Study On Molecular Mechanism Of Long Noncoding RNA LOXL1-AS1 Promoting The Progression Of Retinoblastoma

Background:Retinoblastoma(RB)is an intraocular malignancy that is most common in children,accounting for 60% of malignant paediatric ocular tumors.Although the occurrence of retinoblastoma is relatively rare,the survival rate is still low due to the high degree of malignancy.The vast majority of retinoblastomas initiate with biallelic inactivation of RB1,yet its development is by no means caused by a single gene.Addressing novel biomarkers and therapeutic targets for RB to modulate tumor progression has become a challenge.Molecular biology has matured gradually and systematically,bringing forth theories and technologies that have given impetus to life sciences.In the human genome,approximately 70%–90% of genes are transcribed into RNAs,but only 2% of the total RNAs are translated into proteins while more than 90% of the rest are transcribed into noncoding RNA(nc RNAs).The transcribed content of lncRNAs in nc RNAs is up to 80%.It has been shown that the majority of lncRNAs are localized in the nucleus and the number of human lncRNAs is around3,300 which since identified with its considerable presence has been widely studied.LncRNAs are non-protein-coding RNA molecules longer than 200 nt in length,but it can regulate the expression of target genes at the transcriptional and posttranscriptional level.Increasing evidence has suggested that lncRNAs could play critical roles in almost all the biological processes,including stem cell maintenance,cell proliferation,cell apoptosis,cell invasion,and metastasis.LncRNAs are often dysregulated in cancer specimens and play a complex and precise regulatory roles and progression by acting as oncogenes or tumor suppressors,which is closely related to the development of cancer.Through extensive literature research,LOXL1-AS1 has been proven to affect cell proliferation,cell cycle,migration,and invasion as an oncogenic lncRNA in other cancers.LncRNA LOXL1-AS1 was identified based on the impact of its abnormal expression in serious studies linked with the cellular stress response,we consider that LOXL1-AS1 may be related to the prognosis of tumors.The mechanism of LOXL1-AS1 in retinoblastoma has not been elucidated in current research.Therefore,the aim of the present study was to investigate the function of LOXL1-AS1 in retinoblastoma proliferation and metastasis.To further evaluate the effects of LOXL1-AS1 in retinoblastoma tissue and cells,we employed lentivirus transfection methods to knockdown LOXL1-AS1 expression and through bioinformatics analysis techniques to explore the regulatory mechanisms of downstream target genes and signaling pathways of LOXL1-AS1.Moreover,the role of lncRNAs as novel biomarkers and therapeutic targe for retinoblastoma has provided a new basis for diagnosis,clinical treatment and prognosis in tumor development.Effects of lncRNA LOXL1-AS1 expression and function in retinoblastomaPurpose:To investigate the expression level of LOXL1-AS1 in retinoblastoma and the functionality of LOXL1-AS1 on the proliferation,apoptosis,migration and invasion.Methods:(1)The expression level of LOXL 1-AS1 in retinoblastoma tissues and cells was examined by qRT-PCR experiments;(2)The transfection efficiency was determined using qRT-PCR experiments after the transfection of lentivirus specific sh RNAs and overexpression LOXL1-AS1 plasmid in retinoblastoma cell lines;(3)The detection of stable transfection of lentivirus specific sh RNAs by immunofluorescence staining;(4)CCK-8 assay was used to determine the transfection effects of lentivirus specific sh RNAs and LOXL1-AS1 plasmid on cell viability;(5)Wound scratch assay was used to detect the effect of LOXL1-AS1 overexpression on the horizontal migratory ability of Y79 cells;(6)Soft agar colony formation assay was used to detect the effect of knockdown LOXL1-AS1 on the colony forming ability;(7)Transwell assay was used to evaluate the change in the migration and invasion ability of LOXL1-AS1 knockdown;(8)Annexin Ⅴ-FITC/PI double staining flow cytometry were used to detect cell apoptosis cell cycle;(9)The western blot assay was used to detect the expression of apoptotic related proteins.Results:(1)qRT-PCR experiments suggested that LOXL1-AS1 was overexpressed in retinoblastoma tissues and cells;(2)qRT-PCR assay showed that expression of LOXL1-AS1 were decreased and increased respectively,after transfection of lentivirus specific sh RNAs and overexpression LOXL1-AS1 plasmid in retinoblastoma cell lines;(3)Immunofluorescence staining showed the distribution of fluorescence in the in Y79 cells and WERI-Rb1 cells,suggesting that LOXL1-AS1 expression was stably reduced;(4)CCK-8 assay showed that the knockdown of LOXL1-AS1 obviously induced the proliferative activity,and the overexpression of LOXL1-AS1 increased the cell viability;(5)Wound scratch assay showed that overexpression of LOXL1-AS1 can effectively promote the horizontal migratory ability;(6)Soft agar colony formation assay found that the knockdown of LOXL1-AS1 inhibited colony formation capacity;(7)The Transwell assay revealed that LOXL1-AS1 knockdown cells inhibited cell migration and reduced invasion;(8)Annexin Ⅴ-FITC/PI double staining flow cytometry revealed that LOXL1-AS1 knockdown can promote cells apoptosis and arrested the cell cycle at the G2/M phase;(9)Western blot showed that LOXL1-AS1 knockdown can increase the expression of pro-apoptotic proteins and decrease the expression of anti-apoptotic proteins.Conclusion:In vitro experiment indicated that over expression of LOXL1-AS1 can promote cell proliferation and migration.The knockdown of LOXL1-AS1 can inhibit cell proliferation,cell clone formation,cell migration and invasion,promote cell apoptosis,and arrest cell cycle in G2/M phase.LOXL1-AS1 targets expression of HMGB1 and regulates MAPK signaling pathway in retinoblastoma growthPurpose:To investigate the effects of downstream gene expression and signaling pathway targeted by LOXL1-AS1 on the growth of retinoblastoma.Methods:(1)Prediction of the enrichment of downstream target genes and signal pathways by eukaryotic transcriptome sequencing;(2)According to the transcriptome sequencing results,Western blot was used to detect the expression of key proteins related to MAPK signal pathway after LOXL1-AS1 knockdown;(3)Xenograft models of retinoblastoma were established by subcutaneous tumorigenesis experiment in nude mice.HE and immunohistochemical staining were used to dected the growth and proliferation of knock-down LOXL1-AS1 in vivo.(4)The expression of the target gene HMGB1 in the transcriptome sequencing results was examined by qRT-PCR experiments;(5)Western blot were performed to detect the transfection of HA-HMGB1 plasmid;(6)CCK-8 assay was examined to detect the effect of HMGB1 on cells viability and proliferation after the addition of the autophagy inhibitor chloroquine;(7)Western blot assay was used to detect the changes in the expression of key autophagy-related proteins after co-transfection of LOXL1-AS1 and HMGB1.(8)Immunofluorescence assay was used to access the the expression of GFP-LC3 autophagic puncta.Results:(1)The eukaryotic transcriptome sequencing results showed that the KEGG enrichment of overexpression LOXL1-AS1 was indicated at the MAPK signaling pathway,the expression of HMGB1,SNRPA,UBE2 S were significantly increased;(2)Western blot observed that the phosphorylated levels of MAPK,including p-Erk and p-MEK,were markedly decreased in LOXL1-AS1 knockdown cells;(3)Subcutaneous tumorigenesis in nude mice confirmed that knockdown of LOXL1-AS1 had an inhibitory effect in the growth of retinoblastoma in vivo.HE and immunohistochemical staining showed the sections of tumor after resection,and the expression of important index of tumor proliferation Ki-67 showed knockdown LOXL1-AS1 decreased proliferation ability in vivo;(4)According to the prediction of the sequencing results,qRT-PCR results indicated that the expression of HMGB1 was decreased in knockdown LOXL 1-AS1 cells,and its expression was overexpressed in retinoblastoma cells;(5)Western blot assay of the expression of HA tag protein proved that HA-HMGB1 was successfully transfected;(6)CCK-8 assay showed that HMGB1 can promoted cell viability in retinoblastoma cells and LOXL1-AS1 had an inhibitory effect on HMGB1-mediated autophagy on cell viability after adding autophagy inhibitor chloroquine;(7)Western blot assay showed that LOXL1-AS1 can promote the expressions of autophagy-related key proteins by HMGB 1-mediated;(8)Laser confocal microscopy showed that overexpression LOXL1-AS1 upregulated the the number of GFP-LC3 puncta.Conclusion:According to the eukaryotic transcriptome sequencing results,HMGB1 was identified as a downstream target gene regulated by LOXL1-AS1.LOXL1-AS1 modulates the progression of retinoblastoma through the MAPK singaling pathway in vitro and vivo.LOXL1-AS1 impacts the progression of retinoblastoma by regulating miR-7-5p / HMGB1 axisPurpose:To investigate the effects of LOXL1-AS1 impacts the progression of retinoblastoma as a function of ce RNA regulating downstream miRNA expression.Methods:(1)The results of small RNA informatics analysis was examined to predict the downstream miRNA by LOXL1-AS1 regulated;(2)The expresssion of miR-7-5p in LOXL 1-AS1 knockdown cells as well as in retinoblastoma cell by qRT-PCR experiments;(3)The transfection efficiency of miR-7-5p mimics,miR-7-5p inhibitor and their corresponding control plasmids into Y79 cell by qRT-PCR experiments;(4)Dual luciferase reporter gene detection experiments was used to whether LOXL1-AS1 is directly bound to miR-7-5p;(5)CCK-8 assay was used to determine the effects of miR-7-5p mimics,miR-7-5p inhibitor and their corresponding control plasmids into Y79 cell on the cell viability and LOXL1-AS1;(6)Soft agar colony formation assay was used to detect the effects of LOXL 1-AS1 knockdown on the ability of miR-7-5p to colony formation capacity;(7)Transwell assay was used to dectet the effect of knockdown LOXL 1-AS1 on miR-7-5p in cell migration and invasion;(8)Dual luciferase reporter gene detection experiments was used to whether there is a binding site between miR-7-5p and HMGB1;(9)Western blot assay was used to verified the expression of HMGB 1 protein transfected with miR-7-5p mimics,miR-7-5p inhibitor and their corresponding control plasmids.Results:(1)According to the results of small RNA informatics analysis,the expresions of,miR-103a-3p and miR-451 a were decreased after overexpression LOXL1-AS1;(2)qRT-PCR experiments confirmed that the expression of miR-7-5p was increased in knockdown of LOXL 1-AS1 groups;(3)After transfection of miR-7-5p mimics,miR-7-5p inhibitor and their corresponding control plasmids,qRT-PCR experiments confirmed that the expression of miR-7-5p mimics was increased and miR-7-5p inhibitor was decreased;(4)Dual luciferase reporter gene detection experiments showed that there was a direct binding site between LOXL1-AS1 and miR-7-5p;(5)CCK-8 assay performed that the miR-7-5p inhibitor increased cell viability compared to the control group,while the miR-7-5p mimics decreased cell viability compared to the control group.Mi R-7-5p inhibitor increased the ability of LOXL1-AS1 knockdown to increase cell proliferation,while miR-7-5p mimics showed the opposite effect.(6)Soft agar colony formation assay discovered that miR-7-5p inhibitor can respond to the LOXL1-AS1 knockdown clone formation capacity;(7)Transwell assay revealed that miR-7-5p inhibitor can respond to the cells migration and invasion by knockdown of LOXL1-AS1;(8)Dual luciferase reporter gene detection experiments showed that there was a direct binding site between miR-7-5p and HMGB1;(9)Western blot assay showed that the expression of HMGB1 protein was increased in the miR-7-5p inhibitor group compared to the control group,while the expression of HMGB1 protein was decreased in the miR-7-5p mimic group compared to the control group.Conclusion:LncRNA LOXL1-AS1 can competitivel combine with miR-7-5p to regulate the expression of HMGB1 and promote the proliferation,migration and invasion of retinoblastoma cell lines.