The Effect Of Smad6 On Proliferation Of Human Glioblastoma Cells And Its Regulatory Mechanism

Background:In the human central nervous system,glioma is the most common primary tumor,and glioblastoma is the most malignant form of glioma,it is characterized by its infinite proliferation and high degree of invasiveness.At present,the main method of clinical treatment to glioblastoma is surgery combined with radiotherapy and chemotherapy.However,due to glioblastoma is difficult to completely resect,the existence of blood brain barrier affect the effective effect of drugs,radiotherapy has some toxic side-effects,Therefore,it is very difficult to cure glioblastomas.It is known that glioblastomas are essentially polygenic lesions.Multiple genes and signaling pathways are involved in the occurrence and development of glioblastomas.Signal Transducer and Activator of Transcription 3(STAT3)is one of the key factors.In glioblastomas,STAT3 is abnormally activated and directly or indirectly regulates multiple oncogenes.It affects the occurrence and development,invasion and metastasis of glioblastomas,and has close relationship with resistance to radiotherapy and chemotherapy.Therefore,STAT3 is one of the most important molecules in the study of glioblastomas targeted therapy.It is very important to clarify the molecular mechanism of abnormal activation of STAT3 in glioblastomas.Smads family proteins have great homology in protein structure.Several members have been found to interact with STAT3,and Smad6 is mainly expressed in the nuclear.Therefore,we speculated that Smad6 may be involved in the regulation of sustained activation of STAT3 in glioblastomas.Objective:To make clear whether the nuclear-Smad6 regulates the proliferation of human glioblastoma cells and explore its mechanism.Methods:(1)Western blot was used to detect the expression of nuclear Smad6in primary human glioblastoma cells provided by our clinical research center.(2)Nuclear-Smad6 overexpression or knockdown stable glioblastoma cell lines were constructed by lentivirus infection.(3)Using CCK-8 to detect the effect of Smad6on proliferation of glioblastoma cells.(4)Using the nude mice to test the effect of Smad6 on tumorgenesis in vivo.(5)The SIE luciferase reporter assay was used to detect whether Smad6 affects IL-6-induced STAT3 activation,and western blot was used to detect whether Smad6 regulates STAT3 target gene expression which realted with proliferation.Results:(1)Four primary glioblastoma cells were selected and numbered as GBM-1,GBM-2,GBM-3,and GBM-4.The results of Western blot showed that the expression level of Smad6 in nuclear was different in these cells.(2)The CCK-8 cell proliferation experiment was used to dectet the proliferation rate of these four cells,the absorbance value of these cells incubating after 4 hours was seted as 1,the proliferation rate of these four cells at 24,48,and 72 hours was as follows:GBM-1was 1.20±0.03,1.68±0.09,2.29±0.15;GBM-2 was 1.27±0.05,1.85±0.09,2.76±0.18;GBM-3 was 1.43±0.05,2.20±0.15,4.10±0.27;GBM-4 was 1.32±0.06,2.02±0.10,3.39±0.26.We campared the differences of the proliferation ability of these 4 cells and the results showed they have statistically significant differences(P<0.05~0.001).(3)Pearson correlation analysis was used to calculate the correlation between the expression of nuclear-Smad6 and the cell proliferation in primary glioblastoma cells,and r was 0.968,P<0.05.(4)There are green fluorescent proteins expressed in the stable glioblastoma cell lines with nuclear-Smad6overexpression(Smad6-NLS)and its negative control(Vector),the stable glioblastoma cell lines with Smad6 knockdown(Smad6-KD)and its negative control(NC).And the fluorescence of Samd6-NLS and Vector both accumulate in the nuclear.(5)Western blot showed that the Smad6 expression was significantly higher in the Smad6-NLS than in the Vector.Compared with the NC,the Smad6expression was significantly reduced in the Smad6-KD.(6)The CCK-8 cell proliferation experiment was used to dectet the proliferation rate of these four stable glioblastoma cells,the absorbance value of these cells incubating after 4 hours was seted as 1,the proliferation rate of these four stable glioblastoma cells at 24,48,and72 hours was as follows:Smad6-NLS was 1.57±0.03,2.61±0.23,4.35±0.27;Vector was 1.30±0.05,1.73±0.06,2.52±0.16;Smad6-KD was 1.18±0.04,1.57±0.05,2.24±0.11;NC was 1.41±0.04,2.07±0.14,3.88±0.25.The differences of the proliferation rate of Smad6-NLS and Vector,Smad6-KD and NC at 72 hours were statistically significant(P<0.001).(7)The length and width of the tumor in nude mice was recorded and the volume was calculated.The tumor volume at 7,14,21,and 28 days after injecting was as follows:Smad6-NLS was(25.28±2.61)mm~3,(61.04±6.62)mm~3,(160.63±13.40)mm~3,(304.24±17.07)mm~3;Vector was(24.97±3.54)mm~3,(41.79±6.16)mm~3,(71.48±5.67)mm~3,(134.74±11.66)mm~3;Smad6-KD was(24.44±3.85)mm~3,(32.08±2.82)mm~3,(51.14±4.83)mm~3,(85.69±7.85)mm~3;NC was(25.00±3.01)mm~3,(42.31±3.39)mm~3,(84.56±10.68)mm~3,(164.14±14.51)mm~3.The differences in tumor volume between Smad6-NLS and Vector,Smad6-KD and NC were statistically significant at 28 days after the cells injected into the nude mice(P<0.001).(8)The tumor mass of Smad6-NLS,Vector,Smad6-KD and NC grown 28 days in nude mice were(85.00±20.17)mg,(28.88±10.36)mg,(21.25±7.78)mg,(42.13±11.89)mg.The differences in tumor mass between Smad6-NLS and Vector,Smad6-KD and NC were statistically significant(P<0.01~0.001).(9)Luciferase reporter assays showed that Smad6 can promote IL-6-induced SIE activity.(10)The results of western blot showed that Smad6 can promote STAT3 target genes expression which realted with proliferation.Conclusion:Smad6 is mainly expressed in the nuclear of glioblastoma cells,and its high expression level is conducive to the proliferation and tumor formation in vivo;Mechanism studies have shown that Smad6 can promote STAT3 target genes expression which realted with proliferation by enhancing the transcriptional activity of STAT3.