The Influence Of Kinesin KIF4 On Proliferation Of Gastric Cancer Cells BGC And Migration Of Ovarian Cancer Cells SK-OV-3

Objective:To identify the expression of kinesin KIF4 in gastric caner and explore its effects on cell proliferation of gastric cancer cells BGC. Furthermoer, to investigate its effect on migration of ovarian cancer cells SK-OV-3.Method:一,The research of KIF4 expression in gastric cancer tissue1,The expression condition of kinesin KIF4 in human gastric carcinoma23 patients with the primary gastric cancer included in this study were performed surgery in Qilu Hospital, Shandong University between 2006 and 2008. After surgery, tumor samples and their adjacent tissues from the patients were paraffin-embedded and stored at room temperature. These samples were approved by the local ethics committee to be used in the study. Their clinicalpathological characteristics were obtained from the Qilu Hospital.For immunohistochemical staining assay, paraffin-embedded tumor samples were sectioned at a thickness of 4-5μm and placed on clean slides. After blocking with goat sera, polyclonal anti-KIF4 antibodies were added onto the slides by 1:500 dilution. The immunostaining was carried out using Histostain-Plus Kit following the manufacturer's instructions. Negative control was performed using PBS solution instead of primary antibodies. Each slide was photographed with a digital camera on an invert microscope of Olympusâ…¨81. Five pictures on each slide were randomly taken. The integrated optical density (IOD) of pictures was measured using Image-Pro Plus 6.0 software. 2,The correlation between KIF4 expression and clinical dataStatistical analysis was done using SPSS 11.0 software:The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples. Contingency table was used to analyze the relationship between KIF4 expression and clinicopathologic variables in gastric cancer patients.二,The influence of KIF4 on proliferation of gastric cancer cell BGC1. Select the stable cell line which expressed GFP-KIF4 or GFP(1) Construction of pEGFP-KIF4 expression plasmidsThe cDNAs of full-length coding region of KIF4 was generated by PCR and subcloned into Bglâ…¡-Sal I sites of the mammalian expression vector pEGFP-C1.(2) Transfect pEGFP-KIF4 or pEGFP to BGC cellCell transfection was performed using Lipofectamine 2000 following the manufacturer's instruction. BGC cells stably expressing GFP-KIF4 or GFP were generated using G418-selection methods. In brief,3×10⁴ of BGC cells were plated into each well of 24-well plate and cultured overnight. Then 0.5μg of pEGFP-KIF4 or pEGFP plasmids was transfected into the cells using Lipofectamne 2000. Two days later, cells were split into a 10cm dish and cultured in complete medium contained 1 mg/ml of G418. After two to three weeks, positive cell colonies were picked up under an invert fluorescent microscope and then continued to culture with 1mg/ml of G418 selection medium. Finally, the BGC cells stably expressing GFP-KIF4 (BGC-GFP-KIF4 cells) or GFP (BGC-GFP cells) were grown up and lysed for Western Blot assays or fixed for immunofluorescent staining assays.(3) Western blot analysis for KIF4 protein expressionCells were collected by centrifugation and lysed in lysis buffer.40μg of cell extract was resolved on 12% polyacrylamide gels using minigel apparatus and transferred to PVDF membrane. Affinity-purified rabbit antibodies against KIF4 were diluted 1:1000 to detect endogenous KIF4 and over expressed GFP-KIF4 fusion protein. The anti-a-tubulin monoclonal antibody was used with 1:1000 dilution. Blots were then exposed to HRP-conjugated goat anti-rabbit lgG(1:10000 dilution) followed by development using ECL reagent.2. Immunofluorescence staining show the polynucleation of BGC-GFP-KIF4Cells grown on coverslips were fixed with cold methanol for 5 minutes at room temperature and then blocked with PBS contained 5% goat serum and 0.3% Triton X-100. The staining was performed using mouse anti-a-tubulin (1:10000 dilution) followed by Texas Red conjugated goat anti-mouse antibodies (1:10000 dilution) for 2 hours at room temperature respectively. Finally, DNA was stained with DAPI (1μg/ml) for 5 minutes. After washing with PBS, coverslips were amounted on clean slides with FluoroGuard Antifade Reagent and sealed with Cytoseal 60.3. Cell proliferate counting and MTT assay investigate the influence on proliferation of BGC when overexpressing KIF45000 cells were seeded into each well of 24-well plates and cultured in complete RPMI-1640 medium. In the following 9 days, cells were counting every two day as routine. Then cell growth curves were drew according to the data. Each independent experiment was performed three times.5000 cells were seeded into each well of 96-well plates and cultured in complete RPMI-1640 medium. In the following 9 days, cells were assayed every two day by MTT method as routine. Briefly,20μl of 5mg/ml MTT was added into each well and continued to culture for four hours. Then the cell culture medium was removed followed by adding another 200μl of DMSO.30 minutes later, the plates were placed on a micro-plate auto-reader. Optical density (OD) was read at 570 nm wavelength, then cell growth curves were drew according to the data. Each independent experiment was performed three times.4. Soft-agar colony-formation assay investigate the influence on proliferation of BGC when overexpressing KIF4For cell colony formation assay, 1×104 cells were added to 1 ml of growth medium with 0.3%agar and layered onto 2 ml of 0.6%agar beds in 35mm dishes. After three weeks culture in soft agar, cells were stained with 0.05% crystal violet for half an hour and photographed. Cell colonies were counted using Image-Pro Plus 6.0 software. Assays were conducted in triplicate in three independent experiments.5. Xenograft assays in nude mice investigate the potentiality of tumor formation when overexpressing KIF4 in BGCFemale BABL/c mice (6-8 weeks old) were purchased from SLAC laboratory animal Co. Ltd. A total of 4×10⁶ cells of BGC-GFP-KIF4 or BGC-GFP cells and 100μg of Matrigel per animal were inoculated into the left flank of athymic nude (nu/nu) mice (n=5 for each group). All procedures complied with the protocols approved by the Institutional Animal Care Committee. The situation of tumor growth was recorded every week with a caliper-like instrument. Tumor volume was calculated according to the formula:volume=(lengthxwidth2)/2. At the end of 7 weeks, mice were sacrificed by cervical dislocation. Tumor tissues were removed from mice and the volume and weight of tumor tissues were measured and photographed.6. Statistical analysisStatistical analysis was done using SPSS 11.0 software. The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples.三,The research of KIF4 on ovarian cancer cells SK-OV-3 migration.1. Select the stable cell line which low expressing KIF4(1)Construction of pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC expression plasmidsThe shRNA toward KIF4 was generated by synthesis and subcloned into the mammalian knockdown vector pGPU6/GFP/Neo.(2) Transfect pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC to SK-OV-3 cellCell transfection was performed using Lipofectamine 2000 following the manufacturer's instruction. SK-OV-3 cells stably expressing pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC were generated using G418-selection methods. In brief,3×104 of SK-OV-3 cells were plated into each well of 24-well plate and cultured overnight. Then 0.5μg of pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC plasmids was transfected into the cells using Lipofectamne 2000. Two days later, cells were split into a 10cm dish and cultured in complete medium contained 1 mg/ml of G418. After two to three weeks, positive cell colonies were picked up under an invert fluorescent microscope and then continued to culture with 1 mg/ml of G418 selection medium. Finally, the SK-OV-3 cells stably expressing pGPU6/GFP/Neo-KIF4 or pGPU6/GFP/Neo-NC were grown up and lysed for Western Blot assays or fixed for immunofluorescent staining assays.(3) Western blot analysis for KIF4 protein expressionCells were collected by centrifugation and lysed in lysis buffer.40μg of cell extract was resolved on 12%polyacrylamide gels using minigel apparatus and transferred to PVDF membrane. Affinity-purified rabbit antibodies against KIF4 were diluted 1:1000 to detect endogenous KIF4. The anti-α-tubulin monoclonal antibody was used with 1:1000 dilution. Blots were then exposed to HRP-conjugated goat anti-rabbit lgG(1:10000 dilution) followed by development using ECL reagent.2,Transwell assay investigate the influence on migration of SK-OV-3 when knockdown KIF4(1) Transwell assayMigration of SK-OV-3 cells were measured by Transwell chambers with 8.0μm pore membranes.The cells were seeded into the top chamber of the Transwell with a concentration of 1×10⁵/100μL in FBS free medium.The bottom chamber of the Transwell contained 600μL 10%FBS medium.Cells were allowed to migration for 22 h at 37℃,5% CO₂.Then the Transwell top surface of the membrance was cleared of adherent cells with a cotton swab.Cells that had migrated to the bottom side of the membrance were fixed in methanol for 10min,stained with eosin.The number of migrated cells,counted under a microscope at 200×magnification,is the sum of cells found in the bottom side of the membrance and the lower chamber.(2) Statistical analysisStatistical analysis was done using SPSS 11.0 software. The results of every experiment were depicted as mean±SD. Student's two-sided t test was used to compare values of test and control samples.Result:一,The research of KIF4 on expression in gastric cancer tissue1,Kinesin KIF4 appears reduced expression in human gastric carcinomaKinesin KIF4 is previously report to play an intriguing role in tumorigenesis. To investigate the critical role of kinesin KIF4 in human gastric cancer, we examined 23 gastric carcinoma samples along with corresponding adjacent tissue samples using immunohistochemical analysis. Immunohistochemical staining on the paraffin-embedded samples was performed using anti-KIF4 polyclonal antibodies as previously published. The expression level of kinesin KIF4 in each specimen is classified into eight grades according to immunohistochemical staining assay following the instruction of previous described methods. The result come out that 13 of 23 (56.5%) samples appeared reduced expression of KIF4 in carcinoma compare with their corresponding adjacent tissues. Using Image-Pro Plus 6.0 software, we measured the integrated optical density (IOD) of KIF4 staining in all above samples and found 4-fold lower level of KIF4 expression in total carcinoma than that of adjacent tissues. Immunohistochemical analysis clearly showed KIF4 localized in cytoplasm and nucleus in gastric tissues which is consistent with previous reports in other human tissues or in vitro cultured cells.2,KIF4 appears reduced expression in human gastric carcinoma and associates with the differentiation of gastric carcinoma.We next studied the associativity between expression of KIF4 and clinicopathologic features as well as clinical profiles of these samples. Our results indicated a significant association of low expression of KIF4 with poor differentiation of clinical gastric carcinoma. Among nineteen poorly differentiated carcinoma samples, thirteen samples showed lower expression of KIF4 in carcinoma compare with corresponding adjacent tissues. However none of four moderately differentiated carcinoma samples took place lower level of KIF4 than their adjacent tissues. All these results suggest that kinesin KIF4 is lowly expressed in human gastric cancer and its expression level is closely correlated with the tumor differentiation grade.二,The influence of KIF4 on proliferation of gastric cancer cell BGC1. Successfully select the stable cell line which expressed GFP-KIF4 or GFP(1) Construction of pEGFP-KIF4 expression plasmidsEnzymes digestion, PCR and DNA sequencing confirmed that the combinant vector, pEGFP-KIF4 successfully constructed.(2) Transfect pEGFP-KIF4 or pEGFP to BGC cellThe stable cell lines which expressing GFP-KIF4 or GFP were selected. The ratio which cell expressing green fluorescence protein is about 93%.(3) Western blot analysis for KIF4 protein expressionWestern blot results showed that GFP-KIF4 protein could express in BGC stable cell line. GFP-KIF4 fusion protein was highly expressed in BGC-GFP-KIF4 cells and even higher than endogenous KIF4 protein in the cells as well as in BGC and BGC-GFP cells. 2. Immunofluorescence staining show that over expression of KIF4 in gastric cancer cells resulted in multinucleation.Given that low expression of KIF4 in clinical gastric carcinoma specimens, the effect of over expression of KIF4 in human gastric cancer cells such as BGC cells would be interestingly to survey. To study the critical role of KIF4 in the growth of BGC cells, we stably expressed GFP-KIF4 fusion protein in BGC cells (designated as BGC-GFP-KIF4 cells) as well as GFP in BGC cells (designated as BGC-GFP cells). The Western Blotting assay showed that GFP-KIF4 fusion protein was highly expressed in BGC-GFP-KIF4 cells and even higher than endogenous KIF4 protein in the cells as well as in BGC and BGC-GFP cells. We then performed immunofluorescent staining on the cells to examine morphological differences between these cells. Consistent with previous reports in in vitro cultured cells, GFP-KIF4 predominantly localized to the nucleus in BGC-GFP-KIF4 cells. Strikingly, we found more multinucleation exist in BGC-GFP-KIF4 cells than in BGC-GFP cells. There were 14.5±0.82%of BGC-GFP-KIF4 cells that come out multinucleation. In contrast, only 4.4±0.46% of BGC-GFP cells become multinucleated similar to maternal BGC cells.3. Cell proliferate counting and MTT assay investigate the influence on proliferation of BGC when overexpressing KIF4Over expression of KIF4 in BGC cells resulted in multinucleation and then potentially affected cell proliferation in vitro. To assess inhibition effect of cell proliferation by over expression of KIF4 in gastric cancer cells,we carried out cell proliferate counting and MTT assay in BGC-GFP-KIF4 cells as well as BGC-GFP cells. Assay was performed every two day for 9 days and three independent experiments have been achieved. The result showed that no significant difference occurred in above cells within three days. However, the proliferation of BGC-GFP-KIF4 cells were gradually inhibited from the fifth day when compare with BGC-GFP as well as BGC cells. There was no difference of cell proliferation rate between BGC-GFP and BGC cells indicating that expression of GFP protein in BGC cells did not affect the ability of cell proliferation.4. Soft-agar colony-formation assay investigate the influence on proliferation of BGC when overexpressing KIF4 To examine cell viability in anchorage-independent condition, we performed soft-agar colony-formation assay with BGC-GFP-KIF4 and BGC-GFP cells.104 cells were plated on the soft-agar medium in a 35 mm dish. Three weeks later, the cells were stained with crystal violet and photographed. The cell colonies were scored and results from three independent experiments were analyzed together. Average 143±9 cell colonies were formed in BGC-GFP cells but only 30±9 colonies in BGC-GFP-KIF4 cells. These results suggested that cell growth were significantly inhibited in anchorage-independent condition in BGC cells because of over expression of KIF4.5. Xenograft assays in nude mice investigate the potentiality of tumor formation when overexpressing KIF4 in BGCAs described above, the growth rate of BGC-GFP-KIF4 cells was noticeably lower than that of BGC-GFP cells in vitro. We next determined the ability of these cells giving rise to tumor in nude mice.4×10⁶ of BGC-GFP-KIF4 or BGC-GFP cells were inoculated into the left flank of athymic nude mice (n=5 for each group) and tumor size was recorded weekly for 7 weeks. The tumor derived from BGC-GFP-KIF4 cells grew much slowly compare with BGC-GFP cells during this period. Seven weeks later, all mice were killed and dissected to isolate the final xenografts. Then the tumor samples were weighed and photographed. The weight of tumors derived from BGC-GFP-KIF4 cells (0.9±0.24g) was significantly less than that from BGC-GFP cells (3.0±1.17g). It was about 3-fold lighter of tumors from BGC-GFP-KIF4 cells than that from BGC-GFP cells. These results indicated that the ability of BGC-GFP-KIF4 cells to form tumor in nude mice was much weaker than that of BGC-GFP cells suggesting that over expression of ectopic KIF4 protein in BGC cells can inhibit tumor formation in vivo.三,The research of KIF4 on ovarian cancer cells SK-OV-3 migration.1. Select the stable cell line which low expressing KIF4(1)Construction of pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC expression plasmidsEnzymes digestion, PCR and DNA sequencing confirmed that the combinant vector, pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC successfully constructed.(2) Transfect pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC to BGC cellThe stable cell lines which expressing GFP-KIF4 or GFP were selected. The ratio which cell expressing green fluorescence protein is about 92%.(3) Western blot analysis for KIF4 protein expressionWestern blot results showed that KIF4 protein was lowly expressed in SK-OV-3 cells which was transfected pGPU6/GFP/Neo-KIF4.2,Transwell assay investigate the influence on migration of SK-OV-3 when knockdown KIF4The migration of SK-OV-3 cells was determined by transwell chambers assay.Migratory ability of pGPU6/GFP/Neo-KIF4 transfected SK-OV-3 cells was increased significantly compared to that pGPU6/GFP/Neo-NC transfected and Mock SK-OV-3 cells(P<0.05).Conclusions:1. We successfully constructed one fusion expression vectors pEGFP-KIF4, two knockdown vectors pGPU6/GFP/Neo-KIF4 and pGPU6/GFP/Neo-NC,which could produce a marked effect in vitro efficiently. This can provide requirement for further study of KIF4 function.2. This study confirmed kinesin KIF4 appears reduced expression in human gastric carcinoma and associates with the differentiation of gastric carcinoma.This study provided necessary experimental data for further research on the function of KIF4.3. In this study, both of our in vitro and in vivo studies showed an apparent inhibition of proliferation in BGC cells by way of over expression of KIF4. These results strongly imply that chromokinesin KIF4 functions as an inhibitor for the proliferation of human gastric cancer cells.4. This study confirmed silencing KIF4 in human ovarian cancer cell SK-OV-3 increases its migration capability.