom right) of DU145 cells with either <i>hsa-miR-765</i> mimic or negative-control mimic relative to the control cells with negative-control mimic are presented (n = 3). (F) <i>Hsa-miR-765</i> mimic significantly reduces stress fibers and filopodia formations in DU145 cells. Representative micrographs and the percentages of the cells with intense stress fibers and the filopodial cells (n = 3) are presented. Student's t-test was used for comparisons with a cutoff p value of 0.05. ** p<0.01; bar = S.D.</p

HMGA1 is a direct target of <i>hsa-miR-765</i>.

<p>(A) The 3′UTR of <i>HMGA1</i> from +8910 to +8929 is predicted to be <i>hsa-miR-765</i> binding site. (B) <i>Hsa-miR-765</i> interacts with 3′UTR of <i>HMGA1</i> in a targeting reporter assay. DU145 cells were transfected with either pMIR-empty or pMIR-HMGA1-3UTR in which 3′ UTR of <i>HMGA1</i> (+8026–+9332) was cloned into the 3′ end of luciferase. Reporter activities of the pMIR-HMGA1-3UTR transfected cells treated with <i>hsa-miR-765</i> mimic or negative-control mimic are compared (n = 3). (C) <i>Hsa-miR-765</i> mimic reduced HMGA1 protein expression in DU145 cells. Protein and mRNA levels of HMGA1 in the <i>hsa-miR-765</i> mimic- and negative-control mimic-treated cells were determined by Western blot analysis (upper) and real-time RT-PCR analysis (lower), respectively. Results from <i>miR-765</i> mimic vs negative control mimic are compared (n = 3). (D) Fulvestrant reduces HMGA1 protein expression in DU145 cells. Protein level of HMGA1 and β-actin in the fulvestrant-treated and ethanol-treated control (CTL) cells were determined by Western blot analysis. (E) Ectopic expression of HMGA1 blocks fulvestrant-induced DU145 cell growth inhibition. The relative cell growth was determined after 4 days of treatment with fulvestrant or ethanol after stable transfection of <i>HMGA1</i> (or empty vector for control) for a week. Protein levels of HMGA1 were shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037.s006" target="_blank">Figure S6</a>. The cell growth of fulvestrant-treated cells with HMGA1 overexpression vs empty vector are compared (n = 8). Student's t-test was performed to determine significance between groups using a cutoff p value of 0.05. **p<0.01; bar = S.D.</p

Fulvestrant inhibits DU145 cell growth, migration, and invasion.

<p>(A) Fulvestrant induces growth inhibition of DU145 cells via an ERβ-dependent mechanism. Growth of the fulvestrant-treated DU145 cells with or without ERβ siRNA knockdown for 4 days relative to the ethanol-treated control cells with negative-control siRNA are presented and compared (n = 8). ERβ expression was also knocked down by another siRNA (siRNA#2) and the similar results were obtained (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037.s005" target="_blank">Figure S5</a>). (B) Fulvestrant induces DU145 cell-cycle arrest at G2/M phase. Representative DNA histograms of 48 hrs fulvestrant -or ethanol- (control) treated cells and percentage distributions of the cells at G0/G1 and G2/M phases (n = 3) are presented and compared. (C) Fulvestrant induces expression of G2/M markers. DU145 cells were treated with fulvestrant or ethanol for 2 days (control) and cell cycle markers were determined by Western blot analysis. Two independent experiments were performed and one representative set of data was presented. (D) Fulvestrant suppresses cell migration. A wound-healing assay was performed on the fulvestrant- and ethanol (EtOH)-treated DU145 cells (n = 3). Representative micrographs of the fulvestrant- and ethanol-treated cell cultures with scratches at 0 h and after 16 h are shown. The wound is marked by dotted lines. (E) Fulvestrant inhibits transwell migration (left panel) and invasion (right panel) in DU145 cells (n = 3) after 5 hrs of fulvestrant treatment. (F) Reductions of filopodial cells and cells with intense stress fibers by fulvestrant (treated with 48 hrs) via an ERβ-dependent mechanism. Representative micrographs and the percentages of the cells with intense stress fibers and the filopodial cells (n = 3) are presented. Student t-test was performed to determine significance with a cutoff p value of 0.05. ** p<0.01; bars = S.D.</p

ERβ is involved in fulvestrant-induced upregulation of <i>hsa-miR-765</i> expression.

<p>(A) ERβ siRNA knockdown blocks fulvestrant-induced upregulation of <i>hsa-miR-765</i> expression in DU145 cells. Expression levels of <i>hsa-miR-765</i> determined by qRT-PCR analysis of the fulvestrant-treated cells with ERβ-siRNA (siERβ) or scramble negative-control (siNeg) were compared (n = 3). (B) SiRNA knockdown of ERβ blocks fulvestrant-induced transactivation of the 5′ upstream regulatory region of <i>hsa-miR-765</i> in DU145 cells. 5′ upstream regulatory region of <i>hsa-miR-765</i> was cloned into a luciferase vector. The reporter activities with ERβ-knockdown (siERβ) or scramble negative-control (siNeg) in the presence of fulvestrant were compared (n = 3). (C) Deletion mapping analysis defines a fulvestrant-responsive segment in <i>hsa-miR-765</i> regulatory region in DU145 cells. The 5′ upstream DNA sequence of <i>hsa-miR-765</i> from nt. −3208 to +100 was analyzed using luciferase reporter system. Serial deletions from the 5′ end of the cloned sequence in the vector were conducted. Reporter activities were compared between the fulvestrant-treated (Fulvestrant) and control (ETOH) cells for each reporter vector (n = 3). (D) Fulvestrant-induces recruitment of ERβ onto the putative <i>hsa-miR-765</i> regulatory region. Chromatin-immunoprecipitation revealed the recruitment of ERβ to a sequence in the 5′-regulatory region of <i>hsa-miR765</i>. Mouse IgG and RNA polymerase II serve as negative and positive control, respectively. Fulvestrant induced 17 fold increase in ERβ recruitment when compared with non-ERβ binding region (the 0N promoter of ERβ <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037-Zhu1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0098037#pone.0098037-Fernandes1" target="_blank">[41]</a>). Student's t-test was performed to determine significance of between groups using a cutoff p value of 0.05. ** p<0.01; bar = S.D.</p