Urinary exosomal miRNA-451a can be used as a potential noninvasive biomarker for diagnosis, reflecting tubulointerstitial damage and therapeutic response in IgA nephropathy.

To investigate the potential clinical value of urinary exosomal (uE) miR-451a as a biomarker for IgAN, urinary exosomes were isolated from 40 patients with IgAN, 30 patients with primary renal diseases without IgA as disease controls (non-IgAN group) and 21 healthy controls (HCs). The expression of miR-451a within exosomes was examined by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). uE miR-451a was significantly upregulated in patients with IgAN compared to non-IgAN and HCs. The uE miR-451a level was positively correlated with the change in eGFR and negatively correlated with serum creatinine, urinary macrophage migration inhibitory factor (MIF), interleukin-6 (IL-6) and tumor necrosis factor (TNF-α). A dual-luciferase reporter assay confirmed that MIF was a direct target of miR-451a. Receiver operating characteristic (ROC) curve analysis revealed that the expression of uE miR-451a showed potential diagnostic value for IgAN. Additionally, the uE miR-451a level could distinguish patients with IgAN with mild tubular atrophy/interstitial fibrosis from those with severe tubular atrophy/interstitial fibrosis. After a mean follow-up of 14.2 months, the levels of eGFR loss (ml/min/1.73 m2/year) were negatively correlated with baseline miR-451a. The levels of baseline miR-451a in the complete remission group were significantly higher than those in the non-complete remission group. uE miR-451a expression was significantly elevated in patients with IgA nephropathy and may serve as a potential biomarker for the diagnosis of IgAN and evaluation of tubulointerstitial damage, while the baseline levels of uE miR-451a may be predictors of therapeutic efficacy and disease progression.

Urinary Exosomal MicroRNAs as New Noninvasive Biomarkers of IgA Nephropathy.

Immunoglobulin A nephropathy (IgAN) is the most common type of primary glomerulonephritis. It is very important to find new noninvasive biomarkers for the diagnosis and treatment of IgAN. The purpose of this study was to explore the clinical value of urinary exosomal miRNAs in IgAN. In this study, urinary exosomes were isolated from 29 IgAN patients and 29 healthy controls. The miRNA was analyzed by high-throughput sequencing. The expression of hsa-miR-451a and hsa-let-7d-3p was examined by real-time quantitative polymerase chain reaction (RT-qPCR). The diagnostic value of miRNAs was evaluated using receiver operating characteristic (ROC) curves. Here, hsa-miR-451a and hsa-let-7d-3p were upregulated in IgAN patients compared with healthy controls. We evaluated the diagnostic value of hsa-miR-451a and hsa-let-7d-3p using ROC curves; hsa-miR-451a (AUC = 0.805, p = 0.001), hsa-mir-7d-3p (AUC = 0.76, p = 0.0049), and the combination of hsa-miR-451a and hsa-let-7d-3p (AUC = 0.8125, p = 0.0007). Hsa-miR-451a has correlations with Lee's grades (r = 0.511, p = 0.021), and 24-h urinary protein excretion (UPE; r = 0.557, p = 0.011). Hsa-let-7d-3p showed correlations with Lee's grades (r = 0.6, p = 0.005), UPE (r = 0.518, p = 0.019), serum creatinine (r = 0.564, p = 0.01), and estimated glomerular filtration rate (r = -0.532, p = 0.016). According to the Oxford classification, for hsa-miR-451a, S0 had lower levels than S1 (p = 0.016); for hsa-mir-7d-3p, M0 had lower levels than M1 (p = 0.05). These findings suggest that hsa-miR-451a and hsa-let-7d-3p may serve as noninvasive biomarkers for the evaluation of IgAN.

Differential Expression of Urinary Exosomal miRNA in Idiopathic Membranous Nephropathy and Evaluation of its Diagnostic Value.

Urinary exosomal miRNA is an ideal non-invasive biomarker of renal disease, but little is known about its ability to diagnose idiopathic membranous nephropathy (IMN). The purpose of this study was to explore the clinical value of urinary exosomal miRNAs in IMN. Urine samples were collected from 36 IMN patients and 36 healthy subjects. Some samples were used to analyze the miRNA profiles of urinary exosomes by high-throughput sequencing. The remaining cases were verified by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, the serum of the patients and healthy people was collected, and the clinical parameters were detected. Through high-throughput sequencing of samples, it was found that 20 miRNAs were markedly down-regulated. MiR-9-5p and miR-30b-5p were selected for verification, and the results were consistent with those of high-throughput sequencing. MiR-9-5p was correlated with the level of triglyceride and estimated glomerular filtration rate. MiR-30b-5p was related to the levels of anti-phospholipase A2 receptor antibody, serum albumin, ß 2-microglobulin and the ratio of global sclerosis/observed glomeruli number. The analysis of Receiver Operating Characteristic curves revealed that miR-30b-5p and miR-9-5p showed a potential diagnostic value for IMN. This study showed that there were significant differences in urinary exosome miRNA profiles between IMN patients and healthy persons. MiR-30b-5p and miR-9-5p may become new non-invasive biomarkers of IMN.

Recombinant water stress protein 1 (Re-WSP1) suppresses colon cancer cell growth through the miR-539/ß-catenin signaling pathway.

BACKGROUND: Nostoc commune Vauch. is a nitrogen-fixing blue-green algae that expresses a large number of active molecules with medicinal properties. Our previous study found that a water stress protein (WSP1) from N. commune and its recombinant counterpart (Re-WSP1) exhibited significant anti-colon cancer activity both in vitro and in vivo. This study is to investigate the effects of Re-WSP1 on proliferation of colon cancer cells and to elucidate the relevant mechanisms. METHODS: Real-time quantitative PCR was used to detect the expression of miR-539 in colon cancer HT-29 and DLD1 cells. Colon cancer cells were transfected with miR-539 mimics and negative controls, and cell proliferation were detected by CCK8 and clonogenic assays. The target gene of miR-539 was predicted, and the dual luciferase reporter gene experiment was used to verify the target gene. After colon cancer cells were transfected with miR-539 mimics or inhibitors, the expression of target gene ß-catenin was detected by Western blot. miR-539 inhibitor confirmed cell proliferation. RESULTS: Re-WSP1 inhibited colon cancer cell growth in a dose-dependent manner. Re-WSP1 inhibited the expression of ß-catenin, which was partly reversed by LiCl treatment. Quantitative PCR analysis showed that the expression of miR-539 was significantly upregulated after Re-WSP1 treatment. Moreover, miR-539 negatively regulated the expression of ß-catenin by directly binding to the 3'UTR of ß-catenin mRNA. The cell growth inhibition and the decrease in ß-catenin expression induced by Re-WSP1 were significantly reversed by miR-539 inhibitor. CONCLUSION: Re-WSP1 suppresses colon cancer cell growth via the miR-539/ß-catenin axis.

Baicalin induced colon cancer cells apoptosis through miR-217/DKK1-mediated inhibition of Wnt signaling pathway.

To analyze the anti-tumor mechanism of Baicalin in human colon cancer. The MTT assay and colony formation assay demonstrated that Baicalin treatment inhibits the proliferation of DLD1 and HCT-116 cells. The apoptosis rates were induced upon Baicalin treatment and which was determined by FACS. The qPCR and western blot analysis showed that Baicalin promotes expression of DKK1 (Dickkopf), an important antagonist of Wnt signaling pathway, thereby reduces the expression of its downstream protein ß-catenin and c-Myc. Reduction of DKK1 expression by siRNA attenuates ß-catenin and c-Myc expression. microRNA-217 (miR-217) is decreased upon Baicalin treatment. Moreover, DKK1 is identified as the direct downstream target gene of miR-217 through the dual-luciferase reporter assay. miR-217/DKK1-mediated inhibition of Wnt signaling pathway participate in apoptosis induced by Baicalin.

Metabolomics combined with serum pharmacochemistry discovering the potential effective compounds of Fangji Huangqi Tang against nephrotic syndrome.

Fangji Huangqi Tang (FHT) was first recorded in "Jin Gui Yao Lue," invented by the archaic Chinese medical doctor Zhongjing Zhang, and is a classic medicine that tonifies qi and expels wind, invigorates spleen for diuresis. A large number of literatures indicated that FHT showed a significant effect on Nephrotic Syndrome (NS). A comprehensive strategy was proposed to discover the potential effective compounds and therapeutic targets of FHT against NS as a case study. Serum metabolomics combined with multivariate statistical analysis was employed to analysis and screen the differential endogenous metabolites in serum samples of the control and model rats induced by Adriamycin. The correlation analysis between the efficacy biomarkers and different compounds absorbed in serum of FHT was conducted to explore the potential effective compounds of FHT against NS. With the help of network pharmacology, the therapeutic targets and the possible molecular mechanisms of FHT against NS were further investigated. Fifteen metabolites, including l-phenylalanine, 3-Hydroxybutyric acid and linolenic acid, were associated with renal damage based on the serum metabolomic results. Metabolic pathway analysis indicated that phenylalanine, tyrosine and tryptophan biosynthesis and linoleic acid metabolism were the key pathways associated with NS. Among them, 6 metabolites were defined as efficacy biomarkers such as uric acid, 2-methylbutyrylcarnitine and 10-HDA. The results of correlation analysis suggested that 14 constituents such as fanGhinoline, cycloastragenol, atractylenolide III, and glycyrrhetinic acid were recognized as potential effective compounds, whose potential protein targets participated in the MAPK signaling pathway, GnRH signaling pathway and aldoaterone-regulated sodium reabsorption. This study has clarified the potential effective compounds and therapeutic targets of FHT against NS. The results provided new evidence for the pharmacological mechanism of FHT on NS.

[Effect of CPEB4 on Proliferation and Apoptosis of Chronic Myeloid Leukemia Cells].

OBJECTIVE: To explore the expression of CPEB4 in K562 cells, the biological activity and its possible molecular mechanisms. METHODS: Western blot was used to detect the expression of CPEB4 in normal leukocytes and K562 cells. The overexpression plasmid pcDNA3.1(+)-His-CPEB4 and the silencing plasmid pPLK+Puro-CPEB4 shRNA were transfected into K562 cells to change the expression of CPEB4 in K562 cells, and the transfection efficiency was detected by Western blot. Finally, CCK-8 and flow cytometry were used to detect the proliferation and apoptosis of differently treated cells, and the expression changes of proliferation and apoptosis marker proteins (AKT, p-AKT, caspase-3, BCL-2) were detected by Western blot. RESULTS: Compared with normal leukocytes, the expression of CPEB4 protein in K562 cells was higher (P＜0.01). Compared with the control group, the proliferation of CPEB4-silenced K562 cells significantly increased (P＜0.01), the number of apoptotic cell significantly decreased, and expression of AKT, p-AKT and BCL-2 was significantly increased, the protein expression of caspase-3 was significantly reduced. The proliferation of K562 cells after CPEB4 overexpression was slowed down (P＜0.05), the number of apoptotic cells significantly increased,the expressions of AKT, p-AKT and BCL-2 were significantly down-regulated, and the expression of caspase-3 was up-regulated. CONCLUSION: CPEB4 can inhibit the proliferation and promote the apoptosis of K562 cells, the AKT, p-AKT, BCL-2 and caspase-3 are involved in the regulation mechanism.

Salvianolic acid A inhibits tumor-associated angiogenesis by blocking GRP78 secretion.

Glucose-regulated protein 78 (GRP78) often highly expresses in a wide range of tumors, which plays promotive functions due to its diversity of location in the development of tumor. Particularly, GRP78 can be secreted into microenvironment by tumor cells through the pathway of exosome, which promotes proliferation, angiogenesis, and drug resistance in cancer cells. Hence, we discovered a potential inhibitor to block GRP78 secretion. We screened five small molecules that may interact with the GRP78 from 51 traditional Chinese medicine molecules by molecular docking. By using western blot, we found that one of the molecules can inhibit the secretion of GRP78, which is salvianolic acid A (SAA). Further, SAA could interact with the lysine residue 633 (K633) of GRP78, which inhibited GRP78 secretion. Moreover, SAA-GRP78 interaction can facilitate GRP78 of cytosol sorted into lysosome for degradation rather than exosome. In conclusion, our research revealed that SAA has the novel function of anti-angiogenesis via the tumor environment.

Migration inhibition of water stress proteins from Nostoc commune Vauch. via activation of autophagy in DLD-1 cells.

Water stress proteins (WSP1) from Nostoc commune Vauch. had been proven to selectively induce colon cancer cells apoptosis. In this study, the effect of WSP1 on migration of human colon cancer cells was investigated. It showed that WSP1 inhibited DLD-1 cell migration, but with an insignificant effect on normal human colon epithelial cells. The data further indicated that WSP1 activated autophagy through down regulation of PI3K/AKT/mTOR pathway. Meanwhile, ß­catenin was degraded by autophagy, which then restrained epithelial-mesenchymal transition (EMT) of DLD-1 cell and its migration was subsequently suppressed significantly. The same changes occurred in xenografted nude mice according to the obtained immunohistochemical results. Consistently, the application of autophagy inhibitor largely reversed the inhibited migration by WSP1 treatment. Taken together, WSP1 could suppress migration of DLD-1 cells by autophagy inhibited EMT. The results suggested that WSP1 possessed the potential as a selective therapeutic agent against metastatic colon cancer.

Isolation and antitumor efficacy evaluation of a polysaccharide from Nostoc commune Vauch.

Nostoc commune Vauch. has been traditionally used as a healthy food and medicine for centuries especially in China. It has been demonstrated that the polysaccharides isolated from Nostoc commune Vauch. exhibit strong antimicrobial and antioxidant activities. However, little is known about their anticancer activities and the underlying mechanisms of action. Herein, we report the isolation of a polysaccharide from Nostoc commune Vauch. (NVPS), and its physicochemical properties were analyzed. In an attempt to demonstrate the potential application of NVPS in tumor chemotherapy, the in vitro antitumor activity was determined. NVPS significantly suppressed the growth and proliferation of MCF-7 and DLD1 cells. The molecular mechanism underlying this in vitro antitumor efficacy was elucidated, and the results indicated that NVPS simultaneously triggered intrinsic, extrinsic and endoplasmic reticulum stress (ERS)-mediated apoptotic signaling pathways. Collectively, these findings demonstrate that NVPS could be used as a novel promising source of natural antitumor agents.

Water stress proteins from Nostoc commune Vauch. exhibit anti-colon cancer activities in vitro and in vivo.

Nostoc commune has been traditionally used in China as a health food and medicine. The water stress proteins (WSP) of Nostoc commune are the major component of the extracellular matrix. This study purified and identified the water stress proteins (WSP1) from Nostoc commune Vauch., which could inhibit the proliferation of human colon cancer cell lines. The IC50 values of WSP1 against DLD1, HCT116, HT29, and SW480 cells were 0.19 ± 0.02, 0.21 ± 0.03, 0.39 ± 0.05, and 0.41 ± 0.01 µg/µL, respectively. Notably, it displayed very little effect on the normal human intestinal epithelial FHC cell line. The IC50 value of WSP1 against FHC cells was 0.67 ± 0.05 µg/µL. Moreover, the growth of DLD1 xenografted tumors in nude mice were significantly suppressed in the WSP1 treated group. Mechanistically, the cell-cycle analysis revealed that WSP1 induced growth inhibition by G1/S arrest. Meanwhile, Western blotting and immunohistochemistry assays showed WSP1 could activate caspase-8, -9, and -3, along with subsequent PARP cleavage. Furthermore, the pan-caspase inhibitor, z-VAD-FMK, partly reversed the effect caused by WSP1, confirming that WSP1 induced cell apoptosis through caspase-dependent pathway. Collectively, WSP1 has targeted inhibition for colon cancer proliferation both in vitro and in vivo and it is valuable for future exploitation and utilization as an antitumor agent.