Serum miR371 in testicular germ cell cancer before and after orchiectomy, assessed by digital-droplet PCR in a prospective study.

MicroRNA-371a-3p (miR371) has been suggested as a sensitive biomarker in testicular germ cell cancer (TGCC). We aimed to compare miR371 with the classical biomarkers α-fetoprotein (AFP) and ß-human chorionic gonadotropin (hCGß). Overall, 180 patients were prospectively enrolled in the study, with serum samples collected before and after orchiectomy. We compared the use of digital droplet PCR (RT-ddPCR) with the quantitative PCR used by others for detection of miR371. The novel RT-ddPCR protocol showed high performance in detection of miR371 in serum samples. In the study cohort, miR371 was measured using RT-ddPCR. MiR371 detected CS1 of the seminoma and the non-seminoma sub-types with a sensitivity of 87% and 89%, respectively. The total sensitivity was 89%. After orchiectomy, miR371 levels declined in 154 of 159 TGCC cases. The ratio of miR371 pre- and post-orchiectomy was 20.5 in CS1 compared to 6.5 in systemic disease. AFP and hCGß had sensitivities of 52% and 51% in the non-seminomas. MiR371 is a sensitive marker that performs better than the classical markers in all sub-types and clinical stages. Especially for the seminomas CS1, the high sensitivity of miR371 in detecting TGCC cells may have clinical implications.

Quantitative PCR Measurement of miR-371a-3p and miR-372-p Is Influenced by Hemolysis.

Cell-free microRNAs have been reported as biomarkers for several diseases. For testicular germ cell tumors (GCT), circulating microRNAs 371a-3p and 372-3p in serum and plasma have been proposed as biomarkers for diagnostic and disease monitoring purposes. The most widely used method for quantification of specific microRNAs in serum and plasma is reverse transcriptase real-time quantitative PCR (RT-qPCR) by the comparative Ct-method. In this method one or several reference genes or reference microRNAs are needed in order to normalize and calculate the relative microRNA levels across samples. One of the pitfalls in analysis of microRNAs from serum and plasma is the release of microRNAs from blood cells during hemolysis. This is an important issue because varying degrees of hemolysis are not uncommon in routine blood sampling. Thus, hemolysis must be taken into consideration when working with circulating microRNAs from blood. miR-93-5p, miR-30b-5p, and miR-20a-5p have been reported as reference microRNA in analysis of the miR-371a-373 cluster. We here show how these three microRNAs are influenced by hemolysis. We also propose a new reference microRNA, miR-191-5p, which is relatively stable in serum samples with mild hemolysis. In addition, we show how hemolysis can have effect on the reported microRNA levels in patient samples when these reference microRNAs are used in samples with varying levels of hemolysis.

Circadian variations in clock gene expression of human bone marrow CD34+ cells.

Time-dependent variations in clock gene expression have recently been observed in mouse hematopoietic cells, but the activity of these genes in human bone marrow (BM) has so far not been investigated. Since such data can be of considerable clinical interest for monitoring the dynamics in stem/progenitor cells, the authors have studied mRNA expression of the clock genes hPer1 , hPer2, hCry1, hCry2, hBmal1, hRev-erb alpha, and hClock in human hematopoietic CD34-positive (CD34( +)) cells. CD34(+) cells were isolated from the BM samples obtained from 10 healthy men at 6 times over 24 h. In addition, clock gene mRNA expression was analyzed in the whole BM in 3 subjects. Rhythms in serum cortisol, growth hormone, testosterone, and leukocyte counts documented that subjects exhibited standardized circadian patterns. All 7 clock genes were expressed both in CD34(+) cells and the whole BM, with some differences in magnitude between the 2 cell populations. A clear circadian rhythm was shown for hPer1, hPer2, and hCry2 expression in CD34(+) cells and for hPer1 in the whole BM, with maxima from early morning to midday. Similar to mouse hematopoietic cells, h Bmal1 was not oscillating rhythmically. The study demonstrates that clock gene expression in human BM stem/progenitor cells may be developmentally regulated, with strong or weaker circadian profiles as compared to those reported in other mature tissues.

Circadian expression of clock genes in purified hematopoietic stem cells is developmentally regulated in mouse bone marrow.

OBJECTIVE: Clock genes are known to mediate circadian rhythms in the central nervous system and peripheral organs. Although they are expressed in mouse hematopoietic progenitor and stem cells, it is unknown if they are related to circadian rhythms in these cells. We therefore investigated the 24-hour patterns in the activity of several clock genes in the bone marrow (BM) side population (SP) primitive stem cells, and compared these 24-hour patterns to clock gene variations in the whole BM and liver. METHODS: Cells were obtained from 84 B6D2F(1) mice in three replicate experiments on the second day after release into constant darkness from a standardizing light-dark schedule. mRNA expression of clock genes was measured with quantitative reverse transcriptase polymerase chain reaction. RESULTS: mPer2 displayed circadian rhythms in SP cells, whole BM, and liver cells. mPer1 and mRev-erb alpha showed a circadian rhythm in whole BM and liver, but not SP cells. mBmal1 was not expressed rhythmically in SP cells, nor in the whole BM, contrary to rhythms observed in the liver. CONCLUSIONS: With the exception of mPer2, most clock genes studied in primitive hematopoietic SP stem cells were not oscillating in a fully organized circadian manner, which is similar to immature cells in rapidly proliferating organs, such as the testis and thymus. These findings indicate that circadian clock gene expression variations in BM are developmentally regulated.

Clock gene expression in purified mouse hematopoietic stem cells.

OBJECTIVE: Circadian genes have recently been characterized in many tissues, but not in hematopoietic stem cells. These cells are rare in the bone marrow (BM), which makes it difficult to collect enough cells for detailed molecular analysis in a short period of time without reduced RNA quality. The aim was to improve methodology and reliability of clock gene expression analysis in purified mouse hematopoietic stem cells. METHODS: Stem cells were highly enriched by high-speed flow cytometric cell sorting of the side population (SP) cells from Hoechst 33342 (Hoechst)-stained mouse BM. Total RNA was isolated from sorted SP and whole BM cells and exposed to DNase treatment. The relative mRNA levels of major clock genes mPer1, mPer2, mBmal1, mCry1, mClock, and mRev-erb alpha were measured with real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) and normalized to m36B4, used as a reference gene. The clonogenity of sorted SP cells and whole BM; cells taken before and after sorting, were tested in colony-formation assay. RESULTS: Clock gene activity in sorted SP cells showed pronounced relative differences compared with whole BM for mPer1 and mCry1. The high-speed sorting procedure did not influence clock gene expression or cell clonogenity, even when this was performed with a delay period up to 24 hours. CONCLUSIONS: We demonstrated expression of six clock genes in mouse hematopoietic stem cells. A combination of high-speed flow cytometric sorting and Q-RT-PCR was shown to be useful and reliable for analysis of clock gene activity in small stem cell fractions.

Increased salivary gland tissue expression of Fas, Fas ligand, cytotoxic T lymphocyte-associated antigen 4, and programmed cell death 1 in primary Sjögren's syndrome.

OBJECTIVE: To assess salivary gland tissues obtained from patients with primary Sjögren's syndrome (SS) for the gene expression profile of the candidate genes TNFRSF6 (Fas), TNFSF6 (FasL), SSA1 (Ro52 alpha and the splice variant Ro52 beta), SSB (La), CTLA4, PDCD1 (PD-1), and ORM2, which were selected on the basis of their putative participation in salivary gland inflammation. METHODS: Quantitative real-time reverse transcriptase-polymerase chain reaction (RT-PCR) was used to examine the expression of messenger RNA (mRNA). Tissue localization of the expressed proteins was detected by immunohistochemistry. RESULTS: Expression of mRNA was increased for Fas (5.1-fold; P < 0.001), FasL (8.8-fold; P < 0.05), cytotoxic T lymphocyte-associated antigen 4 (CTLA-4) (11.2-fold; P < 0.01), programmed cell death 1 (PD-1) (15.2-fold; P < 0.05), Ro52 alpha (3.0-fold; P < 0.01), La (2.3-fold; P < 0.05), and orosomucoid 2 (ORM2) (4.4-fold; P < 0.05) in patients compared with controls when GAPDH was used as endogenous standard in duplex runs. In single runs using 2 other endogenous standards (18S and beta-actin), statistically significant differences between patients and controls were confirmed for expression of Fas, FasL, CTLA-4, and PD-1, but this difference was not observed for Ro52 alpha, La, and ORM2. Expression of Ro52 beta mRNA was similar in patients and controls. CONCLUSION: The present study demonstrates a substantial increase in expression of the negative regulator molecules PD-1 and CTLA-4 and the apoptotic signal molecules Fas and FasL in patients with primary SS compared with controls, which corresponded to the immunomorphologic pattern. The results strongly indicate that these molecules have central roles in the inflammatory process in the salivary glands of patients with primary SS.