Comparison of the diagnostic performance of fluorescence in situ hybridization (FISH), nuclear matrix protein 22 (NMP22), and their combination model in bladder carcinoma detection: a systematic review and meta-analysis.

BACKGROUND: Emerging studies reported that combination of fluorescence in situ hybridization (FISH) and nuclear matrix protein 22 (NMP22) could increase the sensitivity and specificity of bladder carcinoma (BC) management. Nevertheless, the reports remain inconsistent. This meta-analysis was undertaken to evaluate the diagnostic performance of FISH, NMP22, and their combination model in BC. MATERIALS AND METHODS: A systematic literature search was carried out in PubMed, Embase, Cochrane Library, Web of Science, Chinese National Knowledge Infrastructure, and Wanfang database dated up to October 2018. Suitable studies were identified and raw data were extracted. Meta-analysis was conducted to calculate the global sensitivities, specificities, likelihood ratio, diagnostic odds ratio (DOR), and the areas under the summary receiver operating characteristic (SROC) curves for FISH, NMP22, and their combination model, separately. All the meta-analysis estimates were derived using STATA (version 12.0) and MetaDisc (version 1.4) software packages. RESULTS: Seven eligible studies were included for analysis. The global sensitivities with 95% CI for FISH, NMP22, and their combination model were 0.79 (95% CI: 0.75-0.83), 0.76 (95% CI: 0.71-0.81), and 0.82 (95% CI: 0.75-0.88); specificities were 0.85 (95% CI: 0.76-0.91), 0.70 (95% CI: 0.55-0.81), and 0.90 (95% CI: 0.70-0.97); DORs were 22.215 (95% CI: 10.695-46.144), 7.365 (95% CI: 3.986-13.610), and 41.940 (95% CI: 13.546-129.853); and the areas under the SROC curves were 0.86 (95% CI: 0.82-0.88), 0.79 (95% CI: 0.76-0.83), and 0.90 (95% CI: 0.87-0.92). CONCLUSION: Our systematic review implied that the diagnostic performance of combination model of FISH plus NMP22 may outperform FISH or NMP22 alone in BC detection.

Integrated microRNA and protein expression analysis reveals novel microRNA regulation of targets in fetal down syndrome.

Down syndrome (DS) is caused by trisomy of human chromosome 21 and is associated with a number of deleterious phenotypes. To investigate the role of microRNA (miRNA) in the regulation of DS, high­throughput Illumina sequencing technology and isobaric tagging for relative and absolute protein quantification analysis were utilized for simultaneous expression profiling of miRNA and protein in fetuses with DS and normal fetuses. A total of 344 miRNAs were associated with DS. Gene Ontology and Kyoto Encyclopedia of Genes and Genomes pathway analyses were used to investigate the proteins found to be differentially expressed. Functionally important miRNAs were determined by identifying enriched or depleted targets in the transcript and the protein expression levels were consistent with miRNA regulation. The results indicated that GRB2, TMSB10, RUVBL2, the hsa­miR­329 and hsa­miR­27b, hsa­miR­27a targets, and MAPK1, PTPN11, ACTA2 and PTK2 or other differentially expressed proteins were connected with each other directly or indirectly. Integrative analysis of miRNAs and proteins provided an expansive view of the molecular signaling pathways in DS.

Quantitative proteomic analysis of Down syndrome in the umbilical cord blood using iTRAQ.

Down sydrome (DS) is a relatively frequent chromosomal disorder, which has no safe and effective method of prenatal diagnosis to date. The present study was designed to identify DS biomarkers. We quantified the changes in the umbilical cord blood protein levels between DS-affected and healthy (control) pregnant females using isobaric tags for relative and absolute quantification (iTRAQ) and Gene Ontology (GO) analysis. A total of 505 proteins were identified, and of these, five proteins showed significantly different concentrations between the DS and the control group. These proteins may thus be relevant to DS and constitute potential DS biomarkers.

Integrated profiling of microRNA expression in membranous nephropathy using high-throughput sequencing technology.

The present study analyzed microRNA (miRNA) expression profiles in peripheral blood lymphocyte cells (PBLCs) from patients with membranous nephropathy (MN) and normal controls (NC), in an effort to improve the understanding of the pathogenesis of MN. High-throughput sequencing was performed on 30 MN patients and 30 healthy individuals (NC group). Known and novel miRNAs were analyzed and the results were confirmed by quantitative reverse transcription PCR (qRT-PCR). In total, 326 miRNAs showed a significant difference in expression between the MN and NC groups. This included 286 downregulated miRNAs and 40 upregulated miRNAs. In addition, there were 6 novel miRNAs that presented differential levels of expression between the MN and NC groups. The miRNAs were mapped to the genome, using a short oligonucleotide alignment program (SOAP), to analyze their expression and distribution. Twenty-five percent of the unique miRNAs in the MN group and 52.1% in the NC group were mapped to the genome. One hundred and eight mismatches were identified. Seventy-seven mismatches were detected in a higher proportion of the MN samples, compared with the NC samples. Twenty-five mismatches were detected in a higher proportion of the NC samples than the MN samples. Differential miRNA expression was also detected between 10 randomly selected pair groups, as depicted in a cluster analysis diagram. These data indicate that differential miRNA expression may be involved in the pathogenesis of MN. In addition, the discrepancies between the MN and NC groups, in the mismatched miRNAs that were mapped to the genome, strongly suggest that miRNAs play an important role in the pathogenesis of human disorders. miRNAs may provide a potential breakthrough in the research of MN and may provide a novel biomarker for the diagnosis and treatment of the disease.

Analysis of microRNA expression profile by small RNA sequencing in Down syndrome fetuses.

Down syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and is associated with numerous deleterious phenotypes, including cognitive impairment, childhood leukemia and immune defects. Five Hsa21­derived microRNAs (i.e., hsa-miR-99a, let-7c, miR-125b-2, miR-155 and miR-802) are involved in variable phenotypes of DS. However, the changes involved in the genome-wide microRNA expression of DS fetuses under the influence of trisomy 21 have yet to be determined. To investigate the expression characteristic of microRNAs during the development of DS fetuses and identify whether another microRNA gene resides in the Hsa21, Illumina high-throughput sequencing technology was employed to comprehensively characterize the microRNA expression profiles of the DS and normal fetal cord blood mononuclear cells (CBMCs). In total, 149 of 395 identified microRNAs were significantly differentially expressed (fold change >2.0 and P<0.001) and 2 of 181 candidate novel microRNAs were identified as residing within the ̔DS critical region̓ of human chromosome 21 (chr21q22.2­22.3). Additionally, 7 of 14 Hsa21-derived microRNAs were detected, although not all seven were overexpressed in DS CBMCs compared with the control. Gene ontology enrichment analyses revealed that a set of abnormally expressed microRNAs were involved in the regulation of transcription, gene expression, cellular biosynthetic process and nucleic acid metabolic process. Significantly, most of the mRNA targets in these categories were associated with immune modulation (i.e., SOD1, MXD4, PBX1, BCLAF1 and FOXO1). Findings of the present study provided a considerable insight into understanding the expression characteristic of microRNAs in the DS fetal CBMCs. To the best of our knowledge, this is the first study to examine genome-wide microRNA expression profiles in the DS fetus. Differentially expressed microRNAs may be involved in hemopoietic abnormalities and the immune defects of DS fetuses and newborns.

Identification of dysregulated microRNAs in lymphocytes from children with Down syndrome.

Given the important roles of miRNAs in post-transcriptional regulation and its implications for the development of immune tissues and cells, characterization of miRNAs promotes us to uncover the molecular mechanisms underlying the pathway of trisomic chromosome 21 that disrupts the disomic genes expression and immunological defects related to Down syndrome (DS). In the present study, we analyzed global changes and chromosome distribution characteristics of miRNAs expression in lymphocytes from children with trisomy 21 by means of the Illumina high-throughput sequencing technology. Two small libraries were constructed using pool RNA of normal and DS children. The results have been further validated by stem-loop quantitative RT-PCR. Comparison between DS and normal profiles revealed that most of identified miRNAs were expressed at similar levels. The chromosome 21 that contributes to the abundantly expressed miRNAs was small, and not all Hsa21-derived miRNAs were over-expressed with ratios significantly ≥ 1.5 in Down syndrome children lymphocytes. Based on the deep sequencing technology, 108 novel candidate miRNAs have been identified, and 2 of them were derived from human chromosome 21. For the 114 significantly differentially expressed miRNAs, function annotation of target genes indicated that a set of highly abundantly and significantly differentially expressed miRNAs were involved in hematopoietic or lymphoid organ development, thymus development, and T/B cell differentiation and activation. Our results indicated that these abnormally expressed miRNAs might be associated with the mechanisms that trisomy 21 results in dysregulation of disomic genes and involved in the immunological defects seen in DS.

Genome-wide analysis of microRNAs expression profiling in patients with primary IgA nephropathy.

The aim of this study was to investigate the differential expression characteristics and the roles of the genome-wide microRNAs (miRNAs) in immunoglobulin A nephropathy (IgAN) kidney tissues. We used Illumina high-throughput sequencing technology to evaluate the miRNAs expression of six biopsy tissues from IgAN and six normal renal cortex specimens from patients with renal cell carcinoma. We observed a total of 85 miRNAs that were differentially expressed in the six IgAN patients, of which 11 miRNAs were up-regulated and 74 miRNAs were down-regulated in patients' tissues compared with control tissues. Additionally, we identified 55 candidate novel miRNAs in our study, which comprised seven candidates who were detected in the IgAN group and 49 candidates who were detected in the control group. Only one candidate (miR-n-9) was expressed in both groups. The bioinformatics showed that the regulated target genes of differentially expressed miRNAs were associated with immune and renal pathological changes. The identification of specific tissue miRNAs in our study not only helped clarify the genetics or immunology mechanisms involved in the pathogenesis of IgAN but also helped explain the pathological changes in the kidney tissues. We hypothesize that some significant miRNAs might potentially serve as novel diagnostic biomarkers in IgAN patients.

1H NMR-based metabolic profiling of human serum before and after renal transplantation.

Renal transplant success is closely tied to the ability to monitor transplant recipients. However, transplant monitoring still depends on relatively dated technologies. Thus, we applied a novel method of proton nuclear magnetic resonance (NMR)-based metabolomics to investigate the altered metabolic pattern in serum, seeking to identify biomarkers involved to different periods of renal transplant patients. Serum was obtained from 28 healthy controls (class 4) and from 20 renal transplant patients in different periods: pretransplant (class 1) and on the 1st (class 2) and the 7th day (class 3) after transplantation. After performing proton NMR spectroscopy, multivariate pattern recognition was applied to cluster the groups and establish disease-specific metabolite biomarker profiles. Compared with class 4, 19 different peaks and 10 potential biomarkers were identified in class 1, class 2, and class 3 (p < 0.0001). Nevertheless, there was no obvious difference between class 1, class 2, and class 3 (p > 0.05). Partial least squares-discriminant analysis models were able to identify patients with sensitivity and specificity of 98.7% and 95.4%, respectively. These results not only indicate that this novel method has sufficient sensitivity to distinguish renal transplant patients from controls but also identify biomarkers to monitor graft function, which could be developed to a clinically useful diagnostic tool.

The effect of mesenchymal stromal cells on doxorubicin-induced nephropathy in rats.

BACKGROUND AIMS: The potential protective effects of mesenchymal stromal cells (MSCs) on some kidney diseases has been reported. However, the effect of MSCs on doxorubicin-induced nephropathy is still poorly understood. METHODS: Rats with doxorubicin-induced kidney injuries were treated with human cord-derived MSCs. Human MSCs were first labeled with 5-bromo-2'-deoxyuridine to track their homing in kidneys after infusion. RESULTS: Alleviation of proteinuria, decreased serum albumin, alleviation of lipid disorders and histologic alterations were found in rats 4 weeks after treatment with MSCs, particularly in rats that were given repeat doses. Decreases in serum levels of interleukin-6, tumor necrosis factor-α and prostaglandin E2 and decreases in messenger RNA levels of kidney tissue cylooxygenase-2 and EP4 were found in MSC-treated rats. MSC-treated rats also displayed an increase in serum interleukin-10 levels. CONCLUSIONS: These results indicate that MSCs ameliorate doxorubicin-induced kidney injuries and inflammation, suggesting a potential clinical treatment for inflammatory kidney diseases.

Generation of systemic lupus erythematosus-specific induced pluripotent stem cells from urine.

Systemic lupus erythematosus (SLE) is the prototype of complex autoimmune diseases characterized by the production of autoantibodies which results in widespread immunologic abnormalities and immune complex formation. The underlying etiology remains largely unknown. When progressing toward kidney failure, it is becoming a serious public health problem. Kidney transplantation is a feasible therapy, but significant limitations were existed, including shortage of donor organs and lack of funding. To find an alternative proposal for kidney replacement, the induced pluripotent stem cells (iPSCs) technology was adopted. We identified typical SLE patients. Lentiviral transduction of OCT4, SOX2, KLF4, and c-MYC, under feeder conditions, resulted in reprogramming of urine-derived renal tubular cells. We investigated the viability of iPSCs generation from patients with SLE by identification of totipotency and pluripotency. SLE patient renal tubular cells-derived iPSCs exhibited properties of human embryonic stem cells, including morphology, growth properties, alkaline phosphatase, expression of pluripotency, genes and surface markers, and teratoma formation. We demonstrated that generation of SLE-specific iPSCs from urine was not only the first time worldwide, but was feasible and efficient. IPSCs from SLE would provide convenient model to study disease pathogenesis, drugs screening, and gene therapy.

microRNA expression profile of peripheral blood mononuclear cells of Klinefelter syndrome.

microRNAs are a type of small non-coding RNAs which play important roles in post-transcriptional gene regulation, and the characterization of microRNA expression profiling in peripheral blood mononuclear cells (PBMCs) from patients with Klinefelter syndrome requires further investigation. In this study, PBMCs were obtained from patients with Klinefelter syndrome and normal controls. After preparation of small RNA libraries, the two groups of samples were sequenced simultaneously using next generation high-throughput sequencing technology, and novel and known microRNAs were analyzed. A total of 9,772,392 and 9,717,633 small RNA reads were obtained; 8,014,466 (82.01%) and 8,104,423 (83.40%) genome-matched reads, 64 and 49 novel microRNAs were identified in the library of Klinefelter syndrome and the library of healthy controls, respectively. There were 71 known microRNAs with differential expression levels between the two libraries. Clustering of over-represented gene ontology (GO) classes in predicted targets of novel microRNAs in the Klinefelter syndrome library showed that the most significant GO terms were genes involved in the endomembrane system, nucleotide binding and kinase activity. Our data revealed that there are a large number of microRNAs deregulated in PBMCs taken from patients with Klinefelter syndrome, of which certain novel and known microRNAs may be involved in the pathological process of Klinefelter syndrome. Further studies are necessary to determine the roles of microRNAs in the pathological process of Klinefelter syndrome in the future.

[Prokaryotic expression and purification of a tandem repeat of ovarian cancer antigen CA125 and preparation of its antiserum].

AIM: To establish a prokaryotic expression system of the tandem repeat of CA125 (CA125R), express and purify the recombinant CA125R protein, prepare its antiserum. METHODS: The full gene sequence of one tandem repeat of CA125 was synthesized and cloned into pET-32a(+) to construct a prokaryotic expression vector of the CA125R protein (pET-CA125R). The pET-CA125R was transformed into E.coli BL21 (DE3) and the soluble expression conditions were optimized; the pure recombinant CA125R protein was prepared by affinity Ni-NTA chromatography and identified by Western blotting. A rabbit was immunized with the pure recombinant CA125R protein to prepare its antiserum. RESULTS: The prokaryotic expression vector of CA125R was successfully constructed. The optimal soluble induction expression conditions were 0.5 mmol/L isopropyl ß-D-1-thiogalactopyranoside (IPTG) at 15DegreesCelsius for 6 h. Western blotting confirmed the pure CA125R recombinant protein of high purity. The prepared antiserum specifically recognized recombinant CA125R protein and natural CA125 glycoprotein. CONCLUSION: We successfully established the efficient prokaryotic expression system of the CA125R, and prepared the recombinant CA125R protein of high purity and its antiserum.

Circulating microRNAs as candidate biomarkers in patients with systemic lupus erythematosus.

Aberrant expression of microRNAs (miRNAs) has been identified in various diseases. Recent studies demonstrated that miRNAs can be detected in the circulation and serve as potential biomarkers of various diseases. Moreover, the detection of circulating miRNAs can provide important novel information concerning diseases. In this study, a miRNA profile was used to determine the aberrantly expressed circulating miRNAs in patients with systemic lupus erythematosus (SLE) compared with patients with rheumatoid arthritis (RA) and healthy controls (HCs). To further confirm the microarray data, we identified 8 miRNAs (miR-126, miR-21, miR-451, miR-223, miR-16, miR-125a-3p, miR-155, and miR-146a) by real-time quantitative PCR (qRT-PCR) in 20 healthy controls and in 55 patients, of whom 30 patients were diagnosed with SLE and 25 were diagnosed with RA. Consistent with the microarray data, miR-126 was specifically enriched only in the blood of the SLE patients, but 4 other miRNAs (miR-21, miR-451, miR-223, and miR-16) were upregulated in the patients with SLE and were also significantly increased in the patients with RA. In contrast, miR-125a-3p, miR-155, and miR-146a showed a trend toward significantly reduced levels in the patients with SLE. In addition, to further estimate the potential roles of these differentially expressed circulating miRNAs in the pathogenesis of SLE, we used a bioinformatics exploratory analysis and identified a number of significantly enriched pathways, which implied that most dysregulated circulating miRNAs might be involved in various signal transduction pathways and cell interactions, particularly the mitogen-activated protein kinase signaling pathway. Based on these findings, we postulate that aberrantly expressed plasma miRNAs could be attractive as candidates for putative biomarkers of SLE and may help elucidate the possible pathogenesis of SLE.

A proton nuclear magnetic resonance-based metabonomics study of metabolic profiling in immunoglobulin a nephropathy.

OBJECTIVES: Immunoglobulin A nephropathy is the most common cause of chronic renal failure among primary glomerulonephritis patients. The ability to diagnose immunoglobulin A nephropathy remains poor. However, renal biopsy is an inconvenient, invasive, and painful examination, and no reliable biomarkers have been developed for use in routine patient evaluations. The aims of the present study were to identify immunoglobulin A nephropathy patients, to identify useful biomarkers of immunoglobulin A nephropathy and to establish a human immunoglobulin A nephropathy metabolic profile. METHODS: Serum samples were collected from immunoglobulin A nephropathy patients who were not using immunosuppressants. A pilot study was undertaken to determine disease-specific metabolite biomarker profiles in three groups: healthy controls (N = 23), low-risk patients in whom immunoglobulin A nephropathy was confirmed as grades I-II by renal biopsy (N = 23), and high-risk patients with nephropathies of grades IV-V (N = 12). Serum samples were analyzed using proton nuclear magnetic resonance spectroscopy and by applying multivariate pattern recognition analysis for disease classification. RESULTS: Compared with the healthy controls, both the low-risk and high-risk patients had higher levels of phenylalanine, myo-Inositol, lactate, L6 lipids ( = CH-CH2-CH = O), L5 lipids (-CH2-C = O), and L3 lipids (-CH2-CH2-C = O) as well as lower levels of ß -glucose, α-glucose, valine, tyrosine, phosphocholine, lysine, isoleucine, glycerolphosphocholine, glycine, glutamine, glutamate, alanine, acetate, 3-hydroxybutyrate, and 1-methylhistidine. CONCLUSIONS: These metabolites investigated in this study may serve as potential biomarkers of immunoglobulin A nephropathy. Point scoring of pattern recognition analysis was able to distinguish immunoglobulin A nephropathy patients from healthy controls. However, there were no obvious differences between the low-risk and high-risk groups in our research. These results offer new, sensitive and specific, noninvasive approaches that may be of great benefit to immunoglobulin A nephropathy patients by enabling earlier diagnosis.

A proton nuclear magnetic resonance-based metabonomics study of metabolic profiling in immunoglobulin a nephropathy

OBJECTIVES: Immunoglobulin A nephropathy is the most common cause of chronic renal failure among primary glomerulonephritis patients. The ability to diagnose immunoglobulin A nephropathy remains poor. However, renal biopsy is an inconvenient, invasive, and painful examination, and no reliable biomarkers have been developed for use in routine patient evaluations. The aims of the present study were to identify immunoglobulin A nephropathy patients, to identify useful biomarkers of immunoglobulin A nephropathy and to establish a human immunoglobulin A nephropathy metabolic profile. METHODS: Serum samples were collected from immunoglobulin A nephropathy patients who were not using immunosuppressants. A pilot study was undertaken to determine disease-specific metabolite biomarker profiles in three groups: healthy controls (N = 23), low-risk patients in whom immunoglobulin A nephropathy was confirmed as grades I-II by renal biopsy (N = 23), and high-risk patients with nephropathies of grades IV-V (N = 12). Serum samples were analyzed using proton nuclear magnetic resonance spectroscopy and by applying multivariate pattern recognition analysis for disease classification. RESULTS: Compared with the healthy controls, both the low-risk and high-risk patients had higher levels of phenylalanine, myo-Inositol, lactate, L6 lipids ( = CH-CH2-CH = O), L5 lipids (-CH2-C = O), and L3 lipids (-CH2-CH2-C = O) as well as lower levels of β -glucose, α-glucose, valine, tyrosine, phosphocholine, lysine, isoleucine, glycerolphosphocholine, glycine, glutamine, glutamate, alanine, acetate, 3-hydroxybutyrate, and 1-methylhistidine. CONCLUSIONS: These metabolites investigated in this study may serve as potential biomarkers of immunoglobulin A nephropathy. Point scoring of pattern recognition analysis was able to distinguish immunoglobulin A nephropathy patients from healthy controls. However, there were no obvious differences between the low-risk and high-risk groups in our research. These results offer new, sensitive and specific, noninvasive approaches that may be of great benefit to immunoglobulin A nephropathy patients by enabling earlier diagnosis.

Tissue factor pathway inhibitor-2 inhibits the growth and invasion of hepatocellular carcinoma cells and is inactivated in human hepatocellular carcinoma.

Human tissue factor pathway inhibitor-2 (TFPI-2) is an extracellular matrix-associated Kunitz-type serine proteinase inhibitor that inhibits the plasmin- and trypsin-mediated activation of matrix metalloproteinases and inhibits tumor progression, invasion and metastasis. Previous studies have shown that TFPI-2 is downregulated in the progression of various tumors. The purpose of this study was to investigate the expression and function of TFPI-2 in hepatocellular carcinoma (HCC). In situ hybridization was used to detect human TFPI-2 mRNA and immunohistochemistry was performed to examine the role of TFPI-2 expression in hepatocarcinoma tissues. Cell proliferation was assessed using MTT assay. In situ hybridization and immunohistochemical analyses revealed that the expression of TFPI-2 in hepatocarcinoma tissues was markedly lower than that in tumor-adjacent normal hepatic tissues. Restored expression of TFPI-2 in HepG(2) cells inhibits cell proliferation and invasion. Taken together, the results suggest that TFPI-2 has a tumor-suppression action and its inactivation may contribute to HCC.