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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 cell proliferation and colony formation.Cellular growth curves of HCT-116, RKO and DLD1 cell lines following transfection with (a) miR-134 or miR-370 mimics; (b) miR-134 or miR-370 inhibitors and (c) siRNAs targeting EGFR and PIK3CA. A total of 1 × 103 cells/cell line were seeded in 96-well plates and proliferation was assessed at 0, 24, 48, 72 and 96 time points using the MTT assay. Colony formation assay was performed with the same cell lines used for cell proliferation. The cell lines were treated with (d) miR-134 or miR-370 mimics; (e) miR-134 or miR-370 inhibitors and (f) siRNAs targeting EGFR and PIK3CA. The upper panels show representative examples of the scanned plates and the lower panels signify the overall quantification of the colony counts. Details are as discussed in the materials and methods (significance was set at p < 0.05 as compared to control group).
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f3: miRs-134 and 370 suppress cell proliferation and colony formation.Cellular growth curves of HCT-116, RKO and DLD1 cell lines following transfection with (a) miR-134 or miR-370 mimics; (b) miR-134 or miR-370 inhibitors and (c) siRNAs targeting EGFR and PIK3CA. A total of 1 × 103 cells/cell line were seeded in 96-well plates and proliferation was assessed at 0, 24, 48, 72 and 96 time points using the MTT assay. Colony formation assay was performed with the same cell lines used for cell proliferation. The cell lines were treated with (d) miR-134 or miR-370 mimics; (e) miR-134 or miR-370 inhibitors and (f) siRNAs targeting EGFR and PIK3CA. The upper panels show representative examples of the scanned plates and the lower panels signify the overall quantification of the colony counts. Details are as discussed in the materials and methods (significance was set at p < 0.05 as compared to control group).

Mentions: After our observation that miRs-134 and -370 significantly alter the EGFR signaling cascade, we evaluated the functional outcome of this deregulation. Increased proliferation is a major phenotype of activated PI3K/AKT/mTOR signaling, and using mimics and inhibitors of miRs-134 and -370, we conducted an MTT assay over a 96 hr time period in HCT-116, RKO and DLD1 cell lines. With the mimics of miR-134 and -370 and siRNAs against EGFR and PIK3CA, we observed a significant decrease in cell proliferation 96 hrs after transfection as compared to the scrambled transfected controls. With the miRNA inhibitors, the reverse pattern was observed but was significant only for miR-134 in HCT116 (Fig. 3a–c). Similarly, clonogenic assays which evaluate the ability of a single cell to grow and proliferate into a colony were additionally carried out with the same transfection conditions for mimics, inhibitors or siRNAs. Also with this assay, we observed significantly fewer colonies with the transfection of miR-134 and -370 mimics compared to scrambled-sequence controls (Fig. 3d–f). The converse was observed with the miRNA inhibitors. Interestingly, in both proliferation and colonogenic assays, transfection with both EGFR and PIK3CA siRNAs did not yield the same degree of suppression as miR-134 or -370 mimics.


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 cell proliferation and colony formation.Cellular growth curves of HCT-116, RKO and DLD1 cell lines following transfection with (a) miR-134 or miR-370 mimics; (b) miR-134 or miR-370 inhibitors and (c) siRNAs targeting EGFR and PIK3CA. A total of 1 × 103 cells/cell line were seeded in 96-well plates and proliferation was assessed at 0, 24, 48, 72 and 96 time points using the MTT assay. Colony formation assay was performed with the same cell lines used for cell proliferation. The cell lines were treated with (d) miR-134 or miR-370 mimics; (e) miR-134 or miR-370 inhibitors and (f) siRNAs targeting EGFR and PIK3CA. The upper panels show representative examples of the scanned plates and the lower panels signify the overall quantification of the colony counts. Details are as discussed in the materials and methods (significance was set at p < 0.05 as compared to control group).
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: miRs-134 and 370 suppress cell proliferation and colony formation.Cellular growth curves of HCT-116, RKO and DLD1 cell lines following transfection with (a) miR-134 or miR-370 mimics; (b) miR-134 or miR-370 inhibitors and (c) siRNAs targeting EGFR and PIK3CA. A total of 1 × 103 cells/cell line were seeded in 96-well plates and proliferation was assessed at 0, 24, 48, 72 and 96 time points using the MTT assay. Colony formation assay was performed with the same cell lines used for cell proliferation. The cell lines were treated with (d) miR-134 or miR-370 mimics; (e) miR-134 or miR-370 inhibitors and (f) siRNAs targeting EGFR and PIK3CA. The upper panels show representative examples of the scanned plates and the lower panels signify the overall quantification of the colony counts. Details are as discussed in the materials and methods (significance was set at p < 0.05 as compared to control group).
Mentions: After our observation that miRs-134 and -370 significantly alter the EGFR signaling cascade, we evaluated the functional outcome of this deregulation. Increased proliferation is a major phenotype of activated PI3K/AKT/mTOR signaling, and using mimics and inhibitors of miRs-134 and -370, we conducted an MTT assay over a 96 hr time period in HCT-116, RKO and DLD1 cell lines. With the mimics of miR-134 and -370 and siRNAs against EGFR and PIK3CA, we observed a significant decrease in cell proliferation 96 hrs after transfection as compared to the scrambled transfected controls. With the miRNA inhibitors, the reverse pattern was observed but was significant only for miR-134 in HCT116 (Fig. 3a–c). Similarly, clonogenic assays which evaluate the ability of a single cell to grow and proliferate into a colony were additionally carried out with the same transfection conditions for mimics, inhibitors or siRNAs. Also with this assay, we observed significantly fewer colonies with the transfection of miR-134 and -370 mimics compared to scrambled-sequence controls (Fig. 3d–f). The converse was observed with the miRNA inhibitors. Interestingly, in both proliferation and colonogenic assays, transfection with both EGFR and PIK3CA siRNAs did not yield the same degree of suppression as miR-134 or -370 mimics.

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