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miRs-134 and -370 function as tumor suppressors in colorectal cancer by independently suppressing EGFR and PI3K signalling.

El-Daly SM, Abba ML, Patil N, Allgayer H - Sci Rep (2016)

Bottom Line: Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis.Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins.Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Germany.

ABSTRACT
Growth factor receptor signalling plays a central and critical role in colorectal cancer. Most importantly, the EGFR signalling cascade involving PI3K/AKT/mTOR and Raf/MEK/ERK pathways are particularly relevant, since they are commonly activated in several cancer entities, including colorectal cancer. In this study, we show that miRs-134 and -370 are both capable of regulating these pathways by targeting EGFR and PIK3CA. In three different colorectal cancer cell lines (DLD1, HCT-116 and RKO), suppression of EGFR and PIK3CA through the enhanced expression of miR-134 or -370 led to a suppression of the key molecules of the PI3K/AKT/mTOR pathway. Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis. Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins. Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

No MeSH data available.


Related in: MedlinePlus

miRs 134 and -370 modulate growth factor signaling by targeting EGFR and PIK3CA.(a) Cell line screening for the endogenous expression of miRs-134 and -370 in a panel of colorectal cancer cell lines, graph shows expression relative to the GEO cell line after normalization to RNU6B. (b) Expression of EGFR and PIK3CA in the same colorectal cancer cell lines screened for miR-134 and miR-370, expression is shown relative to the GEO cell line. (c,d) Evaluation of EGFR and PIK3CA mRNA expression following transfection with miR-134, miR-370 or siRNAs against EGFR or PIK3CA (e) Alignment of seed sequences of miR-134 and miR-370 with binding motifs in the 3′UTRs of their respective target mRNAs and nucleotides which were mutated (underlined) for luciferase assays; (f,g), Reporter gene assays showing a direct regulation of EGFR 3′UTR by miR-134 and -370 and PIK3CA by miR-370. Site directed mutagenesis shows elimination of the observed regulation.
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f1: miRs 134 and -370 modulate growth factor signaling by targeting EGFR and PIK3CA.(a) Cell line screening for the endogenous expression of miRs-134 and -370 in a panel of colorectal cancer cell lines, graph shows expression relative to the GEO cell line after normalization to RNU6B. (b) Expression of EGFR and PIK3CA in the same colorectal cancer cell lines screened for miR-134 and miR-370, expression is shown relative to the GEO cell line. (c,d) Evaluation of EGFR and PIK3CA mRNA expression following transfection with miR-134, miR-370 or siRNAs against EGFR or PIK3CA (e) Alignment of seed sequences of miR-134 and miR-370 with binding motifs in the 3′UTRs of their respective target mRNAs and nucleotides which were mutated (underlined) for luciferase assays; (f,g), Reporter gene assays showing a direct regulation of EGFR 3′UTR by miR-134 and -370 and PIK3CA by miR-370. Site directed mutagenesis shows elimination of the observed regulation.

Mentions: To explore novel oncogenic targets of miRs-134 and -370, we used a combination of in silico prediction algorithms (TargetScan, miRANDA and Ingenuity), and consistently EGFR was found to be a highly predicted target for miRs-134 and -370 and PIK3CA to be a highly predicted target for miR-370 (Supplementary Table 1). As a guide to finding out if this prediction was accurate, we screened a panel of colorectal cancer cell lines for the expression of these two miRs as well as the target mRNAs. We found that the expression of these two mRNAs were higher in cell lines with low miRNA expression and vice-versa (p < 0.05, Fig. 1a,b, Supplementary Table 2). Thereafter we proceeded to confirm if the forced expression of these two miRs had any effect on the mRNA of EGFR and PIK3CA using miRNA mimics and siRNAs, as well as appropriate controls, where we discovered that both miR-134 and -370 led to a significant decrease of EGFR and PI3K mRNA in cultured cells (Fig. 1c,d). Based on these results, we sought to demonstrate that these two genes are direct targets of these miRNAs. The 3′UTR of EGFR has two sites for miR-370 and one for miR-134 while PIK3CA harbors one site for miR-370 (Fig. 1e). Reporter gene assays with the wild type and mutated 3′UTR constructs of these mRNAs showed that EGFR is a valid target for miRs-134 and 370 and PIK3CA is a valid targets of miR-370 (Fig. 1f,g, p < 0.05).


miRs-134 and -370 function as tumor suppressors in colorectal cancer by independently suppressing EGFR and PI3K signalling.

El-Daly SM, Abba ML, Patil N, Allgayer H - Sci Rep (2016)

miRs 134 and -370 modulate growth factor signaling by targeting EGFR and PIK3CA.(a) Cell line screening for the endogenous expression of miRs-134 and -370 in a panel of colorectal cancer cell lines, graph shows expression relative to the GEO cell line after normalization to RNU6B. (b) Expression of EGFR and PIK3CA in the same colorectal cancer cell lines screened for miR-134 and miR-370, expression is shown relative to the GEO cell line. (c,d) Evaluation of EGFR and PIK3CA mRNA expression following transfection with miR-134, miR-370 or siRNAs against EGFR or PIK3CA (e) Alignment of seed sequences of miR-134 and miR-370 with binding motifs in the 3′UTRs of their respective target mRNAs and nucleotides which were mutated (underlined) for luciferase assays; (f,g), Reporter gene assays showing a direct regulation of EGFR 3′UTR by miR-134 and -370 and PIK3CA by miR-370. Site directed mutagenesis shows elimination of the observed regulation.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4837379&req=5

f1: miRs 134 and -370 modulate growth factor signaling by targeting EGFR and PIK3CA.(a) Cell line screening for the endogenous expression of miRs-134 and -370 in a panel of colorectal cancer cell lines, graph shows expression relative to the GEO cell line after normalization to RNU6B. (b) Expression of EGFR and PIK3CA in the same colorectal cancer cell lines screened for miR-134 and miR-370, expression is shown relative to the GEO cell line. (c,d) Evaluation of EGFR and PIK3CA mRNA expression following transfection with miR-134, miR-370 or siRNAs against EGFR or PIK3CA (e) Alignment of seed sequences of miR-134 and miR-370 with binding motifs in the 3′UTRs of their respective target mRNAs and nucleotides which were mutated (underlined) for luciferase assays; (f,g), Reporter gene assays showing a direct regulation of EGFR 3′UTR by miR-134 and -370 and PIK3CA by miR-370. Site directed mutagenesis shows elimination of the observed regulation.
Mentions: To explore novel oncogenic targets of miRs-134 and -370, we used a combination of in silico prediction algorithms (TargetScan, miRANDA and Ingenuity), and consistently EGFR was found to be a highly predicted target for miRs-134 and -370 and PIK3CA to be a highly predicted target for miR-370 (Supplementary Table 1). As a guide to finding out if this prediction was accurate, we screened a panel of colorectal cancer cell lines for the expression of these two miRs as well as the target mRNAs. We found that the expression of these two mRNAs were higher in cell lines with low miRNA expression and vice-versa (p < 0.05, Fig. 1a,b, Supplementary Table 2). Thereafter we proceeded to confirm if the forced expression of these two miRs had any effect on the mRNA of EGFR and PIK3CA using miRNA mimics and siRNAs, as well as appropriate controls, where we discovered that both miR-134 and -370 led to a significant decrease of EGFR and PI3K mRNA in cultured cells (Fig. 1c,d). Based on these results, we sought to demonstrate that these two genes are direct targets of these miRNAs. The 3′UTR of EGFR has two sites for miR-370 and one for miR-134 while PIK3CA harbors one site for miR-370 (Fig. 1e). Reporter gene assays with the wild type and mutated 3′UTR constructs of these mRNAs showed that EGFR is a valid target for miRs-134 and 370 and PIK3CA is a valid targets of miR-370 (Fig. 1f,g, p < 0.05).

Bottom Line: Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis.Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins.Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

View Article: PubMed Central - PubMed

Affiliation: Department of Experimental Surgery, Medical Faculty Mannheim, University of Heidelberg, Germany.

ABSTRACT
Growth factor receptor signalling plays a central and critical role in colorectal cancer. Most importantly, the EGFR signalling cascade involving PI3K/AKT/mTOR and Raf/MEK/ERK pathways are particularly relevant, since they are commonly activated in several cancer entities, including colorectal cancer. In this study, we show that miRs-134 and -370 are both capable of regulating these pathways by targeting EGFR and PIK3CA. In three different colorectal cancer cell lines (DLD1, HCT-116 and RKO), suppression of EGFR and PIK3CA through the enhanced expression of miR-134 or -370 led to a suppression of the key molecules of the PI3K/AKT/mTOR pathway. Furthermore, overexpression of miR-134 or -370 resulted in a significant reduction of cell proliferation, colony formation, migration, invasion and in-vivo tumor growth and metastasis. Concurrent experiments with small interfering RNAs targeting the prime targets show that our selected miRNAs exert a greater functional influence and affect more downstream molecules than is seen with silencing of the individual proteins. Taken together, these data indicate that miRs-134 and -370 are potential tumour suppressor miRNAs and could play a fundamental role in suppressing colorectal cancer tumorigenesis through their ability to co-ordinately regulate EGFR signalling cascade by independently targeting EGFR and PIK3CA.

No MeSH data available.


Related in: MedlinePlus