Supplementary Material for: LncRNA HOXA-AS2 facilitates prostate cancer progression by inhibiting miR-885-5p to upregulate KDM5B

Background: The regulatory network of competing endogenous RNAs (ceRNAs) affects tumorigenesis. In this study, we aimed to investigate the mechanism by which long non-coding RNA (lncRNA) HOXA-AS2 promotes prostate cancer (PCa) progression. Methods: The expression levels of HOXA-AS2, miR-885-5p, and KDM5B in PCa tissues and cell lines were evaluated by qRT-PCR or western blotting. CCK-8 assay, caspase-3 activity assay, flow cytometry, and scratch test revealed changes in cell proliferation, caspase-3 activity, apoptosis, and migration, respectively. Luciferase and radioimmunoprecipitation assays were used to evaluate the correlation among HOXA-AS2, miR-885-5p, and KDM5B expression profiles. Results: HOXA-AS2 expression level was elevated in PCa tissues and cells. Silencing of HOXA-AS2 suppressed proliferation and migration and facilitated apoptosis in PCa cells. HOXA-AS2 competitively adsorbed miR-885-5p, thereby blocking the effect of HOXA-AS2 knockdown by the miR-885-5p inhibitor in PCa cells. Moreover, KDM5B, a target of miR-885-5p, neutralized the function of miR-885-5p in PCa cells. Conclusions: This study revealed a potential ceRNA regulatory pathway in which HOXA-AS2 affects KDM5B expression levels by sponging miR-885-5p to promote PCa development and progression

Erratum: Biventricular Pacing Cardiac Contractility Modulation Improves Cardiac Contractile Function via Upregulating SERCA2 and miR-133 in a Rabbit Model of Congestive Heart Failure

<b><i>Objective: </i></b>To compare the effects of biventricular electrical pacing and conventional single-ventricular pacing for cardiac contractility modulation (CCM) on cardiac contractile function and to delineate the underlying molecular mechanisms. <b><i>Methods</i></b><b><i>: </i></b>Forty rabbits were divided into four groups before surgery: healthy control, HF sham, HF left ventricular pacing CCM (LVP-CCM), and HF biventricular pacing CCM (BVP-CCM) groups with n=10 for each group. A rabbit model of chronic heart failure was established by ligating ascending aortic root of rabbits. Then electrical stimulations during the absolute refractory period were delivered to the anterior wall of left ventricle in the LVP-CCM group and on the anterior wall of both left and right ventricles in the BVP-CCM group lasting six hours per day for seven days. Changes in ventricular structure, cardiac function and electrocardiogram were monitored before and after CCM stimulation. <b><i>Results: </i></b>Compared with the sham-operated group, heart weight, heart weight index, LV end-systolic diameter (LVESD), LV end-diastolic diameter (LVEDD) in the LVP-CCM and BVP-CCM groups were significantly decreased (<i>p</i><0.05), while LV ejection fraction (LVEF) and fractional shortening fraction (FS) were increased (<i>p</i><0.05). Notably all these changes were consistently found to be greater in BVP-CCM than in LVP-CCM. Moreover, plasma BNP levels were highest in the HF sham-control group, followed by the LVP-CCM group, and lowest in the BVP-CCM group (<i>p</i><0.05). Furthermore, sarco/endoplasmic reticulum Ca²⁺-ATPase (SERCA2a) protein levels were upregulated by 1.7 and 2.4 fold, along with simultaneous upregulation of a cardiac-enriched microRNA miR-133 levels by 2.6 and 3.3 fold, in LVP-CCM and BVP-CCM, respectively, compared to sham. <b><i>Conclusions: </i></b>Biventricular pacing CCM is superior to conventional monoventricular pacing CCM, producing greater improvement cardiac contractile function. Greater upregulation of SERCA2 and miR-133 may account, at least partially, for the improvement by BVP-CCM

Supplementary Material for: Two Novel <b><i>HOGA1</i></b> Splicing Mutations Identified in a Chinese Patient with Primary Hyperoxaluria Type 3

<b><i>Background:</i></b> Twenty-six <i>HOGA1</i> mutations have been reported in primary hyperoxaluria (PH) type 3 (PH3) patients with c.700 + 5G>T accounting for about 50% of the total alleles. However, PH3 has never been described in Asians. <b><i>Methods:</i></b> A Chinese child with early-onset nephrolithiasis was suspected of having PH. We searched for <i>AGXT</i>, <i>GRHPR</i> and <i>HOGA1</i> gene mutations in this patient and his parents. All coding regions, including intron-exon boundaries, were analyzed using PCR followed by direct sequence analysis. <b><i>Results:</i></b> Two heterozygous mutations not previously described in the literature about <i>HOGA1</i> were identified (compound heterozygous). One mutation was a successive 2 bp substitution at the last nucleotide of exon 6 and at the first nucleotide of intron 6, respectively (c.834_834 + 1GG>TT), while the other one was a guanine to adenine substitution of the last nucleotide of exon 6 (c.834G>A). Direct sequencing analysis failed to find these mutations in 100 unrelated healthy subjects and the functional role on splicing of both variants found in this study was confirmed by a minigene assay based on the pSPL3 exon trapping vector. In addition, we found a SNP in this family (c.715G>A, p.V239I). There were no mutations detected in <i>AGXT</i> and <i>GRHPR</i>. <b><i>Conclusion:</i></b> Two novel <i>HOGA1</i> mutations were identified in association with PH3. This is the first description and investigation on mutant gene analysis of PH3 in an Asian

Supplementary Material for: A New and Practical Model of Human-like Ascending Aorta Aneurysm in Rats

Introduction: Ascending aortic aneurysm is a serious health risk. In order to study ascending aortic aneurysms, elastase and calcium ion treatment for aneurysm formation are mainly used, but their aneurysm formation time is long, the aneurysm formation rate is low. Thus, this study aimed to construct a rat model of ascending aorta aneurysm with a short modeling time and high aneurysm formation rate, which may mimic the pathological processes of human ascending aorta aneurysm. Methods: Cushion needles with different pipe diameters (1.0, 1.2, 1.4 and 1.6 mm) were used to establish a human-like rat model of ascending aortic aneurysm by narrowing the ascending aorta of rats and increasing the force of blood flow on the vessel wall. The vascular diameters were evaluated using color Doppler ultrasonography after two weeks. The characteristics of ascending aortic aneurysm in rats were detected by Masson’s trichrome staining, Verhoeff’s Van Gieson staining and hematoxylin and eosin staining while RT-PCR were utilized to assess the total RNA of cytokine interleukin-1β, interleukin 6, transforming growth factor-beta1 and metalloproteinase 2. Results: Two weeks after surgery, the ultrasound images and the statistical analysis demonstrated that the diameter of the ascending aorta in rats increased more than 1.5 times, similar to that in humans, indicating the success of animal modeling of ascending aortic aneurysm. Moreover, the optimal constriction diameter of the ascending aortic aneurysm model is 1.4 mm by the statistical analysis of the rate of ascending aortic aneurysm and mortality rate in rats with different constriction diameters. Conclusions: The human-like ascending aortic aneurysm model developed in this study can be used for the studies of the pathological processes and mechanisms in ascending aortic aneurysm in a more clinically relevant fashion

Supplementary Material for: A Novel Tumor-Suppressor, CDH18, Inhibits Glioma Cell Invasiveness Via UQCRC2 and Correlates with the Prognosis of Glioma Patients

<b><i>Background/Aims:</i></b> CDH18 (cadherin 18) is specifically expressed in the central nervous system and associated with various neuropsychiatric disorders. In this study, the role of CDH18 in glioma carcinogenesis and progression was investigated. <b><i>Methods:</i></b> The expression of CDH18 and its prognostic value in patients with gliomas were analyzed in public database and validated by real-time PCR/immunohistochemical staining (IHC) in our cohort. CCK-8 assay, transwell migration assay, wound healing assay, clonogenic assay and tumorigenicity assay were used to compare the proliferation, invasion and migration ability of glioma cells with different expressions of CDH18. iTRAQ-based quantitative proteomic analysis were used to reveal the downstream target of CDH18. Rescue experiments were conducted to further validate the relationship between UQCRC2 and CDH18. <b><i>Results:</i></b> The expression of CDH18 was depressed in a ladder-like pattern from normal tissues to WHO IV gliomas, and was an independent prognostic factor in TCGA (The Cancer Genome Atlas), CGGA (the Chinese glioma genome-atlas) and our glioma cohorts (n=453). Functional experiments <i>in vitro</i> and <i>in vivo</i> demonstrated that CDH18 inhibited invasion/migration, enhanced chemoresistance and suppressed tumorigenicity of glioma cells. UQCRC2 was identified as the downstream target of CDH18 by proteomic analysis. The expression of UQCRC2 was gradually absent as the WHO grades of gliomas escalated and was positively correlated with the expression of CDH18. Furthermore, <i>in vitro</i> assays demonstrated that down-regulation of UQCRC2 partly reversed the inhibition of invasion/migration ability and chemoresistance in CDH18 overexpressed glioma cell lines. Survival analysis demonstrated that combined CDH18/UQCRC2 biomarkers significantly influenced the prognosis of glioma patients. <b><i>Conclusions:</i></b> The present research demonstrated that CDH18 exerted its tumor-suppressor role via UQCRC2 in glioma cells and CDH18 might serve as a therapeutic target for treating gliomas

Supplementary Material for: A Phase 3 Study of Pembrolizumab Versus Placebo for Previously Treated Patients From Asia With Hepatocellular Carcinoma: Health-Related Quality of Life Analysis From KEYNOTE-394

Introduction KEYNOTE-394 showed pembrolizumab significantly improved overall survival, progression-free survival, and objective response rate with manageable safety versus placebo for patients from Asia with previously treated advanced hepatocellular carcinoma. We present results on health-related quality of life (HRQoL). Methods HRQoL was evaluated using the EORTC Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) and EuroQol-5D-3L (EQ-5D-3L) questionnaires. Key HRQoL endpoints were least squares mean (LSM) score changes from baseline to week 12 and time to deterioration (TTD) for EORTC QLQ-C30 global health status (GHS)/QoL. p values were one-sided and nominal without adjustment for multiplicity. Results The HRQoL population included patients randomly assigned to pembrolizumab (n = 298) and placebo (n = 152). From baseline to week 12, a greater decline in EORTC QLQ-C30 GHS/QoL score was observed with placebo (LSM, −8.4; 95% CI: −11.7 to −5.1) versus pembrolizumab (−4.0; 95% CI: −6.4 to −1.6; difference vs placebo: 4.4; 95% CI: 0.5–8.4; nominal p = 0.0142). Similarly, a greater decline in EQ-5D-3L visual analog scale score was observed with placebo (−6.9; 95% CI: −9.4 to −4.5) versus pembrolizumab (−2.7; 95% CI: −4.5 to −1.0; difference vs placebo: 4.2; 95% CI: 1.2–7.2; nominal p = 0.0030). TTD in EORTC QLQ-C30 GHS/QoL score was similar between arms (hazard ratio, 0.85; 95% CI: 0.58–1.25; nominal p = 0.1993). Conclusion Patients receiving placebo showed greater decline in HRQoL than those receiving pembrolizumab. Combined with efficacy and safety data from KEYNOTE-394 and the global KEYNOTE-240 and KEYNOTE-224 trials, our data support the clinically meaningful benefit and manageable tolerability of pembrolizumab as second-line therapy for patients with advanced hepatocellular carcinoma

Supplementary Material for: A Phase 3 Study of Pembrolizumab Versus Placebo for Previously Treated Patients From Asia With Hepatocellular Carcinoma: Health-Related Quality of Life Analysis From KEYNOTE-394

Introduction KEYNOTE-394 showed pembrolizumab significantly improved overall survival, progression-free survival, and objective response rate with manageable safety versus placebo for patients from Asia with previously treated advanced hepatocellular carcinoma. We present results on health-related quality of life (HRQoL). Methods HRQoL was evaluated using the EORTC Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) and EuroQol-5D-3L (EQ-5D-3L) questionnaires. Key HRQoL endpoints were least squares mean (LSM) score changes from baseline to week 12 and time to deterioration (TTD) for EORTC QLQ-C30 global health status (GHS)/QoL. p values were one-sided and nominal without adjustment for multiplicity. Results The HRQoL population included patients randomly assigned to pembrolizumab (n = 298) and placebo (n = 152). From baseline to week 12, a greater decline in EORTC QLQ-C30 GHS/QoL score was observed with placebo (LSM, −8.4; 95% CI: −11.7 to −5.1) versus pembrolizumab (−4.0; 95% CI: −6.4 to −1.6; difference vs placebo: 4.4; 95% CI: 0.5–8.4; nominal p = 0.0142). Similarly, a greater decline in EQ-5D-3L visual analog scale score was observed with placebo (−6.9; 95% CI: −9.4 to −4.5) versus pembrolizumab (−2.7; 95% CI: −4.5 to −1.0; difference vs placebo: 4.2; 95% CI: 1.2–7.2; nominal p = 0.0030). TTD in EORTC QLQ-C30 GHS/QoL score was similar between arms (hazard ratio, 0.85; 95% CI: 0.58–1.25; nominal p = 0.1993). Conclusion Patients receiving placebo showed greater decline in HRQoL than those receiving pembrolizumab. Combined with efficacy and safety data from KEYNOTE-394 and the global KEYNOTE-240 and KEYNOTE-224 trials, our data support the clinically meaningful benefit and manageable tolerability of pembrolizumab as second-line therapy for patients with advanced hepatocellular carcinoma

Supplementary Material for: A Phase 3 Study of Pembrolizumab Versus Placebo for Previously Treated Patients From Asia With Hepatocellular Carcinoma: Health-Related Quality of Life Analysis From KEYNOTE-394

Introduction KEYNOTE-394 showed pembrolizumab significantly improved overall survival, progression-free survival, and objective response rate with manageable safety versus placebo for patients from Asia with previously treated advanced hepatocellular carcinoma. We present results on health-related quality of life (HRQoL). Methods HRQoL was evaluated using the EORTC Quality of Life Questionnaire-Core 30 (EORTC QLQ-C30) and EuroQol-5D-3L (EQ-5D-3L) questionnaires. Key HRQoL endpoints were least squares mean (LSM) score changes from baseline to week 12 and time to deterioration (TTD) for EORTC QLQ-C30 global health status (GHS)/QoL. p values were one-sided and nominal without adjustment for multiplicity. Results The HRQoL population included patients randomly assigned to pembrolizumab (n = 298) and placebo (n = 152). From baseline to week 12, a greater decline in EORTC QLQ-C30 GHS/QoL score was observed with placebo (LSM, −8.4; 95% CI: −11.7 to −5.1) versus pembrolizumab (−4.0; 95% CI: −6.4 to −1.6; difference vs placebo: 4.4; 95% CI: 0.5–8.4; nominal p = 0.0142). Similarly, a greater decline in EQ-5D-3L visual analog scale score was observed with placebo (−6.9; 95% CI: −9.4 to −4.5) versus pembrolizumab (−2.7; 95% CI: −4.5 to −1.0; difference vs placebo: 4.2; 95% CI: 1.2–7.2; nominal p = 0.0030). TTD in EORTC QLQ-C30 GHS/QoL score was similar between arms (hazard ratio, 0.85; 95% CI: 0.58–1.25; nominal p = 0.1993). Conclusion Patients receiving placebo showed greater decline in HRQoL than those receiving pembrolizumab. Combined with efficacy and safety data from KEYNOTE-394 and the global KEYNOTE-240 and KEYNOTE-224 trials, our data support the clinically meaningful benefit and manageable tolerability of pembrolizumab as second-line therapy for patients with advanced hepatocellular carcinoma