Experimental Study On The Effect Of LncRNA-ZNF281 On The "Dry" Characteristics And Invasion Of Glioma Stem Cells

Background and Objective:Glioma is a primary intracranial malignant tumor,and it is still the focus and difficulty of neurosurgical medicine.It has the characteristics of high incidence,high recurrence rate,high mortality and low cure rate.Traditional treatment methods include surgery,radiotherapy and chemotherapy.In recent years,glioma precision localization and combined radiotherapy have been carried out,but the cure rate is still not ideal.Molecular precision treatment has become a new direction of research and a new hope for the cure of glioma.With the continuous development of glioma stem cell research in recent years,more and more researchers believe that glioma stem cell-like cells are the root cause of the development of glioma.The discovery of GSCs dates back to 2002.When the researchers cultured glioma cells in vitro,they found cells resembling neural stem cells in tumor cells.After 2004,researchers used different cell surface markers to obtain GSCs from glioma cells.These surface markers include:CD133,CD15/SSEA-1,L1CAM,A2B5,and integrin 6 and the like.In order to completely cure glioma,the root cause of glioma development must be eradicated,that is,glioma stem cells must be eliminated to cure glioma.In order to inhibit the function of glioma stem cells in tumorigenesis and development,and to obtain more ideal effects,researchers at home and abroad have adopted a variety of targeted strategies,such as inhibition of proliferation,promotion of differentiation,induction of apoptosis and enhancement of radio sensitivity.It is reported in the literature that the maintenance of tumor stem cell function depends on cell signaling pathways such as Wnt,Shh and Notch,human telomerase(hTERT),Oct3/4,Nanog,Sox2 and other factors.Inhibition of the above signaling pathway and human telomerase(hTERT)activity will seriously affect the characteristics of tumor stem cell self-renewal and multi-directional differentiation.The use of exogenous BMP proteins by Piccirillo induces differentiation of GSCs,resulting in a severe decline in the tumorigenic capacity of the entire tumor population.Zhang et al.used the oncolytic adenovirus ONYX-411 to carry siRNA transfecting mutant K-ras to transfect pancreatic cancer and other cancer cells,which can effectively promote tumor cell apoptosis.Bao et al.found that glioma stem cells can initiate DNA damage repair reactions more effectively than non-stem cell-like glioma cells,reducing the damage caused by radiation;further studies have found that by inhibiting cell cycle checkpoint kinase Chkl and The activity of Chk2 effectively weakens the radiotherapy resistance of glioma stem cells and restores sensitivity to radiotherapy.The progress of glioma stem cells and gliomas involves a variety of gene mutations.The most widely studied in recent years is the regulation of non-coding RNAs,especially the role of long-chain non-coding RNA in the growth and development of gliomas.Neglect.Long-chain non-coding RNA(LncRNA)is a nucleotide sequence with a nucleotide sequence of>200 nt.It affects the formation of downstream proteins through epigenetic regulation,transcriptional regulation and post-transcriptional regulation,and participates in the regulation of glioma cells.Changes in proliferative,invasive,and apoptotic capabilities.Some studies have demonstrated that LncRNA plays an important role in the growth of glioma cells:LncRNA HOXA-AS3 is expressed at higher levels in glioma tissues and glioma cells than non-tumor brain tissue.In glioma cells,down-regulation of LncRNA HOXA-AS3 expression can inhibit cell proliferation and promote apoptosis;in glioma cells,down-regulation of LncRNA HOX expression can inhibit cell proliferation and invasion,and promote cell-promoting Apoptotic ability.Long-chain non-coding RNAs such as LncRNA MALAT1,LncRNA HOXA11-AS,LncRNA XIST and LncRNA MEG3 all affect the biological function of glioma cells.LncRNA-ZNF281 is a novel class of long-chain non-coding RNAs with a nucleotide length of 353 nt and located on chromosome 1q32.1.In our previous experimental studies,it was found that lncRNA-ZNF281 can inhibit the invasion of glioma cell lines U87 and SHG139 by regulating the expression of VEGF and MMP2,but the effects of glioma stem cells have not been further studied.In this experiment,we will study the regulation of LncRNA-ZNF281 on glioma stem cell-like cells,and provide a new scientific basis for lncRNA-ZNF281 as a new target for the treatment of glioma.Materials and Method1.Isolation,culture and identification of stem cell characteristics of glioma stem cell U251 s derived from glioma cell line U251.2.Different invasive ability and expression of invasion-related proteins in glioma stem cell U251 s and glioma cell line U251.3.Real-time quantitative PCR was used to detect the expression of lncRNA-ZNF281 in glioma tissues,glioma stem cells U251s,glioma cell line U251 and normal brain tissues.4.The lncRNA-ZNF281 overexpression sequence was designed and synthesized by Suzhou Gemma Biotech Co.,Ltd.and constructed lncRNA-ZNF281 overexpressing lentivirus.5.Immunofluorescence,secondary globization,quantitative PCR,western-blot detection of lncRNA-ZNF281 on the dry characteristics of glioma stem cell-like cells U251s(detection of expression and self-renewal of molecular markers on glioma stem cells)ability).Transwell assay was used to detect the effect of lncRNA-ZNF281 on the invasion ability of glioma stem cell U25 Is in vitro and the effect of IncRNA-ZNF281 on the expression of NF-KB-related protein in glioma stem cell U251s invasion-related signaling pathway was detected by Western blot.7.Construct a subcutaneous xenograft model of nude mice and verify the effect of LncRNA-ZNF281 on tumor growth in nude mice by measuring the size of subcutaneous xenografts in nude mice.8.Intracranial model of nude mice xenografts was constructed,HE staining was used to detect intracranial tumor formation in nude mice.Immunohistochemistry was used to detect the expression of proliferation-related protein Ki-67,NF-KB-related protein and MMP2 in the tumor.Happening;9.Data analysis was performed using SPSS 18.0 software,and the mean ± standard deviation was used to represent the data.A comparison between the two sets of data was performed using a t test.A P value of less than 0.05 was considered statistically significant.Results1.Culture and identification of glioma stem cell U251sIn order to study the characteristics of glioma stem cells,we used the reverse culture method to sort out glioma stem cell-like cells U251s from glioma cell line U251.We detected the expression of protein and mRNA levels of stem cell surface markers CD133,A2B5,Nestin,SOX2,OCT4 and Nanog in glioma stem cell U251s by immunofluorescence and RT-qPCR.We found that our cultured glioma stem cells are highly expressed.Stem cell markers;simultaneous glomerulation experiments showed that glioma stem cell-like cells U251s have self-renewal ability.2.Analysis of invasive ability of glioma stem cellsWe used in vitro and in vivo experiments to verify whether the glioma stem cell U251s has stronger invasive properties than the common glioma cell line U251.We showed that the expression of the invasive protein was significantly increased by detecting the expression of the relevant invasive protein;The intracranial xenograft experiment(using two sets of glioma cells carrying different fluorescence from the laboratory given by Professor Mon,USA)demonstrated that glioma stem cell U251s is comparable to glioma in the nude mouse intracranial xenograft experiment.Cell line U251 has a stronger ability to spread.3.Effect of LncRNA-ZNF281 on the dry characteristics of glioma stem cell-like cellsTo test whether LncRNA-ZNF281 plays a role in the development of glioma and the function of glioma stem cells,we first examined the expression of LncRNA-ZNF281 in glioma tissues,glioma cells and glioma stem cells.The results showed that the expression of LncRNA-ZNF281 in glioma tissues was significantly lower than that in normal brain tissues,and the expression in glioma stem cells was significantly lower than that in glioma cells,suggesting that LncRNA-ZNF281 may be in glia.It plays an important role in tumor stem cells.Subsequently,we designed and constructed LncRNA-ZNF281 overexpressing lentivirus and transfected into glioma stem cell U251s.Then we used immunofluorescence,western-blot and RT-qPCR to detect lncRNA-ZNF281 pair.The expression of glioma stem cell-like cells U251s stem cell surface markers(CD 133,Nestin,OCT4,SOX2,Nanog)showed that lncRNA-ZNF281 can significantly attenuate glioma stem cell markers(CD133,Nestein,OCT4,Nanog).expression.At the same time,we used the second globule assay to detect the effect of LncRNA-ZNF281 on the self-renewal ability of glioma stem cell U251s.We found that the self-renewal rate of glioma stem cell U251s was significantly decreased after LncRNA-ZNF281 overexpression of lentivirus.This indicates that LncRNA-ZNF281 can effectively inhibit the proliferation ability(self-renewal ability)of glioma stem cell U251 s after overexpression in vitro.4.Effect of LncRNA-ZNF281 on the invasion ability of glioma stem cellsGlioma stem cells have a stronger invasive ability than glioma cell lines.They are a major feature of glioma stem cells.To investigate the effect of LncRNA-ZNF281 on the invasion of glioma stem cell-like cells,we used in vitro experiments.The transwell chamber was tested for its invasive ability.The results showed that LncRNA-ZNF281 can effectively inhibit the invasive ability of glioma stem cells in vitro.The expression of related invasive proteins was detected by western-blot,and the mechanism of lncRNA-ZNF281 was explored.It was shown that LncRNA-ZNF281 can inhibit the invasion of glioma stem cells by inhibiting the expression of NF-KB signaling pathway and invasion-related protein MMP2.5.In vivo experiments verify the effect of LncRNA-ZNF281 on invasion and tumorigenic ability of glioma stem cellsThe LncRNA-ZNF281 U251s and LncRNA-NC U251s cells were used to establish a subcutaneous xenograft model to measure the tumor growth rate.The inhibitory effect of LncRNA-ZNF281 on the self-renewal of glioma stem cells was demonstrated.The results showed that LncRNA-ZNF281 can be significantly inhibited.The growth rate of subcutaneous xenografts was also established.The intracranial xenograft model was constructed and the expression of related proteins was detected by immunohistochemistry.The results showed that the expression of MMP2 in LncRNA-ZNF281 U251s group was significantly lower than that in LncRNA-NC U25 Is group.Statistical significance.ConclusionLncRNA is a type of long-chain non-coding RNA with a nucleotide sequence length greater than 200 nt.In recent years,studies have found that LncRNA plays an important regulatory role in various tumors,such as:LncRNA ATB plays an important role in the regulation of proliferation and invasion of osteosarcoma;LncRNA CRNDE plays a role in the growth and development of colon cancer Important regulatory role,LncRNA RGMB-AS1 affects the invasive ability of lung cancer,and so on.In gliomas,a large number of LncRNAs have also been shown to play an important role.LncRNA-ZNF281 is a novel class of long-chain non-coding RNAs with a nucleotide length of 353 nt and located on chromosome 1q32.1.In our previous experimental studies,it was found that lncRNA-ZNF281 can inhibit the invasion of glioma cell lines U87 and SHG139 by regulating the expression of VEGF and MMP2,but the effects of glioma stem cells have not been further studied.In this experiment,we will study the regulation of LncRNA-ZNF281 on glioma stem cell-like cells.To verify the regulation of LncRNA-ZNF281 on glioma stem cells.We first obtained glioma stem cell U251s from glioma cell line U251 by reverse culture,and identified stem cell characteristics and self-renewal ability.At the same time,we examined the invasive ability of glioma stem cell U251s and glioma cell line U251.The results showed that glioma stem cell U251s was more aggressive.Our experimental data demonstrate that the expression level of LncRNA-ZNF281 in glioma stem cell U251s is lower than that of glioma cell line U251;therefore,we speculate that LncRNA-ZNF281 may play an important role in the maintenance and invasion of glioma stem cells..In this experiment,we confirmed in vitro that LncRNA-ZNF281 can inhibit the dry characteristics and invasive ability of glioma stem cell U251s,and detect the expression of invasion-related NF-kB signaling pathway and invasion-related protein MMP2,and found LncRNA-ZNF281 may play a role mainly by affecting the NF-kB signaling pathway.In vivo,it was confirmed that LncRNA-ZNF281 can effectively inhibit the proliferation and invasion of glioma stem cell U251s through NF-kB signaling pathway,which is a new target of lncRNA-ZNF281.Point treatment of glioma provides a new scientific basis.