| OBJECTIVE:To clarify the effects of rapamycin on autophagy and apoptosis of osteosarcoma cells,and to explore the molecular mechanism of lncRNA-FLJ11812 regulating autophagy and apoptosis of osteosarcoma cells treated with rapamycin.METHODS:In order to regulate how rapamycin can the transcription level of lncRNA,lncRNA were analyzed and screened by high-throughput second-generation sequencing technology between osteosarcoma cells treated with rapamycin and osteosarcoma cells in control group.Multiple molecular biological methods,such as qPCR and western blot,were used to verify that rapamycin mediated autophagy and apoptosis of osteosarcoma cells by regulating the transcription of lncRNA and then regulating the genes related to autophagy and apoptosis.ChIRP experiment of lncRNA-FLJ11812 was carried out to analyze the interacting proteins and target genes of lncRNA-FLJ 11812,and to study the mechanism of the transcription level of lncRNA-FLJ11812.RESULTS:1.Rapamycin MG63 cells were incubated at 1 nM,5 nM,10 nM and 20 nM for 48 h,respectively,and it was found that 20 nM could promote apoptosis of osteosarcoma cells,with statistical difference(p<0.001).2.To add 12 per hole orifice MG63 cells to 20 nM rapamycin solution,the control group to join the corresponding volume DMSO,cultivate found after 48 hours,compared with the control group to join in the group of rapamycin Beclin 1,ATG1,ATG13,Bax,Bcl2 and LC3Ⅱ/Ⅰ mRNA and protein levels were elevated(p<0.01,p<0.001),p53 and p62 mRNA level decreased,and the result was statistically significant(p<0.05).3.MG63 cells were added into 20 nM rapamycin solution and 1μl DMSO solution in the control group.After 48 h of culture,it was found that lncRNA-FLJ 11812 RNA levels in the rapamycin-treated group were significantly increased compared with the control group,and the results were statistically significant(p<0.01).4.PCDNA3.1-ha-lncRNA-FLJ11812 plasmid and pGL3.0-basi-Bax promoter reporter gene plasmid were constructed to detect the cell cycle and apoptosis of the overexpressed lncRNA-FLJ11812 plasmid in MG63 cells,and the results showed that the proportion of G0/G1 phase cells was significantly increased and the proportion of S/G2 phase cells was decreased in the overexpressed group compared with the control group(p<0.05).5.lncRNA-FLJ11812 plasmid,NC siRNA and lncRNA-FLJ11812 siRNA were transfected into 12-well MG63 cells,respectively,and the cells were collected 48h later.The results showed that the Bax mRNA level in the overexpressed lncRNA-FLJ11812 group was significantly increased(p<0.001).The mRNA level of Bax in lncRNA-FLJ11812 siRNA transfected group was significantly decreased(p<0.05).6.lncRNA-FLJ11812 siRNA was transfected into 12-well plate MG63 cells.After 24 h inhibition of lncRNA-FLJ11812,20 nM rapamycin was added for further culture for 48 h.Cells were collected to prepare samples,and the levels of Bax mRNA and protein were detected by qPCR and Western blot.The results showed that the levels of Bax mRNA and protein were increased in the 20 nM rapamycin group(p<0.001),while the lncRNAFLJ11812 group was inhibited by rapamycin,and the levels of Bax mRNA and protein were not changed(p>0.05).7.According to the analysis of pathologists,tumors in patients No.7,6,2,1 and 5 showed high necrosis rate,while tumors in patients No.9,4,8 and 3 showed low necrosis rate(p<0.05).The level of lncRNA-FLJ11812 in all samples was detected by qPCR method,and we found that tumors of patients 7,6,2,1,and 5 showed high expression,while tumors of patients 9,4,8,and 3 showed low expression(p<0.05).Therefore,patients numbered 7,6,2,1 and 5 were classified into the high expression group(OS),and patients numbered 9,4,8 and 3 were classified into the low expression group(Con).CONCLUSIONS:Rapamycin can promote the transcription of lncRNA-FLJ11812 in osteosarcoma cells,then induce the increase of autophagy level and promote the apoptosis of osteosarcoma cells.Bax is the target gene of lncRNA-FLJ11812 to regulate apoptosis.Rapamycin regulates the expression of Bax gene by regulating the level of lncRNAFLJ11812.High expression of lncRN A-FLJ 11812 in osteosarcoma samples indicates a high rate of tumor necrosisOBJECTIVE:To further study the miRNA interaction between lncRNA and autophagy and apoptosis-related genes in osteosarcoma cells,and to explore the mechanism of lncRNA-FLJ 11812 regulating the miRNA.METHODS:RAP experiments were conducted for lncRNA-FLJ11812 to analyze the miRNA interacting with lncRNA-FLJ11812 and to study the mechanism of action at the post-transcriptional level.Through the website,we predicted that lncRNA-FLJ11812 might regulate the expression of miR-141-3p.We transfected pcDNA3.1-3xHA,pcDNA3.13xHA-lncRNA-FLJ11812 plasmid,NC siRNA and pcDNA3.1-3xHA-lncRNA-FLJ11812 siRNA into MG63 cells,and detected the changes in the transcription level of miR-141-3p by qPCR method.In order to prove that lncRNA-FLJ11812 can regulate the expression of miR-141-3p,dual luciferase reporter gene assay was used in this study.We constructed plasmid pDNA3.1-3xHA-lncRNA-FLJ11812 and pGL3.0-basic-miR-141-3p promoter reporter gene plasmid,and carried out in vitro experiments in 293T cells.In order to confirm whether rapamycin regulates the expression of miR-141-3p by regulating the level of lncRNA-FLJ 11812,we transfected lncRNA-FLJ11812 siRNA in MG63 cells to inhibit lncRNA-FLJ 11812 and NC siRNA,and then added rapamycin with a final concentration of 20 nM.Cells were collected and samples were prepared,and the expression level of miR141-3p was detected by qPCR.RESULTS:1.The expression level of miR-141-3p was decreased when the pcDNA3.1-3xHA-lncRNA-FLJ11812 plasmid was overexpressed in MG63 cells(p<0.05);When lncRNA-FLJ 11812 was inhibited in MG63 cells,the expression level of miR-141-3p was increased(p<0.001).2.Transient co-transfection of pcDNA3.1-3xHA,pcDNA3.1-3xHA-lncRNAFLJ11812,pGL3.0-basic-miR-141-3p promoter reporter gene plasmid and Renilla plasmid showed that lncRNA-FLJ 11812 could inhibit the increase of photolinase activity(p<0.001).Inhibition of lncRNA-FLJ 11812 could enhance luciferase activity(p<0.05).3.Adding rapamycin and inhibiting lncRNA-FLJ11812 group significantly decreased the expression level of miR-141-3p(p<0.05).4.Finally,the above results were verified in osteosarcoma tissue samples,and the same results were obtained.CONCLUSIONS:lncRNA-FLJ11812 binding with miR-141-3p promoter negatively regulates transcription in rapamycin-treated osteosarcoma cells.OBJECTIVE:To further study the transcription factor targeting the regulation of lncRNA-FLJ11812,and to identify the specific binding sites of the regulation of lncRNA by this transcription factor.METHODS:By DNA pulldown technique,the protein factors that regulate the expression of lncRNA-FLJ11812 were screened to interfere with the expression of transcription factors in osteosarcoma cells,and the regulation of transcription factors on the expression level of lncRNA-FLJ11812 was studied.The sites of lncRNA-FLJ11812 regulated by transcription regulatory proteins were analyzed by ChIP-PCR,reporter genes and EMSA to further clarify the specific binding sites of lncRNA regulated by transcription factors.We used NCBI and UCSC websites to predict the promoter region of lncRNAFLJ11812.In order to verify that the transcription factor GATA1 regulates the expression of lncRNA-FLJ11812,we overexpressed the plasmid pcDNA3.1-3xHA-GATA 1 in MG63 cells and transfected the GATA1 siRNA knockdown GATA1,respectively,and detected the changes in the expression level of lncRNA-FLJ11812 by qPCR.According to lncRNAFLJ11812 promoter sequences,analysis of transcription factor binding sites(http://jaspar.genereg.net),transcription factor GATA1 in lncRNA-FLJ11812 promoter regions with binding sites(TTTTCATCTCT),in order to further prove GATA1 can directly on the lncRNA-FLJ11812 promoter,we mutated the TTTTCATCTCT sequence in the plasmid of pGL3.0-basic-lncRNA-FLJ11812 reporter gene and repeated the double luciferase reporter gene experiment.At the same time,we transfected pGL3.0-basiclncRNA-FLJ11812 promoter reporter plasmid using the expression of GATA1 siRNA knockdown transcription factor GATA1 gene in 293T cells,and detected the activity of reporter luciferase using dual luciferase reporter assay.In order to verify that rapamycin is involved in the expression of transcription factor GATA1 and can regulate the transcription of lncRNA-FLJ11812,we first transfected lncRNA-FLJ11812 siRNA into 12-well plate MG63 cells and inhibited the addition of lncRNA-FLJ11812 with 20 nM rapamycin for further culture for 48h.The mRNA and protein levels of GATA1 were detected by qPCR and western blot.Then,rapamycin was added after GATA1 inhibition in MG63 cells,and the expression of lncRNA-FLJ11812 was detected by qPCR method.RESULTS:1.Using the website,we predicted that the transcription factor GATA1 could bind to the promoter region of lncRNA-FLJ 11812.2.Overexpression of pcDNA3.1-3xHA-GATA1 plasmid lncRNA-FLJ11812 in MG63 cells increased the transcription level(p<0.001).GATA1 was inhibited by GATA1 siRNA transfection in MG63 cells,and the expression level of lncRNA-FLJ11812 was detected by qPCR,and the transcription level of lncRNA-FLJ11812 was decreased(p<0.05).3.PCDNA3.1-3xHA vector,pcDNA3.1-3xHA-GATA1 plasmid and pGL3.0-basiclncRNA-FLJ11812 promoter reporter gene plasmid were co-transfected into 12-well plate 293T cells using RENILLA as internal reference.The results showed that GATA1 could enhance the activity of photolinase(p<0.001),and inhibition of GATA1 could decrease the activity of luciferase(p<0.01).GATA1 siRNA and pGL3.0-basic-lncRNA-FLJ11812 reporter gene mutated plasmid were co-transfected with Renilla as internal reference,and the results showed that GATA1 could not reduce the activity of photolinase(p>0.05).4.Rapamycin promoted the increase of GATA1 protein and mRNA levels in MG63 cells(p<0.001).In MG63 cells,the level of lncRNA-FLJ11812 decreased when rapamycin was added after GATA1 inhibition(p<0.05).5.Finally,the above results were verified in osteosarcoma tissue samples,and the same results were obtained.CONCLUSIONS:GATA1 directly binding to lncRNA-FLJ11812 promoter positively regulates the expression of lncRNA-FLJ 11812 gene at the transcriptional level in rapamycintreated osteosarcoma cells. |
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