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MicroRNA-330-5p as a Putative Modulator of Neoadjuvant Chemoradiotherapy Sensitivity in Oesophageal Adenocarcinoma.

Bibby BA, Reynolds JV, Maher SG - PLoS ONE (2015)

Bottom Line: MiRNAs have also been identified as modulators of key cellular pathways associated with resistance to CRT.In pre-treatment biopsies miR-330-5p was the most downregulated miRNA in patients who subsequently failed to respond to neo-CRT.However, miR-330-mediated alterations to the E2F1/p-AKT pathway were insufficient to significantly alter cellular sensitivity to chemotherapy (cisplatin and 5-flurouracil).

View Article: PubMed Central - PubMed

Affiliation: School of Biological, Biomedical and Environmental Sciences, University of Hull, Hull, United Kingdom.

ABSTRACT
Oesophageal adenocarcinoma (OAC) is the sixth most common cause of cancer deaths worldwide, and the 5-year survival rate for patients diagnosed with the disease is approximately 17%. The standard of care for locally advanced disease is neoadjuvant chemotherapy or, more commonly, combined neoadjuvant chemoradiation therapy (neo-CRT) prior to surgery. Unfortunately, ~60-70% of patients will fail to respond to neo-CRT. Therefore, the identification of biomarkers indicative of patient response to treatment has significant clinical implications in the stratification of patient treatment. Furthermore, understanding the molecular mechanisms underpinning tumour response and resistance to neo-CRT will contribute towards the identification of novel therapeutic targets for enhancing OAC sensitivity to CRT. MicroRNAs (miRNA/miR) function to regulate gene and protein expression and play a causal role in cancer development and progression. MiRNAs have also been identified as modulators of key cellular pathways associated with resistance to CRT. Here, to identify miRNAs associated with resistance to CRT, pre-treatment diagnostic biopsy specimens from patients with OAC were analysed using miRNA-profiling arrays. In pre-treatment biopsies miR-330-5p was the most downregulated miRNA in patients who subsequently failed to respond to neo-CRT. The role of miR-330 as a potential modulator of tumour response and sensitivity to CRT in OAC was further investigated in vitro. Through vector-based overexpression the E2F1/p-AKT survival pathway, as previously described, was confirmed as a target of miR-330 regulation. However, miR-330-mediated alterations to the E2F1/p-AKT pathway were insufficient to significantly alter cellular sensitivity to chemotherapy (cisplatin and 5-flurouracil). In contrast, silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to clinically relevant doses of radiation. This study highlights the need for further investigation into the potential of miR-330-5p as a predictive biomarker of patient sensitivity to neo-CRT and as a novel therapeutic target for manipulating cellular sensitivity to neo-CRT in patients with OAC.

No MeSH data available.


Related in: MedlinePlus

Alterations in E2F1 protein expression with miR-330 overexpression and silencing.Transient miR-330 overexpression (A and B) significantly decreased E2F1 protein expression when compared to the miR-VC (vector control). Densitometry was used to analyse western blot images. Analysis was performed using the one sample t-test; miR-VC 72 h vs. miR-330 72 h ***p < 0.001. Silencing miR-330-5p (miRZIP-330-5p) (C and D) did not alter E2F1 protein expression when compared to the miRZIP-VC (vector control). Data are presented as the mean ± SEM.
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pone.0134180.g002: Alterations in E2F1 protein expression with miR-330 overexpression and silencing.Transient miR-330 overexpression (A and B) significantly decreased E2F1 protein expression when compared to the miR-VC (vector control). Densitometry was used to analyse western blot images. Analysis was performed using the one sample t-test; miR-VC 72 h vs. miR-330 72 h ***p < 0.001. Silencing miR-330-5p (miRZIP-330-5p) (C and D) did not alter E2F1 protein expression when compared to the miRZIP-VC (vector control). Data are presented as the mean ± SEM.

Mentions: To manipulate miR-330 expression in vitro, OAC cell lines were transfected with a miRNA precursor construct encoding the miR-330 precursor sequence (for overexpression) or a plasmid encoding an anti-sense miR-330-5p sequence (for silencing). The overexpression plasmid construct produces both miR-330-3p and miR-330-5p. Therefore, overexpression refers to a general miR-330 overexpression, as it is not possible to discriminate between the contributions of miR-330-3p and -5p in this model. In line with the previous findings in prostate, overexpression of miR-330 significantly downregulated E2F1 protein levels (Fig 2A and 2B), and subsequently the levels of p-Akt also decreased (Fig 3) [27]. These data also confirmed the biological activity of the vector. Interestingly, E2F1 mRNA levels did not decrease with the overexpression of miR-330, suggesting that miR-330 represses post-transcriptional E2F1 mRNA translation, rather than degradation of the message (S3 Fig). The silencing vector encodes the complementary antisense sequence to miR-330-5p, which was specifically downregulated in the patient tumours. The antisense RNA produced by the plasmid binds specifically and irreversibly to endogenously expressed miR-330-5p in the cells. Silencing miR-330-5p did not alter expression of the E2F1 protein (Fig 2C and 2D).


MicroRNA-330-5p as a Putative Modulator of Neoadjuvant Chemoradiotherapy Sensitivity in Oesophageal Adenocarcinoma.

Bibby BA, Reynolds JV, Maher SG - PLoS ONE (2015)

Alterations in E2F1 protein expression with miR-330 overexpression and silencing.Transient miR-330 overexpression (A and B) significantly decreased E2F1 protein expression when compared to the miR-VC (vector control). Densitometry was used to analyse western blot images. Analysis was performed using the one sample t-test; miR-VC 72 h vs. miR-330 72 h ***p < 0.001. Silencing miR-330-5p (miRZIP-330-5p) (C and D) did not alter E2F1 protein expression when compared to the miRZIP-VC (vector control). Data are presented as the mean ± SEM.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519309&req=5

pone.0134180.g002: Alterations in E2F1 protein expression with miR-330 overexpression and silencing.Transient miR-330 overexpression (A and B) significantly decreased E2F1 protein expression when compared to the miR-VC (vector control). Densitometry was used to analyse western blot images. Analysis was performed using the one sample t-test; miR-VC 72 h vs. miR-330 72 h ***p < 0.001. Silencing miR-330-5p (miRZIP-330-5p) (C and D) did not alter E2F1 protein expression when compared to the miRZIP-VC (vector control). Data are presented as the mean ± SEM.
Mentions: To manipulate miR-330 expression in vitro, OAC cell lines were transfected with a miRNA precursor construct encoding the miR-330 precursor sequence (for overexpression) or a plasmid encoding an anti-sense miR-330-5p sequence (for silencing). The overexpression plasmid construct produces both miR-330-3p and miR-330-5p. Therefore, overexpression refers to a general miR-330 overexpression, as it is not possible to discriminate between the contributions of miR-330-3p and -5p in this model. In line with the previous findings in prostate, overexpression of miR-330 significantly downregulated E2F1 protein levels (Fig 2A and 2B), and subsequently the levels of p-Akt also decreased (Fig 3) [27]. These data also confirmed the biological activity of the vector. Interestingly, E2F1 mRNA levels did not decrease with the overexpression of miR-330, suggesting that miR-330 represses post-transcriptional E2F1 mRNA translation, rather than degradation of the message (S3 Fig). The silencing vector encodes the complementary antisense sequence to miR-330-5p, which was specifically downregulated in the patient tumours. The antisense RNA produced by the plasmid binds specifically and irreversibly to endogenously expressed miR-330-5p in the cells. Silencing miR-330-5p did not alter expression of the E2F1 protein (Fig 2C and 2D).

Bottom Line: MiRNAs have also been identified as modulators of key cellular pathways associated with resistance to CRT.In pre-treatment biopsies miR-330-5p was the most downregulated miRNA in patients who subsequently failed to respond to neo-CRT.However, miR-330-mediated alterations to the E2F1/p-AKT pathway were insufficient to significantly alter cellular sensitivity to chemotherapy (cisplatin and 5-flurouracil).

View Article: PubMed Central - PubMed

Affiliation: School of Biological, Biomedical and Environmental Sciences, University of Hull, Hull, United Kingdom.

ABSTRACT
Oesophageal adenocarcinoma (OAC) is the sixth most common cause of cancer deaths worldwide, and the 5-year survival rate for patients diagnosed with the disease is approximately 17%. The standard of care for locally advanced disease is neoadjuvant chemotherapy or, more commonly, combined neoadjuvant chemoradiation therapy (neo-CRT) prior to surgery. Unfortunately, ~60-70% of patients will fail to respond to neo-CRT. Therefore, the identification of biomarkers indicative of patient response to treatment has significant clinical implications in the stratification of patient treatment. Furthermore, understanding the molecular mechanisms underpinning tumour response and resistance to neo-CRT will contribute towards the identification of novel therapeutic targets for enhancing OAC sensitivity to CRT. MicroRNAs (miRNA/miR) function to regulate gene and protein expression and play a causal role in cancer development and progression. MiRNAs have also been identified as modulators of key cellular pathways associated with resistance to CRT. Here, to identify miRNAs associated with resistance to CRT, pre-treatment diagnostic biopsy specimens from patients with OAC were analysed using miRNA-profiling arrays. In pre-treatment biopsies miR-330-5p was the most downregulated miRNA in patients who subsequently failed to respond to neo-CRT. The role of miR-330 as a potential modulator of tumour response and sensitivity to CRT in OAC was further investigated in vitro. Through vector-based overexpression the E2F1/p-AKT survival pathway, as previously described, was confirmed as a target of miR-330 regulation. However, miR-330-mediated alterations to the E2F1/p-AKT pathway were insufficient to significantly alter cellular sensitivity to chemotherapy (cisplatin and 5-flurouracil). In contrast, silencing of miR-330-5p enhanced, albeit subtly, cellular resistance to clinically relevant doses of radiation. This study highlights the need for further investigation into the potential of miR-330-5p as a predictive biomarker of patient sensitivity to neo-CRT and as a novel therapeutic target for manipulating cellular sensitivity to neo-CRT in patients with OAC.

No MeSH data available.


Related in: MedlinePlus