Study On Expression, Function And Mechanism Of WISP-2 Gene In Human Brain Astrocytomas

Objective:To investigate the expression and significance of Wnt-1 induced secreted protein 2 (WISP-2) gene in human brain astrocytomas and its relationship with the genesis and development of human brain astrocytomas.Methods:The expression of WISP-2 mRNA were determined by reverse transcription-polymerase chain reaction (RT-PCR) in 47 samples of human brain astrocytomas tissues, as well as 10 normal brain tissues.The correlation between WISP-2 expression levels of astrocytomas and its clinical indicators was analysed.Results:RT-PCR results showed that normal brain tissues had no WISP-2 mRNA expression.The relative expression level in astrocytomas was 0.677±0.445. Compared with the normal group there were statistically significant differences (t= 4.783, P<0.05). The relative expression levels of WISP-2 mRNA in high grade group (gradeⅢ-Ⅳ) and low grade group (gradeⅠ-Ⅱ) were 0.924±0.438,0.478±0.344, respectively.The difference was significant (t=3.909, P<0.05).The increase of WISP-2 correlated positively with tumor grade (r=0.448, P=0.002). The WISP-2 mRNA expression had no significant relationship with age, sex, tumor location (all P>0.05), but had a significant relationship with tumor size (P<0.05).Conclusion:WISP-2 mRNA could expresse in human brain astrocytomas and the expression was correlated with the malignant degree of human astrocytomas. WISP-2 mRNA may play an important regulatory role in the occurrence and development of human astrocytomas, which contributes to human brain astrocytomas malignant progression. Objective To investigate the expression and significance of WISP-2 protein in human brain astrocytomas and and its clinicopathologic significance.Methods The expression of WISP-2 protein of 54 human astrocytomas and 10 human normal brain tissues that were as control group was respectively examined. The expression of WISP-2 protein in these samples was detected using immunohistochemical staining. Statistical analysis was performed to investigate the ralation between WISP-2 protein expression and the clinicopathologic features.Results Immunohistochemical staining showed that the protein expression was undetected in normal brain tissues (0/10),but the positive rate of WISP-2 protein expression was 64.8%(35/54) in astrocytomas. There was significant difference between the positive rate of WISP-2 expression in astrocytomas and that of normal tissues(P<0.05). The positive rates of WISP-2 protein in high-grade group(gradeⅢ-Ⅳ) and low-grade group (gradeⅠ-Ⅱ) were 80.8%(21/26) and 50.0%(14/28), respectively.The significant difference was seen between high-grade (gradeⅢ-Ⅳ) and low-grade (gradeⅠ-Ⅱ) (P<0.05). With the combination of histopathological data, we found that the expression level of WISP-2 protein was closely correlated the differentiation degrees and tumor diameters of astrocytomas, but not with patients' age, gender, and tumor location(all P>0.05).Conclusion The expression of WISP-2 is closely related to the malignant degree of human brain astrocytoms., and WISP-2 may be one of the malignant biomarkers in the pathogenesis and progression of human brain astrocytoms. Objective:To construct siRNA silencing human WISP-2 gene expression and filter out effective siRNA sequence.Methods:The expression of WISP-2 in the U251 was checked by RT-PCR first. The sequences targeting WISP-2 gene were designed and synthesized, then cloned into siRNA expression vectors. There combinant plasmids were transiently transfected into U251 cells via LipofectamineTM2000. The RT-PCR was used to examine mRNA expression of WISP-2 gene.Western Blot was used to detect expression of WISP-2 protein.And inverted fluorescence microscope was used to observe the effect of transfection.Results:WISP-2 mRNA expressed in U251 cell lines. RT-PCR and Western blot used to detect that WISP-2-siRNA-289 sequence inhibiting WISP-2 expression and had the best silencing effect. Observed by Inverted fluorescence microscope the transfection efficiency reached (87.03±11.56)%.Conclusion:The siRNA targeting WISP-2 gene can inhibit the expression ofKEYWORDS:siRNA, WISP-2, transfectionObjective:The present study was carried out to investigate the effect on the bionomics of U251 by the silence of WISP-2. Then we could figure out the function of WISP-2 during the proliferation and apoptosis of human astrocytomas.Methods:There were three groups in the experiment:blank control group(used the normal U251), negative control group(the inclusion of non-specific siRNA/ Lipofectamine complexes in U251 cell lines) and siRNA interference group (WISP-2-siRNA-289-liposome-transfected U251 cell line, which was contracted in the Part 3). Human astrocytic glioma cell line U251 was cultured in vitro, then MTT method, Transwell assay, and flow cytometer (FCM) analysis and Western Blot were applied to measure cell growth,cell invasiveness,apoptosis,expression of WISP-2,Bcl-2,and Bax protein.Results:Detected at the same time by MTT, the siRNA interference group OD was significantly higher than that of blank control group and negative control group (P<0.05). The result of Transwell assay exhibited that cell number through the artificial polycarbonate basal membrane became decreased compared with the control group after 48 hours(P<0.05). By the flow cytometry's test, we found that the mean apoptotic rate of siRNA interference group was (16.59±1.40)%. Compared with the apoptotic rate of blank control group (3.13±0.34)% and the apoptotic rate of negative control group (3.42±0.48)%, the difference was statistically significant (P<0.01). The expression of WISP-2 protein in siRNA interference group(18.67±1.40) % was significantly lower than blank control group (70.18±1.82)% and negative control group (49.51±1.71)%(P<0.01). The expression of Bcl-2 protein in siRNA interference group(29.67±1.47)% was significantly lower than blank control group (49.51±1.71)% and negative control group (49.07±1.35)%(P<0.01). The expression of Bax protein in siRNA interference group(31.62±1.32)%was significantly higher than blank control group (23.98±1.47)% and negative control group (24.06±1.55)% (P<0.01).Correlation analysis showed that the expression of WISP-2 had a positive correlation with the expression of Bcl-2 protein. after siRNA silencing (r=0.995, P<0.05), and it had a negative correlation with the expression of Bax protein. after siRNA silencing (r=-0.944, P<0.05).Conclusion:The siRNA silencing expression of WISP-2 gene inhibits the proliferation and migration of U251 cells. and it increases the apoptosis of U251 cells. And it may by inhibiting expression of WISP-2 protein downregulate the ratio of Bcl-2/Bax to induce cell apoptosis. Objective:To evaluate the effect of combined therapy of Etoposide and WISP-2 siRNA on growth inhibition of human glioma U251 cells.Methods:U251 cells were transfected with WISP-2 siRNA via LipofectaminTM2000,and at the same time etoposide (VP16) was added into U251 cells medium. The proliferation and apoptosis of U251 cells were evaluated after 48 hours by MTT assay and flow cytometry.Results:Inhibitory effects of VP16 on proliferation of U251 cells were observed by MTT colorimetric survival assay. was which were in a time-and dose-dependent manner and the IC50 was 13.43μg/mL for 48 hours.Combined therapy of WISP-2 siRNA with VP16 inhibited proliferation of U251 cells.MTT assay showed that the the OD value of blank control group, negative control group, siRNA interference group, VP16 group and siRNA interference+VP16 group was 0.257±0.011,0.252±0.015,0.166±0.011,0.155±0.012,0.076±0.014, respectively. We find that the OD value of siRNA interference group and VP16 group was significantly lower comparing with that of blank control group and negative control group (P<0.05).The OD value of siRNA interference+VP16 group was significantly lower compared with that of siRNA interference group and the VP16 group (P<0.05), There were no significant difference between the blank control group and the negative control group (P>0.05).Combining therapy of WISP-2 siRNA and etoposide (VP16) significantly reduced the U251 cell proliferation. By the flow cytometry's test, we found that the apoptotic rate of blank control group, negative control group,siRNA interference group, VP16 group and siRNA interference+VP16 group was (3.06±0.39)%, (3.28±0.57)%, (15.72±1.55)%, (14.76±1.41)%, (33.09±2.13)%, respectively. Statistical analysis showed the apoptotic rate of siRNA interference group and VP16 group was statistically significant higher than that of blank control group and negative control group.(P<0.01), and the apoptotic rate of siRNA interference+ VP16 group was statistically significant higher than that of siRNA interference group and VP16 group (P<0.05).There were no significant difference between the negative control group and the blank control group (P=0.848).Conclusion:The combination therapy of WISP-2 siRNA and etoposide could effectively improve thesensitivity of U251 cells to etoposide by inhibiting U251 cells' proliferation and promoting U251' apoptosis.