LINC00665 Sponge Adsorption Of MiR-1249-5p Up-regulated WNT2B Promotes The Proliferation,Invasion,Metastasis And Emt Of Osteosarcoma Cells

Osteosarcoma(OS,osteosarcoma),also known as osteosarcoma,is a malignant tumor with frequent occurrence among teenagers.It is developed from bone mesenchymal cell line and is characterized by rapid growth,high degree of malignancy,easy recurrence,and remote metastasis.At present,the pathogenesis of osteosarcoma is not completely clear,so it is of great significance to study the pathogenesis and biological process of osteosarcoma for the treatment of osteosarcoma.Long non-codingRNA(lncRNA)has been reported to play a pro-cancer role in many tumors.We found that the expression level of LINC00665 in osteosarcoma cells was significantly higher than that in normal osteoblasts,but the mechanism of LINC00665 in the occurrence of osteosarcoma is still unclear.This study focused on effect of LINC00665on the proliferation,invasion and metastasis of osteosarcoma cells and EMT,and explored the role and regulatory mechanism of LINC00665 in the occurrence and development of osteosarcoma.The results of in vitro experiments showed that interfering LINC00665expression in osteosarcoma cells could effectively inhibit the proliferation,invasion and metastasis of osteosarcoma cells,inhibit the EMT of osteosarcoma cells,and effectively promote the apoptosis of osteosarcoma cells,thus inhibiting the malignant phenotype of osteosarcoma cells.The above results indicate that LINC00665 is highly expressed in osteosarcoma cells,has an influence on the occurrence of osteosarcoma and the formation of malignant phenotype,and is a regulatory gene that plays a pro-cancer role in osteosarcoma.A large number of studies have reported the regulatory role and mechanism of lncRNA in tumor genesis and development,among which the mechanism of ceRNA is a common model of lncRNA in tumor,which has been widely studied and reported.Because there are a rich miRNA in the lncRNA sequence on the binding sites,and the miRNA can be combined by means of complementary base pairing,in cell lncRNA can play the role of molecular sponge competitively combined with miRNA,simultaneously blocking the miRNA inhibition of mRNA,regulating the mRNA expression level rise,indirectly promote the mRNA protein translation,thus affect the development of the tumor.The results of the nuclear and cytoplasmic separation test and fluorescence pictures ofRNA FISH showed that INC00665 mainly locate in the cytoplasm,combined with the existing about LINC00665 research reported in other tumors,bioinformatics prediction combined with LINC00665 existence possible microRNAs,and through the MS2-RIP experiment confirm LINC0066 in osteosarcoma cells combined with miR-1249-5p,further validation LINC00665 and miR-1249-5p by RISC complex interactions.At the molecular level,LINC00665 can inhibit the expression of miR-1249-5p,and cell function experiments have also confirmed that LINC00665 can promote the occurrence of osteosarcoma by inhibiting miR-1249-5p.There is a major correlation between WNT signaling pathway and tumor occurrence and development,and a large number of studies have shown that abnormal activation of WNT signaling pathway plays a role in promoting tumor occurrence.In further study,we found that LINC00665 markedly affected the activity of WNT signaling pathway,and the activity of WNT signaling pathway decreased notably after interfering with the expression of LINC00665 in osteosarcoma cells.To explore LINC00665 control targets in the WNT signaling pathways,we obtained by bioinformatics methods to predict 10 WNT family proteins are potential target genes of miR-1249-5p,and screen out 10 WNT gene family is regulated by the miR-1249-5p is the most significant downstream target genes WNT2B,preliminary obtained LINC00665/miR-1249-5p/WNT2B signal axis,and verify the three at the molecular level by ceRNA mechanisms play a role of regulation.At the cell level,the signal axis was verified to be effective in promoting the proliferation,invasion and metastasis of osteosarcoma cells and inhibiting the apoptosis of osteosarcoma cells.We verified that the LINC00665/miR-1249-5p/WNT2B signaling axis can activate the WNT signaling pathway,and the WNT signaling pathway can also affect the expression of LINC00665,and the activation of the WNT signaling pathway can effectively enhance the promoter activity of LINC00665.LEF/TCF transcription complex is WNT signaling pathways downstream of regulatory factors,through activation of downstream target genes expression to promote the development of tumor,bioinformatics prediction results show that the LEF/TCF transcription complex on the promoter sequence of LINC00665 potential binding sites,chromatin immune coprecipitation experiment and promoter luciferase reporter gene also confirmed LEF/TCF transcription complex combination on LINC00665promoter region.We concluded that the signal axis of LINC00665/miR-1249-5p/WNT2B activated the WNT signaling pathway to promote the proliferation,invasion,metastasis and EMT of osteosarcoma cells,and the activated WNT signaling pathway fed back to promote the expression of LINC00665 to form a closed-loop loop,playing a pro-cancer role in the formation of osteosarcoma.This research demonstrates LINC00665 at the molecular level in the high expression of osteosarcoma and promote the occurrence mechanism of action of osteosarcoma,enrich the regulation network of osteosarcoma occurred,at the same time LINC00665,WNT2B and WNT signaling pathway is the key control nodes in osteosarcoma occurred,the discovery of these nodes the clinical target therapy of osteosarcoma is of great significance.purpose1.The expression level of LINC00665 in osteosarcoma cell lines and normal osteoblasts was detected by real-time quantitative PCR,and it was clear that LINC00665was significantly highly expressed in osteosarcoma cells.The expression of interference LINC00665 cells in vitro experiments,using CCK8,EDU detection,the clone formation experiment LINC00665 expression level of osteosarcoma cell proliferation,the influence of using caspase 3 activity detection and Tunel experiment LINC00665 expression level influence on osteosarcoma cells apoptosis,LINC00665 expression in the scratches and transwell experiments testing level influence to the invasion and metastasis of osteosarcoma cells,immunofluorescence test and western blot were used to detect the effect of LINC00665 on the occurrence of EMT in osteosarcoma cells,and to verify that LINC00665 promotes the occurrence of osteosarcoma.2.Explore the molecular mechanism by which LINC00665 plays a pro-cancer role in osteosarcoma,and reveal that LINC00665,as a molecular sponge in osteosarcoma cells,regulates the miR-1249-5p/WNT2B signaling axis to activate the WNT signaling pathway,promotes the proliferation,invasion,metastasis and EMT of osteosarcoma cells,and plays a pro-cancer role in the occurrence and development of osteosarcoma.3.Revealed that the WNT signaling pathway up-regulated the expression of LINC00665 at the transcription level,and formed a positive feedback loop with the LINC00665/miR-1249-5p/WNT2B signaling axis.Materials and methods1.The expression levels of LINC00665 in osteosarcoma cell lines and normal osteoblasts were detected:four osteosarcoma cell lines(U20S,MG-63,SAOS-2,143B)and one human fetal osteoblastic cell line(HFOB)with good cell status in the laboratory were used as the normal control.The cells were inoculated in DMEM medium containing10%FBS,and then placed in a cell incubator at 37?and 5%CO₂ for expanded culture.TotalRNA and RT-qPCR of cells were extracted for detection:the cells in good condition were centrifuged and the cell masses were collected,and the totalRNA of each cell was obtained by Trizol method.The totalRNA extracted from each group was reversely transcribed,and the specific qPCR primer of LINC00665 was added to detect the expression levels of LINC00665 in different cell samples.2.Cell function assay:the effect of LINC00665 expression level on the proliferation activity of osteosarcoma cells transfected with sh-LINC00665 interfering expression vector and sh-NC negative control vector was detected by CCK8 assay,EDU assay and clone formation assay,respectively.The effect of LINC00665 expression level on apoptosis of osteosarcoma cells was detected by caspase 3 activity assays and Tunel assay.The effect of LINC00665 expression level on the invasion and metastasis ability of osteosarcoma cells was detected by wound healing and trasnwell test.The effect of LINC00665 expression level on EMT in osteosarcoma cells was detected by immunofluorescence test and western blot.3.Testing LINC00665 positioning:in osteosarcoma cells using the nucleus and cytoplasm separation technology on osteosarcoma cell nuclear transfer separation processing,using Trizol method respectively to extractRNA andRNA in the cytoplasm of the nucleus,with GAPDH as positive control of cytoplasm,U6 as the nucleus of positive control group,after RT-qPCR method to detect LINC00665 expression level in the cytoplasm and nuclei.LINC00665 was labeled with a specific probe with fluorescent groups,and the distribution of LINC00665 in osteosarcoma cells was observed under fluorescence microscope.4.Bioinformatics predicted the possible miRNA binding with LINC000665:online website DIANA tools was used to obtain the miRNA binding with LINC00665,and the top five ranked miRNA were:miR-1249-5p,miR-6891-5p,miR-6785-5p,miR-4728-5p,and miR-6780a-5p.5.The MS2-rip experiment verified the binding of LINC00665 to miRNA:the LINC00665 sequence is built into the carrier MS2 and transfection on osteosarcoma cells,for 48 hours after the cells of pyrolysis process,and the specificity of MS protein antibody capture MS2 carrier and LINC00665 sequence of microRNAs,RNA product after purification on test five microRNAs in MS2 system combined with the quantity,select the combination of the most closely miR-1249-5p.6.RT-qPCR was used to detect the expression level of miR-1249-5p in osteosarcoma cells and the effect of LINC00665 on the expression level of miR-1249-5p.7.MiRNA pull-down assay reversely verified the binding of miR-1249-5p to LINC00665:miRNA probes with biogenic markers were prepared according to the gene sequence of miR-1249-5p,andRNA bound to miR-1249-5p was fully mixed with lysates of osteosarcoma cells to extract,and the adsorption of LINC00665 was detected after the extraction and purification ofRNA products.8.AGO2-RIP assay was used to detect the contents of LINC00665 and miR-1249-5p in RISC complex:the IP-level antibody of AGO2,the core protein of RISC complex,was selected for RIP assay,andRNA in RISC complex was captured,afterRNA products were extracted and purified,the contents of LINC00665 and miR-1249-5p in RISC complex were detected by RT-qPCR.9.Bioinformatics prediction miR-1249-5p binding sites on LINC00665 sequence:get LINC00665 gene sequences in the NCBI database,from miRBase database miR-1249-5p gene sequence,based on the principle of complementary base pairing and the conservatism of microRNAs seed area,looking for LINC00665 sequences and miR-1249-5p complementary matching area is the miR-1249-5p potential binding sites on LINC00665sequence.10.Luciferase reporter gene analysis:building contains LINC00665 sequence of wild type and mutant miRNA sequences of binding sites of luciferase reporter gene carrier,respectively,the wild type and mutant report gene carrier and miR-1249-5p simulation and the negative control miR-NC transfection to osteosarcoma cell line MG-63,SAOS-2 cells,was detected in 48 hours after the luciferase activity in groups of cells.11.Rescue experiment confirmed that LINC00665/miR-1249-5p plays a pro-cancer role in osteosarcoma cells:CCK8,EDU,clone formation assay,Caspase 3 activity test,tunel,scratch,transwell test and protein immunofluorescence test were conducted in the rescue group of sh-NC,sh-LINC00665,sh-LINC00665+miR-1249-5p inhibitor,to detect the effect of LINC00665/miR-1249-5p signal axis on the proliferation,apoptosis,invasion,metastasis and EMT of osteosarcoma cells.12.Detection of the effect of LINC00665 on signaling pathways in osteosarcoma cells:Cignal Finder cancer array chips were used to detect the activity of signaling pathways closely related to tumor occurrence in osteosarcoma cells transfected with sh-LINC00665and sh-NC,and to search for the signaling pathways most significantly affected by the expression level of LINC00665 in osteosarcoma cells.13.Search for the downstream target genes of miR-1249-5p:according to the chip results of Cignal Finder cancer array,it was confirmed that the expression level of LINC00665 was closely correlated with the activity of WNT signaling pathway.The downstream target genes of miR-1249-5p were obtained from Target Scan,an online database,and 10 WNT family genes were screened out,among which the binding score of miR-1249-5p and the target gene WNT2B was the highest.14.WNT2B was confirmed to be the downstream target gene of miR-1249-5p:mRNA and protein expression levels of WNT2B in osteosarcoma cell lines were detected by RT-qPCR and western blot;the effect of miR-1249-5p on the expression level of target gene WNT2B was detected.15.Bioinformatics prediction of potential binding and loci of miR-1249-5p in WNT2B 3'UTR region of target gene:WNT2B 3'UTR region sequence was obtained according to Ensemble database.Based on the principle of base complementary pairing and the conservation of miRNA seed region,the potential binding site of WNT2B 3'UTR region with miR-1249-5p was searched,that is,the potential binding site of miR-1249-5p in WNT2B 3'UTR region.16.MiRNA pull-down assay detected the binding site of miR-1249-5p on the 3'UTR sequence of WNT2B:RNA bound by miR-1249-5p with biotin labeledRNA probe was fully mixed with lysates of osteosarcoma cells to fish theRNA bound by miR-1249-5p probe,and the enrichment of potential binding sites on the 3'UTR sequence of WNT2B was detected after the extraction and purification ofRNA products.17.Luciferase reporter gene experiment prove that miR-1249-5p in potential binding sites and WNT2B 3'UTR sequences:building contains WNT2B 3'UTR sequence of wild type and mutant miR-1249-5p binding site sequence of luciferase reporter gene carrier,respectively,the wild type and mutant report gene carrier and miR-1249-5p simulation and the negative control miR-NC transfection to osteosarcoma cell line MG-63,SAOS-2 cells,was detected in 48 hours after the luciferase activity in groups of cells.18.AGO2-RIP experiment verified that LINC00665,miR-1249-5p and WNT2B3'UTR bind to RISC complex:the enrichment of LINC00665,miR-1249-5p and WNT2B3'UTR was detected simultaneously in theRNA products of AGO2-RIP,verifying that all three bind to RISC complex.19.Rescue experiment verified that LINC00665 adsorbs miR-1249-5p to regulate the expression of WNT2B:in the rescue experimental group of sh-NC,sh-LINC00665,sh-LINC00665+pc DNA-WNT2B,RT-qPCR assay detected the mRNA expression level of WNT2B,and western blot assay detected the protein expression level of WNT2B.20.Cell level rescue experiment verified that LINC00665/miR-1249-5p/WNT2B signaling axis promoted tumor genesis in osteosarcoma:the effects of LINC00665/miR-1249-5p/WNT2B signal axis on the proliferation,apoptosis,invasion,metastasis and EMT of osteosarcoma cells were detected by CCK8,EDU,clone formation assay,Caspase3 activity assay,tunel,scratch,transwell assay and protein immunofluorescence assay in the rescue group of osteosarcoma cells divided into sh-NC,sh-LINC00665 and sh-LINC00665+pc DNA-WNT2B.21.TOP/FOP luciferase reporter gene verification LINC00665/miR-1249-5p/WNT2B signaling axis activated the WNT signaling pathway:the activity of TOP/FOP luciferase reporter gene was detected in osteosarcomas grouped as sh-NC,sh-LINC00665,and sh-LINC00665+pc DNA-WNT2B cells in the rescue experiment to verify the effect of LINC00665/miR-1249-5p/WNT2B signaling axis on the activity of WNT signaling pathway.22.Detection of LINC00665/miR-1249-5p/WNT2B signaling axis activation of WNT signaling pathway and downstream gene expression:In osteosarcoma cells transfected with LINC00665 interfering expression vector sh-LINC00665 and negative control vector sh-NC,the mRNA and protein expression levels of WNT signaling pathway genes WNT2B,beta-catenin,LEF1 and downstream genes c-MYC,CCND1,CDK4,MMP2and Snail were detected by RT-qPCR and western blot.23.Detection of the effect of WNT signaling pathway on the expression level of LINC00665:WNT signaling pathway agonists Li Cl and DMSO were added to osteosarcoma cells,respectively,and the expression level of LINC00665 in osteosarcoma cells under different treatment conditions was detected by RT-qPCR.24.Bioinformatics predicted binding sites of TCF/LEF transcription complex in LINC00665 promoter region:TCF/LEF transcription complex from JASPAR database motif of the DNA sequence,from UCSC database LINC00665 TSS loci of the DNA sequence of length 2000 bp upstream area(namely the promoter region of LINC00665),the TCF/LEF motif of the DNA sequence matching with LINC00665 promoter sequences,obtain TCF/LEF transcription complex potential binding sites on LINC00665 promoter region,and according to the specificity of the binding site design qPCR primers,for the detection of subsequent chromatin immunoprecipitation products.25.The chromatin immune co-precipitation experiment TCF/LEF transcription complex combination LINC00665 promoter region:through chromatin immune coprecipitation experiment obtained LEF1 and transcription complex pieces of DNA,after the extraction and purification of DNA fragments,using qPCR experimental detection DNA production LINC00665 potential binding sites in the promoter region of fragments of enrichment.26.The binding site of the LEF/TCF transcription complex in the LINC00665promoter region was verified by luciferase activity test of the promoter:build LINC00665promoter the wild type sequence and TCF/LEF transcription complex binding site area sequence mutation type,connect the sequence PGL3 luciferase reporter gene vector,respectively,after DMSO treatment and Li Cl transfection of osteosarcoma cells LINC00665 wild type and mutant luciferase reporter gene,validation of predicting binding sites is TCF/LEF transcription complex on LINC00665 promoter region of the actual binding sites.Result1.The expression level of LINC00665 in osteosarcoma cell line was significantly higher than that in normal osteoblasts,and LINC00665 was the most highly expressed in MG-63 and 143B.2.Expression levels of LINC00665 were significantly decreased in MG-63 and 143B osteosarcoma cells transfected with sh-LINC00665,and the results of cell function test showed that:interfering LINC00665 expression can significantly inhibit the proliferation,invasion,metastasis and EMT of osteosarcoma cells,and markedly increase the apoptosis level of osteosarcoma cells after interfering LINC00665 expression.Cell transfection showed that LINC00665 was down-regulated in MG-63 and HOS cells.MTT assay and traswell assay showed that the knockdown of LINC00665 significantly inhibited cell proliferation,migration and invasion in MG-63 and HOS cell lines.3.The results of cytoplasmic separation andRNA FISH assay showed that LINC00665 was expressed in both the nucleus and cytoplasm,mainly in the cytoplasm.4.Bioinformatics predicted the possible miRNA binding with LINC000665,among which miR-1249-5p,miR-6891-5p,miR-6785-5p,miR-4728-5p and miR-6780a-5p ranked the first five.5.The MS2-RIP experiment confirmed that among the top five miRNA predicted by bioinformatics,miR-1249-5p most closely bound to LINC00665 in osteosarcoma cells.6.The expression level of miR-1249-5p in osteosarcoma cells was significantly lower than that in normal osteoblasts.After interfering with LINC00665,the expression level of miR-1249-5p was significantly increased,and the experimental results showed that LINC00665 inhibited the expression level of miR-1249-5p in osteosarcoma cells.7.The miRNA pull-down assay reversely verified that miR-1249-5p could bind to LINC00665.8.AGO2-RIP assay was used to detect the contents of LINC00665 and miR-1249-5p in RISC complex:IP level antibody of RISC complex core protein AGO2 was selected for RIP experiment,andRNA in RISC complex was captured and obtained.RNA products were extracted and purified,and the contents of LINC00665 and miR-1249-5p in RISC complex were detected by RT-qPCR.9.Bioinformatics prediction results showed that miR-1249-5p had potential binding sites on LINC00665 sequence.10.The detection results of luciferase reporter gene showed that the wild-type reporter gene of LINC00665 was affected by miR-1249-5p mimics,resulting in decreased activity,while the mutant had no significant change,indicating that the binding site predicted by bioinformatics was the actual binding site of miR-1249-5p on the LINC00665 sequence.11.The detection results of the cell function rescue experiment showed that LINC00665/miR-1249-5p signaling axis promoted the proliferation,invasion,metastasis and EMT of osteosarcoma cells,inhibited the apoptosis of osteosarcoma cells,and played a pro-cancer role in osteosarcoma.12.The detection results of Cignal Finder cancer array showed that the activity of WNT signaling pathway,p RB-E2F signaling diagram and MYC signaling pathway in osteosarcoma cells was significantly reduced after interfering with the expression level of LINC00665,among which the activity of WNT signaling pathway was the most significant.13.Through downstream target genes of miR-1249-5p were obtained with online database Target Scan,and 10 WNT family genes were screened out,among which miR-1249-5p had the highest binding score with target gene WNT2B.14.RT-qPCR and western blot results showed that the mRNA and protein expression levels of WNT2B in osteosarcoma cells were significantly higher than those in normal osteoblasts,miR-1249-5p significantly inhibited the mRNA and protein expression levels of WNT2B.15.The results of bioinformatics prediction showed that there were 4 potential binding sites of miR-1249-5p in the target gene WNT2B 3'UTR region.16.The enrichment of 4 potential binding sites of WNT2B 3'UTR sequence was detected in theRNA products of miRNA pull-down assay,and it was found that the first predicted site was highly enriched in theRNA products,indicating that miR-1249-5p bound WNT2B 3'UTR sequence most significantly at the first predicted site.17.Luciferase reporter gene experimental test results show that Mi R-1249-5p mimics significantly inhibited the luciferase activity of the wild-type reporter gene at the first predicted site,but had no significant effect on the luciferase activity of the mutant luciferase reporter gene,indicating that the first predicted site was the real binding site of miR-1249-5p in the WNT2B 3'UTR region of the target gene.18.The detection results of RT-qPCR showed that LINC00665,miR-1249-5p and WNT2B 3'UTR showed significant enrichment in theRNA products of AGO2-RIP experiment,indicating that all three were bound to the RISC complex.19.Rescue test results at the molecular level showed that LINC00665 adsorbs miR-1249-5p to promote the mRNA and protein expression of WNT2B.20.The rescue test results at the cell level showed that LINC00665/miR-1249-5p/WNT2B signaling axis promoted the proliferation ability of osteosarcoma cells,inhibited the apoptosis of osteosarcoma cells,and also promoted the invasion,metastasis and EMT of osteosarcoma cells.21.The test results of TOP/FOP luciferase reporter gene showed that the activity of WNT signaling pathway decreased after interfering with the expression of LINC00665,while the activity of WNT signaling pathway increased after the overexpression of WNT2B under the interference of LINC00665,indicating that the LINC00665/miR-1249-5p/WNT2B signaling axis could effectively activate the activity of WNT signaling pathway.22.In osteosarcoma cells that interfere with the expression of LINC00665,mRNA and protein expression levels of genes WNT2B,beta-catenin,LEF1 and downstream genes c-MYC,CCND1,CDK4,MMP2 and Snail in the WNT signaling pathway were reduced,further verifying the activation effect of LINC00665 on the WNT signaling pathway.23.The detection results of RT-qPCR showed that the expression level of LINC00665in osteosarcoma cells after adding the WNT signaling pathway agonist Li Cl was significantly higher than that in DMSO cells,suggesting that the WNT signaling pathway plays a role in promoting the expression of LINC00665.24.Bioinformatics predicted that the TCF/LEF transcription complex had two possible binding sites in the LINC00665 promoter region.25.The enrichment of the first prediction site and the second prediction site was detected in the DNA products of chromatin immunoprecipitation assay,and the results of qPCR showed that significant binding signals were detected at the second prediction site,indicating that the TCF/LEF transcription complex may bind to the promoter region of LINC00665 at the second prediction site.26.LINC00665 promoter luciferase report gene in the experimental test results show that adding WNT signaling pathways agonist Li Cl can significantly enhance the binding sites of wild type luciferase reporter gene promoter activity,while the second binding site mutation of luciferase reporter gene activity there was no significant change,shows the second site is TCF/LEF transcription complex actual binding sites on LINC00665promoter region.Conclusion1.LINC00665 was significantly highly expressed in osteosarcoma cell lines.After knocking down LINC00665,the proliferation,migration and EMT of osteosarcoma cells were significantly inhibited,and the apoptosis level of the cells was significantly increased,and LINC00665 plays a role in promoting cancer in osteosarcoma cells.2.LINC00665,as a miRNA molecular sponge in osteosarcoma,activates the WNT signaling pathway through the LINC00665/miR-1249-5p/WNT2B signaling axis to play a pro-cancer role.3.The downstream TCF/LEF transcription complex of the WNT signaling pathway can promote the transcription of LINC00665 at the transcription level and form a positive feedback loop with the LINC00665/miR-1249-5p/WNT2B signal axis.4.This topic demonstrates LINC00665 at the molecular level in the high expression of osteosarcoma and promote the occurrence mechanism of action of osteosarcoma,enrich the regulation network of osteosarcoma occurred,at the same time LINC00665,WNT2B and WNT signaling pathway is the key control nodes in osteosarcoma occurred,the discovery of these nodes the clinical target therapy of osteosarcoma is of great significance.