MicroRNA biogenesis and mechanismThe mainly of miRNAs are encoded within the genome and are transcribed by RNA polymerase II (Pol II) or RNA polymerase III (pol III) as long precursor transcripts, known as primary miRNAs (pri-miRNAs) of several kilobases (kb) in length (14). Mature miRNAs are produced from pri-miRNAs by sequential processing strategies.

Conventionally animal pri-miRNAs demonstrate nearly 33 bp stem and a terminal loop structure with flanking segments (15). Precursor miRNAs are commonly the microprocessor complex in the nucleus, whose core components are the RNase-III enzyme Drosha and its binding partner DiGeorge syndrome critical region 8 (DGCR8) (16). Approximately 31% of miRNAs are processed from introns of protein-coding genes, whereas many other miRNAs are expressed from committing miRNA gene loci. An own pri miRNA can either produce a single miRNA or contain clusters of two or more miRNAs that are changed from a regular primary transcript (17). However, these long pri-miRNAs are cleaved by Microprocessor, comprising the double-stranded RNase III enzyme DROSHA and its crucial cofactor, the double-stranded RNA binding protein DGCR8 (18).

In this pathway, the nuclear splicing machinery, supplies pre-miRNA from introns. miRNAs produced by this process are appropriately termed mirtrons. These molecules enclose a small class of miRNAs but are identified in many organisms (19). Preliminary, thenuclear microprocessorcomplex identifiesthe miRNA hairpins in the original transcript and cleaves each hairpin roughly 11 nucleotides from its base (20). Recent manifestation suggests that some miRNAs that survive within introns, the so-called mirtrons, bypass Drosha cleavage and depend on the proceeding of the pre-mRNA splicing machinery to originate an approximately 60 nt precursor miRNA (pre-miRNA) hairpin (21). Regardless of whether the pre-miRNA hairpin is excised from the primary transcript by canonical Drosha cleavage or through the mirtron pathway, the next stage in the miRNA biogenesis is recognition of the nearly 60 nt pre-miRNA by exportin-5 and export to the cytoplasm in  a  ran guanine triphosphatase-dependent manner (22). Mirtrons encompass a small cluster of miRNAs but are found in numerous organisms.

Precursor miRNAs are exported from the nucleus to the cytoplasm by the exportin-5 ?RanGTP heterocomplex (23). Complex in cytoplasm compromised of Dicer RNase III endonuclease, TAR RNA-binding protein, and protein kinase Re-activating. Protein further cleaves the precursor miRNA generating a short double-stranded miRNA:miRNA complex intermediate (24).  In the cytoplasm, pre-miRNAs are processed by RNase III enzyme Dicer.

Dicer is thought to react with dsRBDs- containing partner proteins, HIV TAR RNA-binding protein (TRBP) and ? or PKR activating protein (PACT) (25). Then, Dicer cleaves pre-miRNAs into 21–25 nt long miRNA? miRNA duplexes, each strand of which bears 5′ monophosphate, 3′ hydroxyl group and a 3′ 2-nt overhang. It preferentially incorporates one of the duplexes strands into the RNA-induced silencing complex (RISC). Of a miRNA? miRNA duplex, only one strand, designated the miRNA strand, is selected as the guide of mature RISC, whereas the other strand, the miRNA strand, is discarded during RISC assembly (26).

Such biased strand selection depends on the stability of at least three properties of a miRNA?miRNA duplex: the structure; the 5′ nucleotide identity; and the thermodynamic asymmetry (27). These findings propose that miRNA processing by Dicer, During RISC loading, and target RNA cleavage by Ago2 is coupled (28). Paralleled to the RNA cleavage process by Ago2, the translational repression mechanism by miRNAs has been poorly understood Recently, it was revealed that the target mRNAs bindings to RISC through partial base pairing are accumulated in the cytoplasmic foci referred to as processing bodies (P-bodies). P-bodies, in which the mRNAs are stored or degraded by the decapping enzymes and exonucleases, do not contain the translational machinery (29) (Fig. 1). DICERDicer is a critical RNaseIII endonuclease of the microRNA processing pathway, performs a role in carcinogenesis and various types of human malignancies (30). Dicer is a large multi-domain protein its principal function for the last step of microRNA and short-interfering RNA biogenesis.

In human and mouse cell lines, Dicer has been disclosed to be considered in the nuclear clearance of dsRNA as well as the foundation of chromatin agreements (31). The endoribonuclease Dicer is identified for its essential role in the biogenesis of eukaryotic small RNAs/miRNAs although it’s required; Dicer target transcripts have not been directly mapped. Interestingly, mainly Dicer-binding sites remain on mRNAs/ lncRNAs and are not substantially processed into small RNAs. These passive sites normally harbor small, Dicer-bound hairpins within intact transcripts and generally stabilize target expression (32). Dicer, single of the proteins implicated in the synthesis miRNA, is involved in the biogenesis of miRNAs and is influential in carcinogenesis and cancer development and progression (33). Low expressions of Dicer gene and protein correlated with poor prognosis and recurrence of cervical cancer.In addition; investigation revealed that decreased of Dicer gene expression and protein levels were associated with patients with metastatic relapse and tumor stage.

Sufferers with decrease Dicer miRNA and protein expression displayed a shorter 5-year disease-free survival and overall survival. Moreover, low expressions of Dicer befit to be a significant prognostic factor for cervical malignancy. According to these findings in patients with cervical cancer are consistent with the other results, which have been reported in the written works in other tumors (34). Previous studies have shown that inhibition of Dicer in von Hippel-Lindau deficient clear cell renal cell carcinoma contributed to the high levels of the hypoxia-inducible factors -2? and a cancer phenotype, which suggests Dicer, could be a useful therapeutic target for managing this disease (35). MiR-200a and miR-31 are targeted for Dicer and are involved in the carcinogenesis, migration, and behavior of castration-resistant prostate cancer, showing that they could be possible biomarkers for scanning prostate cancer progression (36). The Dicer protein expression is significantly associated with hormone receptor condition and cancer subtype in breast tumors (37). Drosha and Dicer1 mutations impair expression of tumor-suppressing miRNAs, including the let-7 family, significant regulators of MYCN, LIN28 and other Wilms tumor oncogenes. Current findings insights to the mechanisms through which mutations in miRNA biogenesis components reprogrammed miRNA expression in human malignancies and propose that these defects clarify a distinct subclass of Wilms tumors (38).

However, there are several investigations have been revealed that dysregulation of  Dicer gene correlated to the many disorders, including up-regulation in Dicer1 gene expression compared with tumor stages and progression malignancy of Prostate cancer and Smooth muscle tumors, respectively (89, 39).  In addition, preliminary investigations revealed that Up-regulation of Dicer1 is correlated with gastric tumor subtype and advanced tumor stages in Gastric cancer and serous ovarian cancer (40, 41). However, in other studies showed that increases of Dicer1 expression levels are required for proliferation with Oral cancer (42). Faber et al evaluated Dicer1 expression levels in colorectal cancer, and findings revealed an increased level, which is associated with tumor stage and poor survival patients (43).

Chiosea et al disclosed that Up-regulation Dicer1 expression levels are associated with histological subtypes and stages with precursor lesions of lung cancer (44). Ma et al showed that Up-regulation Dicer1 expression levels correlated with clinical stage with Cutaneous melanoma (45). In general, Dicer expression levels are different in cancer types, so that, Witkowski et al revealed that Down-regulation of Dicer1 expression levels could be altered miRNA profile in patients with Bladder cancer (46). Pampalakis et al. revealed that decreased of Dicer1 expression levels are associated with advanced tumor stage and poor patient survival in individuals with ovarian cancer (47). Contradictory to the other findings, Torres et al showed that Down-regulation of Dicer1 gene is not associated with histological grade detected in patients with endometrial cancer (48). Guo et al was observed to decrease expression of Dicer1, which was correlated with shorter patient survival with nasopharyngeal carcinoma (49).

In addition, Lin et al revealed that of Dicer1 expression, levels were significantly lower and associated with global downregulation of miRNAs and poor outcome with Neuroblastoma (50). In breast cancer investigation, Khoshnaw et al. revealed that Dicer1 expression levels decreased are associated with cancer progression and recurrence malignancy (51). Furthermore, investigations displayed that decrease of Dicer1 levels are correlated with metastasis, invasion and poor prognosis and shortened survival in the individuals with Gallbladder adenocarcinoma and Non-small cell lung cancer (52, 53). Wu et al. revealed that down-regulation of Dicer1 is not associated with clinical characteristics with Hepatocellular carcinoma (54). Findings in other studies revealed that decreased of Dicer1 expression levels are associated with progression, prognosis and tumor stage and shorter survival patients with Chronic lymphocytic leukemia, Colorectal cancer, breast cancer, and Papillary Thyroid Carcinoma (55-59) (Table 1).

 DROSHADROSHA involves two RNase III domains, which performs a vital role in miRNA biogenesis both together; Drosha and its double-stranded RNA-binding partner protein Pasha/DGCR8 likely identify and cut miRNA precursor RNAs or pri-miRNA hairpins cotranscriptionally (60). Long pri-miRNAs are cleaved by Microprocessor complex comprising of DROSHA, a type III ribonuclease (RNase-III), and an RNA-binding protein DGCR8 (DiGeorge syndrome critical region gene 8), is so called because a DROSHA/DGCR8 complex is essential and sufficient to process a primary microRNA (pri-miRNA) into a precursor microRNA (pre-miRNA) hairpin in vitro. Dicer then cleaves the pre-miRNA hairpin at the loop to form the mature microRNA.  Fidelity of DROSHA cleavage in the pri-miRNA is critical to the production of a functional microRNA (61). Drosha One of the outstanding factors in microRNA biogenesis and as such obligatory for cellular homeostasis and developmental processes. In collaboration with its cofactor DGCR8, changes the pri-miRNA into the pre-miRNA in the nucleus. Whilst the middle and the C-terminal domain are important for pri-miRNA processing and DGCR8 binding, the activity of the N-terminus remains mysterious.

Different investigations have linked this region to the subcellular localization of Drosha, stabilization, and response to tension (62). The repression occurs solely in mature miRNAs and not in primary miRNA transcripts, proposing that the DROSHA E1147K mutation affects processing of primary miRNAs. The pivotal role of the miRNA biogenesis pathway showed in Wilms tumor (WT) tumorigenesis, particularly the major miRNA processing gene DROSHA (63). Decrease expression of DROSHA, which also highlights a perturbed miRNA biogenesis pathway in melanoma. Furthermore, the irregular subcellular location of DROSHA reveals potential deregulation in the procedures responsible for its proper localization in the nucleus (64). The new results show that, the gastric cancer that cytoplasmic Drosha potentially plays a role in blocking carcinogenesis and tumor progression, and maybe an independent prognosticator of patient consequence (65).

The copy number discrepancy of Dicer1 and DROSHA correlates well with their expression and survival of non-small-cell lung cancer (NSCLC) and alternative cancer patients. The raised expression of DROSHA and Dicer1 decreases and increases the survival, respectively. As a result, copy number variation may be a significant mechanism of up-regulation/downregulation of miRNAs in malignancy and propose an oncogenic role for DROSHA (66). Muralidhar et al. revealed that Up-regulation of DROSHA expression levels could be altered miRNA profile; associated with neoplastic progression with cervical squamous cell carcinoma (SCC) (39).

In addition, Sugito et al. have been shown that increase of DROSHA expression was associated with poor patient survival with Oesophageal malignancy (67). The findings of several investigations were disclosed that Up-regulation of DROSHA was associated with tumor progression, patient survival in, advanced tumor stages and poor prognosis in the patients with various cancer types (37-39, 51). Moreover, Sand et al.

revealed that Up-regulation of DROSHA expression levels was not determined with SCC and basal cell carcinoma (BCC) (68). Avery-Kiejda et al. revealed that Up-regulation of DROSHA expression levels were no significant clinical correlation with triple-negative breast cancer (69).

Similarly, in others investigations, findings showed that Down-regulation of DROSHA expression levels could be altered miRNA profile and correlated with poor patient survival, histological grade, metastasis, invasion and poor prognosis in many cancers (48-50, 52, 64, 70, 71). Findings revealed that altered of DROSHA expression levels are not associated with clinical featuers in  colorectal cancer, breast cancer, and papillary thyroid carcinoma (57-59 ) (Table 1).  DGCR8 Mirtrons pathways include a small class of miRNAs although miRNAs are prepared by the miRtron pathway rather than by Drosha, the synthesis of main miRNAs looks to be Drosha dependent.

The prominent stem-loop in pri-miRNAs is identified through Drosha together with its partner Pasha/DGCR8 Indeed, Pasha/DGCR8 is thought to bind preferentially at the junction between the stem and the more inflexible loop, and this process can be co-transcriptional. This binding then positions Drosha midway up to the stem so that it is correctly positioned to make a pair of staggered breaks to generate the nearly 70 bp pre-miRNA (72). The microprocessor complex mediates intranuclear biogenesis of precursor miRNAs from the primary miRNA transcript. Extranuclear, mature miRNAs are merged into the RISC before interaction with completing target mRNA that leads to protein translationally repression or mRNA destabilization. The DGCR8 is a portion of microprocessor complex and has been disclosed to be crucial for miRNA maturing.

The argonaute 2 Ago2 proteins are a component of a complex protein indicated as RISC. A previous investigation has proven that DGCR8 mRNA expression level is down-regulated in prostate cancer. Up-regulated by the mRNA expression level of DGCR8 has been manifested in epithelial skin cancer and pleomorphic adenomas of the salivary gland.

It has been documented that the Ago2 mRNA expression level is up-regulated in epithelial skin cancer (73). Firstly, the DGCR8 mRNA expression level was up-regulated in colorectal cancer, recommending its influential role in the pathobiology of the colorectal carcinogenesis (74). DGCR8 was considerably expressed in ovarian cancer.   MiR-27b was detected as the mainly highly down-regulated miRNA in DGCR8 knockdown cells and endorsed cell proliferation in ovarian cancer cells (75). Preliminary studies disclosed that expression levels of Drosha in AGS and HepG2 cells lines were stronger than the controls, whereas, Drosha expression level in KYSE-30 cell line was lesser. The Dicer expression levels in AGS and HepG2 cells were increased, whereas, its expression level in KYSE-30 cell was lower.

The DGCR8 expression levels in all three cell lines were significantly higher than the control samples (76). In addition, Sugito et al. revealed that up-regulation of DGCR8 expression levels were associated with poor patient survival with Oesophageal cancer (67). Catto et al.

showed that increases of DGCR8 expression levels have altered the miRNA profile with Bladder cancer (70). Sand et al. displayed that up-regulation of DGCR8 expression levels was not determined with squamous cell carcinoma and Bladder cancer, additionally, revealed that Up-regulation of DGCR8 expression levels were associated with dysregulated miRNA with Prostate cancer (68). Kim et al. revealed that Up-regulation of DGCR8 expression levels were not associated with any clinical parameters with colorectal carcinoma (73). Furthermore, Guo et al. revealed that increases of DGCR8 expression levels were required for cell proliferation, migration, and invasion with ovarian cancer (49).

Findings revealed that altered of DGCR8 expression levels are not associated with clinical featuers in  papillary thyroid carcinoma (59). AGO Pre-miRNAs exported into the cytoplasm are processed by another RNase III enzyme, Dicer into nearly 21bp double-stranded miRNA-miRNA duplexes and moved into the groove of Ago. Afterwards, the miRNA strand dissociation, mature single-stranded miRNA remains loaded into and stabilized by Ago1.

Ago was subsequently separated from of Dicer to bind TNRC64, an essential co-factor in the miRNA-induced silencing complex (77). AGO2 is a major part of the RISC that can directly deteriorate mRNA through slicing Ago2 amasses in cytoplasmic processing bodies (P-bodies), where additional binding interactions promote translational inhibition and mRNA decay. Ago2 also couples with MVEs in structures that have been called “GW-bodies” because of the presence of GW182 but lack of other P-body components. Current publishes to have been demonstrated that Ago2 binds to miRNAs to generate Ago2-miRNA complexes that are found in the extracellular space. Although the majority of descriptions illustrate Ago2 as being present in the extracellular space as a free protein, other investigations have shown that Ago2 and other RNA-processing proteins are contained in the secreted exosomes (78). The genetic polymorphism in Ago2 may be a risk factor for the progressive lymph node metastasis of nasopharyngeal carcinoma (NPC) in Chinese populations, and Ago2 acts as an oncogene in the development of NPC (79).

Sand  et al. revealed that up-regulation Ago1 and Ago2 expression levels were not determined with actinic keratoses, SCC and BCC also revealed that Up-regulation Ago1 and Ago2 expression levels were associated with advanced tumor stages with Serous ovarian carcinoma (68). Findings revealed that the Ago2 expression levels was significantly lower in neoplastic tissues compared to the nonneoplastic tissues in  papillary thyroid carcinoma ( 59) (Table 1).  XPO5XPO5 is a component of the importin-b family of proteins that consist of one major class of nucleo-cytoplasmic transporters. XPO5 binds directly to its pre-miRNA cargo in a Ran-GTP- dependent behavior. As well, XPO5 be capable of recognizing and export structured RNAs that are unrelated to pre-miRNAs, involving viral mini-helix RNA and tRNA, along with certain other proteins, such as STAU2, ILF3, and JAZ.

It has also been indicated that XPO5 is important in siRNA biogenesis and therefore, is a basic point of intersection between the siRNA and miRNA pathways (80). XPO5 is a transporter protein regularly mediating pre-microRNAs’ nuclear export. Recent investigations have displayed that XPO5 May be important and crucial roles in a few of cancers. Anyway, little is known about XPO5 in hepatocellular carcinoma (HCC) (81). The XPO5 genetic defect traps pre-miRNAs in the nucleus decreases miRNA processing and diminishes miRNA-target inhibition (82). XPO5 knockdown promoted HCC cell migration and decreased the expression of E-cadherin and p53. Furthermore, the other hand, after treatment with DAC and TSA, the mRNA level of XPO5 was up-regulated in HCC cells tested; implicating that epigenetic modulation may be involved in the transcription of XPO5. Generally, our findings suggest that XPO5 functions as a potential tumor suppressor in the development and progression of HCC as well as a promising molecular target for HCC therapy (83).

Catto et al, revealed that Up-regulation XPO5 expression levels were associated with altered miRNA profile with Bladder cancer (70) (Table 1). TRBP MiRNAs are next sold abraded from the nucleus to the cytoplasm by exportin-5 mediated pathway, where they are bound via Dicer and the transactivation-responsive RNA-binding protein (TRBP). Further changed taken place by two proteins process pre-miRNAs into mature double-stranded miRNAs. The active strand of this mature miRNA is retained in the Dicer-TRBP complex, which then will be added the endonuclease Ago2.

One strand of the mature miRNA (the guide strand) is loaded into the RISC purposes mRNAs that are complementary to the miRNA (84). Huang et al, revealed that Up-regulation TRBP expression levels were amplified in breast cancer protein with breast cancer (85). TRBP is multifunctional and mediates cross-talk between different pathways.

It is activities at the molecular-level impact the cellular function from normal development to cancer and the response to infections. The amplified in breast cancer-3 protein (AIB3, also known as ASC-2, RAP250, PRIP, TRBP, and NCR) is a newly recognized nuclear receptor co-activator that is amplified and over-expressed in breast cancers (86). Lin et al, revealed that frequently up-regulated and the breast cancer patients with cytoplasm TARBP2 expression had poorer disease free survival and overall survival ( 87). Sand et al. showed that TARBP2 was significantly up-regulated in benign melanocytic nevi (BMN) compared to primary cutaneous malignant melanoma (PCMM) (88) (Table 1).

In previously, we were applied a gold standard method for lymphoma malignances detetion (90-96), whereas its conceivable that using TARBP2 gene expression analysis opened a new window in lymphoma diagnosis. Although, Caramuta et al, revealed that the expression of TARBP2 was significantly higher in diffuse large B cell lymphomas than with lymph nodes (97). In addition, Caramuta et al, assessed the expression level of TARBP2 and showed that significantly elevated in the adrenocortical carcinomas compared with adenomas or normal adrenal cortices. Whereas, TARBP2 expression was not correlated with histopathological and clinical parameters (98). De Vito et al, were revealed that deregulation of TARBP2 in the Ewing sarcoma ( 99).

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