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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 suppress in-vivo tumor growth and metastasis.miR-134, miR-370 (mimic and/or inhibitor) or scrambled -transfected RKO and DLD1 cell lines were inoculated on the upper CAM of 10 day old embryos and following a further 7 days of incubation, tumor growth was evaluated. (a) shows in-situ control and miRNA-treated tumors on the upper CAM at the site of inoculation, (b) shows the resected tumors from control and miRNA-treated groups arranged alongside each other, n = 7 eggs were used in each arm, (c) Average tumor weight in the three groups. (d) lung and e) liver metastasis in the CAM model were evaluated quantitatively by RT-PCR using genomic DNA isolated from the harvested organs; p < 0.05 (*) and p < 0.01 (**).
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f5: miRs-134 and -370 suppress in-vivo tumor growth and metastasis.miR-134, miR-370 (mimic and/or inhibitor) or scrambled -transfected RKO and DLD1 cell lines were inoculated on the upper CAM of 10 day old embryos and following a further 7 days of incubation, tumor growth was evaluated. (a) shows in-situ control and miRNA-treated tumors on the upper CAM at the site of inoculation, (b) shows the resected tumors from control and miRNA-treated groups arranged alongside each other, n = 7 eggs were used in each arm, (c) Average tumor weight in the three groups. (d) lung and e) liver metastasis in the CAM model were evaluated quantitatively by RT-PCR using genomic DNA isolated from the harvested organs; p < 0.05 (*) and p < 0.01 (**).

Mentions: Finally, we sought to assess if these miRs have any effect on tumor growth and distant metastasis in vivo. Towards this end, we used our well established CAM assay model1518. 7 days after seeding the miRs-transfected cells on the upper chorioallantoic membrane, the chicken embryos were sacrificed, the growing tumors on the upper CAM resected, and the lungs and livers harvested. The resected tumor masses were weighed and evaluated (Fig. 5a,b, Supplementary Fig. 1). Distant metastasis was assessed with human-specific Alu-PCR comparing the miR- with the scrambled control groups (Fig. 5c–e). We observed a significant reduction in the tumor weight with miRNA mimics and enhanced tumor growth with the inhibitors and a 3–4 fold decrease in lung and liver metastasis in the miR-134 and -370 transfected groups compared to the controls (p < 0.05, two tailed Mann-Whitney test, Fig. 5a–e). Enhanced expression and repression of miRNAs was monitored at regular intervals in-vitro over the 7 day period (Supplementary Fig. 2). Furthermore, the expression of the key targets, EGFR and PI3KCA in the CAM tumors were also evaluated at the mRNA and protein levels (Supplementary Fig. 3).


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 suppress in-vivo tumor growth and metastasis.miR-134, miR-370 (mimic and/or inhibitor) or scrambled -transfected RKO and DLD1 cell lines were inoculated on the upper CAM of 10 day old embryos and following a further 7 days of incubation, tumor growth was evaluated. (a) shows in-situ control and miRNA-treated tumors on the upper CAM at the site of inoculation, (b) shows the resected tumors from control and miRNA-treated groups arranged alongside each other, n = 7 eggs were used in each arm, (c) Average tumor weight in the three groups. (d) lung and e) liver metastasis in the CAM model were evaluated quantitatively by RT-PCR using genomic DNA isolated from the harvested organs; p < 0.05 (*) and p < 0.01 (**).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4837379&req=5

f5: miRs-134 and -370 suppress in-vivo tumor growth and metastasis.miR-134, miR-370 (mimic and/or inhibitor) or scrambled -transfected RKO and DLD1 cell lines were inoculated on the upper CAM of 10 day old embryos and following a further 7 days of incubation, tumor growth was evaluated. (a) shows in-situ control and miRNA-treated tumors on the upper CAM at the site of inoculation, (b) shows the resected tumors from control and miRNA-treated groups arranged alongside each other, n = 7 eggs were used in each arm, (c) Average tumor weight in the three groups. (d) lung and e) liver metastasis in the CAM model were evaluated quantitatively by RT-PCR using genomic DNA isolated from the harvested organs; p < 0.05 (*) and p < 0.01 (**).
Mentions: Finally, we sought to assess if these miRs have any effect on tumor growth and distant metastasis in vivo. Towards this end, we used our well established CAM assay model1518. 7 days after seeding the miRs-transfected cells on the upper chorioallantoic membrane, the chicken embryos were sacrificed, the growing tumors on the upper CAM resected, and the lungs and livers harvested. The resected tumor masses were weighed and evaluated (Fig. 5a,b, Supplementary Fig. 1). Distant metastasis was assessed with human-specific Alu-PCR comparing the miR- with the scrambled control groups (Fig. 5c–e). We observed a significant reduction in the tumor weight with miRNA mimics and enhanced tumor growth with the inhibitors and a 3–4 fold decrease in lung and liver metastasis in the miR-134 and -370 transfected groups compared to the controls (p < 0.05, two tailed Mann-Whitney test, Fig. 5a–e). Enhanced expression and repression of miRNAs was monitored at regular intervals in-vitro over the 7 day period (Supplementary Fig. 2). Furthermore, the expression of the key targets, EGFR and PI3KCA in the CAM tumors were also evaluated at the mRNA and protein levels (Supplementary Fig. 3).

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