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Downregulation of LIMK1 Inhibit Migration and Invasion in Human Colon Cancer SW480 Cells Induced by Diallyl Disulfide

Author: ShiLing
Tutor: SuZuo
School: Nanhua University
Course: Pathology and Pathophysiology
Keywords: Human colon cancer SW480 cells Diallyl disulfide Migrate Attack microRNA The Rac1-LIMK1 signal path uPA uPAR
CLC: R735.35
Type: Master's thesis
Year: 2011
Downloads: 18
Quote: 0
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Abstract


Objective: To study diallyl disulfide (diallyl disulfide, DADS) on human colon cancer SW480 cells LIMK1 expression and the role of migration and invasion, and silence the LIMK1 the impact of uPA / uPAR expression observed DADS and LIMK1 explore DADS suppression molecular mechanisms of SW480 cell migration and invasion. Methods: MTT detection DADS proliferation of SW480 cells; RNA interference technology to establish a stable LIMK1-miRNA/SW480 cell lines; RT-PCR, Western blot, immunocytochemistry, scratches experiment and invasion assays were used to detect the DADS and RNA interference of SW480 cells of LIMK1 uPA / uPAR, migration and invasion and Rac1-LIMK1 pathway signaling molecules. Results: 1 DADS can inhibit human colon cancer SW480 cell proliferation, migration and invasion of MTT results showed, DADS was time - dose-dependent inhibition of proliferation of SW480 cells (P lt; 0.05). Migration experiments showed that after 48 hours, DADS group in a concentration-dependent inhibition of cell migration rate (P lt; 0.05). Invasion assay penetrating cells, DADS treatment compared with the control group showed a concentration-dependent manner significantly reduce (P lt; 0.05). Showed that DADS a concentration-dependent inhibition of SW480 cell migration and invasion. 2. DADS down the SW480 cell LIMK1 expression RT-PCR results show that the the DADS was time-dependent inhibition of LIMK1 mRNA (P lt; 0.01). The immunocytochemical found LIMK1 expression was strongly positive in the untreated group, DADS different times after treatment, LIMK1 expression was significantly reduced (P lt; 0.05). Western blot showed that, compared with the control group, DADS dose - time-dependent reduction LIMK1 expression (P lt; 0.05). 3. Silent LIMK1 DADS inhibit SW480 cell migration and invasion immunofluorescence, Western blot, immune cells chemical testing proved successful build of stable the LIMK1 gene's silence LIMK1-miR/SW480 cells strains. Western blot, the the DADS group interference group LIMK1 and p-LIMK1 group compared with the control group and the empty vector were significantly lowered, of LIMK1 silent group before and after DADS treatment, LIMK1 expression was no significant change. That DADS can inhibit LIMK1 expression and phosphorylation. Migration experiments, 48h after the interference group and DADS-treated cells mobility compared with the control group and the empty vector were significantly lower (P lt; 0.05), empty vector group and control group had no significant change (P gt; 0.05). Show that the LIMK1 positively correlated with the SW480 cell migration; DADS by down-regulating LIMK1 and phosphorylated inhibit SW480 cell migration. Interference cell migration inhibition after dosing group most significantly, indicating that DADS may also inhibit cell migration through other means. Invasion assay show, interference group and DADS group to wear mesangial cells than the untreated group and empty vector group significantly reduce (P lt; 0.05); empty vector group compared with the control group had no significant with change (P gt; 0.05), showed that LIMK1 and SW480 cells invasive ability was positively correlated, DADS by down-regulating LIMK1 phosphorylation inhibit the invasion of SW480 cells. Interference group dosing cell invasion inhibition is more pronounced, indicating that DADS also inhibit cell invasion through other mechanisms. 4. DADS the Rac1-LIMK1 signaling pathway RT-PCR and Western blot results show that the the DADS was time-dependent inhibition of Rac1, Rock1, PAK1, LIMK1, Destrin mRNA and protein expression (P lt; 0.05). cofilin1 mRNA and protein expression was no significant difference (P gt; 0.05) in a time-dependent inhibition of p-LIMK1, p-cofilin1 expression (P lt; 0.05). 5. Of DADS and silence LIMK1 pair of uPA / uPAR protein expression in the Western blot results show DADS treatment group and LIMK1 interference group of of uPA, uPAR expression significantly with lowered DADS treatment interference group of uPA / uPAR expression down more significantly (P lt; 0.05). That DADS can inhibit the uPA / uPAR expression silence LIMK1 negative regulation regulation of uPA / uPAR expression the DADS directly or by down-regulating LIMK1 lowered uPA / uPAR. Conclusion: DADS through negative regulation of Rac1-LIMK1 signaling pathway, causing LIMK1 expression inhibition of phosphorylation blocked inhibition of human colon cancer SW480 cell migration and invasion. 2. Success to build LIMK1-miR/SW480 recombinant cell lines. Silence LIMK1 inhibits SW480 cell migration and invasion. 4. DADS LIMK1 regulation of uPA / uPAR expression. 5. DADS, either directly or indirectly through LIMK1 lowered uPA / uPAR inhibit SW480 cell migration and invasion.

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CLC: > Medicine, health > Oncology > Gastrointestinal Cancer > Intestinal neoplasms > Colon tumor
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