Application Of Mouse Fluorescence Imaging In A Research Of Zinc Finger Protein 146 Promoting Glioma Proliferation

BACKGROUNDThe accumulation of DNA damage is an important factor leading to cell carcinogenesis[1-2],which is closely related to proliferation of tumors.DNA damage repair genes,known as one of three major tumor genes,coexit in normal cells and participate in the regulation of cell growth,differentiation and proliferation.Abnormalities in DNA damage repair genes,such as XRCC1,XRCC5 and CCN3 gene mutations[3-5],which play great role in tumorigenesis,metastasis and resistance to radiotherapy and chemotherapy.Zinc finger protein 146(RNF146)is a factor related to DNA damage and repair.It participates in pathological process of many malignant tumor[6-12]by regulating the ubiquitin degradation of many tumor-related proteins,such as Axin[8],β-catenin[8-9]and PARP1[10],etc.However,the relationship between RNF146 and glioma is rarely reported.To confirm the research value of RNF146 in gliomas,in Part l,we used the TCGA database[13-14]to analyze the relationship between the expression level of RNF146 and gliomas.The immunohistochemical staining of glioma tissue chip was used to confirm the retrieval results of TCGA database from the protein level,which laid a foundation for the follow-up mechanism research.In vivo fluorescence imaging of small animals is an important tool for tumor dynamic research.At present,the commonly used fluorescence imaging principles are mainly focused on direct imaging under fluorescent protein or fluorescent dye labeling or indirect imaging on tumor cells labeled with luc catalytic substrate.Because of its high sensitivity,strong penetration,non-invasion and easy operation,so the latter is widely used in the study of tumor models[15-17].In order to clarify the function of RNF146 in glioma proliferation,we used the GL261-luc labeled with Fluc as the object to replicate the tumor-bearing mouse model,knocked down the expression of RNF146 in GL261-luc by gene modification in vitro,and study the correlation between RNF146 and glioma according to phenotypic changes(part Ⅲ).However,the risk of miss or other important gene dysfunction often occurs in the process of exogenous gene introduction or target gene knockout.The expression of luciferase or fluorescent protein in tumor cells that should be stably expressed is often unstable,with decreased enzyme activity,abnormal protein expression or loss of important luminescent substances after many passages[49].It will seriously affects the efficiency of in vivo fluorescence detection.Therefore,it is necessary to detect the fluorescence imaging ability of genetically modified tumor cells in vitro before implantation in vivo.To ensure the in vivo biofluorescence imaging effect,we identified the luciferase expression efficiency,activity and stability of luciferase-labeled glioma cell GL261-luc in advance(Part 2).The above work not only understands the role of RNF146 in proliferation of glioma,but also provides a theoretical supplement for the study of the effect of gene modification on flu labeled cell imaging in vitro.Part 1 The role of DNA damage repair factor RNF146 in the occurrence and development of human gliomaObjective To research the correlation separately between the expression of DNA damage repair factor RNF146 and the diffenent ages,pathological grades and prognosis of human gliomas.Methods The RNA-seq Level3 data of 515 cases of low grade glioma(LGG)(grade Ⅰ-Ⅲ)and 152 cases of malignant glioma(Glioblastoma,GBM)(grade IV)were selected from TCGA database.The different expression of RNF146 gene among different age groups(≤46 years old and>46 years old)was studied by Mann-Whitney U test.Moreover,the glioma samples are from different genders and pathological grades(Ⅰ-Ⅲ grade and GBM).The expression level of RNF146 in cancer tissues and its correlation with clinical data was analyzed by Spearman’s test.While the effect of RNF146 expression level on survival time was detected by Kaplan-Meier method.Results The immunohistochemical analysis of RNF146 was performed on brain glioma tissue microarray(3 cases of normal brain tissue,23 cases of low grade glioma and 64 cases of high grade glioma)(P<0.001).According to the diffenent IHC scores in the expression of RNF 146,two groups which called high expression and low expression were divided.The high RNF 146 expression accounted for 87%of low-grade gliomas and the opposite one accounted for 91%of high-grade gliomas.The risk of death in glioma patients with low expression of RNF 146 was significantly higher than that with high expression of RNF 146 through Kaplan-Meier analysis(P<0.001).Conclusion Through the search of TCGA and tissue microarray immunohistochemical staining of RNF 146,we propose for the first time that RNF 146 is an independent negative factor which relates to high grades of glioma,advanced age and survival time of patients who are suffering from glioma.The decrease of RNF 146 expression rate indicates that the prognosis of glioma patients is poor,and it is also an important reason for the malignant degree of gliomas and the high risk of death.Part 2 The effect of RNF146 Knockdown on Activity and Stability of Luciferase in GL261-luc cells.Objective To confirm the influence of knocking down of RNF146 expression by interfering lentivirus on the stability and activity of luciferase in mouse glioma cell line GL261-luc transfected with luciferase.Methods Lentivirus infection(ShRNA scramble virus infection as negative control)was used to down-regulate the expression of RNF146 in glioma cell line GL261-luc,and real-time PCR detected the mRNA expression of RNF146(internal reference:GAPDH)and Western-blot detected the protein expression of RNF146 and luciferase(internal reference:β-tubulin).The activity and stability of luciferase was tested by luciferase reporter gene detection system after down-regulation of RNF146.Results Compared with the ShRNA-Scramble and negative control groups,RNF146 expression in GL261-luc cells which was infected by RNF146 interference lentivirus significantly decreased(P<0.001).The luciferase protein in GL261-luc cells had no changes before and after infection.The luciferase activity of GL261-luc was increased with the increase of numbers of cells,and the activity and stability after passages of GL261-luc had no differences after down-regulating the expression of RNF146(P>0.05).Conclusion Down-regulation of RNF146 expression in GL261-luc cells by interfering with virus infection does not affect the expression of protein,activity of luciferase and its stability after passage.The cell model can be used for follow-up bioluminescence imaging of small animals.Part 3 The Application of Mouse In Vivo Biofluorescence Imaging Based on Firefly Luciferase in the study of RNF146 Promoting Proliferation of Glioma.Objective To confirm promoting effects of RNF146 on tumor proliferation in mice bearing glioma(GL261-luc)by using in vivo biofluorescence imaging based on firefly luciferase.Methods Female BALB/c Nude mice were divided into GL261-luc,ShRNA-scramble/GL261-luc,ShRNA-RNF146/GL261-luc and sham operation group.Three groups above in logarithmic phase were injected on the right back of mouse,PBS was injected on the last group of mouse.One week later,the animals were imaged in vivo and injected intraperitoneally with D-fluorescein potassium salt(D-luciferin).The fluorescence area and fluorescence intensity per unit area were recorded to evaluate the effect of RNF 146 on the proliferation rate and activity of glioma in vivo.The effect was detected once a week and dynamically observed for 4 weeks.The relationship between RNF 146 and life time of tumor-bearing mice were confirmed.The morphological change of tumor cells were found by HE staining,and the effects of RNF 146 and GFAP immunohistochemical staining on the expression of glioma-specific protein GFAP were confirmed.Results The fluorescence area(P<0.001)and fluorescence intensity per unit area(P<0.001)in shRNA-RNF146/GL261-luc were significantly higher than those in GL261-luc and shRNA-scramble/GL261-luc groups.The survival time of it was shorter than the other two groups(P<0.001).The tumor shape and size between GL261-luc and shRNA-scramble/GL261-luc groups were same(P>0.05).The level of RNF146 and GFAP in shRNA-RNF146/GL261-luc group were lower than that in GL261-luc and shRNA-scramble/GL261-luc(P<0.001).Conclusion The proliferation of glioma can be promoted by the low expression of RNF146 and the risks of death in down-regulation of RNF146 group also be shorterned.