CLEC19A overexpression inhibits tumor cell proliferation/migration and promotes apoptosis concomitant suppression of PI3K/AKT/NF-κB signaling pathway in glioblastoma multiforme.

BACKGROUND: GBM is the most frequent malignant primary brain tumor in humans. The CLEC19A is a member of the C-type lectin family, which has a high expression in brain tissue. Herein, we sought to carry out an in-depth analysis to pinpoint the role of CLEC19A expression in GBM. METHODS: To determine the localization of CLEC19A, this protein was detected using Western blot, Immunocytochemistry/Immunofluorescence, and confocal microscopy imaging. CLEC19A expression in glioma cells and tissues was evaluated by qRT-PCR. Cell viability, proliferation, migration, and apoptosis were examined through MTT assay, CFSE assay, colony formation, wound healing assay, transwell test, and flow cytometry respectively after CLEC19A overexpression. The effect of CLEC19A overexpression on the PI3K/AKT/NF-κB signaling pathway was investigated using Western blot. An in vivo experiment substantiated the in vitro results using the glioblastoma rat models. RESULTS: Our in-silico analysis using TCGA data and measuring CLEC19A expression level by qRT-PCR determined significantly lower expression of CLEC19A in human glioma tissues compared to healthy brain tissues. By employment of ICC/IF, confocal microscopy imaging, and Western blot we could show that CLEC19A is plausibly a secreted protein. Results obtained from several in vitro readouts showed that CLEC19A overexpression in U87 and C6 glioma cell lines is associated with the inhibition of cell proliferation, viability, and migration. Further, qRT-PCR and Western blot analysis showed CLEC19A overexpression could reduce the expression levels of PI3K, VEGFα, MMP2, and NF-κB and increase PTEN, TIMP3, RECK, and PDCD4 expression levels in glioma cell lines. Furthermore, flow cytometry results revealed that CLEC19A overexpression was associated with significant cell cycle arrest and promotion of apoptosis in glioma cell lines. Interestingly, using a glioma rat model we could substantiate that CLEC19A overexpression suppresses glioma tumor growth. CONCLUSIONS: To our knowledge, this is the first report providing in-silico, molecular, cellular, and in vivo evidences on the role of CLEC19A as a putative tumor suppressor gene in GBM. These results enhance our understanding of the role of CLEC19A in glioma and warrant further exploration of CLEC19A as a potential therapeutic target for GBM.

InterOpt: Improved gene expression quantification in qPCR experiments using weighted aggregation of reference genes.

qPCR is still the gold standard for gene expression quantification. However, its accuracy is highly dependent on the normalization procedure. The conventional method involves using the geometric mean of multiple study-specific reference genes (RGs) expression for cross-sample normalization. While research on selecting stably expressed RGs is extensive, scant literature exists regarding the optimal approach for aggregating multiple RGs into a unified RG. In this paper, we introduce a family of scale-invariant functions as an alternative to the geometric mean aggregation. Our candidate method (weighted geometric mean minimizing standard deviation) demonstrated significantly better results compared to other proposed methods. We provide theoretical and experimental support for this finding using real data from solid tumors and liquid biopsies. Moreover, the closed form and regression-based solution enable efficient computation and straightforward adoption on various platforms. All the proposed methods have been implemented within an easy-to-use R package with graphics processing unit (GPU) acceleration.

Synthetic miR-21 decoy circularized by tRNA splicing mechanism inhibited tumorigenesis in glioblastoma in vitro and in vivo models.

Glioblastoma multiforme (GBM) is the deadliest primary central nervous system tumor. miRNAs (miRs), a class of non-coding RNAs, are considered pivotal post-transcriptional regulators of cell signaling pathways. miR-21 is a reliable oncogene that promotes tumorigenesis of cancer cells. We first performed an in silico analysis on 10 microarray datasets retrieved from TCGA and GEO databases to elucidate top differentially expressed miRs. Furthermore, we generated a circular miR-21 decoy, CM21D, using the tRNA-splicing mechanism in GBM cell models, U87 and C6. The inhibitory efficacy of CM21D with that of a linear form, LM21D, was compared under in vitro conditions and an intracranial C6 rat glioblastoma model. miR-21 significantly overexpressed in GBM samples and confirmed in GBM cell models using qRT-PCR. CM21D was more efficient than LM21D at inducing apoptosis, inhibiting cell proliferation and migration, and interrupting the cell cycle by restoring the expression of miR-21 target genes at RNA and protein levels. Moreover, CM21D suppressed tumor growth more effectively than LM21D in the C6-rat GBM model (p < 0.001). Our findings validate miR-21 as a promising therapeutic target for GBM. The introduced CM21D by sponging miR-21 reduced tumorigenesis of GBM and can be considered a potential RNA-base therapy to inhibit cancers.

Stimulation of spinal cord according to recorded theta hippocampal rhythm during rat move on treadmill.

OBJECTIVES: Several studies have revealed that after spinal cord injury (SCI), in acute and sub-acute phase the spinal cord neurons below the injury are alive and could stimulate by use of electrical pulses. Spinal cord electrical stimulation could generate movement for paralyzed limbs and is a rehabilitation strategy for paralyzed patients. An innovative idea for controlling spinal cord electrical stimulation onset time is presented in current study. METHODS: In our method, the time of applying electrical pulse on the spinal cord is according to rat behavioral movement and two movements behaviors are recognized only based on rat EEG theta rhythm on the treadmill line. Briefly, 5 rats were placed on the treadmill and the animals experienced zero or 12 m/min speeds. RESULTS: These speeds were recognized based on EEG signals and off-line periodogram analysis. Finally, the electrical stimulation pulses had been applied to the spinal cord if the results of the EEG analysis had detected running behavior. CONCLUSIONS: These findings may guide future research in utilizing theta rhythms for the recognition of animal motor behavior and designing electrical stimulation systems based on it.

Dopamine Receptors Gene Expression Pattern and Locomotor Improvement Differ Between Female and Male Zebrafish During Spinal Cord Auto Repair.

The dopaminergic system, a spinal cord (SC) motor circuit regulator, is administrated by sexual hormones and evolutionary conserved in all vertebrates. Accordingly, we hypothesized that the dopamine receptor (DAR) expression pattern may be dissimilar in female and male zebrafish SC auto repair. We implemented an uncomplicated method to induce spinal cord injury (SCI) on fully reproductive adult zebrafish, in both genders. SCI was induced using a 28-gauge needle at 9th-10th vertebra without skin incision. Thereupon, lesioned SC was harvested for DAR gene expression analysis; zebrafish were tracked routinely for any improvement in swim distance, speed, and their roaming capabilities/preference. Our findings revealed discrepancies between drd2a, drd2b, drd3, drd4a, and drd4b expression patterns at 1, 7, and 14 days postinjury (DPI) between female and male zebrafish. The receptors were mostly upregulated at 7 DPI in both genders, whereas drd2a and drd2b were mostly maximized in females. Surprisingly, drd3 was measured greater even in intact SC in males. In addition, female zebrafish were able to swim farther distances more accelerated, in multiple directions, by engaging more caudal muscles compared with males, of course with no statistical significance. Indeed, females were able to generate whole-body rotation and move forward using the muscles downstream to the lesion site, whereas the coordinated movement in males was accomplished by rostral muscles. In conclusion, there are differences in DAR gene expression pattern throughout SC autonomous recovery between adult female and male zebrafish, and also, female locomotion seems to ameliorate more rapidly.

Expression and Function of C1orf132 Long-Noncoding RNA in Breast Cancer Cell Lines and Tissues.

miR-29b2 and miR-29c play a suppressive role in breast cancer progression. C1orf132 (also named MIR29B2CHG) is the host gene for generating both microRNAs. However, the region also expresses longer transcripts with unknown functions. We employed bioinformatics and experimental approaches to decipher C1orf132 expression and function in breast cancer tissues. We also used the CRISPR/Cas9 technique to excise a predicted C1orf132 distal promoter and followed the behavior of the edited cells by real-time PCR, flow cytometry, migration assay, and RNA-seq techniques. We observed that C1orf132 long transcript is significantly downregulated in triple-negative breast cancer. We also identified a promoter for the longer transcripts of C1orf132 whose functionality was demonstrated by transfecting MCF7 cells with a C1orf132 promoter-GFP construct. Knocking-out the promoter by means of CRISPR/Cas9 revealed no alterations in the expression of the neighboring genes CD46 and CD34, while the expression of miR-29c was reduced by half. Furthermore, the promoter knockout elevated the migration ability of the edited cells. RNA sequencing revealed many up- and downregulated genes involved in various cellular pathways, including epithelial to mesenchymal transition and mammary gland development pathways. Altogether, we are reporting here the existence of an additional/distal promoter with an enhancer effect on miR-29 generation and an inhibitory effect on cell migration.

miR-361-5p as a promising qRT-PCR internal control for tumor and normal breast tissues.

BACKGROUND: One of the most widely used evaluation methods in miRNA experiments is qRT-PCR. However, selecting suitable internal controls (IC) is crucial for qRT-PCR experiments. Currently, there is no consensus on the ICs for miRNA qRT-PCR experiments in breast cancer. To this end, we tried to identify the most stable (the least expression alteration) and promising miRNAs in normal and tumor breast tissues by employing TCGA miRNA-Seq data and then experimentally validated them on fresh clinical samples. METHODS: A multi-component scoring system was used which takes into account multiple expression stability criteria as well as correlation with clinical characteristics. Furthermore, we extended the scoring system for more than two biological sub-groups. TCGA BRCA samples were analyzed based on two grouping criteria: Tumor & Normal samples and Tumor subtypes. The top 10 most stable miRNAs were further investigated by differential expression and survival analysis. Then, we examined the expression level of the top scored miRNA (hsa-miR-361-5p) along with two commonly used ICs hsa-miR-16-5p and U48 on 34 pairs of Primary breast tumor and their adjacent normal tissues using qRT-PCR. RESULTS: According to our multi-component scoring system, hsa-miR-361-5p had the highest stability score in both grouping criteria and hsa-miR-16-5p showed significantly lower scores. Based on our qRT-PCR assay, while U48 was the most abundant IC, hsa-miR-361-5p had lower standard deviation and also was the only IC capable of detecting a significant up-regulation of hsa-miR-21-5p in breast tumor tissue. CONCLUSIONS: miRNA-Seq data is a great source to discover stable ICs. Our results demonstrated that hsa-miR-361-5p is a highly stable miRNA in tumor and non-tumor breast tissue and we recommend it as a suitable reference gene for miRNA expression studies in breast cancer. Additionally, although hsa-miR-16-5p is a commonly used IC, it's not a suitable one for breast cancer studies.

Postendodontic Pain after Pulpotomy or Root Canal Treatment in Mature Teeth with Carious Pulp Exposure: A Multicenter Randomized Controlled Trial.

This equivalence, randomized, clinical trial aimed to compare the postoperative pain of root canal therapy (RCT) with pulpotomy with mineral trioxide aggregate (PMTA) or calcium-enriched mixture (PCEM) in permanent mature teeth. In seven academic centers, 550 cariously exposed pulps were included and randomly allocated into PMTA (n = 188), PCEM (n = 194), or RCT (n = 168) arms. Preoperative "Pain Intensity" (PI) on Numerical Rating Scale and postoperative PIs until day 7 were recorded. Patients' demographic and pre-/intra-/postoperative factors/conditions were recorded/analysed. The arms were homogeneous in terms of demographics. The mean preoperative PIs were similar (P=0.998), the mean sum PIs recorded during 10 postoperative intervals were comparable (P=0.939), and the trend/changes in pain relief were parallel (P=0.821) in all study arms. The incidences of preoperative moderate-severe pain in RCT, PMTA, and PCEM arms were 56.5%, 55.7%, and 56.7%, which after 24 hours considerably decreased to 13.1%, 10.6%, and 12.9%, respectively (P=0.578). The time span of endodontic procedures was statistically different; RCT = 69.73, PMTA = 35.37, and PCEM = 33.62 minutes (P < 0.001). Patients with greater preoperative pain, symptomatic apical periodontitis, or presence of PDL widening suffered more pain (P=0.002, 0.035, and 0.023, resp.); however, other pre-/intra-/postoperative factors/conditions were comparable. Pulpotomy with MTA/CEM and RCT demonstrate comparable and effective postoperative pain relief.