Characterization of the first microRNA in human CDH1 that affects cell cycle and apoptosis and indicates breast cancers progression.

The E-cadherin protein (Cadherin 1, gene: CDH1), a master regulator of the human epithelial homeostasis, contributes to the epithelial-mesenchymal transition (EMT) which confers cell migratory features to the cells. The EMT is central to many pathophysiological changes in cancer. Therefore, a better understanding of this regulatory scenario is beneficial for therapeutic regiments. The CDH1 gene is approximately 100 kbp long and consists of 16 exons with a relatively large second intron. Since none microRNA (miRNA) has been identified in CDH1 up to now we screened the CDH1 gene for promising miRNA hairpin structures in silico. Out of the 27 hairpin structures we identified, one stable RNA fold with a promising sequence motive was selected for experimental verification. The exogenous validation of the hairpin sequence was performed by transfection of HEK293T cells and the mature miRNA sequences could be verified by quantitative polymerase chain reaction. The endogenous expression of the mature miRNA provisionally named CDH1-i2-miR-1 could be confirmed in two normal (HEK293T, HUVEK) and five cancer cell lines (MCF7, MDA-MB-231, SW480, HT-29, A549). The functional characterization by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay showed a suppression of HEK293T cell proliferation. A flow cytometry-based approach showed the ability of CDH1-i2-miR-1 to arrest transfected cells on a G2/M state while annexin staining exemplified an apoptotic effect. BAX and PTEN expression levels were affected following the overexpression with the new miRNA. The in vivo expression level was assessed in 35 breast tumor tissues and their paired nonmalignant marginal part. A fourfold downregulation in the tumor specimens compared to their marginal controls could be observed. It can be concluded that the sequence of the hub gene CDH1 harbors at least one miRNA but eventually even more relevant for the pathophysiology of breast cancer.

TrkC-miR2 regulates TGFß signaling pathway through targeting of SMAD3 transcript.

TrkC, neurotrophin receptor, functions inside and outside of the nervous system and has a crucial effect on the regulation of cardiovascular formation. Recently, we introduced TrkC-miR2 as a novel microRNA located in TrkC gene, which is a regulator of the Wnt signaling pathway. Here, we presented a lot of evidence showing that TrkC-miR2 also regulates the transforming growth factor-beta (TGFß) signaling pathway. Bioinformatics studies predicted SMAD3 as one of the bona fide TrkC-miR2 target genes. Quantitative reverse transcription PCR (RT-qPCR), Western blot analysis, and dual luciferase assay analysis confirmed that SMAD3 is targeted by TrkC-miR2. On the other hand, overexpression of TrkC-miR2 in cardiosphere-derived cells (CDCs) rendered downregulation of TGFßR1, TGFßR2, and SMAD7 detected by RT-qPCR. Consistently, an inverse correlation of expression between TrkC-miR2 and SMAD3 genes was detected during the course of CDC differentiation, and also during the course of human embryonic stem cells differentiation to cardiomyocytes. Overall, we conclude that TrkC-miR2 downregulates the expression of SMAD3 and potentially regulates the TGFß signaling pathway. Knowing its approved effect on Wnt signaling, TrkC-miR2 here is introduced as a common regulator of both the Wnt and TGFß signaling pathways. Therefore, it may be a potential key element in controlling both of these signaling pathways in cell processes like colorectal cancer and cardiogenesis.

Alternative splicing of the OCC-1 gene generates three splice variants and a novel exonic microRNA, which regulate the Wnt signaling pathway.

The Wnt signaling pathway is hyperactivated in most colorectal cancers (CRC). Finding new regulators of this pathway represents the potential for cancer diagnosis or treatment. OCC-1 was initially reported as an up-regulated gene in colon carcinoma, without knowing its mechanism of action. Here, two novel transcript variants and an exonic microRNA that originated from the OCC-1 gene are reported, showing positive effects on Wnt activity. Up-regulation of the known OCC-1 variant (assigned as OCC-1A/B) was limited to CRC, and its overexpression increased survival of CRC-originated SW480 cells (Wnt+), while resulting in apoptosis of Wnt-suppressed SW480 cells or HeLa cells (Wnt-) detected by PI staining. Immunocytochemistry showed that the OCC-1A/B-encoded peptide was localized to the nucleus, where its overexpression resulted in Wnt signaling up-regulation, detected by TOP/FOPflash assay. The noncoding portion of the OCC-1A/B transcript had a suppressive effect on Wnt activity and had a negative correlation with APPL2 neighboring gene expression. Unlike OCC-1A/B, the novel OCC-1C splice variant had no expression alteration in CRC, and it seemed to encode a smaller peptide with cytoplasmic localization. A 60-nucleotide (nt) fragment containing an AUG start codon is spliced out to produce an OCC-1D noncoding RNA variant. The 60-nt RNA was validated as the precursor of a novel microRNA, which we named miR-ex1 Both OCC-1D and miR-ex1 were coordinately up-regulated in CRC. MiR-ex1 functional analysis revealed that it is targeting the APC2 tumor suppressor gene and is an activator of the Wnt signaling pathway. Overall, the OCC-1 gene is now introduced as a novel Wnt signaling regulator and as a potential therapeutic target.

Electrochemical-based biosensors for microRNA detection: Nanotechnology comes into view.

Nanotechnology plays an undeniable significant role in medical sciences, particularly in the field of biomedicine. Development of several diagnostic procedures in medicine has been possible through the beneficial application of nano-materials, among which electrochemical nano-biosensors can be mentioned. They can be employed to quantify various clinical biomarkers in detection, evaluation, and follow up stages of the illnesses. MicroRNAs, a group of regulatory short RNA fragments, added a new dimension to the management and diagnosis of several diseases. Mature miRNAs are single-stranded RNA molecules approximately 22 nucleotides in length, which regulate a vast range of biological functions from cellular proliferation and death to cancer development and progression. Recently, diagnostic value of miRNAs in various diseases has been demonstrated. There are many traditional methods for detection of miRNAs including northern blotting, quantitative real time PCR (qRT-PCR), microarray technology, nanotechnology-based approaches, and molecular biology tools including miRNA biosensors. In comparison with other techniques, electrochemical nucleic acid biosensor methods exhibit many interesting features, and could play an important role in the future nucleic acid analysis. This review paper provides an overview of some different types of nanotechnology-based biosensors for detection of miRNAs.

A novel microRNA located in the TrkC gene regulates the Wnt signaling pathway and is differentially expressed in colorectal cancer specimens.

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis, differentiation, and tumorigenesis. TrkC diverse functions might be attributed to the hypothetical non-coding RNAs embedded within the gene. Using bioinformatics approaches, a novel microRNA named TrkC-miR2 was predicted within the TrkC gene capable of regulating the Wnt pathway. For experimental verification of this microRNA, the predicted TrkC-premir2 sequence was overexpressed in SW480 cells, which led to the detection of two mature TrkC-miR2 isomiRs, and their endogenous forms were detected in human cell lines as well. Later, an independent promoter was deduced for TrkC-miR2 after the treatment of HCT116 cells with 5-azacytidine, which resulted in differential expression of TrkC-miR2 and TrkC host gene. RT-quantitative PCR and luciferase assays indicated that the APC2 gene is targeted by TrkC-miR2, and Wnt signaling is up-regulated. Also, Wnt inhibition by using small molecules along with TrkC-miR2 overexpression and TOP/FOP flash assays confirmed the positive effect of TrkC-miR2 on the Wnt pathway. Consistently, TrkC-miR2 overexpression promoted SW480 cell survival, which was detected by flow cytometry, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays, and crystal violate analysis. RT-qPCR analysis revealed that TrkC-miR2 is significantly up-regulated (∼70 times) in colorectal tumor tissues compared with their normal pairs. Moreover, the TrkC-miR2 expression level discriminated grades of tumor malignancies, which was consistent with its endogenous levels in HCT116, HT29, and SW480 colorectal cancer cell lines. Finally, an opposite expression pattern was observed for TrkC-miR2 and the APC2 gene in colorectal cancer specimens. In conclusion, here we introduce TrkC-miR2 as a novel regulator of Wnt signaling, which might be a candidate oncogenic colorectal cancer biomarker.

Experimental verification of a predicted novel microRNA located in human PIK3CA gene with a potential oncogenic function in colorectal cancer.

PI3K/AKT signaling is involved in cell survival, proliferation, and migration. In this pathway, PI3Kα enzyme is composed of a regulatory protein encoded by p85 gene and a catalytic protein encoded by PIK3CA gene. Human PIK3CA locus is amplified in several cancers including lung and colorectal cancer (CRC). Therefore, microRNAs (miRNAs) that are encoded within the PIK3CA gene might have a role in cancer development. Here, we report a novel microRNA named PIK3CA-miR1 (EBI accession no. LN626315), which is located within PIK3CA gene. A DNA segment corresponding to PIK3CA-premir1 sequence was transfected in human cell lines that resulted in generation of mature exogenous PIK3CA-miR1. Following the overexpression of PIK3CA-miR1, its predicted target genes (APPL1 and TrkC) were significantly downregulated in the CRC-originated HCT116 and SW480 cell lines, detected by qRT-PCR. Then, dual luciferase assay supported the interaction of PIK3CA-miR1 with APPL1 and TrkC transcripts. Endogenous PIK3CA-miR1 expression was also detected in several cell lines (highly in HCT116 and SW480) and highly in CRC specimens. Consistently, overexpression of PIK3CA-premir1 in HCT116 and SW480 cells resulted in significant reduction of the sub-G1 cell distribution and apoptotic cell rate, as detected by flowcytometry, and resulted in increased cell proliferation, as detected by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. PIK3CA-miR1 overexpression also resulted in Wnt signaling upregulation detected by Top/Fop assay. Overall, accumulative evidences indicated the presence of a bona fide novel onco-miRNA encoded within the PIK3CA oncogene, which is highly expressed in colorectal cancer and has a survival effect in CRC-originated cells.

Experimental verification of a conserved intronic microRNA located in the human TrkC gene with a cell type-dependent apoptotic function.

Tropomyosin receptor kinase C (TrkC) is involved in cell survival, apoptosis induction and tumorigenesis. We hypothesized that, similar to p75(NTR) receptor, some of the diverse functions of TrkC could be mediated by a microRNA (miRNA) embedded within the gene. Here, we experimentally verified the expression and processing of two bioinformatically predicted miRNAs named TrkC-miR1-5p and TrkC-miR1-3p. Transfecting a DNA fragment corresponding to the TrkC-premir1 sequence in HEK293t cells caused ~300-fold elevation in the level of mature TrkC-miR1 and also a significant downregulation of its predicted target genes. Furthermore, endogenous TrkC-miR1 was detected in several cell lines and brain tumors confirming its endogenous generation. Furthermore, its orthologous miRNA was detected in developing rat brain. Accordingly, TrkC-miR1 expression was increased during the course of neural differentiation of NT2 cell, whereas its suppression attenuated NT2 differentiation. Consistent with opposite functions of TrkC, TrkC-miR1 overexpression promoted survival and apoptosis in U87 and HEK293t cell lines, respectively. In conclusion, our data report the discovery of a new miRNA with overlapping function to TrkC.

The oncogene Musashi1 encodes novel miRNAs in breast cancer.

RNA-binding protein Musashi1 (MSI1) shows an increased expression level in several cancers and has been introduced as a prognostic marker in some malignancies. It is expected that if any miRNA is encoded by this gene, it might have a role in cancer development or could be considered as a prognostic biomarker. Accordingly, in this study, we aimed to find novel miRNA(s) inside the intronic regions of the MSI1 gene. Here, we report two novel miRNAs within intron 4 of MSI1 gene, named MSM2 and MSM3, which were selected among several miRNA precursors predicted by bioinformatic studies. For experimental analysis, corresponding precursor miRNAs were transfected into HEK293T cells and exogenous expression of the mature miRNAs were detected. Two mature miRNAs, MSM3-3p and MSM3-5p were generated by MSM3 precursor and one, MSM2-5p was derived from MSM2. Besides, endogenous expression of MSM2-5p and MSM3-3p was detected in MCF-7 and SH-SY5Y cell lines. Expression of both mature miRNAs was also detected in clinical samples of breast cancer. Additionally, the interaction between the MSM3-3p and 3'UTR region of PDE11A was confirmed by dual luciferase assay. Overall, our data demonstrated that MSI1 gene encodes two novel miRNAs in breast cancer cells.

Up-Regulation of Hsa-miR-11181 in Glioblastoma Multiforme as A Regulator of AKT2 and TGFBR1 Signalling.

OBJECTIVE: MicroRNAs (miRNAs) are short non-coding RNAs that play a role in post-transcriptional regulation of gene expression. Hsa-miR-11181 was originally introduced as a regulator of genes involved in some brain tumours. Due to the high expression of Hsa-miR-11181 in limited glioblastoma brain tumours, in this study we intend to assess the expressions of Hsa-miR-11181 and Has-miR11181-3p in brain tumour tissues and attribute new target genes to these miRNAs. MATERIALS AND METHODS: In this experimental study, total RNA from brain tissue samples was extracted for real-time quantitative polymerase chain reaction (RT-qPCR) analysis after cDNA synthesis. In order to confirm a direct interaction of Hsa-miR-11181 with two target genes, the 3' UTR of AKT2 and transforming growth factor-beta receptor 1 (TGFBR1) were cloned separately for assessment by the dual luciferase assay. RESULTS: RT-qPCR analysis indicated that both Hsa-miR-11181-5p and Has-miR11181-3p specifically up-regulated in higher grades of glioma tumours versus other brain tumour types. Consistently, lower expression levels of AKT2 and TGFBR1 were detected in higher grade gliomas compared to other types of brain tumours, which was inverse to the level of expression detected for the heparin-binding EGF-like growth factor (HBEGF) gene. The results of the dual luciferase assay supported a direct interaction of Hsa-miR-11181 with the 3' UTR sequences of the AKT2 and TGFBR1 genes. CONCLUSION: Overall, our data suggest that miR-1118 is a potential molecular biomarker for discrimination of glioma brain tumours from other brain tumour types.

LOC646329 long non-coding RNA sponges miR-29b-1 and regulates TGFß signaling in colorectal cancer.

Non-coding RNAs (ncRNAs) are reported to be regulators of signaling pathways that are involved in colorectal cancer (CRC) progression. Aiming at finding ncRNAs (miRNAs) that are differentially expressed in tumor versus normal colorectal tissue samples, online RNA-seq data were analyzed. Of between 18 candidate miRNAs, hsa-miR-29b-1 (miR-29b-1) represented the highest fold change of expression level. Hsa-miR-29b-1 is encoded from the third intron of LOC646329 long ncRNA gene. Surprisingly, two miR-29b sponging sites were predicted within exons of LOC646329 gene. Then, dual luciferase assay supported the interaction of miR-29b-1 with LOC646329-variant D transcript. Also, a direct indication of miR-29b-1 with 3'UTR sequence of SMAD3 gene was verified through dual luciferase assay and RT-qPCR analysis. Furthermore, a reverse pattern of expression was detected between miR-29b-1 and LOC646329-variant D transcript in about 25 pairs of CRC tumor samples, detected by RTqPCR. Consistently, overexpression of LOC646329-variant D transcript was followed by increased SMAD3 and p21 genes expression level and downregulation of CyclinD1 genes in HCT116 cells, detected by RT-qPCR, and western analysis. Also, overexpression of it was followed by increased G1 cell population of HCT-116 cells. All of these data suggested a tumor suppressor effect for LOC646329-variant D in CRC tumor tissue samples, consistent to its reduced expression level at late stages of CRC progression. Data also indicated that LOC646329-variant D exerts its suppression effect on CRC progression through sponging miR-29b, which in turn regulates Wnt and TGFB signaling pathways. This makes LOC646329-variant D transcript as a novel potential therapy target.

hsa-miR-766-5p as a new regulator of mitochondrial apoptosis pathway for discriminating of cell death from cardiac differentiation.

Dispose of unnecessary cells in multicellular organism take place through apoptosis as a mode of programmed cell death (PCD). This process is triggered through two main pathway including extrinsic pathway or death receptor pathway and intrinsic or mitochondrial pathway. An alternative role for mitochondrial pathway of cell death is its involvement in cell differentiation. Biochemistry of cell differentiation indicates a common origin for differentiation and apoptosis. miRNAs are a group of small non coding mediator RNAs in regulation of many routes such as apoptosis and differentiation. By using bioinformatics tools hsa-miR-766-5p was predicted to target the BAX, BAK and BOK genes involved in mitochondrial apoptosis pathway. RT-qPCR and dual luciferase assay showed targeting of BAX, BAK and BOK 3'UTRs via hsa-miR-766, detected in SW480 and HEK293T cell lines. Caspases 3/7 and 9 activity assay revealed the involvement of hsa-miR-766-5p in mitochondrial apoptosis pathway regulation detected following overexpression and downregulation of this miRNA, detected in SW480 cells treated with 1 µM doxorubicin. Flow cytometry and MTT assay indicated cell death reduction and viability elevation effect of hsa-miR-766 in SW480 cells after its overexpression. Endogenous expression of hsa-miR-766 during the course of human embryonic stem cells (hESCs) differentiation into cardiomyocytes revealed an inverse expression status of this miRNA with BOK. However, the expression of this miRNA was inversely related to BAX and BAK for some time points of differentiation. Overall this results show the involvement of hsa-miR-766 in regulation of mitochondrial apoptosis pathway.

Hsa-miR-11181 regulates Wnt signaling pathway through targeting of APC2 transcripts in SW480 cell line.

Wnt signaling plays important roles in differentiation, morphogenesis and development. This signaling pathway is highly regulated at all levels and microRNAs are small noncoding RNAs regulating Wnt signaling. Here, we intended to investigate hsa-miR-11181 (a novel miRNA located in TrkC gene) effect on Wnt signaling pathway in SW480 cell line. TOP/FOP flash assay indicated up-regulation of Wnt signaling, following the overexpression of hsa-miR-11181, verified through RT-qPCR. Bioinformatics analysis predicted APC1, APC2 and Axin1 might be targeted by hsa-miR-11181. Then, RT-qPCR analysis indicated that APC2 and Axin1 have been significantly down-regulated following the hsa-miR-11181 overexpression. However dual luciferase assay analysis supported only APC2 3'-UTR is directly targeted by this miRNA. Then, treatment of SW480 cells with Wnt-inhibitory small molecules supported the effect of hsa-miR-11181 at the inhibitory complex level containing APC2 protein. Consistently, viability of SW480 cells overexpressing hsa-miR-11181 was significantly elevated, measured through MTT assay. Overall, these results suggest that hsa-miR-11181 may play a crucial role in Wnt signaling regulation and confirmed that APC2 3'-UTR is targeted by hsa-miR-11181 and propose the presence of its recognition sites in the promoter or coding regions of Axin1 gene.

Overexpression of hsa-miR-939 follows by NGFR down-regulation and apoptosis reduction.

Neurotrophin receptors play a crucial role in neuronal survival, differentiation and regeneration. Nerve growth factor receptor (NGFR) or P75NTR is a neurotrophin receptor that is involved in many pathological conditions including cancers. Genetic factors that are involved in regulation of neurotrophin receptors are under intense investigation. MiRNAs are novel regulators of signalling pathways that are candidates for regulation of neurotrophin receptors. Computational programs predicted that NGFR gene is a bona fide target for hsa-miR- 939. RT-qPCR, Western analysis and dual luciferase assay evidences indicated that NGFR transcript is targeted by hsa-miR-939. Also, hsa-miR-939 overexpression brought about down-regulation of NGFR expression in U87 cell line, followed by cell death rate reduction, detected by flow cytometry. Taken together, here for the first time, hsa-miR-939 is introduced as a novel key regulator of NGFR expression and its involvement in cell death/survival processes is suggested.

Experimental evidences for hsa-miR-497-5p as a negative regulator of SMAD3 gene expression.

The SMAD family comprises of transcription factors that function as signal transducers of transforming growth factor (TGFß) superfamily members. MiRNAs are a class of small noncoding RNAs that may play a major role in post transcriptional regulation of SMAD genes. Here, we intended to investigate if hsa-miR-497-5p is capable of regulating SMAD3 gene expression. Hsa-miR-497-5p was bioinformatically predicted as a candidate regulator of SMAD3 gene expression and then, hsa-miR-497-5p expression status was analyzed in different cell lines using RT-qPCR. Overexpression of hsa-miR-497-5p in HEK293t cells resulted in downregulation of SMAD3 which was detected by RT-qPCR and western analysis. Further, dual luciferase assay results supported direct interaction of hsa-miR-497-5p with 3'-UTR sequences of SMAD3 transcript. Overexpression of hsa-miR-497-5p in HEK293t cells resulted in cell cycle arrest in G0/G1 phase, detected by flow cytometry. Overall, accumulative results indicated that hsa-miR-497-5p by targeting SMAD3 is potentially one of the regulators of the TGFß signaling pathway.