The Effect And Mechanism Of Kif2a Gene Silencing On The Biological Behavior Of Glioma Cells

ForewordGlioma is one of the most common human tumors of the central nervous system. In adults and children,50-60 percent of intracranial tumors are gliomas with an incidence of about 6.42 cases/100,000.5-year survival of glioma patients is less than 30 percent. In recent years, although a variety of treatment methods adopted to treat gliomas have made great progress, such as surgery, radiotherapy and chemotherapy, therapeutic efficacy for high grade glioma hasn’t been significantly improved. It remains to be a poor prognosis with an average survival time of less than 1 year. Glioma recurrence, metastasis are the main causative factor in the prognosis of glioma patients. Therefore, it is of significance to further study and explore new mechanisms of glioma invasion and metastasis so that deep and comprehensive treatment for glioma patients can be exploited and improve the prognosis.Previous studies have shown that the cytoskeleton plays an important role in tumor invasion and metastasis. Reduction and depolymerization of microtubule is related to metastasis of malignant tumors. The kinesin play an crucial role in the process of microtubule depolymerization and polymerization. Kinesin superfamily proteins have a motor catalytic domain and ATP binding region where chemical energy from ATP hydrolysis can be converted into mechanical energy. Kinesin superfamily can be involved in the important life processes, including intracellular transport, chromosome segregation, mitosis, spindle formation and so on. With scientists’ efforts, so far we have found 45 kinds of kinesins, divided into 14 families. Kinesin-13 family members are mainly involved in cell mitosis, especially in the process of spindle formation, chromosome and nucleus motion and cytokinesis. One study confirmed that Kif2a, one of Kinesin-13 family member, plays a key role in the regulation of the formation of the bipolar spindle, spindle length and direction and inhibiting the extension of neuron deputy collateral by depolymerization of tubulin. Excessive activation or inhibition of Kif2a can affect the microtubule length between chromosomes, disorder chromosome center, resulting in abnormal mitosis. In recent years, our laboratory and other research institutions have found Kif2a upregulated in oral cancer, breast cancer and colorectal cancer tissues. And it plays an important role in the metastasis and invasion of oral cancer, breast cancer and other tumor cells. However, the relationship between Kif2a expression and tumor metastasis and invasion in glioma remains unclear.The present study is the first to explore the expression Kif2a in gliomas and study the role of Kif2a in glioma metastasis and invasion and its possible mechanism. Our results imply that Kif2a plays an important role in glioma invasion, which can serve as one effective molecular target to determine the prognosis of glioma.Part ⅠExpression of Kif2a and its clinical significanceObjectiveTo examine the relationship between the expression level and clinical significance of Kif2a in gliomas.MethodsCollection of Specimens1.A total of 35 patients with glioma by pathological diagnosis who underwent surgery at the First Affiliated Hospital of Liaoning Medical College between 2008 and 2013 were enrolled in this study. All patients suffered from primary tumors. None of them had received any induction chemotherapy or radiotherapy or other therapy before surgery. All patients or guardians signed the informed consent form to agree on the experimental research.20 cases of normal brain autopsy tissue samples were set as controls. All specimens into groups were classified according to the WHO standard (2000).2.35 cases of fresh glioma tissues obtained from surgical treatment were collected at the neurosurgical inpatients(the Qilu Hospital, Shandong University) from 2013 to 2014. All specimens were pathologically diagnosed with human brain glioma and prepared for paraffin sections. All tumors were primary types and none of these patients had received any induction chemotherapy or radiotherapy or other therapy before surgery. After the patient or guardian signed the informed consent form, the experimental research was conducted. All diagnosed tissue samples were graded in accordance with the WHO standard(2000). All participants included 15 cases (Ⅰ-Ⅱ grade),20 cases (Ⅲ-Ⅳ grade) consisted of 23 males and 12 females, ranging in age from 4 to 70 years.Immunohistochemistry analysis of Kif2a expression in glioma tissuesKif2a protein expression level was examined using the immunohistochemical staining method in the high grade glioma tissues,different grades of brain glioma and normal brain tissues. To investigate the relationship among the expression levels of Kif2a protein of high grade gliomas, normal brain tissues and the low grade brain gliomas.mRNA expression level detection by Real-time PCRmRNA expression level of Kif2a was examined using Real-time PCR in fresh glioma tumor tissues. The relationship between the expression level of Kif2a and the clinicopathological features was also investigated in different grade gliomas tissues.ResultsImmunohistochemistrical analysis showed that Kif2a was expressed both in 35 cases of high grade gliomas and 20 cases of normal brain tissues. Compared with normal brain tissues, the expression level of Kif2a was significantly higher in high grade glioma tissues than in normal brain tissues (P=0.001). In 35 cases of glioma tissues with different expression levels of Kif2a, the expression level of Kif2a was significantly higher in high grade glioma tissues than in low grade gliomas (P=0.044).As for the expression area of Kif2a, it was mainly expressed in the cytoplasm and nucleus and stained brownish. In high grade gliomas cells, it had strong cytoplasmic staining, while stained lighter in low grade gliomas and normal brain tissues. The mRNA expression level of Kif2a was detected in fresh glioma tissues using Real-time qPCR method. With the increase of the glioma degree, the expression level of Kif2a was higher (P= 0.033).ConclusionsKif2a had the high expression level in high grade gliomas, while low in low grade gliomas and normal brain tissue. The expression level of Kif2a was strongly correlated with the pathological grade and the clinical staging. Kif2a can be served as an important predictor in evaluating the clinical therapy.Part ⅡEffects of knockdown of Kif2a on the proliferation, apoptosis, invasion and migration in the glioma cell line A172ObjectiveTo investigate the impact of knockdown of Kif2a mediated by siRNA on the proliferation, apoptosis, invasion and migration in the glioma A172 cell line; To explore the underlying molecular mechanism that the knockdown of Kif2a inhibits human glioma cell growth, invasion and metastasis in glioma cell line A172.Methods1 Screening of the human glioma cell line:The human glioma cell lines A172 and U251 went through the routine resuscitation, passage to obtain appropriate cells. A172 and U251 were grown in DMEM supplemented with 10% heat-inactivated fetal bovine serum (FBS) at 37℃in 5% CO2. Total RNA was extracted in A172 and U251 cells, then reverse transcribed into cDNA, amplied by PCR to determine the human glioma cell line A172 for study.2 Determination of plasmid interference ratio:Cells were divided into the Kif2a siRNA group and the scrambled siRNA group.2 × 105 A172 cells in the exponential growth phase were seeded in 6-well plates. Transfect cells using Lipofectamine 2000 with Kif2a siRNA and the scrambled siRNA, respectively. After 48h of transfection, cells were collected to extract total cellular RNA and total protein. The mRNA expression level of Kif2a was measured by RT-qPCR in different experimental groups. The protein expression level of Kif2a was detected using Western-blot method. mRNA and protein expression level were analyzed in A172 cells in different experimental groups to evaluate the interference efficiency of the Kif2a siRNA group.3 Evaluate the impact of Kif2a gene silencing on cell proliferation in human glioma cell line A172:After digestion and cell counting, A172 cells in the exponential growth phase of the Kif2a siRNA group and the scrambled siRNA cells were seeded in 96-well cell culture plate by 1×104 cells/well. Cells were cultured for 3 days and counted at the 0,24,48 and 72 hours using CCK8 method to detect the cell proliferation.4 Use the flow cytometry to determine the effects of Kif2a gene silencing on cell apoptosis in human glioma cell line A172:After digestion and cell counting,2×105 cells/well were seeded in 12-well plates. After cells grew to fuse approximately 80% of the plate, cells were collected for Annexin V and PI staining to conduct the flow cytometry for apoptosis rate changes.5 Use the transwell chamber tumor invasion assay to determine the effects of Kif2a gene silencing on cell migration and invasion in human glioma cell line A172:100μl serum-free A172 cell suspension of each group was added into the small upper chamber and the lower chamber was added 600μl DMEM culture medium containing 10% FBS. After the culture for 16h, count the cell number of penetrating to the back of membranes to evaluate the ability of cell invasion and migration under the light microscope.6 Use the gelatin zymography assay to detect the activities of MMP2 and MMP9 after Kif2a gene silencing in human glioma cell line A172.7 Use the Western blot method to determine the effects of Kif2a gene silencing on PI3K/AKT and MAPK/ERK signaling pathway in human glioma cell line A172.8 Use the Western blot assay to determine the effects of Kif2a gene silencing on the expression level of MMP2,MMP9in human glioma cell line A172.Results1.siRNA targeted Kif2a can effectively down regulate the expression of Kif2a. Use the Real Time-qPCR assay to detect the mRNA expression level of Kif2a after 24h, 48h.Results indicated that it had a high knockdown efficiency after 48 hours (P= 0.003). Then we used the Western blot assay to further verify that result, found that it also had the high efficiency of knockdown the protein expression level of Kif2a at 48h in both the Kif2a siRNA group and control group.2.Use CCK8 assay to examine the effects of Kif2a knockdown on A172 cells. Our results showed that the proliferation rate of A172 cells in Kif2a siRNA knockdown group was significantly lower than the control group.3.We used Flow Cytometry assay to detect the cell apoptosis rate between the Kif2a siRNA group and the control group at 48h after transfection. Our results showed that in contrast to the control siRNA group, the Kif2a knockdown group increased the apoptosis rate of A172 cells significantly (P<0.0001).4.Tanswell chamber tumor invasion assay results showed that the ability of migration of Kif2a siRNA knockdown group cells was significantly lower than the control group.5. The gelatin zymography study found that the MMP2, MMP9 activity of the supernatant in the experiment group was significantly lower than the control group.6.Western blot analysis revealed that in the Kif2a siRNA knockdown group, the phosphorylation levels was down regulated in the AKT signaling pathway associated with glioma cell invasion and migration. Moreover, MMP2 protein expressed less in the experiment group. The phosphorylation levels of the p38/MAPK signaling pathway related to glioma cell proliferation were also down regulated.Conclusions1.siRNA targeted Kif2a could effectively inhibit the expression of Kif2a in A172 cells. After Kif2a gene silencing, the abilities of cell proliferation and invasion in A172 cells significantly reduced,but apoptosis level improved a lot.2.Kif2a plays an important role in the malignant biological behaviors of glioma cells through the PI3K/AKT and MAPK/ERK signaling pathway.3. Kif2a regulates the ability of glioma cell invasion and metastasis by MMPs.Part ⅢExpression and clinical significance of HIFla and relationship with Kif2a.ObjectiveTo determine the expression level and clinical significance of HIF1α in glioma tissues, and analyze the relationship with Kif2a.MethodsCollection of specimens35 cases of fresh glioma tissues obtained from surgical treatment were collected at the neurosurgical inpatients(the Qilu Hospital, Shandong University) from 2013 to 2014. All specimens were pathologically diagnosed with human brain glioma and prepared for paraffin sections. All tumors were primary types and none of these patients had received any induction chemotherapy or radiotherapy or other therapy before surgery. After the patient or guardian signed the informed consent form, the experimental research was conducted. All diagnosed tissue samples were graded in accordance with the WHO standard(2000). All participants included 15 cases (I-II grade),20 cases (Ⅲ-Ⅳ grade) consisted of 23 males and 12 females, ranging in age from 4 to 70 years old.Immunohistochemistry stainingTo detect the expression levels of HIF1α in different grades of glioma tissues, the immunohistochemical staining was conducted. We analyzed the expression levels of HIFla in different grades of glioma tissues and investigated the correlation of Kif2a and HIFla in glioma tissues.Results1.The relationship between HIF1α expression and the clinical characteristics of glioma patients.Immunohistochemical staining results showed that HIF1α mainly expressed in the nucleus and cytoplasm. HIF1α positive expression rate in 35 cases with brain glioma was 68.6%. In 20 cases of Ⅲ-Ⅳ glioma sections,17 (85.3%) samples tested were HIF1α positive,3 (14.7%) samples HIF1α negative. In 15 cases of Ⅰ-Ⅱ glioma sections,7 (46.7%) samples tested were HIFla positive,8 (53.3%) samples HIF1α negative. The expression level of HIFla in low grade (Ⅰ-Ⅱ) glioma and high grade (III-IV) glioma had significant differences (P=0.027). HIF1α expressed significantly higher in high grade gliomas than in low grade gliomas (P<0.05), and moreover, the expression level increased with the tumor grade rising in glioma tumors. In addition, the expression level of HIFla in glioma was unrelated with the age, gender, tumor size and occurrence sites.2.The correlation between Kifia and HIF1α in glioma tissues.Immunohistochemistry results indicated that the expression level of Kif2a and HIF1α in gliomas had no significant difference (P= 0.33).ConclusionsHIF1α expresses highly in high-grade gliomas, weakly in low grade gliomas. Expression levels of HIF1α are associated with the pathological grade and clinical staging in gliomas. HIF1α protein can serve as an important predictor to evaluate the clinical manifestations. Kif2a and HIF1α are likely to play a positive regulatory role in the metastasis process of gliomas. However, the expression level of these two protein factors have no correlation. Therefore, the underlying mechanism needs to be further studied.