The Investigation Of Significance And Its Function Of Kinesin Kif2a In Breast Cancer

Breast cancer is the most common malignant tumor in women. In the USA,40, 000 American female died from breast cancer by statistics in 2008.In Asian and many developping country, morbility rate is increasing every year. Breast cancer is already the leading cause of cancer in Southeast Asian women, and is second only to gastric cancer in East Asian women. Breast Cancer is also the leading cause of death among women aged 40 to 79 years. Nowadays, the incidence of breast cancer is increasing by 3～4% per year, greater than that of the worldwide increasing speed in China. In some areas, breast cancer is the most common cause of death among women such as Beijing and Shanghai.The causes of breast cancer is unclear now. In the 90 s of twenty century, microtubule kinesin family was found. These kinesins play an important role in intracellular transport, cell division and the process of bipolar spindle assembly among cells during spindle formation. In current years, researchers found that some kinesins expression had close relationship with the evolution and progression in carcinoma. A few members of the kinesin family of motor proteins have been identified, especially, kinesin-13 proteins family including Kif2a, Kif2b and Kif2c/MCAK. The correlation between Kif2c/MCAK and carcinoma was reported. So far, there is no report on the effects of Kif2a in carcinoma evolution and progression and correlation with prognosis. This study has been divided into three parts to investigate the relationship PART I The clinicopathologic significance of Kif2a in breast cancerObjectiveTo identify the expression of protein and mRNA of kinesin Kif2a in breast cancer and adjacent tissue for investigation relation between the expression of Kif2a and clinicopathologic characteristics in breast cancer. Furthermore, it may be helpful in improving cancer therapy to explore new biological target therapy of breast cancer.Methods1. Patients and tissue samples collectionOne hundred sixteen female patients with breast cancer who underwent surgery at the Qilu hospital of Shandong University were involved in our study. All the patients did not receive any chemotherapy, endocrine therapy or radiotherapy before surgery. Among the 116 cases,60 female breast cancer patients were obtained entire information from following-up, and the median age was 45 years (range from 20 to 65 years) who were followed for more than 10 years after that the patients discharged from the hospital.2. Immunocytochemical stainingOne hundred sixteen paraffin-embedded specimens were routinely stained with antibody of Kif2a according to protocol. The staining results of Kif2a were evaluated by modified system of Harvey to estimate cancer cells immunohistochemistry staining.3. Real time RT-PCRAnother parts of breast cancer and adjacent tissue were collected and stored at-196℃liquid nitrogen at the same time when the tissue were obtained for paraffin embedding. Frozen tissue specimens were homogenized in guanidium thiocyanate, and total RNA was obtained. Then we used reverse transcription, DNA amplification method for detection of mRNA expression.4. Statistic analysisStatistical analysis were performed using the SPSS16.0 software package for Windows. The results of every experiment were depicted as mean±SD. Student's t test, Log-rank were used to analyse the data. Differences were considered significant at P< 0.05.Results1. The expression of Kif2a in primary breast carcinoma was higher than that in adjacent tissueWe identified Kif2a staining in the cytoplasm and nucleus of cancer cells, whose expression was stronger in the great majority of primary tumor than the weak staining in the adjacent tissues epithelium. The score of Kif2a expression on primary cancer and adjacent tissue was 7.0517±0.74419 and 6.2931±0.97817, respectively. The cases whose Kif2a expression was higher in primary cancer than that in adjacent tissue accounted for 68.2%. There was a significant difference between the primary cancer and their corresponding adjacent tissue (P<0.05).2. The expression of Kif2a in human breast carcinoma on mRNA was higher than that in adjacent tissueFurthermore, to investigate the high level of Kif2a expression in the primary cancer samples,4 pairs clinical cases initial tumors and adjacent tissues were submitted to real time RT-PCR. Four representative clinical breast cancer samples demonstrated unequivocal Kif2a mRNA expression in primary cancer samples compared with protein expression in the paired samples of adjacent tissue. The expression level of Kif2a in the primary cancer samples was higher than that in the adjacent epithelium. The four paired tissues that also exhibited Kif2a significantly higher in the cancer tissue than that in the adjacent tissue at mRNA expression level. The result was consisted with Kif2a IHC staining which were higher expression in initial tumors and lower expression in adjacent tissues.3. Kif2a expression was overexpression in breast cancer tissue and correlated with clinicopathologic characteristicsIn 116 cases, overexpression of Kif2a was associated with clinicopathologic characteristics of patients with breast cancer. There was a significant difference between the primary cancer with axillary lymph node metastasis and those without axillary lymph node metastasis. Majority samples showed stronger Kif2a expression in the primary carcinoma tissues with lymph node metastasis than those tissues without axillary lymph node metastasis(7.3043±0.6486 vs.6.6809±0.7255), P<0.05. However, the Kif2a expression of patients had no relativity with age, tumor size and pathologic grade. There were no difference between these groups (P>0.05)4. Kif2a expression was correlated with unfavorable prognosisSixty out of 116 patients were followed up for more than 10 years. Overexpression of Kif2a was associated with prognosis for patients with breast cancer. The patients with high expression of Kif2a had 51.1% survival rate and low expression group had 93.3% survival rate. The survival difference between these two groups was statistically significant (P<0.05), which indicated that there was relationship between the level of Kif2a expression in cancer cells and the patient's survival rate. The Kif2a expression of patients with liver, lung or bone metastasis usually had higher score.Conclusions1. This study showed that Kif2a was overexpression in human breast carcinoma and weak staining in adjacent tissues at protein and mRNA level.2. The overexperession of Kif2a in human breast carcinoma tissues had no relationship with age, pathological grade and tumor size except lymph node metastasis.3. Kif2a expression was up-regulated in breast cancer tissue and correlated with unfavorable prognosis. PARTⅡEffects of Kif2a-siRNA transfection onbiological behavior of breast cancer MCF-7 cellsObjectiveAfter silencing Kif2a gene with siRNA, to identify the alterations on cell proliferation, migration, invasion and apoptosis of breast cancer MCF-7 cells. To explore the effects of kinesin Kif2a on cell proliferation, migration, invasion and apoptosis of breast cancer MCF-7 cells. Furthermoer, it may be helpful in improving cancer therapy to explore new biological target therapy of breast cancer.Methods1. Transfection of siRNAKif2a-siRNA transfection was done according to the protocol supplied by Invitrogen Company. Briefly,2×105 cells were seeded into six-well plates and incubated 16h. For each well,5μl siRNA was mixed with 200μl OPTI-MEM. The mixture was then combined with a solution of 5μl lipofectamine in 200μl OPTI-MEM, after a 20-minute incubation at room temperature, the mixture was applied to the cells in a final volume of OPTI-MEM, making the final dose 50 nM Kif2a-siRNA for each well. After incubation for 4-6 h at 37℃,5%CO2, RPMI1640 supplemented with 10% serum was added. Cells were then cultured for an additional time before analysis.2. Cell MTT assayThe 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was employed to determine cell proliferation. Breast cancer MCF-7 cells were seeded at 5.0×103 cells in 96-well flat-bottomed microtitre plates, and incubated in a humidified atmosphere (37℃and 5%CO2). Kif2a silencing with siRNA was performed as described above. After incubation 24h,48 h and 72 h respectively,20μl (5mg/ml) of MTT labeling reagent was added to each well, and the plate was incubated for 4 h, the stain was eluted into 150μl of DMSO after discarding liquid supernatant. After the purple formazan crystals were completely solubilised, the optical densities were quantified on a multiwell spectrophotometer with a test wavelength of 570 nm and a reference wave length of 630 nm.3. Cell migration assayMigration of MCF-7 cells was measured by 24-well Boyden chambers containing polycarbonate filters with a pore size of 8.0-μm. The different cell groups, mock, nonsense-siRNA and Kif2a-siRNA groups, were seeded into the top chamber of the Transwell with 2×104 cells/100μl per chamber. Cells were allowed to migrate for 16 hours at 37℃,5%CO2. Cells that had migrated to the bottom side of the membrane were fixed in methanol 10 min, stained with eosin. The number of migrated cells were counted under 20×microscope.4. Cell invasion assayThe filters of 24-well Boyden chambers were coated with 40μl of Matrigel. Cells were allowed to invade for 24 hours at 37℃,5%CO2. The other methods of experiment were the same as the cell migration assay's.5. The apoptosis ratio was detected by flow cytometry analysisThe cells of mock group, nonsense-siRNA control group and Kif2a-siRNA group were harvested. After extraction of cellular pellet, it was resuspended in 100μl Annexin V buffer Kit. After the addition of 5μl of Annexin-V-FITC and 10μl of PI, cells were incubated during 15 minutes at room temperature in the dark. Finally,400μl of Annexin V buffer Kit were added. The flow cytometry analysis was performed after infiltration.Results1. Proliferation activity was inhibited after Kif2a silencing in breast cancer MCF-7 cellsThe absorbance of the breast cancer MCF-7 cells was detected at 24h,48h, and 72 h after transfection with Kif2a-siRNA was significantly lower than those of the mock and nonsense-siRNA group as assessed by the MTT assay. They were 0.4198±0.0616 (mock),0.4076±0.0439 (nonsense-siRNA) and 0.2864±0.0230 (Kif2a-siRNA) at 24h; 0.7287±0.1318 (mock),0.7019±0.05225 (nonsense-siRNA),0.5054±0.1011 (Kif2a-siRNA) at 48 h; 0.7948±0.0668 (mock),0.6689±0.0951 (nonsense-siRNA), 0.4745±0.0849 (Kif2a-siRNA) at 72h, (P<0.05). There was a significant difference in the growth rate between the mock, nonsense-siRNA and Kif2a-siRNA groups. Between mock cells and nonsense-siRNA transfected cells, there was no significant difference in the proliferation assays. These results clearly showed that cell proliferation was obviously inhibited in the transfected Kif2a-siRNA group.2. Migratory ability was inhibited after Kif2a silencing in breast cancer MCF-7 cellsMigratory ability of breast cancer MCF-7 cells transfected Kif2a-siRNA was decreased significantly compared to the mock group and transfected nonsense-siRNA group. Silencing Kif2a resulted in a decrease in cell migration of MCF-7 cells transfected Kif2a-siRNA group compared to the two control groups. The migrated number of breast cancer MCF-7 cells interfered with Kif2a-siRNA that were less significantly compared with that of the mock group and nonsense-siRNA group at 16h (P<0.05). This result consisted with the finding of clinical samples in the partⅠ. That was to say, Kif2a expression in the primary carcinoma tissues were obvious correlation with axillary lymph node metastasis.3. Invasive ability was inhibited after Kif2a silencing in breast cancer MCF-7 cellsInvasive ability of MCF-7 cells transfected Kif2a-siRNA was decreased significantly compared to the nonsense-siRNA and mock-transfected MCF-7 cells. The number of MCF-7 cells interfered with Kif2a-siRNA that invaded the Matrigel membrane decreased significantly compared with that of the nonsense-siRNA and mock at 24 h (P<0.05). This result also consisted with the finding of clinical samples in partⅠ, meant Kif2a expression in the primary carcinoma tissues were obvious correlation with axillary lymph node metastasis.4. Apoptosis ratio had no change after Kif2a silencing in breast cancer MCF-7 cellsIn this study, breast cancer cells MCF-7 were treated with Kif2a-siRNA. The apoptosis ratio had no obvious difference between mock and Kif2a-siRNA groups at different time (P>0.05). They were 7.19%±3.48%(mock),9.21%±3.60% (Kif2a-siRNA) at 24h; 9.51%±3.14%(mock),16.65%±5.5212% (Kif2a-siRNA) at 48h; 9.32%±5.34%(mock),13.88%±5.65%(Kif2a-siRNA) at 72h, respectively. Apoptosis test indicated that Kif2a-siRNA group might have no inductive effect to apoptosis of MCF-7 cells.Conclusions1. Proliferation activity was inhibited after Kif2a silencing in breast cancer MCF-7 cells.2. Migratory and invasive ability was inhibited after Kif2a silencing in breast cancer MCF-7 cells.3. Apoptosis ratio had no change after Kif2a silencing in breast cancer MCF-7 cells.PARTⅢRelationship Between the CEACAM1 and Kif2a Expression and Prognosis in Breast CancerObjectiveTo identify the expression of kinesin Kif2a and CEACAM1 in breast cancer tissue and adjacent tissue for investigation relationship between the expression of Kif2a and CEACAM1. To make clear the effects of Kif2a and CEACAM1 in carcinogenesis and the relativity with patients'prognosis.MethodsSixty female breast cancer patients with integrate information of more than 10-year follow-up were involved in our study. The protein expression of Kif2a and CEACAM1 in 60 cases of breast cancer specimens and adjacent tissues were detected using immunohistochemical staining assay. Statistical analysis were performed using the SPSS 16.0 software package for correlation between Kif2a, CEACAM1 expression and survival rate. Results1. The expression of Kif2a in primary breast carcinoma was higher than that in adjacent tissueImmunohistochemical staining assay was applied on 60 samples. In the 60 cases, the cases whose Kif2a expression was higher in primary cancer than that in adjacent tissue accounted for 57%. There was a significant different expression of Kif2a between the primary cancer and their corresponding adjacent tissue (P<0.05).2. CEACAM1 gene expression in primary cancer and adjacent tissueIHC staining was applied on 60 breast cancer tissues. The CEACAM1 expression in breast cancer tissues was lower than that in adjacent tissues. The cases whose CEACAM1 expression was lower in primary cancer than that in adjacent tissue accounted for 65%. The expression of CEACAM1 in adjacent tissue was higher than that in primary tumors (P<0.05).3. Relativity of Kif2a and CEACAM1 gene expression in cancer tissueThe expression of Kif2a in breast cancer tissues was investigated compared with expression of CEACAM1 in the same tissues. The results indicated that both of the expression of Kif2a and CEACAM1 were negative relativity each other (P<0.05), R=-0.295. The higher Kif2a expressed, the lower CEACAM1 expressed.Vice versa, The lower Kif2a expressed, the higher CEACAM1 expressed.4. The relation of CEACAM1 expression and patients' prognosisThe patients were followed up for a period of 10-year. The 5-year overall survival rate was 85%, and 10-year overall survival rate 65%. The group of moderate/high expression had 80% of 10-years survival comparison with absent/weak expression 44%. There was significant difference on overall-survival rate between the two groups(P<0.05). At the same time, in this group of the patients, age, tumor size, grade of tumor differentiation, and lymph node status had no significant influence on the expression of CEACAMI (P>0.05). Conclusions1. The expression of Kif2a was higher in breast cancer than that in adjacent tissue. But the expression of CEACAM1 was lower in breast cancer than that in adjacent tissue.2. The expression of Kif2a and CEACAM1 in breast cancer were negative correlation. The higher Kif2a expressed, the lower CEACAM1 expressed. Vice versa, the lower Kif2a expressed, the higher CEACAM1 expressed.3. There was relationship between the level of Kif2a expression in cancer cells and the patient's survival rate. The patients with high expression of Kif2a and low or absent CEACAM1 expression had poor prognosis. Detection of Kif2a and CEACAM1 will be important factors to predict prognosis.