MicroRNA-145 overexpression inhibits neuroblastoma tumorigenesis in vitro and in vivo

Abstract

Neuroblastoma (NB) is responsible for 15% of all childhood cancer deaths. Despite advances in treatment and disease management, the overall 5-year survival rates remain poor in high-risk disease (25-40%). It is well known that miR-145 functions as a tumor suppressor in several types of cancer. However, the impact of miR-145 on NB is still ambiguous. Our aim was to investigate the potential tumor suppressive role and mechanisms of miR-145 in high-risk neuroblastoma. Expression levels of miR-145 in tissues and cells were determined using RT-qPCR. The effect of miR-145 on cell viability was evaluated using MTT assays, apoptosis levels were determined using TUNEL staining, and the MTDH protein expression was determined using western blot and RT-PCR. Luciferase reporter plasmids were constructed to confirm direct targeting for MTDH. The results showed that miR-145 expression was significantly lower in high-risk MYCN amplified (MNA) tumors and low miR-145 expression was associated with worse EFS and OS in our cohort. Over-expression of miR-145 reduced cell viability and increased apoptosis in SH-SY-5Y cells. We identified MTDH as a direct target for miR-145 in SH-SY-5Y cells. Targeting MTDH has the similar results as miR-145 overexpression. Our findings suggest that low miR-145 expression was associated with poor prognosis in patients with NB, and the overexpression of miR-145 inhibited NB cells growth by down-regulating MTDH, thus providing a potential target for the development of microRNA-based approach for NB therapy.

Conservation of Zebrafish MicroRNA-145 and Its Role during Neural Crest Cell Development

“…Acridine Orange assays show that high levels of miR-145 increase apoptosis of zebrafish embryonic cells, suggesting that NCCs may die before differentiation ( Figure 6 ). This finding agrees with data gathered in embryonic and cancer cultured cell lines, wherein miR-145 overexpression promotes apoptosis and reduces cell viability [ 16 , 34 , 69 , 77 , 78 ]. These observations as a whole could suggest that miR-145 participates in the control of cell death regulation in both normal and pathological conditions.…”

The clinical significance of microRNA-409 in pancreatic carcinoma and associated tumor cellular functions

“…Different types of cancer have different miRNA expression profiles, which can distinguish adjacent normal tissues [19][20][21]. The discovery of miRNA not only provides new insights into the regulation mechanism of cell proliferation, differentiation, apoptosis but also promotes the development of diagnosis and treatment of diseases [22,23]. The biological characteristics of PC are closely related to the up-regulation and down-regulation of miRNA expression in PC [11,24].…”

Optimization of N-hydroxysuccinimide ester coupling with aminoallyl-modified RNA for fluorescent labeling

“…Post-synthetic labeling of RNA is applied for preparing fluorescently modified RNAs to alleviate the synthetic bottleneck due to steric inhibition from bulky fluorophores to RNAs [6][7][8]. And coupling reaction of N-hydroxysuccinimide ester (NHS ester) with primary amino group is a useful strategy for post-synthetic modification of RNAs [5,9,10]. Natural RNAs lack reactive primary amino groups, and site-specific introduction of primary amino to RNAs can trigger the conjugation between NHS ester and the specific site [7].…”

“…The aminoally-modified rbA71 (aa-rbA71) was synthesized by incorporation of 5-aminoally-UTP into Site 22 of rbA71 (Site 22 is labeled in red in Figure 1(a)) to obtain aa-rbA71 via PLOR (Position-specific Labeling of RNA) (the detailed synthesis is listed in Material and Methods), which is a novel platform for sitespecific labeling of RNA [15,21]. aa-rbA71 reacted with NHS ester-linked Tide Fluor 3 (NHS-TF3) to generate fluorescent-labeled rbA71 following the reported condition [7,[10][11][12][13][14][15][16][17][22][23][24][25][26]. Various conditions for NHS coupling reaction were reported.…”

“…Studies on protein or peptides labeling indicated that the NHS ester coupling reaction has been influenced by buffer pH greatly, while the buffer pH for RNA conjugation with NHSreagent narrows in 7.0-9.0 [7,[10][11][12][13][14][15][16][17][22][23][24][25][26]. Besides, the reaction time in some protocols may be shortened to drop the potential degradation of RNAs.…”