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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 growth. (A) Proliferation curves of human CRC HCT 116 cells analyzed by xCelligence real time cellular analysis following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). Red, green and blue indicate the SYK(L) overexpression, SYK(S) overexpression and negative control groups, respectively. (B) Cells transfected with recombinant lentivirus vector with SYK(L) or SYK(S) were EdU-labeled and compared with control cells. Red and blue indicate EdU- and Hoechst 33342-labeled cells, respectively. (C) Cell cycle progression analysis in HCT 116 cells following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). 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; EdU, 5-ethynyl-2-deoxyuridine; CRC, colorectal cancer.
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f2-ol-0-0-4858: Effects of ectopic SYK(L) and SYK(S) expression on CRC tumor growth. (A) Proliferation curves of human CRC HCT 116 cells analyzed by xCelligence real time cellular analysis following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). Red, green and blue indicate the SYK(L) overexpression, SYK(S) overexpression and negative control groups, respectively. (B) Cells transfected with recombinant lentivirus vector with SYK(L) or SYK(S) were EdU-labeled and compared with control cells. Red and blue indicate EdU- and Hoechst 33342-labeled cells, respectively. (C) Cell cycle progression analysis in HCT 116 cells following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). 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; EdU, 5-ethynyl-2-deoxyuridine; CRC, colorectal cancer.

Mentions: To investigate the potential effects of SYK isoforms in CRC cells in vitro, transfected HCT 116 cells underwent RTCA and EdU assay. As shown in Fig. 2A, the RTCA revealed that the SYK(L) overexpression group clearly suppressed cell proliferation compared with the negative control group (P=0.004), whereas the SYK(S) overexpression group did not significantly affect HCT 116 cell proliferation (P=0.063). These findings were further confirmed by an EdU assay (Fig. 2B); the percentage of EdU-positive cells was significantly lower in cells expressing SYK(L) (P=0.020). However, there was no significant difference between SYK(S) overexpression group and the negative control group (P=0.054). Overall, these findings suggest that SYK(L), and not SYK(S), suppress the cell proliferation capability of HCT 116 cells.


Alternative splicing of spleen tyrosine kinase differentially regulates colorectal cancer progression
Effects of ectopic SYK(L) and SYK(S) expression on CRC tumor growth. (A) Proliferation curves of human CRC HCT 116 cells analyzed by xCelligence real time cellular analysis following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). Red, green and blue indicate the SYK(L) overexpression, SYK(S) overexpression and negative control groups, respectively. (B) Cells transfected with recombinant lentivirus vector with SYK(L) or SYK(S) were EdU-labeled and compared with control cells. Red and blue indicate EdU- and Hoechst 33342-labeled cells, respectively. (C) Cell cycle progression analysis in HCT 116 cells following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). 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; EdU, 5-ethynyl-2-deoxyuridine; CRC, colorectal cancer.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f2-ol-0-0-4858: Effects of ectopic SYK(L) and SYK(S) expression on CRC tumor growth. (A) Proliferation curves of human CRC HCT 116 cells analyzed by xCelligence real time cellular analysis following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). Red, green and blue indicate the SYK(L) overexpression, SYK(S) overexpression and negative control groups, respectively. (B) Cells transfected with recombinant lentivirus vector with SYK(L) or SYK(S) were EdU-labeled and compared with control cells. Red and blue indicate EdU- and Hoechst 33342-labeled cells, respectively. (C) Cell cycle progression analysis in HCT 116 cells following transfection with recombinant lentivirus vector with SYK(L) or SYK(S). 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; EdU, 5-ethynyl-2-deoxyuridine; CRC, colorectal cancer.
Mentions: To investigate the potential effects of SYK isoforms in CRC cells in vitro, transfected HCT 116 cells underwent RTCA and EdU assay. As shown in Fig. 2A, the RTCA revealed that the SYK(L) overexpression group clearly suppressed cell proliferation compared with the negative control group (P=0.004), whereas the SYK(S) overexpression group did not significantly affect HCT 116 cell proliferation (P=0.063). These findings were further confirmed by an EdU assay (Fig. 2B); the percentage of EdU-positive cells was significantly lower in cells expressing SYK(L) (P=0.020). However, there was no significant difference between SYK(S) overexpression group and the negative control group (P=0.054). Overall, these findings suggest that SYK(L), and not SYK(S), suppress the cell proliferation capability of HCT 116 cells.

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