Secretome Profiling and Computational Biology of Human Leiomyoma Samples Unravel Molecular Signatures with Potential for Diagnostic and Therapeutic Interventions.

In recent years, significant advancements have been made in the way the complex proteome samples are compared but the ultimate goal of routine biomarker discovery has yet to be achieved. Based on reverse genetic strategy, our study involved the spotting of genes showing expressional variability in uterine leiomyoma females. Serum samples were taken from uterine leiomyomas subjects (n=6) and healthy control subjects (n=6) for proteomic studies. Additionally, leiomyoma tissue samples (n=25) and normal myometrium samples (n=25) were taken for validation studies. In this study, we profiled the proteomes of uterine leiomyoma patient's serum and healthy control, along with relative quantification using Nano LC-MS/MS analysis. A total of 146 proteins were reported to be significantly differentially expressed (P value less than 0.05) in case and control sample. Statistical analysis identified a number of molecular signatures distinguishing healthy from diseased serum. Among these, five proteins lumican, ficolin, MASP2, EMSY, and kallistatin were further chosen according to their function for validation. Kallistatin was downregulated while ficolin, MASP2, lumican, and EMSY were found to be upregulated in the diseased sample. The expression modulations in the identified genes were further validated in twenty-five cases. Interactions among the differentially expressed proteins were identified followed with network analysis. Network analysis emphasized important pathways that are highly deregulated in myoma, and functional significance of these pathways in the pathology of the disease was discussed. Comparative expression analysis reveals distinct molecular signatures and their probable role in diagnosis of the disease.

Differential levels of CHMP2B, LLPH, and SLC25A51 proteins in secondary renal amyloidosis.

OBJECTIVES: Renal amyloidosis (RA) is a rare disease, typically manifested with proteinuria, nephrotic syndrome, and ultimately leads to renal failure. The present study aims to profile the proteomes of renal amyloidosis patient's serum and healthy controls, along with relative quantification to find out robust markers for RA. METHODS: In this study, 12 RA patients and their corresponding age and gender-matched healthy controls were recruited from the Nephrology department of Max Super Specialty Hospital, New Delhi. We employed gel-based proteomic approach coupled with MALDI-TOF MS to compare protein expression patterns in RA patients and controls. Furthermore, validation of differential proteins (selected) was done using bio-layer interferometry. RESULTS: Eleven proteins showed remarkably altered expression levels. Moreover, expression modulation of three proteins (LLPH, SLC25A51, and CHMP2B) was validated which corroborated with two-dimensional gel electrophoresis (2-DE) results showing significant upregulation (p < 0.05) in RA patients followed by ROC analysis which demonstrated the diagnostic potential of these proteins. A protein-protein master network was generated implicating the above identified proteins along with their interactors, fishing out the routes leading to amyloidosis. CONCLUSION: This study indicates that the identified serum proteomic signatures could improve early diagnosis and lead to possible therapeutic targets in RA.

Differential expression of Lumican, Mimecan, Annexin A5 and Serotransferrin in ectopic and matched eutopic endometrium in ovarian endometriosis: a case-control study.

AIM: Endometriosis is a debilitating disease marked by recurrent gynecological proliferations. The present study aimed at performing differential proteomic analysis of matched eutopic and ectopic endometrium from women with ovarian endometriosis. MATERIALS AND METHODS: Proteomes were resolved using nano LC-MS and further identified and quantified using ProteinLynx Global SERVER (PLGS) software. Selected proteins were further chosen for validation by real time-polymerase chain reaction (RT-PCR). RESULTS: The protein profiles uncovered several differentially expressed proteins in the diseased sample (ectopic endometrium) as compared to the reference sample (eutopic endometrium). The study involved an advanced proteomic approach, nano LC-MS, and validates for the first time the upregulation of Mimecan and Lumican proteins in endometriosis. CONCLUSIONS: These proteins may hence prove as potentially useful tools in the search for diagnostic markers for early detection of the disease.

Differential expression of MAP3K7 and TROPONIN C proteins and related perturbations in renal amyloidosis.

OBJECTIVES: Renal amyloidosis (RA) is a rare protein misfolding disorder that prompts progressive renal insufficiency. This study aimed to decipher proteomic changes in human sera to understand the pathophysiology and molecular mechanisms underlying the disease development, hence assisting in the diagnosis of RA. METHODS: Serum proteomic analysis was performed using a gel-based approach followed by MALDI-TOF MS. RA patients with age and sex matched healthy volunteers were recruited from Max Super Speciality Hospital, New Delhi, India. RESULTS: Proteome profiles of serum revealed eight differentially expressed proteins namely, Zinc finger protein 624, Protein FAM183A, Calcium-binding mitochondrial carrier protein Scamc-3, V-type proton ATPase 116 kDa subunit A isoforms 2, Protein TXNRD3NB, ATP - dependent RNA helicase, Troponin C and Mitogen-activated protein kinase kinase kinase 7. These proteins were reported first time in RA. The increased levels of MAP3K7 and TROPONIN C were validated by bio-layer interferometry and their diagnostic accuracy was evaluated by ROC curve analysis. The differentially expressed proteins were predominantly associated with vesicular trafficking, transcriptional regulation, metabolic processes, apoptotic process and mitochondrial metabolism. CONCLUSION: The results indicate that these proteomic signatures may be considered as potential molecular targets for RA diagnostics and therapeutics subject to validation on large sample size. Abbreviations: AßP= Amyloid-beta protein, Aß=Amyloid-beta, AL= Light chain amyloidosis, AA= Amyloid A, ALECT2= LECT2 amyloidosis, APS= Ammonium persulfate CKD= Chronic Kidney Diseases, EBRT= external beam radiation therapy, ESRD= End-Stage Kidney Disease, Glis2= Gli-similar 2, JNK= c-Jun NH 2-terminal kinase, MAPK= Mitogen-Activated Protein Kinase, MM=Multiple Myeloma, PHD= Prolyl hydroxylase, RA = Renal Amyloidosis, SAA= Serum Amyloid A, SD= Standard Deviation, Sepp= Selenoprotein, SCC= Squamous cell carcinoma, SDS= Sodium dodecyl sulfate, TEMED = tetramethyl ethylenediamine, TGF-Beta-1=Transforming growth factor- Beta-1, Trx = Thioredoxin, TrxR= Thioredoxin reductase.

Transcriptome Analysis of mRNA in Uterine Leiomyoma Using Next-generation RNA Sequencing.

BACKGROUND: Uterine leiomyoma is a benign smooth muscle tumor of monoclonal nature in the female reproductive tract and is one of the major health problems. More than 70% of the female population suffers from uterine leiomyoma in their lifetime and in the advanced condition, it is associated with pregnancy complications and infertility. OBJECTIVE: Characterization and relative expression of mRNA transcripts through transcriptome profiling in uterine leiomyoma and adjacent normal myometrium. METHODS: Uterine leiomyoma tissue of an Indian female, age 32 years, with a family history of leiomyoma (evident from mother's hysterectomy for the same pathology) was used. Patient showed 9 multiple large lesions appearing heterogeneously, deforming the uterine contour and causing distortion and splaying of the endometrial cavity showing disease aggressiveness was taken for Next-generation sequencing (NGS) to develop whole transcriptome profile along with the adjacent normal myometrium as control. The validation of the relative expression of the selective transcripts was done using Real-Time PCR. RESULTS: The transcriptome profile indicated 128 genes up-regulated and 98 down-regulated, with the Log2 fold change ≥ 2 and P ≤ 0.05, highlighting the molecular network closely associated with focal adhesion, hyaluronan and MAPK-signaling pathways. The mean relative fold change obtained from quantitative PCR as well as the P-values of 10 selected transcripts evaluated from student's t-test were as follows: BCAN: 7.93 fold (p-value =0.0013); AAK1: 2.2 fold (p-value =0.0036); PCBP3: 3.4 fold (p-value =0.0197); MOV10L1: 3.4 fold (p-value =0.0062); TWISTNB: 1.8 fold (p-value =0.006); TMSB15A: 2.1 fold (p-value =0.0023); SMAD1: 0.8 fold (p-value =0.0112); ANXA1: 0.6 fold (p-value =0.0012); FOS: 0.6 fold (p-value =0.0191); SLFN11: 0.56 fold (p-value =0.0001). CONCLUSION: The present study provides a roadmap, towards the analysis of genes and their roles in corresponding pathways throwing light on their possible involvement in the pathology of the disease.

Interactome Analysis of the Differentially Expressed Proteins in Uterine Leiomyoma.

BACKGROUND: Recent advances in proteomics present enormous opportunities to discover proteome related disparities and thus understanding the molecular mechanisms related to a disease. Uterine leiomyoma is a benign monoclonal tumor, located in the pelvic region, and affecting 40% of reproductive aged female. OBJECTIVE: Identification and characterization of the differentially expressed proteins associated with leiomyogenesis by comparing uterine leiomyoma and normal myometrium. METHODS: Paired samples of uterine leiomyoma and adjacent myometrium retrieved from twenty-five females suffering from uterine leiomyoma (n=50) were submitted to two-dimensional electrophoresis (2-DE), matrixassisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) and to reverse transcription polymerase chain reaction (RT-PCR). RESULTS: Comparison of protein patterns revealed seven proteins with concordantly increased spot intensities in leiomyoma samples. E3 ubiquitin-protein ligase MIB2 (MIB2), Mediator of RNA polymerase II transcription subunit 10 (MED10), HIRA-interacting protein (HIRP3) and Fatty acid binding protein brain (FABP7) were found to be upregulated. While, Biogenesis of lysosome-related organelles complex 1 subunit 2 (BL1S2), Shadow of prion protein (SPRN) and RNA binding motif protein X linked like 2 (RMXL2) were found to be exclusively present in leiomyoma sample. The expression modulations of the corresponding genes were further validated which corroborated with the 2-DE result showing significant upregulation in leiomyoma. We have generated a master network showing the interactions of the experimentally identified proteins with their close neighbors and further scrutinized the network to prioritize the routes leading to cell proliferation and tumorigenesis. CONCLUSION: This study highlights the importance of identified proteins as potential targets for therapeutic purpose. This work provides an insight into the mechanism underlying the overexpression of the proteins but warrants further investigations.