The impact of preanalytical variables on the analysis of cell-free DNA from blood and urine samples.

Cell-free DNA (cfDNA), a burgeoning class of molecular biomarkers, has been extensively studied across a variety of biomedical fields. As a key component of liquid biopsy, cfDNA testing is gaining prominence in disease detection and management due to the convenience of sample collection and the abundant wealth of genetic information it provides. However, the broader clinical application of cfDNA is currently impeded by a lack of standardization in the preanalytical procedures for cfDNA analysis. A number of fundamental challenges, including the selection of appropriate preanalytical procedures, prevention of short cfDNA fragment loss, and the validation of various cfDNA measurement methods, remain unaddressed. These existing hurdles lead to difficulties in comparing results and ensuring repeatability, thereby undermining the reliability of cfDNA analysis in clinical settings. This review discusses the crucial preanalytical factors that influence cfDNA analysis outcomes, including sample collection, transportation, temporary storage, processing, extraction, quality control, and long-term storage. The review provides clarification on achievable consensus and offers an analysis of the current issues with the goal of standardizing preanalytical procedures for cfDNA analysis.

MicroRNA-144 Suppresses Prostate Cancer Growth and Metastasis by Targeting EZH2.

BACKGROUND: Prostate cancer is a common malignant tumor with a high incidence. MicroRNAs (miRNAs) have been shown to be important post-transcriptional regulators during tumorigenesis. This study aimed to explore the effect of miR-144 on PCa proliferation and apoptosis. MATERIAL AND METHODS: The expression of miR-144 and EZH2 were examined in clinical PCa tissues. PCa cell line LNCAP and DU-145 was employed and transfected with miR-144 mimics or inhibitors. The correlation between miR-144 and EZH2 was verified by luciferase reporter assay. Cell viability, apoptosis and migratory capacity were detected by CCK-8, flow cytometry assay and wound healing assay. The protein level of EZH2, E-Cadherin, N-Cadherin and vimentin were analyzed by western blotting. RESULTS: miR-144 was found to be negatively correlated to the expression of EZH2 in PCa tissues. Further studies identified EZH2 as a direct target of miR-144. Moreover, overexpression of miR-144 downregulated expression of EZH2, reduced cell viability and promoted cell apoptosis, while knockdown of miR-144 led to an inverse result. miR-144 also suppressed epithelial-mesenchymal transition level of PCa cells. CONCLUSION: Our study indicated that miR-144 negatively regulate the expression of EZH2 in clinical specimens and in vitro. miR-144 can inhibit cell proliferation and induce cell apoptosis in PCa cells. Therefore, miR-144 has the potential to be used as a biomarker for predicting the progression of PCa.

Hydroxychloroquine Blocks Autophagy and Promotes Apoptosis of the Prostate after Castration in Rats.

Autophagy is an important pro-survival mechanism and closely related to apoptosis. The aim of this study was to investigate whether hydroxychloroquine (HCQ) blocks autophagy and promotes apoptosis of the prostate after castration. METHODS: Thirty-six male SD rats were randomly divided into 3 groups (n = 12): control group (sham operation), castration group, and HCQ group (castrated and treated with HCQ). On day 7, all mice were executed and prostates were isolated. The morphological changes of prostates were observed by light microscope, and the ultrastructure changes were observed under scanning electron microscope (SEM). The protein expression of Beclin-l, P62, caspase-3, Bcl-2, and Bax was assessed by immunohistochemical analyses. The mRNA expression of microtubule-associated protein light chain 3 (LC3) and autophagy-related gene 5 (Atg5) was detected by RT-PCR. RESULTS: Prostates of castration group shrank remarkably and prostates of HCQ group shrank more remarkably than castration group. Cytolysosomes were visible in the prostates of the castration group under SEM. Immunohistochemistry showed that the protein of Beclin-1 increased in the castration group compared to the control group, while decreased in the HCQ group compared to the castration group. While P62 protein moderately dyed in the control group and weakly dyed in the castration group, it strongly dyed in the HCQ group. Caspase-3 and Bax protein were weakly dyed in the control group but moderately dyed in the castration group and strongly dyed in the HCQ group. The expressions of apoptosis suppressor Bcl-2 were reduced in the castration group and further reduced in the HCQ group compared to the castration group. RT-PCR revealed that the mRNA of LC3 and Atg5 in the castration group increased compared to the control group, while decreased after treated with HCQ. CONCLUSION: Autophagy increased after castrated in prostates, while decreased after treated with HCQ; all these indicated that HCQ blocked autophagy and then promoted prostate apoptosis of castrated mice.

Sorghumol triterpene inhibits the growth of circulating renal cancer cells by promoting cell apoptosis, G2/M cell cycle arrest and downregulating m-TOR/PI3K/AKT signalling pathway.

PURPOSE: To investigate the growth inhibitory effect of Sorghumol on the circulating renal cancers cells and to investigate the underlying mechanisms including its effects on apoptosis, cell cycle phase distribution and m-TOR/PI3K/AKT signalling pathway. METHODS: The antiproliferative effects were assessed by WST-1 and colony formation assay. Apoptosis was detected by the Hoechst and AO/EB staining using fluorescence microscopy. Cell cycle analysis was carried out by flow cytometry. Protein expression was checked by western blotting. RESULTS: The results revealed that Sorghumol inhibited the growth of the renal cancer cell (RCC) line A498 and circulating RCCs. However, more profound effects were observed on the RCC cells. The anticancer effects were found to be due to induction of apoptosis. Moreover, Sorghumol could also caused G2/M cell cycle arrest of the RCC cells. Besides, examination of the effect of Sorghumol on m-TOR/PI3K/AKT revealed that Sorghumol inhibited the expression of p-mTOR, p-PI3K and p-AKT in a concentration-dependent manner. CONCLUSION: Taken together, we conclude that Sorghumol inhibited the proliferation of circulating RCCs and may therefore prove to be an important lead molecule for the treatment of renal cancer.

Puerarin attenuates renal fibrosis by reducing oxidative stress induced-epithelial cell apoptosis via MAPK signal pathways in vivo and in vitro.

Puerarin (PR) is an isoflavonoid isolated from the root of the plant Pueraria lobata and has been widely used in traditional Chinese herbal medicine for the treatment of various diseases. Oxidative stress and epithelial cell apoptosis play important roles in the renal fibrotic process. The present study aimed to determine whether or not PR inhibits renal fibrosis by reducing oxidative stress induced-epithelial cell apoptosis. In vivo, unilateral ureteral obstruction (UUO) induced renal fibrosis, and epithelial cell apoptosis. A total of 24 mice were randomly assigned to four experimental groups: sham, UUO alone, UUO +50 mg/kg PR, and UUO +100 mg/kg PR. In vitro, 200 µM hydrogen peroxide (H2O2) induced epithelial cell apoptosis. The experiments were dived into four groups: control, H2O2 alone, H2O2+50 µM PR, and H2O2+100 µM PR. Tubular injury was measured in the renal cortex of the mice through periodic acid-Schiff (PAS) staining, and the extracellular matrix (ECM) was measured through Sirius red (SR), immunohistochemistry (IHC) staining, and Western blot. Renal epithelial cell apoptosis was measured through terminal deoxynucleotidyl transferase-mediated dUTP Nick-End labeling (TUNEL), flow cytometry (FCM), and Hoechst assays. The protein expression of NOX4, caspase3, ERK, P38, and JNK was assessed through Western blot. PAS staining showed that PR decreased renal tubular injury in UUO mice. SR and IHC staining demonstrated that PR decreased the accumulation of ECM. PR treatment significantly inhibited epithelial cell apoptosis according to the results of TUNEL, FCM, Hoechst, and Western blot. Furthermore, NOX4 increased in UUO mice and decreased with PR treatment. H2O2-derived oxidative stress activated epithelial apoptosis and mitogen-activated protein kinases (MAPK), and PR treatment significantly reversed it. These results suggest that PR treatment ameliorates renal fibrosis by inhibiting oxidative stress induced-epithelial cell apoptosis through MAPK signaling.

Slit2 ameliorates renal inflammation and fibrosis after hypoxia-and lipopolysaccharide-induced epithelial cells injury in vitro.

Hypoxic acute kidney injury (AKI) is often incompletely repaired and leads to chronic kidney disease (CKD), which is characterized by tubulointerstitial inflammation and fibrosis. The Slit2 family of secreted glycoproteins is expressed in the kidney, it has been shown to exert an anti-inflammatory activity and prevent ischemic renal injury in vivo. However, whether Slit2 reduces renal fibrosis and inflammation after hypoxic and inflammatory epithelial cells injury in vitro remains unknown. In this study, we aimed to evaluate whether Slit2 ameliorated fibrosis and inflammation in two renal epithelial cells line challenged with hypoxia and lipopolysaccharide (LPS). Renal epithelial cells were treated with hypoxia and LPS to induce cell injury. Hoechst staining and Western blot analysis was conducted to examine epithelial cells injury. Immunofluorescence staining and Western blot analysis was performed to evaluate tubulointerstitial fibrosis. Real-time polymerase chain reaction (PCR) tested the inflammatory factor interleukin (IL)-1ß and tumor necrosis factor (TNF)-α, and Western blot analysis determined the hypoxia-inducible factor (HIF)-1α, Toll-like receptor 4 (TLR4) and nuclear factor (NF)-κB. Results revealed that hypoxia induced epithelial cells apoptosis, inflammatory factor IL-1ß and TNF-α release and tubulointerstitial fibrosis. LPS could exacerbate hypoxia -induced epithelial cells apoptosis, IL-1ß and TNF-α release and fibrosis. Slit2 reduced the expression of fibronectin, the rate of epithelial cell apoptosis, and the expression of inflammatory factor. Slit2 could also inhibit the expression of TLR4 and NF-κB, but not the expression of HIF-1α. Therefore, Slit2 attenuated inflammation and fibrosis after LPS- and hypoxia-induced epithelial cells injury via the TLR4/NF-κB signaling pathway, but not depending on the HIF-1α signaling pathway.

Effect of Obesity on Outcomes of Percutaneous Nephrolithotomy in Renal Stone Management: A Systematic Review and Meta-Analysis.

BACKGROUND: Percutaneous nephrolithotomy (PCNL) has been widely used to treat renal stones. The application of PCNL in obese patients results in the emergence of a number of challenges. This study compared the effect of obesity on the outcomes of PCNL in kidney stone treatment. METHODS: Eligible studies were searched in PubMed, Web of Science, and Cochrane Library databases. Data were analyzed using RevMan statistical software, weighted mean differences, ORs, and 95% CIs were calculated. RESULTS: Seven studies involving 2,720 normal-weight, 1,686 obese, and 286 super-obese individuals were included in this meta-analysis. A pooled analysis of safety revealed that no obvious differences in terms of complication rates after treatment existed between obese and normal-weight individuals (OR 0.97, 95% CI 0.80-1.16, p = 0.73), and between super-obese and normal-weight individuals (OR 0.88, 95% CI 0.61-1.27, p = 0.49). A pooled analysis of effectiveness revealed that no obvious difference in terms of stone-free rate after treatment existed between obese and normal-weight individuals (OR 0.98, 95% CI 0.84-1.15, p = 0.79), and between super-obese and normal-weight individuals (OR 1.20, 95% CI 0.88-1.63, p = 0.25). Moreover, no obvious differences in terms of length of hospital stay after treatment existed between super-obese and normal-weight individuals (95% CI -0.15 to 0.37, p = 0.39). Additionally, no obvious differences in terms of operation time existed between obese and normal-weight individuals (95% CI -3.36 to 1.17, p = 0.34). However, the operation time was longer among super-obese individuals than among normal-weight individuals (95% CI -22.64 to -1.40, p = 0.03), and the length of hospital stay was shorter among obese patients than among normal-weight patients (95% CI 0.04-0.34, p = 0.01). No publication bias was observed in this work. CONCLUSION: The PCNL performed in normal-weight, obese, and super-obese individuals for kidney stone treatment showed similar outcomes, except that operation time was longer among super-obese individuals and the hospital stay was shorter in obese individuals than in other groups. Thus, PCNL is a safe and efficacious treatment for renal stones in patients of all sizes.

Astragaloside IV from Astragalus membranaceus ameliorates renal interstitial fibrosis by inhibiting inflammation via TLR4/NF-кB in vivo and in vitro.

Renal fibrosis is characterized by infiltration of inflammatory cells, activation and proliferation of fibroblasts, and accumulation of extracellular matrix (ECM). Astragalus membranaceus (AM) is traditional Chinese medicine and has a range of pharmacological effects. Astragaloside IV (As IV) is the main compound of AM and has anti-inflammation activities. Whether As IV ameliorates renal interstitial fibrosis by inhibiting inflammation remains unknown. Accordingly, this study investigated the ameliorating effect of As IV on renal fibrosis. Renal fibrosis was induced in vivo using the unilateral ureteral obstruction (UUO) model. UUO mice were administered intragastrically with As IV (20 and 40mg/kg/day). After a week, ECM including fibronectin and collagen I was examined by Immunohistochemistry and Western blot, inflammatory cells (CD68 and CD3) were detected by Immunohistochemistry, the release of inflammatory cytokines (tumor necrosis factor-α and interleukin-1ß) was inspected by polymerase chain reaction, and signaling pathway was determined by Western blot. In vitro, 100ng/ml lipopolysaccharide (LPS) stimulated epithelial cells to construct the inflammatory model; these cells were treated by As IV (10 and 20µM) with or without TAK-242 (1µM) for 48h. The released inflammatory cytokines were assayed by enzyme-linked immunosorbent assay, and signaling pathway was evaluated by Western blot. As IV decreased accumulation of ECM and infiltration of inflammatory cells in UUO-induced renal fibrosis. Furthermore, As IV markedly attenuated pro-inflammatory cytokines in UUO mouse and LPS-induced epithelial cells. As IV also inhibited the TLR4 and nuclear factor (NF)-кB signaling pathway in vivo and vitro. These results demonstrate that As IV protects against the progression of renal fibrosis by inhibiting inflammation via the TLR4/NF-кB signaling pathway.

[Feasibility of muscle-derived cell autotransplantation as a treatment for post-prostatectomy urinary incontinence].

OBJECTIVE: To explore the feasibility of muscle-derived cell autotransplantation in the treatment of post-prostatectomy urinary incontinence. METHODS: Skeletal muscle-derived cells (MDC) were isolated and purified by replate technique from 6 female SD rats, and then transduced with adenovirus carrying Lac-Z gene. About 5 x 10(6) of the transduced cells were injected autologously into the bladder neck of the animals. Tissues were harvested after 5 and 15 days for histological examination and X-gal staining. RESULTS: At 5 and 15 days after the autologous MDC transplantation, histological examination revealed no apparent sign of inflammation and inflammatory cell invasion, and X-gal staining showed a large number of cells dyed blue, indicating the survival of the autologous cells. CONCLUSION: Autotransplanted MDCs can survive permanently. Autologous muscle stem cell injection can be an effective treatment for post-prostatectomy urinary incontinence.

[Experimental study of contralateral testicular changes after unilateral testicular torsion in rats].

OBJECTIVE: To study the changes of the contralateral testicular histology and germ cell apoptosis after unilateral testicular torsion (UTT) and to determine whether the contralateral testis is injured or not. METHODS: Sixty SD male rats were divided into control group (12 rats) and experimental group(48 rats). The former underwent sham operation of the left testis under general anaesthesia. The latter underwent left testis torsion(720 degrees) for 6 h, and then 4 of them were sacrificed and the other 44 were subdivided into the torsed testis untwisted group (22 rats) and the torsed testis removal group (22 rats), 7-8 rats were sacrificed and both testes (twisted and untwisted) were removed 1 day, 1 week and 4 weeks after surgery. All testes underwent histological and germ cell apoptosis examination. RESULTS: There were significant histological changes in the contralateral testis, and the germ cell apoptosis was increased greatly in the contralateral testis. CONCLUSIONS: UTT can cause contralateral testicular injury, whose mechanism may be related to reperfusion, and torsed testis removal can prevent or reduce damage to the contralateral testis.