Long Non-coding RNA LINK-A Promotes Glioma Cell Growth And Invasion Via Lactate Dehydrogenase A

Background and objectiveGliomas account for 30 to 40% of all primary intracranial tumors.Approximately half of all gliomas in adults are glioblastoma(GBM),the most aggressive subtype with a 5-year survival rate of less than 5%.Currently,some advances have been achieved in regards to multimodal treatments,including surgical extirpation,local irradiation and conventional chemotherapy.However,the overall survival rate of most patients with glioma is still low,and the treatment prospects are not optimistic,especially for GBM patients.Research on molecules and signaling pathways for glioma cell proliferation,migration and invasion have been underway.However,the mechanisms are still poorly understood,and the identification of key molecules that show a potential effect on glioma development is still imperative.Long non-coding RNAs(lncRNAs)are defined as endogenous cellular RNAs more than 200 nucleotides long,which lack a functional open reading frame.Accumulating evidence suggests that lncRNAs are pivotal regulatory molecules that are implicated in diverse biological processes,including epigenetic,transcriptional and post-transcriptional regulatory mechanisms.It has been found that numerous lncRNAs play central roles in the tumor-related gene regulatory system,and dysregulation of lncRNAs expression is thought to contribute to tumor cell proliferation,invasion and metastasis.Several studies showed that dysregulated lncRNAs,including CRNDE,CASC2,HOTAIR,GAS5 and MEG3,were observed in glioma,.Recently,Lin et al reported that long intergenic noncoding RNA for kinase activation(LINK-A)is critical to the growth factor-induced normoxic HIF1? signaling pathway in triple-negative breast cancer.However,the role of LINK-A in glioma as well as the underlying mechanisms is largely unknown.Therefore,this study first studied the malignant biological function of LINK-A in glioma cells,and further elucidated the molecular mechanism of LINK-A affecting the malignant biological behavior of glioma cells.The purpose of this study is to clarify the role of LINK-A in the proliferation and invasion of glioma cells,thus providing a new theoretical basis for the treatment of glioma.Methods 1.RT-qPCR analysis of LINK-A expression in glioma cell lines(U87 and U251)and normal human astrocytes(HAs).A lentivirus carrying a specific shRNA against LINK-A(shRNA-LINK-A)to knockdown its expression was infected into U87 and U251 glioma cells.U87 and U251 cells infected non-target shRNA(sh-control)served as the control.RT-qPCR analysis was performed to examine its knockdown efficiency.2.ShRNA(shRNA-LINK-A)group and negative control(shRNA-control)group,were infected with U87 and U251 glioma cell lines,respectively.Cell proliferation were examined by MTT assay,and the ability of cell clone formation were detected by plate coloning assay.3.ShRNA(shRNA-LINK-A)group and negative control(shRNA-control)group,were infected with U87 and U251 glioma cell lines,respectively.The migration ability were detected by healing and wound.The invasion ability were detected by transwell assay.4.ShRNA(shRNA-LINK-A)group and negative control(shRNA-control)group,were infected with U87 and U251 glioma cell lines,respectively.RT-qPCR assay were performed to investigate the effects of LINK-A knockdown on LDH-A mRNA expression.Western blot assay were performed to investigate the effects of LINK-A knockdown on LDH-A protein expression.5.LDH-A expressing vector or the empty vector was transfected into U87 and U251 glioma cells,respectively.RT-qPCR and western blot analysis were performed to test its over-expression efficiency.Levels of glucose consumption and lactate production were measured in U87 and U251 cells transfected with the LDH-A expression vector or empty vector.MTT assay was performed to determine the proliferative effect of U87 and U251 cells transfected with the LDH-A expression vector or empty vector.6.ShRNA-LINK-A and LDH-A overexpression plasmids were co-transfected into U87 and U251 glioma cells,and the experiment was divided into four groups: shRNA-control group,shRNA-LINK-A group,LDH-A over-expression group,shRNA-LINK-A + LDH-A over-expression group.The proliferation of U251 and U87 glioma cells were measured by MTT and Colony formation assay.The invasion of U251 and U87 glioma cells were measured by Transwell assay.Results 1.LINK-A is upregulated in glioma cells.RT-qPCR was used to detect the expression of LINK-A in glioma cell lines(U87 and U251)and normal human astrocytes(HAs).The results showed that the expression of LINK-A was significantly increased in U87 and U251 glioma cell lines compared with HAs.2.LINK-A knockdown inhibits cell growth in glioma cells.The control and shRNA(shRNA-LINK-A)group and negative control(shRNA-control)group,were infected with U87 and U251 glioma cell lines,respectively.As a result,LINK-A in the shRNA(shRNA-LINK-A)group was significantly decreased as compared with the control group and the negative control(shRNA-control)group.The results of MTT assay showed that knockdown of LINK-A significantly inhibited the proliferation of U87 and U251 glioma cells compared with the negative control group.The results of cell clone formation experiments showed that knockdown of LINK-A significantly inhibited the clonality of U87 and U251 glioma cells compared with the negative control group.3.LINK-A knockdown inhibits glioma cell migration and invasion.Knockdown of LINK-A inhibits the migration and invasion of glioma cells in vitro.Wound healing assay to evaluate the effect of LINK-A on cell migration in U87 and U251 cells.Invasion assay in U87 and U251 cells was performed to determine cell invasiveness after infection with shRNA-LINK-A and sh-control.4.Suppression of LDH-A by LINK-A knockdown.To explore the molecular mechanism by which LINK-A plays a biological role in glioma cells,we examined the effect of LINK-A on LDH-A.RT-qPCR results showed that knockdown of LINK-A significantly inhibited LDH-A mRNA expression in U87 and U251 glioma cells compared with the negative control group.In addition,western blot results showed that knockdown of LINK-A significantly decreased LDH-A protein expression compared with the negative control group.These results suggest that LDH-A may be involved in LINK-A-mediated proliferation and invasion of glioma cells.5.LDH-A promotes glycolysis and cell proliferation.To explore the biological role of LDH-A gene in glioma,vector and LDH-A overexpression plasmid were constructed and transfected into U87 and U251 glioma cell lines.The results of RT-qPCR and western blot showed that the LDH-A overexpression significantly increased the mRNA and protein levels of LDH-A compared with vector group.The glucose consumption test results showed that LDH-A overexpression significantly increased glucose uptake compared to vector group.The results of lactic acid production experiments showed that the LDH-A overexpression significantly increased the production of lactic acid compared with vector group.MTT assay showed that the LDH-A overexpression significantly increased cell proliferation compared with vector group.6.LDH-A mediates the tumor-suppressive effects of sh-LINK A in glioma cells.MTT assay showed that transfection of shRNA-LINK-A inhibited U87 and U251 glioma cell proliferation compared with sh-control group,however,co-transfection of shRNA-LINK-A and LDH-A overexpression plasmid group partially reversed knockdown of LINK-A mediated proliferation of glioma cells.The results of plate cloning experiments showed that knockdown of LINK-A inhibited the clonal formation of U87 and U251 glioma cells compared with sh-control group,and overexpression of LDH-A partially reversed the knockdown of LINK-A-mediated inhibition of clonal formation.Transwell chamber experiments showed that knockdown of LINK-A inhibited the invasion of U87 and U251 glioma cell compared with sh-control group,and overexpression of LDH-A partially reversed LINK-A-mediated inhibition of glioma cell invasion.These results suggest that LINK-A promotes the growth and invasion of glioma cells through LDH-A.Conclusion LINK-A is highly expressed in glioma cells,and knockdown of LINK-A inhibits proliferation,migration and invasion of glioma cells.Further studies have found that knockdown of LINK-A inhibits the expression of LDH-A,while overexpression of LDH-A promotes lactic acid production,glucose uptake,and cell proliferation in glioma cells.Furthermore,LDH-A overexpression partially reverses the inhibitory effect of LINK-A knockdown-mediated glioma malignant biological behavior.Therefore,this study revealed that LINK-A promotes the growth and invasion of glioma cells through LDH-A in glioma cells,which will provide a theoretical basis for the treatment of glioma.