The Effect Of LKB1on Proliferation And Migration And The Signaling Pathway Involved In Colorectal Cancer Cells

Background and ObjectiveLKB1gene, known as a tumor suppressor gene, has been identified to be deficient in most patients with PJS and to be responsible for the accidence of PJS, and also to be somatically mutated in non-small cell pulmonary carcinomas, invasive ductal carcinomas of breast, cervical carcinomas, squamous cell carcinomas and other cancers. LKB1gene, highlighted as a tumor suppressor, plays a role in cell cycle arrest, signaling transduction pathway, cell polarity and energy metabolism. Recent studies reveal that deficiency or inactivation of LKB1gene, which results in uncontrolled cell proliferation and promotes the ability of cell migration, is associated with the tumorgenesis in a variety of sporadic cancers. However, the underlying mechanism of how LKB1gene contributes to the progression of malignant tumor remains unclear.Herein, we focus on determination the role of LKB1gene in colorectal carcinoma cells proliferation and migration, and attempted to elucidate the potential mechanism of the progress. Materials and Methods Regents and MaterialsSW1116,SW480,SW620,Colo205and HCT116colorectal cancer cells, LKB1expression plasmid, small interference RNA fragment, plasmid extraction kits, cell proliferation kit, cell invasion kit, LKB1antibody,β-catenin antibody, cyclinDl antibody, Claudin-1antibody, MMP-7antibody,β-actin antibody.MethodsIdentification of LKB1expression plasmidThe LKB1recombinant plasmid was identified by PCR amplification and DNA sequencing. The PCR was initiated by5min incubation at95℃,31cycles for denaturation at95℃for30s, annealing at60℃for45s, and72℃for50s, ended after a7min extension at72℃using a PCR kit.Detecting gene expression of LKB1in colorectal tumor cellsThe LKB1gene expression in cells was detected by semi-quantitative RT-PCR. Total RNA was extracted using Trizol (TAKARA). RNA samples (3μg) were subjected to reverse transcription using a cDNA Synthesis Kit (Invitrogen). The PCR was performed as above using a PCR kit.Detecting protein expression of LKB1in colorectal tumor cellsThe LKB1protein expression in cells was detected by Western blotting. Cells lysed with ice-cold buffer and PMSF, equal amounts of protein samples were electrophoretically separated by SDS-PAGE in10%acrylamide gel and transferred to PVDF members. Following transferring, the members were blocked for1.5h at room temperature with5%skimmed milk in0.05%TBST, and then incubated with primary antibody at4℃, shaking for night. After removing primary antibody, they were incubated for lh at room temperature with a proper horseradish peroxidase conjugated secondary antibody. Members were detected by using a the enhanced ECL system.β-actin was also detected as internal control. Cell culture and transient transfectionThe cell lines were cultured in RMPI-1640with10%FBS and set in5%CO2,37℃, humidity sufficient conventional culture conditions. Cultured SW1116cells were transfected with the recombinant plasmid and the empty vector using lipofectamine2000(Invitrogen) according to the manufacture’s instructions. And the expression of LKB1was detected by RT-PCR and Western blotting.Examination and selection of LKB1small interfering RNACultured SW480cells were transfected with three small interfering RNA fragments (named as siRNA1455, siRNA1391, siRNA927) and a negative control one, instructed as above. And the interfering effect was estimated by RT-PCR and Western blotting.Cell proliferation assaysCells were seeded into the96-well plates at the density of3000cells (cell growth) or5000cells (cell growth inhibition), cultured for24h and then transiently transfected. And the medium was replaced with100μl of fresh medium and10μl of CCK-8regant in each well at24,48,72, and96h, respectively. After the plate was returned to incubator for1-2h, the absorbency of A450was measured using an enzyme-linked immunosorbent assay plate reader.Cell migration assayWe used the transwell chamber to assess the ability of cell migration. After the cells were treated according to experiment purposes, we prepared the cell suspension at a density of2.0×107/ml by using1640medium with1%FBS.600μl of1640medium containing20%FBS was added into the lower chamber, incubated for1h at37℃, and then100μl of the cell suspension was added into the upper chamber. After48h, we used a cotton swab to wipe the cells staying on the upper chamber,4%paraformaldehyde to fix the cells and0.1%crystal violet for cell staining, the chambers were washed clearly by using the PBS buffer. Microscope (200×) were used to select the5random views for the cell counting.Research of the signaling pathway involved in colorectal cancer cellsRT-PCR and Western blotting were used to analyzed expression changes of signaling pathway proteins after the overexpression or downregulation of LKB1.The involved Wnt/β-catenin signaling pathway proteins include key gene---β-catenin, and associated target gene---cyclinDl,Claudin-1and MMP-7.Statistical analysis.Data were compiled with the software package SPSS13.0. Factorial analysis ANOVA was employed to determine whether the results of proliferation assays had statistical significance. Two-sample t test was used applied to analyze the difference of the cell counting in migration assays. The level of significance was defined as P<0.05.ResultsExpression of CD24was cell type-dependent in colorectal cancer cell linesRT-PCR was used to detect LKB1expression at mRNA level in the colorectal cancer cell lines. Of six cell lines, SW1116maintained low expression level, while in other cancer cells, the expression of LKB1gene was high. Western blotting showed that the LKB1protein expression was consistent with its mRNA level.Identification of LKB1recombinant plasmidThe LKB1recombinant plasmid was identified by PCR and DNA sequencing, and the inserted fragment sequence was consistent with than in Genebank.The effect of up-regulation LKB1expression on cell proliferation and migrationFollowing the instruction of lipofectamine2000, the LKB1recombinant plasmid was transfected into SW1116cells, empty plasmid served as negative control, SW480as blank control. Then the cells were named as SW1116-LKB1, SW1116-Vector and SW1116.CCK-8assay was performed at24h,48h,72h and96h, respectively. Compared with the SW1116-Vector and SW1116cells, SW1116-LKB1cells grew much slowly (F=89.396, P=0.008), which indicates up-regulating LKB1expression could inhibit cell proliferation. The results in migration assay showed that compared with the SW1116-Vector cell, the number of the cell penetrated through the member in the SW1116-LKB1group was reduced (t=43.819, P=0.000). Selection out the most efficient LKB1small interfering RNAThree small interfering RNA oligonucleotides targeting LKB1and a negative control one were synthesized, and then were transfected into SW480cells according to the lipo2000’s instructions. Expression of LKBl mRNA and protein were down regulated by siRNA1455most efficiently.The effect of down-regulation LKBl expression on cell proliferation and migrationWe transfected the siRNA1455oligonucleotides and the NC oligonucleotide into cells, performed as above, while SW480served as blank control. The cells was named as LKB1-siRNA, NC-siRNA and SW40, respectively. Then we used CCK-8assay to assess the velocity of cell growth in various group. Compared with other two cells, down-regulation LKB1expression in SW480resulted a significant increase of cell proliferation (F=711.856, P=0.000). In cell migration assay, there was notable difference between LKB1-siRNA and NC-siRNA. The migration ability was increased significantly in the former group (t=35.034, P=0.000).The effect of the changes of LKBl expression level on the Wnt/p-catenin signaling pathwayTo elucidate the mechanism underlying the effect of LKB1on cell proliferation and migration, we examined expression level of P-catenin gene, results showed that up-regulating LKB1expression led to substantial reduction in the level of β-catenin. Consistent with this reduction inβ-catenin, the expression of some target genes---cyclinDl, Claudin-1and MMP-7, playing roles in cell proliferation and migration, significantly decreased. In order to further examine the effect of LKB1gene on Wnt/p-catenin signaling pathway, we detected the expression levels of genes mentioned above in cells transfected with LKB1siRNA.Results showed that with down-regulation LKB1expression, the levels of those genes were increased, which indicated that suppression of LKB1expression could promote the transcription activity of Wnt/β-catenin signaling pathway in colorectal cancer cells. ConclusionsIn the present study, we found that the expression of LKB1was different in colorectal cancer (CRC) cells, and we evaluated the role of LKB1in the proliferation and migration of the CRC cells and the potential mechanism. Our data showed that LKB1expression could suppress the velocity of cell proliferation and the ability of cell migration, and up-regulation of LKB1expression could resulted in a negative regulation effect on the Wnt/β-catenin signaling pathway, while using RNAi technology down-regulated the LKB1expression that could promote the transcription activity of the signaling pathway. Our results suggested that tumor suppressor gene LKB1played a key role in the tumorgenesis of colorectal carcinoma, and the interaction with the Wnt signaling pathway may be the underlying mechanism. The linkage of LKB1and Wnt signaling pathway paved a new way in regulation of the development of CRCs.