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Alternative splicing of spleen tyrosine kinase differentially regulates colorectal cancer progression

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ABSTRACT

Spleen tyrosine kinase (SYK) has been reported as a potential tumor suppressor in colorectal cancer (CRC). However, the role of alternative splicing of SYK in carcinogenesis remains unclear. In the present study, SYK isoforms were overexpressed in the human CRC HCT 116 cell line using lentiviral expression vectors to investigate the biological functions of full length SYK [SYK(L)] and short form SYK [SYK(S)] in CRC. Real-time cellular analysis and the 5-ethynyl-2-deoxyuridine assay were used to detect the effects of SYK(L) and SYK(S) on cell proliferation. Cell cycle progression and migration were assessed via flow cytometry and Transwell assays, respectively. The results revealed that the recombinant lentivirus with SYK(L) overexpression significantly suppressed the proliferation and metastasis of CRC cells, while SYK(S) overexpression did not. In addition, MTS assays demonstrated that SYK(L) and SYK(S) increased the cellular sensitivity to 5-fluorouracil (5-FU), suggesting that SYK(L) and 5-FU produce a significant synergistic effect on CRC cell proliferation, while SYK(S) has an effect on modulating CRC 5-FU sensitivity. Furthermore, quantitative polymerase chain reaction results revealed that SYK(L) was downregulated in 69% of 26 pairs of CRC and adjacent non-cancerous tissues, whereas SYK(S) exhibited no significant differences between tumor and normal tissues. Overall, the present data provides evidence that SYK(L) is a tumor suppressor in CRC, and both SYK(L) and SYK(S) may serve as important predictors in the chemotherapeutic treatment of CRC.

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Effects of ectopic SYK(L) and SYK(S) expression on CRC tumor metastasis and 5-FU resistance. (A) Cell migration and invasion assays in human CRC HCT 116 cells following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). Representative microscopy images of invaded and migrated cells of three independent experiments. (B) Effects of SYK(L) and SYK(S) on 5-FU resistance in CRC cells by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay. 5-FU half maximal inhibitory concentrations were calculated from three independent experiments performed in quadruplicate. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. empty vector transfected cells. SYK, spleen tyrosine kinase; SYK(L), SYK full length; SYK(S), SYK short form; 5-FU, .5-fluorouracil; CRC, colorectal cancer.
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f3-ol-0-0-4858: Effects of ectopic SYK(L) and SYK(S) expression on CRC tumor metastasis and 5-FU resistance. (A) Cell migration and invasion assays in human CRC HCT 116 cells following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). Representative microscopy images of invaded and migrated cells of three independent experiments. (B) Effects of SYK(L) and SYK(S) on 5-FU resistance in CRC cells by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay. 5-FU half maximal inhibitory concentrations were calculated from three independent experiments performed in quadruplicate. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. empty vector transfected cells. SYK, spleen tyrosine kinase; SYK(L), SYK full length; SYK(S), SYK short form; 5-FU, .5-fluorouracil; CRC, colorectal cancer.

Mentions: Subsequently, whether SYK(L) and SYK(S) had an effect on the migration and invasion ability of HCT 116 cells was analyzed by Transwell assay. Cells on the lower side of the Transwell membrane were the migrating/invading cells. Representative photomicrographs of Transwell assays were obtained at magnification ×100 (Fig. 3A). Subsequently, the number of cells were counted in nine independent visual fields under the microscope (magnification, ×200) from three independent experiments. Overexpression of SYK(L) significantly decreased the migration and invasion capacity by 84% (P=0.003) and 61% (P=0.044), respectively, in HCT 116 cells compared with negative control cells (Fig. 3A). However, overexpression of SYK(S) did not significantly affect the motility (P=0.325) or invasive ability (P=0.271) of the cells. Therefore, the results revealed that SYK(L), and not SYK(S), is involved in CRC cell migration and invasion.


Alternative splicing of spleen tyrosine kinase differentially regulates colorectal cancer progression
Effects of ectopic SYK(L) and SYK(S) expression on CRC tumor metastasis and 5-FU resistance. (A) Cell migration and invasion assays in human CRC HCT 116 cells following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). Representative microscopy images of invaded and migrated cells of three independent experiments. (B) Effects of SYK(L) and SYK(S) on 5-FU resistance in CRC cells by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay. 5-FU half maximal inhibitory concentrations were calculated from three independent experiments performed in quadruplicate. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. empty vector transfected cells. SYK, spleen tyrosine kinase; SYK(L), SYK full length; SYK(S), SYK short form; 5-FU, .5-fluorouracil; CRC, colorectal cancer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4998349&req=5

f3-ol-0-0-4858: Effects of ectopic SYK(L) and SYK(S) expression on CRC tumor metastasis and 5-FU resistance. (A) Cell migration and invasion assays in human CRC HCT 116 cells following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). Representative microscopy images of invaded and migrated cells of three independent experiments. (B) Effects of SYK(L) and SYK(S) on 5-FU resistance in CRC cells by 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium cell viability assay. 5-FU half maximal inhibitory concentrations were calculated from three independent experiments performed in quadruplicate. Data are presented as the mean ± standard deviation. *P<0.05, **P<0.01 vs. empty vector transfected cells. SYK, spleen tyrosine kinase; SYK(L), SYK full length; SYK(S), SYK short form; 5-FU, .5-fluorouracil; CRC, colorectal cancer.
Mentions: Subsequently, whether SYK(L) and SYK(S) had an effect on the migration and invasion ability of HCT 116 cells was analyzed by Transwell assay. Cells on the lower side of the Transwell membrane were the migrating/invading cells. Representative photomicrographs of Transwell assays were obtained at magnification ×100 (Fig. 3A). Subsequently, the number of cells were counted in nine independent visual fields under the microscope (magnification, ×200) from three independent experiments. Overexpression of SYK(L) significantly decreased the migration and invasion capacity by 84% (P=0.003) and 61% (P=0.044), respectively, in HCT 116 cells compared with negative control cells (Fig. 3A). However, overexpression of SYK(S) did not significantly affect the motility (P=0.325) or invasive ability (P=0.271) of the cells. Therefore, the results revealed that SYK(L), and not SYK(S), is involved in CRC cell migration and invasion.

View Article: PubMed Central - PubMed

ABSTRACT

Spleen tyrosine kinase (SYK) has been reported as a potential tumor suppressor in colorectal cancer (CRC). However, the role of alternative splicing of SYK in carcinogenesis remains unclear. In the present study, SYK isoforms were overexpressed in the human CRC HCT 116 cell line using lentiviral expression vectors to investigate the biological functions of full length SYK [SYK(L)] and short form SYK [SYK(S)] in CRC. Real-time cellular analysis and the 5-ethynyl-2-deoxyuridine assay were used to detect the effects of SYK(L) and SYK(S) on cell proliferation. Cell cycle progression and migration were assessed via flow cytometry and Transwell assays, respectively. The results revealed that the recombinant lentivirus with SYK(L) overexpression significantly suppressed the proliferation and metastasis of CRC cells, while SYK(S) overexpression did not. In addition, MTS assays demonstrated that SYK(L) and SYK(S) increased the cellular sensitivity to 5-fluorouracil (5-FU), suggesting that SYK(L) and 5-FU produce a significant synergistic effect on CRC cell proliferation, while SYK(S) has an effect on modulating CRC 5-FU sensitivity. Furthermore, quantitative polymerase chain reaction results revealed that SYK(L) was downregulated in 69% of 26 pairs of CRC and adjacent non-cancerous tissues, whereas SYK(S) exhibited no significant differences between tumor and normal tissues. Overall, the present data provides evidence that SYK(L) is a tumor suppressor in CRC, and both SYK(L) and SYK(S) may serve as important predictors in the chemotherapeutic treatment of CRC.

No MeSH data available.


Related in: MedlinePlus