Effect Of LINC00473 On Biological Behavior Of Glioma By Regulating Autophagy Mediated By MiR-210-3p And ATG7 Pathway

Objective: Glioma is one of the most common primary malignant tumors in the central nervous system,and its overall incidence rate accounts for about 40%-60% of all brain malignant tumors.Seeing from the relevant standards of WHO and the malignancy of tumor cells,gliomas can be divided into four different grades,including Grade I,II,III and IV.And grade IV glioblastoma(GBM)has the highest malignant degree.At present,for gliomas including GBM,the main treatment methods include the largest range of surgical resection,as well as postoperative adjuvant radiotherapy and chemotherapy.Even if most patients receive this treatment,gliomas are easy to relapse and have poor prognosis.Considering the complex specificity and strong invasive ability of gliomas and the limited effect of traditional drug treatment methods such as surgery,radiotherapy and chemotherapy,it is important to deeply research the molecular mechanism of gliomas’ occurrence and development in tumors.That’s how we can identify the causes of the cancer and improve its therapeutic effect.micro RNA(micro RNA)is an endogenous class of small non-coding RNA composed of about 21-23 nucleotide sequences,encoding the target gene by transcriptional regulation by binding to the 3’non coding region of messenger RNA.A large number of related studies have proved that miRNA is an important regulator of tumor development,and its role can not be ignored.Some miRNAs can be detected with obvious high expression in tumor,which can play a clear role in promoting tumor growth by promoting the proliferation and invasion of tumor cells.It has been confirmed that miR-9,miR-196,miR-155 and other miRNAs have a clear role in promoting tumor growth.On the contrary,such as miR-181 family,miR-200 family and so on,the expression level in tumor is decreased,and plays a role in inhibiting tumor progression by inhibiting the expression of oncogenes.Similarly,the mechanism of miRNA is very complex,and it can affect multiple signaling pathways in downstream regulation,so its influence mechanism is not a single linear regulation.Similar to miRNA,long non coding RNA(lncrna)is more upstream in cell regulation,which can play a wide and complex role.Linc00473,also known as chromosome 6 open reading frame 176(c6orf176)or LINC00473,is an lncrna located on chromosome 6q27,which is involved in the regulation of synaptic activity.Reitmair et al.Found that PGE2 receptor subtypes EP2 and EP4 specific agonists can significantly up regulate the expression of linc00473 in human ciliary smooth muscle cells,suggesting that they may regulate camp mediated gene expression.Z.Chen,Li,et al.Found for the first time that linc00473 was upregulated by c AMP / c AMP response element binding protein(CREB)in lung cancer and tumor growth.Overexpression of LINC00473 is related to many human malignant tumors,including hepatocellular carcinoma,breast cancer,esophageal squamous cell carcinoma,colorectal cancer,glioma,osteosarcoma,pancreatic cancer,gastric cancer,cervical cancer,mucoepidermoid carcinoma,head and neck squamous cell carcinoma and cholangiocarcinoma.Our previous study found that linc00473 expression level in glioma is higher than that in peritumoral tissue(NBT),and can independently evaluate the prognosis of high-grade glioma patients.In addition,subsequent cell functional experiments confirmed that lncrna can regulate ATG series proteins to mediate autophagy.The main purpose of this research is to further unveil the important role LINC00473 may play in clinical regulation of biological behavior of malignant glioma,further study and explore its clinical effect and drug action transformation mechanism,and provide new clinical molecular targets and markers for early clinical diagnosis and early treatment of glioma.Methods: 1.cellculture:humangliomacelllines(U251,A172,U87,SHG44)were collected from the American type culture collection(atcc,rockville,MD).The normal human astrocyte Heb cell line was purchased from Shanghai(atcc0459;Shanghai beno Biotechnology Co.,Ltd.,Shanghai,China).The cells were incubated with RPMI-1640(Invitrogen)+10% fetal bovine serum(FBS)(Invitrogen)at 37℃ with 100μ U/ ml penicillin,100μ g / ml streptomycin(GIBCO,Rockville,MD)and 5% CO2.2.Transfection: The prepared cells were seeded on a Thermo Fisher plate.Before transfection,1×10^6 cells were seeded on a 6-well plate and cultured in 3 ml medium containing 10% FBS for 24 hours until 60% confluence.Pc DNA3.1-LINC00473,hsa-miR-210-3p mimetic and negative reference(NC)were purchased from Gene Pharma(Shanghai,China).Lipo3000(Invitrogen)transfected cells were cultured in Opti-MEM serum-free medium.3.Real-time PCR: Total RNA of cells was extracted with TRIzol reagent(Invitrogen).200 ng of that extract RNA was reverse transcribed into c DNA use a Rever Tra-Ace-q PCR-RT kit(TAKARA,Japan).GAPDH and U6 were used to normalize the expression of target genes.CCK8 assay: CCK8 assay(sigma)was used to detect cell viability.The transfected cells were seeded on 96 well plates and co cultured with 10 UL CCK8 for 20 min.The absorbance was measured at 450 nm.5.Western blotting: the total protein was extracted with Ripa reagent.For gel electrophoresis,the total protein extracted from each sample was separated by 10-12% twelve alkyl sulfate polyacrylamide gel(SDS-PAGE)(50 g)and then transferred to PVDF(Billerica Millipore,Massachusetts).Primary antibodies: LC3,ATG7 and β-actin were from Abcam(Shanghai,China).After washing with PBS three times,they were cultured with secondary antibody(Abcam,China).6.Immunofluorescence: Immunofluorescence method: The cells were fixed with 4% paraformaldehyde(Sigma,Beijing)at room temperature for 15 min,then infiltrated with 0.4% tritonx-10 for 5-10 min,and blocked with 5% BSA.Next,the cells were washed with PBS for three times and cultured overnight with primary antibody at 4 ℃.The next day,the cells were co-cultured with secondary antibody for 1h.Finally,it was washed with PBS three times.Then the nuclei were cultured with DAPI and cultured at room temperature for 3min.Fluorescence was observed using a confocal microscope(Olympus,USA).7.Luciferase reporter analysis:the wild type(WT)3’-UTR sequence of ATG7 gene and a mutant(MUT)sequence,which contains the putative binding site of mir-210-3p predicted by the star base(http://http :// www.sysu.edu.cn/).The putative mir-210-3p target binding sequence in LINC00473 was synthesized and cloned into pmirglo dual luciferase reporter vector(Promega,USA)as a wild-type and Starbase predicted mutant(MUT).After transfection of lipo3000(Invitrogen),the luciferase activity was determined by double luciferase method.Renin luciferase activity was used as the standard.8.The localization of lncrna in cells was detected by fluorescence in situ hybridization(FISH).CC cells were seeded on 6 wells.After the fusion rate reached 70%,they were washed with PBS for 3 times and fixed with 4% paraformaldehyde.After that,the cells were cultured in pre hybridizing medium and hybridized with rna-fish solution(servicebio,China)containing LINC00473 probe.The nuclei were stained with 6-diamino-2-phenylindole.Fluorescence was observed by Olympus.9.Statistical analysis: All data were analyzed by spss22.0 statistical software.The measurement results are expressed as average ±standard deviation.T-test or analysis of variance was used to identify the obvious differences among the groups.Result: 1.LINC00473 is a malignant gene in glioma tissue,mainly located in the cytoplasm.Many studies have shown that LINC00473 is a new lncrna,which is related to the low survival rate of glioma patients.However,the mechanism of LINC00473 in glioma remains unclear.Studies have shown that isocitrate dehydrogenase 1(IDH1)mutants usually mark a better survival rate.So,we compared the expression of LINC00473 in different groups(IDH1 mutant or wild type)by TCGA and CGGA,as a way to further identify the relationship between the expression of LINC00473 and the development of glioma.It can be found that the expression of LINC00473 in IDH1wild-type group was higher than that of mutant(P=0.007).Because IDH1 wild type is associated with low survival rate,LINC00473 may be involved in the malignant process of glioma.In addition,we also used CGGA data set to explore the relationship between the expression of LINC00473 and WHO classification according to IDH1 status.The results revealed that the expression of LINC00473 in wild type group was higher than that in mutant group(P <0.001).What’s more,researches have unveiled that age is a risk factor for glioma recurrence.Therefore,we compared the expression of LINC00473 in different age groups by CGGA.We found that LINC00473 was highly expressed in elderly patients(P =0.007).The results above mean that LINC00473 may get involve in the progression of glioma.In order to better explore the role of LINC00473 in glioma progression,we compared the expression of LINC00473 in glioma cell lines,and constructed LINC00473 overexpression cell lines through pcdna3.1-LINC00473,and detected the expression of LINC00473 by PCR,which proved that LINC00473 overexpression cell lines(U251 and U87,respectively)were successfully constructed.Next,we performed CCK8 analysis to evaluate the proliferation of glioma cells and normal cells(U251 and U87,respectively).The results showed that LINC00473 could significantly promote the proliferation of glioma cells.These results provide a clue that LINC00473 overexpression can promote glioma progression.In addition,fish analysis was performed to observe the location of LINC00473.The results showed that it was mainly distributed in the cytoplasm LINC00473 promotes autophagy in glioma cells: autophagy is a highly conserved process in cells,which can protect cells from stress,such as hypoxia,drugs and even immune escape.However,the role of LINC00473 in autophagy remains unclear.So,the study wants to find the mechanism of LINC00473 in autophagy.Western blot analysis was applied to detect the expression of LC3 in the two cell lines.The dot like structure of LC3 was evaluated by immunofluorescence.We found that the lc3 ii / lc3 i ratio of LINC00473 overexpression cells was higher than that of pcDNA3.1.Immunofluorescence also confirmed the similar results.This suggests that LINC00473 promotes autophagy in glioma.LINC00473 promotes autophagy through ATG7: in order to further explore the important role of LINC00473 in glioma cells,we modified the endogenous expression of LINC00473 by transfecting pcdna-LINC00473.We detected the expression of ATGs m RNA by PCR,which indicated that the expression of ATG7 m RNA was enhanced in both cell lines.Subsequently,we compared the expression of ATG7 in tissues using CGGA data sets.The results showed that the expression of ATG7 in wild-type group was higher than that in mutant group(P=0.00017).What’s more,mutant(P=0.0024)is lower than the expression of ATG7 in the WHO IV wild-type group.And the expression of ATG7 in the WHO IV wild-type group was positively correlated with age(P=0.039).Overexpression of LINC00473 is able to enhance the expression of ATG7 m RNA.Additionally,the expression of ATG7 protein in U251 and U87 was detected by Western-blot,and the expression of ATG7 was quantified by image J,which proved that LINC00473 promoted the expression of ATG7.These results indicate that LINC00473 can promote the autophagy of glioma cells through ATG7.ATG7 is the target of mir-210-3p: lncrna is widely involved in autophagy and other processes as Cerna.Therefore,in order to further explore the mechanism of action of LINC00473,we analyzed the relationship between ATG7 and miRNAs(target binding sites)through Starbase.We found that mir-210-3p can target ATG7 m RNA.Studies have shown that mir-210 is a protective factor in the development of glioma.Subsequently,we constructed mir-210-3p overexpression cell lines,and detected the m RNA expression of ATG7(U251 and U87,respectively)by PCR.In addition,we also detected the protein expression of ATG7 in two cell lines.The results showed that mir-210-3p overexpression could reduce the m RNA and protein expression of ATG7,suggesting that mir-210-3p could inhibit the expression of ATG7.Importantly,we constructed a dual luciferase reporter vector to detect the binding between ATG7 m RNA and mir-210-3p.As a standardization,we compared the luciferase activity of different groups in the two cell lines.Mir-210-3p decreased the luciferase activity of ATG7-wt group.These results indicate that ATG7 is the target of mir-210-3p.LINC00473 promotes autophagy through mir-210-3p / ATG7 axis: These results indicate that LINC00473 mediates autophagy through ATG7,and mir-210-3p is the target of ATG7.Therefore,we aim to further explore the LINC00473/ mir-210-3p /ATG7 axis.First,we predicted the target between LINC00473 and mir-210-3p,constructed LINC00473 overexpression cell line,and then detected mir-210-3p in glioma cells by PCR.Then we measured the luciferase activity according to the predicted binding sites(WT and MUT)of the website(Fig.5c-d).In U251 cells,mir-210-3p decreased luciferase activity in LINC00473 wt group compared with mut group(P<0.01),and similar results in U87 were shown in(P <0.05).At last,protein expression of different groups is able to be detected by Western blot.LINC00473 strengthens ATG7 expression and can be blocked by miR-210-3p mimetics.Luciferase activity analysis showed that LINC00473 combined with mir-210-3p.These results indicated that LINC00473 enhanced autophagy through mir-210-3p / ATG7 axis.Conclusions: 1.LINC00473 can promote the autophagy of glioma cells through ATG7,and then affect the proliferation and invasion of glioma.There is a positive regulatory relationship between them.2.LINC00473 mediates autophagy through ATG7,and mir-210-3p is the target of ATG7.In the regulation process,LINC00473 and miR-210-3p play a synergistic role,and miR-210-3p plays a negative role in the regulation process.