Experimental Verification of a Predicted Intronic MicroRNA in Human NGFR Gene with a Potential Pro-Apoptotic Function

Neurotrophins (NTs) are a family of secreted growth factor proteins primarily involved in the regulation of survival and appropriate development of neural cells, functioning by binding to their specific (TrkA, TtkB, and TrkC) and/or common NGFR receptor. NGFR is the common receptor of NTs, binding with low-affinity to all members of the family. Among different functions assigned to NGFR, it is also involved in apoptosis induction and tumorigenesis processes. Interestingly, some of the functions of NGFR appear to be ligand-independent, suggesting a probable involvement of non-coding RNA residing within the sequence of the gene. Here, we are reporting the existence of a conserved putative microRNA, named Hsa-mir-6165 [EBI accession#: FR873488]. Transfection of a DNA segment corresponding to the pre-mir-6165 sequence in Hela cell line caused the generation of mature exogenous mir-6165 (a ∼200,000 fold overexpression). Furthermore, using specific primers, we succeeded to detect the endogenous expression of mir-6165 in several glioma cell lines and glioma primary tumors known to express NGFR. Similar to the pro-apoptotic role of NGFR in some cell types, overexpression of pre-mir-6165 in U87 cell line resulted in an elevated rate of apoptosis. Moreover, coordinated with the increased level of mir-6165 in the transfected U87 cell line, two of its predicted target genes (Pkd1 and DAGLA) were significantly down-regulated. The latter findings suggest that some of the previously attributed functions of NGFR could be explained indirectly by co-transcription of mir-6165 in the cells

Down regulation of hsa-mir-6165 target genes following its precursor overexpression.

<p>Down regulation of DAGLA and Pkd1 predicted target genes following the overexpression of Hsa-mir-6165 compared to the scrambled negative controls. Data of expression were globally normalized against U48, U6 and B2m as endogenous controls.</p

mir-6165 overexpression in U87 cell line induces apoptosis.

<p>A) PI staining of U87 cells 34 hours post transfection was done to investigate the effect of mir-6165 on cell cycle. A dramatic change was observable toward sub-G1 stage in the cells overexpressing mir-6165 compared to negative controls (a′- d′).B) Annexin-PI staining of the U87 cells overexpressing mir-6165 shows, the most of the cells have entered early apoptosis stage compared to negative control and the result is consistent with PI staining in the previous section (a″- d″). The gate setting distinguished between living (bottom left), necrotic (top left), early apoptotic (bottom right), and late apoptotic (top right) cells. Repeated Measures ANOVA analysis shows that the changes observed in flow cytometry of U87 cells is extremely significant (p<0.05) between negative controls and the cells overexpressing mir-6165 (e″).</p

Prediction of pre-mir-6165 within the 4^th intron of human NGFR gene.

<p>A) Position of predicted hairpin structure within the human NGFR gene is shown in the 4^th intorn. This hairpin is predicted to produce Hsa-mir-6165 which is shown as red colored sequence on the stem loop. B) Prediction of Drosha enzyme 5' and 3' cutting sites on the sequence of stem loop by Microprocessor SVM. C) Blat search result shows a strong conservation of Hsa-mir-6165 between human, rhesus, dog and elephant.</p

mir-6165 overexpression in Hela cell line.

<p>A) PI staining of Hela cells overexpressing Hsa-mir-6165 did not show any significant change in the stages of cell cycle after 34 hours post transfection (a′- c′). B) Annexin-PI staining of the Hela cells shown in figures a″- d″. Repeated Measures ANOVA analysis shows that the changes observed in annexin test of Hela cells were not significant between negative controls (scramble) and the test group (e″).</p

Top ten predicted targets for novel Hsa-mir-6165 according to DIANAmicro T v.3.

<p>The Signal to noise ratio (SNR) is calculated by the DIANA-microT algorithm and is based on a comparative analysis of the real miRNA versus a set of mock miRNAs. Higher miTG scores correspond to higher possibility of correct prediction. Greater values of SNR correspond to better distinction from the mock background <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0035561#pone.0035561-Maragkakis1" target="_blank">[38]</a>.</p

Detection of Hsa-mir-6165 in the brain derived cell lines and biopsies.

<p>A) NGFR and mir-6165expression profile in some glioma cell lines is compared to non-glioma NT2 cell line Daoy, 1321N1, U87 (glioma cell lines) and NT2 (non glioma cell line) were used for detection of Hsa-mir-6165 expression. U48 small neucleolar RNA was used as internal control for the amplifications. In glioma cell lines Hsa-mir-6165 expression level was higher than NT2 cell line. B) Relative, Hsa-mir-6165 and its precursor expression levels in various human glioma tissue samples. The expression level of Hsa-mir-6165 in the tumor samples were compared to the lowest grade of tumors. U48 small nucleolar RNA gene (SNORD48) was used for normalizing the expression levels. Error bars indicate standard deviation (SD) of duplicate experiments. Pearson’s test confirmed a positive correlation between NGFR and its intronic miRNA (p = 0.0065). In all of the high grades (HG) tissue samples, the level of NGFR and mir-6165 were higher than the low grad (LG) samples.</p

Primers and oligos used in this research.

<p>Primers and oligos used in this research.</p

pre-mir-6165 overexpression in the Hela cells and detection of Hsa-mir-6165 mature form.

<p>A) Schematic presentation of pre-mir-6165 cloning, overexpression and its RNA adenylation followed by cDNA synthesis, using a universal anchored-oligo-dT primer. For the amplification of precursor, first strand cDNA was PCR amplified using precursor specific F-primer and reverse anchor primer on the oligo-dT tail. For the amplification of mature miRNA, predicted mir-6165 sequence was used as the forward primer. B) Hsa-mir-6165 increased production (200,000x) following transfection of Hela cells with its precursor. In the untransfected cells (U) or scrambled control (M), the level of this miRNA was lower compared to the transfected cells (T). C) Four sequencing result of TA vector clones containing mir-6165 real time PCR products, are compared to the precursor sequence. The sequences between the laboratory added polyA and the upstream vector sequence are considered as mature miRNA. Sequencing of clones #2, #3 and #4 shows that prediction of Hsa-mir-6165 sequence has been correct. Clone#1 also shows the similar sequence plus AGG extra nucleotides which is considered an iso-mir for the miRNA.</p