Neuroblastoma is a paediatric cancer which originates from precursor cells of the sympathetic nervous system and accounts for 15% of childhood cancer mortalities. With regards to the role of miRNAs ...in neuroblastoma, miR-34a, mapping to a chromosome 1p36 region that is commonly deleted, has been found to act as a tumor suppressor through targeting of numerous genes associated with cell proliferation and apoptosis.
A synthetic miR-34a (or negative control) precursor molecule was transfected into NB1691luc and SK-N-ASluc neuroblastoma cells. Quantitative PCR was used to verify increased miR-34a levels in NB1691luc and SK-N-ASluc cell lines prior to in vitro and in vivo analysis. In vitro analysis of the effects of miR-34a over expression on cell growth, cell cycle and phosphoprotein activation in signal transduction pathways was performed. Neuroblastoma cells over expressing miR-34a were injected retroperitoneally into immunocompromised CB17-SCID mice and tumor burden was assessed over a 21 day period by measuring bioluminescence (photons/sec/cm²).
Over expression of miR-34a in both NB1691luc and SK-N-ASluc neuroblastoma cell lines led to a significant decrease in cell number relative to premiR-negative control treated cells over a 72 hour period. Flow cytometry results indicated that miR-34a induced cell cycle arrest and subsequent apoptosis activation. Phosphoprotein analysis highlighted key elements involved in signal transduction, whose activation was dysregulated as a result of miR-34a introduction into cells. As a potential mechanism of miR-34a action on phosphoprotein levels, we demonstrate that miR-34a over-expression results in a significant reduction of MAP3K9 mRNA and protein levels. Although MAP3K9 is a predicted target of miR-34a, direct targeting could not be validated with luciferase reporter assays. Despite this fact, any functional effects of reduced MAP3K9 expression as a result of miR-34a would be expected to be similar regardless of the mechanism involved. Most notably, in vivo studies showed that tumor growth was significantly repressed after exogenous miR-34a administration in retroperitoneal neuroblastoma tumors.
We demonstrate for the first time that miR-34a significantly reduces tumor growth in an in vivo orthotopic murine model of neuroblastoma and identified novel effects that miR-34a has on phospho-activation of key proteins involved with apoptosis.
The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The ...MYCN transcription factor, whose gene is amplified in ∼30% of high‐risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high‐risk subtypes of neuroblastoma lacking MYCN amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non‐MYCN amplified tumour cells, an SK‐N‐AS subline (SK‐N‐AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse‐selection process. High resolution aCGH analysis of SK‐N‐AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (NAIP). Significant overexpression of NAIP mRNA and protein was documented, while experimental modulation of NAIP levels in both SK‐N‐AsCis24 and in parental SK‐N‐AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the NAIP targeting microRNA, miR‐520f, was also demonstrated to be partially responsible for increased NAIP levels in SK‐N‐AsCis24. Interestingly, miR‐520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance in vivo, potentially through decreased NAIP targeting. Our findings provide biological novel insight into neuroblastoma drug‐resistance and have implications for future therapeutic research.
What's new?
Just under one‐third of high‐risk neuroblastomas, in which drug resistance is a central feature, involve amplification of the MYCN gene. Resistance in remaining high‐risk tumors may be determined by any of several non‐MYCN amplification mechanisms. Here, analysis of an SK‐N‐AS subline (SK‐N‐AsCis24) lacking MYCN amplification but resistant to cisplatin and etoposide reveals a link between the development of drug resistance via apoptotic inhibition and the neural apoptosis inhibitory protein (NAIP) and its regulatory microRNA, miR‐520f. NAIP upregulation was associated with DNA copy number gain on chromosome 5 and down‐regulation of miRNA‐520f. MiR‐520f was discovered to be down‐regulated post‐chemotherapy.
The acquisition of multidrug resistance is a major impediment to the successful treatment of neuroblastoma, a clinically heterogeneous cancer accounting for ∼15% of all pediatric cancer deaths. The ...MYCN transcription factor, whose gene is amplified in ∼30% of high‐risk neuroblastoma cases, influences drug resistance by regulating a cadre of genes, including those involved with drug efflux, however, other high‐risk subtypes of neuroblastoma lacking
MYCN
amplification, such as those with chromosome 11q deletions, also acquire multidrug resistance. To elucidate additional mechanisms involved with drug resistance in non‐MYCN amplified tumour cells, an SK‐N‐AS subline (SK‐N‐AsCis24) that is significantly resistant to cisplatin and cross resistant to etoposide was developed through a pulse‐selection process. High resolution aCGH analysis of SK‐N‐AsCis24 revealed a focal gain on chromosome 5 containing the coding sequence for the neural apoptosis inhibitory protein (
NAIP
). Significant overexpression of
NAIP
mRNA and protein was documented, while experimental modulation of NAIP levels in both SK‐N‐AsCis24 and in parental SK‐N‐AS cells confirmed that NAIP was responsible for the drug resistant phenotype by apoptosis inhibition. Furthermore, a decrease in the
NAIP
targeting microRNA, miR‐520f, was also demonstrated to be partially responsible for increased NAIP levels in SK‐N‐AsCis24. Interestingly, miR‐520f levels were determined to be significantly lower in postchemotherapy treatment tumours relative to matched prechemotherapy samples, consistent with a role for this miRNA in the acquisition of drug resistance
in vivo
, potentially through decreased
NAIP
targeting. Our findings provide biological novel insight into neuroblastoma drug‐resistance and have implications for future therapeutic research.
What's new?
Just under one‐third of high‐risk neuroblastomas, in which drug resistance is a central feature, involve amplification of the
MYCN
gene. Resistance in remaining high‐risk tumors may be determined by any of several non‐
MYCN
amplification mechanisms. Here, analysis of an SK‐N‐AS subline (SK‐N‐AsCis24) lacking
MYCN
amplification but resistant to cisplatin and etoposide reveals a link between the development of drug resistance via apoptotic inhibition and the neural apoptosis inhibitory protein (NAIP) and its regulatory microRNA, miR‐520f.
NAIP
upregulation was associated with DNA copy number gain on chromosome 5 and down‐regulation of miRNA‐520f. MiR‐520f was discovered to be down‐regulated post‐chemotherapy.
The purpose of this research was to explore parental perspectives on the impact of parent restrictions imposed in response to the COVID-19 pandemic across Canadian Neonatal Intensive Care Units ...(NICUs). A co-designed online survey was conducted targeting parents (n = 235) of infants admitted to a Canadian NICU from March 1, 2020, until March 5, 2021. Parents completed the survey from 38 Canadian NICUs. Large variation in the severity of policies regarding parental presence was reported. Most respondents (68.9%) were classified as experiencing high restrictions, with one or no support people allowed in the NICU, and felt that policies were less easy to understand, felt less valued and respected, and found it more challenging to access medicine or health care. Parents reported gaps in care related to self-care, accessibility, and mental health outcomes. There is significant variation in parental restrictions implemented across Canadian NICUs. National guidelines are needed to support consistent and equitable care practices.
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