Long Intergenic Non-coding RNA For Kinase Activation Research Articles

M6A demethylase FTO stabilizes LINK-A to exert oncogenic roles via MCM3-mediated cell-cycle progression and HIF-1α activation

RNA N6-methyladenosine (m6A) modification is implicated in cancer progression, yet its role in regulating long noncoding RNAs during cancer progression remains unclear. Here, we report that the m6A demethylase fat mass and obesity-associated protein (FTO) stabilizes long intergenic noncoding RNA for kinase activation (LINK-A) to promote cell proliferation and chemoresistance in esophageal squamous cell carcinoma (ESCC). Mechanistically, LINK-A promotes the interaction between minichromosome maintenance complex component 3 (MCM3) and cyclin-dependent kinase 1 (CDK1), increasing MCM3 phosphorylation. This phosphorylation facilitates the loading of the MCM complex onto chromatin, which promotes cell-cycle progression and subsequent cell proliferation. Moreover, LINK-A disrupts the interaction between MCM3 and hypoxia-inducible factor 1α (HIF-1α), abrogating MCM3-mediated HIF-1α transcriptional repression and promoting glycolysis and chemoresistance. These results elucidate the mechanism by which FTO-stabilized LINK-A plays oncogenic roles and identify the FTO/LINK-A/MCM3/HIF-1α axis as a promising therapeutic target for ESCC.

Inhibition of LINK-A lncRNA overcomes ibrutinib resistance in mantle cell lymphoma by regulating Akt/Bcl2 pathway.

Ibrutinib, a bruton tyrosine kinase (BTK) inhibitor which suppresses B-cell receptor signaling, has remarkably improved the outcome of patients with mantle cell lymphoma (MCL). However, approximately 33% of MCL patients have primary Ibrutinib resistance, and acquired Ibrutinib resistance is nearly universal. Long intergenic non-coding RNA for kinase activation (LINK-A) exerts oncogenic role in different types of tumors, but the role of LINK-A in intrinsic ibrutinib resistance in MCL is still unclear. Here, LINK-A expression level was first assessed using quantitative Real-time PCR (qPCR) and immunofluorescence analysis in five MCL cell lines. The effect of LINK-A on regulating MCL cells viability and apoptosis was assayed using CCK-8 and TdT-mediated dUTP nick end labeling (TUNEL) assay, respectively. The association of LINK-A with AKT activation and B cell lymphoma 2 (Bcl2)expression was evaluated using qPCR and western blot analysis. We found that LINK-A level was elevated in Ibrutinib-resistant MCL cell lines (Mino, REC-1, MAVER-1, and Granta-519) compared to Ibrutinib-sensitive MCL cell lines (Jeko-1). Functionally, LINK-A overexpression in Jeko-1 cells enhanced cell viability and repressed Ibrutinib-induced cell apoptosis. LINK-A knockdown in MAVER-1 cells decreased cell viability and further accelerated Ibrutinib-induced cell apoptosis. LINK-A overexpression enhanced Bcl2 expression in Jeko-1 cells, and Bcl2 inhibition blocked the effect of LINK-A on increasing cell viability in the presence of Ibrutinib. On the contrary, LINK-A knockdown reduced Bcl2 expression in MAVER-1 cells, and Bcl2 overexpression damaged the role of LINK-A inhibition in regulating cell viability. Mechanistically, LINK-A positively regulated the activation of AKT signaling, and inhibition of AKT signaling destroyed LINK-A-induced increased of Bcl2 and resulted in a subsequent suppression of cell viability. Taken together, the current results demonstrate that LINK-A inhibition overcomes Ibrutinib resistance in MCL cells by regulating AKT/Bcl2 pathway.

The role of long intergenic non-coding RNA for kinase activation (LINK-A) as an oncogene in non-small cell lung carcinoma

The oncogenic role of long intergenic non-coding RNA for kinase activation (LINK-A) has been appraised in triple-negative breast cancer. However, the molecular function of LINK-A is still unclear in most cancers including lung cancer. The present study aimed to evaluate the impact of down-regulation of LINK-A in A549 and Calu-3 cell lines as cellular models of non-small cell lung carcinoma (NSCLC). We used the RNA interference system to knock down LINK-A. LINK-A expression was significantly reduced by siRNA transfection in A549 and Calu-3 cell lines. LINK-A down-regulation significantly reduced cell viability, colony-forming ability and cell migration, as measured by MTT, colony formation and invasion assays. Finally, cell cycle analysis and Annexin-V/7AAD staining indicated that apoptosis was influenced by LINK-A silencing. Taken together, LINK-A can be proposed as an oncogene in NSCLC.

LINK-A lncRNA is upregulated in osteosarcoma and regulates migration, invasion and stemness of osteosarcoma cells.

The present study aimed to investigate the involvement of long intergenic non-coding RNA for kinase activation (LINK-A) long non-coding RNA (lncRNA) in osteosarcoma. Plasma levels of LINK-A lncRNA and transforming growth factor β1 (TGF-β1) were measured by reverse transcription-quantitative polymerase chain reaction and ELISA, respectively. Correlation between LINK-A lncRNA and TGF-β1 was analyzed by Pearson correlation coefficient. LINK-A lncRNA and TGF-β1 were upregulated in patients with osteosarcoma compared with healthy controls. LINK-A lncRNA and TGF-β1 were positively correlated in the two groups. LINK-A lncRNA short hairpin RNAs (shRNAs) were transfected into osteosarcoma cell lines and Transwell migration assay, Matrigel invasion assay and flow cytometry were used to evaluate cell migration, invasion and stemness, respectively. Effects of LINK-A lncRNA silencing and overexpression on TGF-β1 expression were analyzed by western blotting. LINK-A lncRNA shRNA silencing inhibited, whereas TGF-β1 treatment promoted cell migration, invasion and stemness. LINK-A lncRNA silencing inhibited TGF-β1 expression, whereas TGF-β1 treatment had no effects on LINK-A lncRNA expression. TGF-β1 reduced the inhibitory effects of LINK-A lncRNA knockdown on cancer cell migration, invasion and stemness. These data indicated that LINK-A lncRNA is upregulated in osteosarcoma and may regulate migration, invasion and stemness of osteosarcoma cells through TGF-β1.

LINK-A long non-coding RNA and VEGF RNA expression in epithelial ovarian cancer patients.

LINK-A (long intergenic non-coding RNA for kinase activation) is a newly identified long non-coding RNA with oncogenic function, which leads to the hyperactivation of AKT and HIF1α. thereby, fosters cell proliferation, mobility and metastasis. VEGF (vascular endothelial growth factor), a well-known cytokine has an important role in angiogenesis. In this study, we quantified RNA expression of LINK-A and VEGF on 45 tumor specimens obtained from Iranian patients diagnosed with Epithelial Ovarian Cancer (EOC). Our goal was to evaluate expression of LINK-A lncRNA and VEGF mRNA in ovarian cancer tissues and find the probable correlation of LINK-A with VEGF as a major transcription target for HIF1α. LINK-A and VEGF were remarkably overexpressed in EOC tissues compared to normal tissues (P value: 0.004, 0.0001, respectively), but we did not find correlation between LINK-A and VEGF RNA expressions in this study. LINK-A was significantly overexpressed in higher stages of cancer and tumor grades. VEGF was only significantly elevated in higher stages. This study confirms importance of novel lncRNA of LINK-A in Iranian EOC patients.

LncRNA LINK-A regulates ROCK1 expression in early-stage pancreatic adenocarcinoma.

Long intergenic non-coding RNA for kinase activation (LINK-A) has been characterized as an oncogenic long non-coding (lnc)RNA in triple-negative breast cancer and ovarian carcinoma, but its involvement in other malignancies remains elusive. In the present study, it was determined that the plasma levels of LINK-A lncRNA and Rho-associated protein kinase 1 (ROCK1) were significantly increased in patients with pancreatic adenocarcinoma compared with those in healthy controls. The plasma levels of LINK-A lncRNA were positively correlated with the plasma levels of ROCK1 in pancreatic adenocarcinoma patients, but not in healthy controls. Silencing of LINK-A led to inhibition of pancreatic adenocarcinoma cell proliferation, migration and invasion. Simultaneous overexpression of ROCK1 attenuated the inhibitory effect of LINK-A silencing on cancer cell proliferation, migration and invasion. Overexpression of LINK-A lncRNA led to upregulation of ROCK1 expression, while overexpression of ROCK1 had no significant effect on LINK-A lncRNA expression. It may therefore be concluded that LINK-A lncRNA may have a role in pancreatic adenocarcinoma, at least in part, by promoting ROCK1 expression.

LINK-A lncRNA is upregulated in metastatic non-small cell lung cancer and is associated with poor prognosis.

Long intergenic non-coding RNA for kinase activation (LINK-A) has been characterized as an oncogenic long non-coding RNA (lncRNA) in triple-negative breast cancer. However, its involvement in non-small cell lung cancer (NSCLC) remains unknown. The aim of the present study was to investigate the involvement of LINK-A in NSCLC. Expression of LINK-A lncRNA in the plasma of patients with NSCLC collected on the day of admission and the day of discharge, and in the plasma of healthy controls, was detected by reverse transcription-quantitative PCR. Diagnostic values of plasma LINK-A for metastatic NSCLC were evaluated by receiver operating characteristic curve analysis. A LINK-A lncRNA expression vector was constructed and transfected into human NSCLC cell lines, and the effects on cell migration and invasion, and Akt activation were detected by Transwell and Matrigel assays, and western blotting, respectively. Plasma levels of LINK-A were found to be significantly higher in patients with different types of metastatic NSCLC than in patients with non-metastatic NSCLC and healthy controls. Plasma levels of LINK-A were lower in patients with metastatic NSCLC on the day of discharge than on the day of admission. Patients with high plasma LINK-A had a higher mortality rate and lower progression-free survival rate within 2 years of discharge. In conclusion, LINK-A is overexpressed in metastatic NSCLC, and may promote the migration and invasion of NSCLC by activating Akt signaling.

LINK‑A lncRNA participates in the pathogenesis of glioma by interacting with survivin

Long intergenic non-coding RNA for kinase activation (LINK-A) long non-coding RNA (lncRNA) has been characterized in triple negative breast cancer, but its potential involvement in glioma has not been investigated. In the present study, serum levels of LINK-A lncRNA and survivin in patients with glioma and healthy controls were determined by RT-qPCR and ELISA, respectively. The diagnostic value of serum LINK-A lncRNA for glioma was evaluated by receiver operating characteristic (ROC) curve analysis. Potential correlations between serum levels of LINK-A lncRNA and survivin were analyzed by Pearson correlation coefficient. LINK-A lncRNA siRNA, LINK-A lncRNA-carrying expression vector and survivin-carrying expression vector were transfected into glioma cells, and the effects on LINK-A lncRNA expression, survivin expression and cell apoptosis were explored by RT-qPCR, western blot analysis and annexin V/propidium iodide staining. It was observed that the serum levels of LINK-A lncRNA and survivin were significantly higher in patients with glioma compared with healthy controls. Increased levels of LINK-A lncRNA distinguished glioma patients from healthy controls, based on ROC curve analysis. Serum levels of LINK-A lncRNA and survivin were positively correlated in glioma patients, but not in healthy controls. Overexpression of LINK-A lncRNA led to increased survivin protein expression, while survivin overexpression had no effect on LINK-A lncRNA expression. LINK-A lncRNA and survivin overexpression each reduced glioma cell apoptosis, but LINK-A lncRNA siRNA-mediated knockdown increased apoptosis. Survivin overexpression attenuated the inducing effects of LINK-A lncRNA knockdown on apoptosis. In conclusion, LINK-A lncRNA inhibited glioma cell apoptosis potentially by the upregulation of survivin. The present study revealed that LINK-A may serve as possible diagnostic marker for glioma.

LINK-A lncRNA activates HIF1α signaling and inhibits podocyte cell apoptosis in diabetic nephropathy.

Previous studies have revealed that long intergenic non-coding RNA for kinase activation (LINK-A), a long non-coding RNA (lncRNA) promotes disease progression in triple-negative breast cancer by activating hypoxia-inducible factor 1α (HIF1α). However, the activation of HIF1α has also been demonstrated to improve diabetic nephropathy. It is therefore reasonable to expect that LINK-A may also participate in diabetic nephropathy. In the current study, the expression of LINK-A lncRNA and HIF1α was determined in renal biopsies of patients with diabetic nephropathy. LINK-A lncRNA and HIF1α expression levels were detected by reverse transcription quantitative (RT-q) PCR and ELISA in diabetic patients without complications and used as controls. Correlations between LINK-A lncRNA and HIF1α expression were analyzed using Pearson's correlation coefficient. Effects of lncRNA and HIF1α overexpression on LINK-A lncRNA expression, HIF1α expression and cell apoptosis were assessed using RT-qPCR, western blotting and a cell apoptosis assay. The results revealed that LINK-A lncRNA and HIF1α were downregulated in patients with diabetic nephropathy, as well as in diabetic patients without complications. The lowest expression of LINK-A lncRNA and HIF1α were observed in healthy controls. A positive correlation was identified between LINK-A lncRNA and HIF1α in both patients groups, but not in the control group. LINK-A lncRNA and HIF1α overexpression inhibited the apoptosis of mouse podocyte cells under a high glucose treatment. LINK-A lncRNA overexpression also promoted HIF1α expression in mouse podocyte cells, while HIF1α overexpression did not significantly affect LINK-A lncRNA expression. In conclusion, LINK-A lncRNA may activate HIF1α signaling resulting in the improvement of diabetic nephropathy treatment.

LINK-A lncRNA functions in the metastasis of osteosarcoma by upregulating HIF1α.

Long intergenic non-coding RNA for kinase activation (LINK-A) long non-coding RNA (lncRNA) is an oncogenic lncRNA in triple-negative breast cancer. The involvement of LINK-A lncRNA in other diseases is unknown. The present study aimed to investigate the possible involvement of LINK-A lncRNA in osteosarcoma. The results demonstrated that plasma levels of LINK-A lncRNA were significantly higher in patients with metastatic osteosarcoma (MO) compared with healthy controls and patients with non-metastatic osteosarcoma (NMO). LINK-A lncRNA overexpression significantly promoted cancer cell migration and invasion in osteosarcoma cell lines MG-63 and U20S. Upregulated expression of hypoxia-inducible factor 1α (HIF1α) was observed in cancer cells following LINK-A lncRNA overexpression. Exogenous HIF1α treatment did not significantly affect the expression of LINK-A lncRNA in cancer cells, whereas treatment with an HIF1α inhibitor significantly attenuated the effects of LINK-A lncRNA overexpression on cancer cell migration and invasion. Based on the results it was concluded that LINK-A lncRNA participated in the metastasis of osteosarcoma by upregulating HIF1α; upregulation of LINK-A lncRNA may serve as a potential diagnostic biomarker for patients with MO but not in those with NMO.

LINK-A promotes cell proliferation through the regulation of aerobic glycolysis in non-small-cell lung cancer.

PurposeNon-small-cell lung cancer (NSCLC) is the one of the most common malignancies worldwide, and occurs at a higher frequency in male individuals. Little is known about the role of the long intergenic noncoding RNA for kinase activation (LINK-A) in NSCLC, so in the present study we assessed its potential role on cell proliferation in NSCLC.MethodsExpression levels of LINK-A in NSCLC tissues and cell lines were detected by quantitative reverse-transcription polymerase chain reaction. LINK-A was knocked down and overexpressed separately in A549 cells and NCI-H1299 cells. The effect of LINK-A expression on cell proliferation was determined by MTT assay. The correlation between LINK-A and hexokinase II (HKII) expression was investigated by Western blot and HKII Activity Assay. Glucose consumption and lactate production assay were used to investigate the aerobic glycolysis in NSCLC cells. The effect of LINK-A in vivo was determined by xenograft assay.ResultsLINK-A expression levels were increased in NSCLC tissues compared with normal tissues. Moreover, LINK-A expression was positively correlated with NSCLC clinicopathological characteristics and survival rate, while knockdown of LINK-A reduced NSCLC cell proliferation. LINK-A expression was also positively correlated with HKII, and NSCLC cells with low LINK-A expression were found to have significantly reduced HKII protein expression, accompanied by a reduction in enzyme activity levels. Both in vitro and in vivo experiments showed that LINK-A expression affected glucose consumption and lactate production through regulation of HKII expression.ConclusionThese data suggest that the functions of LINK-A in NSCLC might play a key role in tumor progression and that LINK-A could be a promising predictive biomarker and potential therapeutic target for NSCLC.

Long non-coding RNA LINK-A promotes glioma cell growth and invasion via lactate dehydrogenaseA.

Long non-coding RNAs (lncRNAs) have recently been identified in mammals as new modulators of cancer origin and progression participating in various cellular pro-cesses. Long intergenic non-coding RNA for kinase activation (LINK-A), belonging to one of the intergenic lncRNAs, was reported to regulate signaling pathways correlated with triple-negative breast cancer. However, the expression and the functional role of LINK-A in glioma are still unclear. In the present study, we demonstrated that LINK-A was upregulated in human glioma cells compared with the expression noted in normal astrocytes. Knockdown of LINK-A inhibited cell proliferation, migration and invasion in U87 and U251 glioma cells. In addition, we found that lactate dehydrogenaseA (LDH-A) was regulated by LINK-A, and enforced expression of LDH-A promoted glycolysis and proliferation in glioma cells. More importantly, we found that LDH-A was involved in the LINK-A-mediated proliferation and invasion of glioma cells. Collectively, these results provide new evidence of an important role for LINK-A in the development of glioma.

The LINK-A lncRNA interacts with PtdIns(3,4,5)P3 tohyperactivate AKTand confer resistance to AKTinhibitors.

Phosphatidylinositol-3,4,5-trisphosphate (PIP3) mediates signaling pathways as a second messenger in response to extracellular signals. Although primordial functions of phospholipids and RNAs have been hypothesized in the “RNA world”, physiological RNA-phospholipid interactions and their involvement in essential cellular processes has remained a mystery. We explicate the contribution of lipid-binding long non-coding RNAs (lncRNAs) in cancer cells. Among them, Long Intergenic Noncoding RNA for Kinase Activation (LINK-A) directly interacts with AKT pleckstrin homology domain and PIP3 at the single nucleotide level, facilitating AKT-PIP3 interaction and consequent enzymatic activation. LINK-A-dependent AKT hyperactivation leads to tumorigenesis and resistance to AKT inhibitors. Genomic deletions of the LINK-A PIP3-binding motif dramatically sensitized breast cancer cells to AKT inhibitors. Furthermore, meta-analysis showed the correlation between LINK-A expression and incidence of a SNP (rs12095274: A>G), AKT phosphorylation status, and poor outcomes for breast and lung cancer patients. PIP3-binding lncRNA modulates AKT activation with broad clinical implications.

The LINK-A lncRNA activates normoxic HIF1α signalling in triple-negative breast cancer.

Although long noncoding RNAs (lncRNAs) predominately reside in nuclear and exert their functions in many biological processes, their potential involvement in cytoplasmic signal transduction remains unexplored. Here, we identified a cytoplasmic lncRNA, Long-Intergenic Noncoding RNA for Kinase Activation (LINK-A), which mediates HB-EGF triggered, EGFR:GPNMB heterodimer-dependent HIF1α phosphorylation at Tyr565 and Ser797 by BRK and LRRK2 respectively. These events cause HIF1α stabilization, HIF1α-p300 interaction, and activation of HIF1α transcriptional programs under normoxic conditions. Mechanistically, LINK-A facilitates the recruitment of BRK to EGFR:GPNMB complex and BRK kinase activation. The BRK-dependent HIF1α Tyr565 phosphorylation interferes with Pro564 hydroxylation, leading to normoxic HIF1α stabilization. Both LINK-A and LINK-A-dependent signaling pathway activation correlate with TNBC, promoting breast cancer glycolysis reprogramming and tumorigenesis. Our findings illustrate the magnitude and diversity of cytoplasmic lncRNAs in signal transduction and highlight the important roles of lncRNAs in cancer.