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.

Cancer is the second leading cause of mortality worldwide 1 . Lung cancer has the highest rank of incidence and global death rate among all cancers at present 2 . Non-small cell lung cancer (NSCLC) comprises approximately 85% of all kinds of lung cancer cases 3 . If being diagnosed in the late stage of the disease, NSCLC patients have a poor prognosis with 17% overall survival rate despite administration of radiotherapy, chemotherapy and other novel therapeutic tools 4,5 . Therefore, identification of molecular mechanisms involved in the pathogenesis of this cancer and recognition of biomarkers for early diagnosis and therapeutic targets are necessary. Long noncoding RNAs (lncRNA) are RNA transcripts longer than 200 nucleotides with limited coding potential, which have received enormous attention in cancer studies in the last years 6,7 . Dysregulation of their expressions influences cell proliferation, metastasis, and invasion of tumor cells [8][9][10] . They are implicated in a variety of biological processes by regulating gene expression in cis or trans modes by various mechanisms including epigenetics, transcriptional, and posttranscriptional regulatory mechanisms 11 . Long intergenic non-coding RNA for kinase activation (LINK-A) is as an oncogenic cytoplasmic non-coding RNA with approximately 1.5 kbp length. This lncRNA provides a scaffold-like structure in the cytoplasm which interacts with some proteins. In addition, it mechanistically facilitates the recruitment of protein kinase 6 (BRK) to the epidermal growth factor receptor EGFR: GPNMB complex (transmembrane tyrosine receptor and NMB glycoprotein, respectively) and provokes the conformational change of BRK for kinase activation. On the other hand, LINK-A interacts with leucine-rich repeat kinase 2 and subsequently causes hypoxia-inducible factor 1 (HIF)-1α phosphorylation and stabilization in normoxic conditions. Ultimately, the consequences of these events are glycolytic reprogramming and tumorigenesis in triple-negative breast cancers cell lines 12 . HIF1α is not exclusively regulated by hypoxia and, in some cases, other stimulators influence its activity even more. Considering its crucial role in cancer progression, HIF1α as a transcription factor is involved in several signaling pathways and overlapping molecular mechanisms, each of which could be an encouraging target to be investigated in cancer studies 13,14 . In addition to its roles in breast cancer, LINK-A has oncogenic role in ovarian cancer 15 , glioma 16 , and lung cancer 17,18 . LINK-A acts as a cytoplasmic scaffold in triple-negative breast cancer cell lines (MDA-MB-231 and MDA-MB-468) leads to the

Materials and methods
Cell culture and transfection. The A549 and Calu-3 human lung adenocarcinoma cell lines were obtained from Pasture Institute (Tehran, Iran). The cells were cultured in Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco) and 1% penicillin-streptomycin in a 98% humidified atmosphere with 5% CO 2 incubator (binder) at 37 °C. Two siRNAs hitting the LINK-A, Hs-cell death control siRNA as positive control and scrambled siRNA as negative control were purchased from Qiagen Company. LINK-A targeting siRNAs were 21 nucleotide length and had 5′-Fluorescein (6 FAM) modification on sense strand for monitoring the efficiency of siRNA delivery into transfected cells by observing under the fluorescent inverted microscope. For siRNA transfection, 4 × 10 5 cells were seeded in each well of 6-well tissue culture plates one day before transfection. Moreover, transfection was conducted by Lipofectamine 2000 according to the manufacturer's instructions in reduced FBS (5%) and free antibiotics media. All experiments were conducted in triplicates.
RNA extraction, cDNA synthesis, and qPCR. After 48 hours from transfection, the total cellular RNA was extracted by the TriPure Isolation Reagent (Roche, Germany) according to the standard procedure defined by the manufacturer's protocol. Additionally, cDNA was synthesized by RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Inc) using two micrograms of total RNA treated by DNaseI (Thermo Fisher Scientific, Inc). Then, the real-time polymerase chain reaction (PCR) was conducted by the SYBR Green PCR kit (Roche) for the quantitative expression analysis of genes examined in this study with specific primers listed in Table 1. Next, thermal cycling was implemented in the Magnetic Induction Cycler system in the specific scheduled program for each primer pairs. Before siRNA transfection, the amplified PCR product of LINK-A was purified and cloned into the compatible site of the Ptg19-T PCR cloning vector (Cinnagene Company, IRAN) and then sequenced with M13 forward and reverse primers by BigDye technology on an AB13700 XL sequencer applied biosystem. Finally, the blast program was used to confirm the accuracy of the detected sequence by matching the sequence with the published NCBI sequences for LINK-A.
MTT assay. Cell viability was assayed by 3-(4, 5-dimethylthiazol-2-yl)-2 5-diphenyl-tetrazolium bromide (MTT) colorimetric assay (Sigma-Aldrich, St. Louis, Mo, USA). Briefly, 3 × 10 4 cells were seeded per well of 96-well plates and, as described previously, transfected with various concentrations of LINK-A-siRNAs. After 24 and 48 hours, 10 µl MTT solution (5 mg/ml) was added into the each well and left into the incubator for further four hours in order to complete the reaction. Then, the media was replaced with 100 µl dimethyl sulfoxide (DMSO, Sigma-Aldrich) in order to dissolve formazan crystals, followed by measuring the absorbance of each well using a microplate photometer (LabSystem Multiscan). The test was performed in triplicates.
Colony formation assay. The colony formation potential of down-regulated LINK-A cells was monitored by the routine colony formation assay after 24 hours from transfection. A total of 500 and 200 cells of A549 www.nature.com/scientificreports/ and Calu-3 cells were seeded in each well of 6-well tissue culture plates with a complete medium, respectively. Cells were maintained under the routine cell culture condition (37 °C, 5% CO 2 , and humidified air) for 7 days by medium replacement every three days. Next, the colonies were washed twice with phosphate-buffered saline (PBS), fixed with cold 95% methanol for 10 minutes, and then stained by 1% trypan blue for 10 minutes. Subsequently, the dye was removed and the plates were washed with PBS before counting and imaging under the microscope. Colonies containing less than 50 cells and smaller than 1 mm were excluded from the counting. The experiment for each group was performed in three replicates.
Wound healing assay. The two-dimensional cell migration was calculated by the simple wound healing assay. After 24 hours, transfected cells were serum starved (0.5% FBS) for an overnight to minimize cell proliferation. Furthermore, the cells were almost 80-90% confluent when a yellow tip was used for creating scratches across cell monolayers. Then, the cellular debris was removed and washed twice with sterile PBS. Then, fresh media was added into the cells. Moreover, the wounded region was examined and photographed by light microscopy immediately, 24, and 48 hours later. The wound edges and migrated cell numbers in the gap areas for the same fields in the photographs were determined and calculated with ImageJ software I, version 1.4.
Transwell assay. Transwell chambers with a pore size of 8.0 µm were used to assess cell migration. Briefly, after 24 hours from transfection, 5 × 10 4 cells were suspended in 250 µl serum free DMEM medium and seeded in the upper chamber of the inserts in the transwell plates (SPL). Then, 750 µl complete medium (10% FBS) was added in the lower chamber as a chemotactic factor. After 48 hours incubation in cell culture condition, cells that were retained on the upper surface of the transwells were wiped off using cotton swab, while the migrated cells to the bottom side were fixed with methanol and stained with Giemsa solution. For invasion assay, the upper 8 µm chambers were coated with Matrigel and dried an overnight in the incubator. The next day, 5 × 10 4 cells were seeded on the matrigel coated chambers following aforementioned the procedure for the migration assay. The number of penetrating cells was counted in five randomly selected fields in the photographs with 200 magnification taken by an inverted microscope.

Significant down-regulation of LINK-A in transfected cells with LINK-A siRNAs. The real-time
PCR approach was utilized to examine endogenous RNA expression in the transfected cells after 48 h of transfection. The efficiency of the transfection was confirmed by observing massive cell death following transfection with HS-cell death control siRNAs as a positive control, along with the experiments. In addition, the expression of the LINK-A in the cells transfected with siRNAs targeting LINK-A was significantly declined compared with the control groups which included the cells that were transfected with scrambled siRNA or treated only with Lipofectamine 2000 (Mock group). There was no significant difference between the mock group and scrambled-siRNA transfected cells. LINK-A siRNAs were fluorescent-labeled (5′-Fluorescein 6 FAM) to determine efficient siRNA delivery into the cells and Student's t-test analysis was conducted to evaluate statistically significant differences (Fig. 1). LINK-A contribution in colony-forming ability was confirmed by using pool 100 nM concentration of siRNAs as well (Fig. 2). The cells with suppressed LINK-A expression exhibited a dramatic decrease in the size and number of colonies seven days after transfection (*P < 0.05, ***P < 0.001 in A549 and Calu-3 cell lines, respectively by two-tailed t-test).

Inhibition of the metastatic potential of A549 and Calu-3 cells by down-regulation of LINK-A
lncRNA. Cell migration plays a critical role in cancer progression and metastasis, thus in the present study,  www.nature.com/scientificreports/ the wound healing assay was performed to investigate the possible impact of LINK-A on the motility and migration capability of A549 (Fig. 3A) and Calu-3 cells at a two-dimensional surface (Fig. 3B).
A remarkable decline of migrated cell numbers in the wound gaps was observed for the LINK-A down-regulated Calu-3 (***P = 0.0002), and A549 cells (*P = 0.01). In addition, we conducted migration and invasion assay using transwells and matrigel coated transwells, respectively, which indicated significantly reduced migration and invasion capability of investigated cells influenced by LINK-A down-regulation (Fig. 4). Therefore, these results represent the probable function of LINK-A in cell mobility or invasion, emphasizing the precise evaluation of this topic in future studies.

Induction of apoptosis by LINK-A silencing. Based on cell cycle analysis, the silencing of LINK-A
affected cell viability, proliferation and caused cell death. Furthermore, Annexin-V-PE/7AAD staining was conducted to determine LINK-A role in the apoptosis and necrosis. The obtained data from A549 revealed an elevated rate of late apoptotic cells in LINK-A siRNA transfected cells (pool 100 nM) related to the controls. In the A549 cell line, the late apoptotic cells were 1.99% and 6.58% in the control and LINK-A silenced groups 24 hours after transfection, respectively. Moreover, the extent of the late apoptotic cell was increased from 2.27 to 22.8%, 48 hours after transfection. The rate of the late apoptotic cells in the Calu-3 were 0.74% and 3.32% in the control and LNK-A down-regulated cells, 24 hours later from transfection, respectively. Furthermore, 48 hours after transfection, the percentage of late apoptotic cells was almost doubled compared to the control group. We also observed 17% cell death in the LINK-A silenced Calu-3 cells (Annexin-v negative, while 7AAD positive). Additionally, acridine orange (AO) and ethidium bromide staining showed occurrence of apoptosis in LINK-A down-regulated cells and obvious cellular morphology differences between control and LINK-A siRNA treated cells (Fig. 6). The green cells with the normal morphology of the nucleus represent the living cells which are stained only with AO. In addition, the green and orange cells with condensed chromatin and blebbing cell membrane characterize apoptotic cells and are stained with both AO/ET because of a moderate alteration in cell membrane permeability. The present study also examined the RNA expression analysis of Bax and Bcl2 genes involved in cell death. The findings showed that the transfection of A549 cells with siRNAs hitting LINK-A causes significant up-regulation of Bax (*P = 0.0234) and noticeable down-regulation of Bcl2 expression (****P < 0.0001). The graphs are shown in Fig. 7.
Altered mRNA expression of BHLHE40, ANGPTL4, and VEGF. Considering earlier discoveries, which highlight the influence of LINK-A lncRNA on some genes and pathways, the study examined BHLHE40, ANGPTL4, and VEGF expressions after the silencing of LINK-A. The real-time PCR was conducted in LINK-A siRNA transfected cells (100 nM Pool siRNA) and the acquired data indicated noticeable down- www.nature.com/scientificreports/ regulation for BHLHE40 (***P = 0.0002) and ANGPTL4 (**P = 0.0074), along with the reduced expression of VEGF (*P = 0.01) (Fig. 7).

Discussion
The findings of the present study revealed that LINK-A down-regulation could induce apoptosis and reduce cell survival and proliferation in the A549 and Calu-3 cell lines. Moreover, the induction of apoptosis was confirmed by the cell cycle and Annexin-V/PI assay with flow cytometry, as well as acridine orange and ethidium bromide staining. The quantification of the mRNA expression levels of apoptosis-related genes in LINK-A silenced cells indicated significant up-and down-regulation of Bax and Bcl2, respectively. High Bcl2 expression levels have been reported in a variety of cancers [19][20][21] . Bcl2 plays a vital role in protecting cancerous cells and fosters the progression www.nature.com/scientificreports/ of cancer by regulating apoptosis 22 , while Bax acts as a tumor suppressor in human malignancies through mediating the apoptosis in response to genotoxic stresses 23 . The impact of LINK-A silencing on the expression of these crucial genes and the consequent induction of apoptosis propose the oncogenic role of LINK-A in the A549 and Calu-3 cell lines and designate LINK-A as a probable therapeutic potential in lung cancer. This study did not scrutinize the molecular mechanism of LINK-A contribution to the regulation of Bcl2 and Bax expressions. However, it is supposed that LINK-A acts as a mediator between the signaling pathways, therefore, the alteration of expression patterns probably is the consequence of multiple events concerning the indirect upstream system of nuclear expression regulatory machine. In addition, Lin et al. have informed the antiapoptotic function of LINK-A lncRNA in MDA-MB-231 of the breast cancer cell line 24 . Considering these observations, this study was the first one to propose that LINK-A lncRNA has a role in the regulation of apoptosis in A549 and Calu-3 cell lines as representatives for NSCLC. Based on the findings of the present study and previous knowledge about LINK-A and HIF1α hyper-activation, a similar function was supposed for LINK-A in lung cancer and the RNA expression change of some imperative genes which could be influenced by the silencing of LINK-A was examined. ANGPTL4, BHLHE40, and VEGF are considered as critical genes in cancer studies, which play pivotal roles in biological processes and are somehow regulated by HIF1α. Further, the RNA expression alteration of the mentioned genes was evaluated by the real-time PCR and the findings represented that all genes were significantly down-regulated in LINK-A siRNA transfected cells. ANGPTL4 belongs to the VEGF family and is structurally related to the proteins modulating angiogenesis known as angiopoietins (ANG). This protein has manifold roles such as lipid metabolism, angiogenesis, cell differentiation, tumorigenesis, and metastasis 25 . Zhu et al. have demonstrated that ANGPTL4 is associated with NSCLC prognosis and its down-regulation could impede tumor cell proliferation and migration ability by the extracellular signal-regulated kinase signaling pathway. They have also found the significant up-regulation of ANGPTL4 in NSCLC tissues related to non-tumor tissues 26 . The potential pro-metastatic function for ANGPTL4 was confirmed in breast cancer 27 , hepatocellular carcinoma cells 28 , and colorectal cancer 29 . In contrast, some studies have found no precise function for ANGPTL4 in cancer progression or even some of them discovered that the elevated expression of ANGPTL4 prevents tumor growth, invasion, and angiogenesis in melanoma and colorectal cancers 30 . HIF1-α is considered as one of the regulatory transcription www.nature.com/scientificreports/ factors of ANGPTL4 and hypoxia is one potential stimulator for the tumorigenic function of ANGPTL4 [31][32][33] . Accordingly, the present study evaluated the expression change of ANGPTL4 after LINK-A down-regulation as a probable ultimate target and the findings revealed a significant decrease. The alteration of the RNA expression of BHLHE40, as another HIF1α target gene, was examined in this study. BHLHE40 role in cancer is unclear although some studies reported the activation of its expression directly by HIF1α in a variety of cancer cells 34,35 . Furthermore, the elevated expression of BHLHE40 was correlated with hypoxia activation, increased metastatic potential, and poor prognosis of various cancer types including hepatocellular carcinoma, pancreatic cancer, and invasive breast cancer [36][37][38] 12 . VEGF is a predominant angiogenic factor, which is overexpressed in an array of malignancies. The circulating amounts of VEGF are upraised in various cancer patients, including those suffering from lung cancer 40,41 and the overexpression of VEGF has an adverse influence on the survival of patients with NSCLC 42,43 . HIF1α is one of the multiple inducers of VEGF production, which binds to its regulatory promoter in LINK-A down-regulated A549 cells. Data are presented as mean ± Sd. Two-tailed student's t-test analysis was performed for each gene separately (*P < 0.05, **P < 0.01, ***P < 0.001).  44 . Based on the findings of this study, significantly reduced expression of VEGF mRNA was observed in LINK-A down-regulated cells. This result is important considering the importance of VEGF in the angiogenesis and progression of cancer. Accordingly, future studies are recommended to investigate LINK-A and angiogenesis correlation deeply. Lin et al. have found another remarkable oncogenic role of LINK-A in the hyper-activation of AKT and conferring resistance to targeted therapy against the AKT pleckstrin homology domain. The direct interaction of LINK-A with PIP3 (phosphatidylinositol-3,4,5-trisphosphate) and Akt facilitates the AKT-PIP3 interface and subsequent AKT activation, which leads to tumorigenesis and extensive clinical implications 24 . According to Wu et al., LINK-A is up-regulated in human glioma cells and LDH-A is regulated by LINK-A, which mediates the proliferation and invasion of glioma cells 16 . In addition, Zhang et al. have recently reported that LINK-A is higher in ovarian carcinoma patients compared with healthy controls, which leads to the metastasis of ovarian carcinoma by up-regulation of HIF1α 15 . In another study conducted by our group, we observed elevated expression of LINK-A in Iranian epithelial ovarian cancer patients, which also was correlated with higher stages and grades of cancer 45 . Zhao et al. have reported increased expression levels of LINK-A in NSCLC tissues compared with normal tissues. Besides, they have indicated its positive correlation with clinicopathologiacal features and the survival rate of NSCLC patients. Moreover, they have demonstrated that LINK-A promotes proliferation of NSCLC cells 18 , which is consistent with our results. In another study conducted by Liu et al., overexpression of LINK-A in metastatic NSCLC has been reported. They have observed that NSCLC patients with higher expression of LINK-A had worse survival rates and higher mortality. They have suggested that LINK-A has a role in the promotion of migration and invasion ability of NSCLC cells (H1993, H1581) by activating AKT signaling 17 . We also indicated that reduction of LINK-A expression in A549 and Calu-3 cell lines significantly inhibited their migration and invasion ability. All these results emphasize the importance of LINK-A in NSCLC. LINK-A, as a novel lncRNA with oncogenic function in multiple cancers through interaction with various molecular pathways, should be precisely appraised for a therapeutic perspective. This study aimed to provide an overview of the LINK-A role in A549 and Calu-3 as NSCLC cell lines in order to find a clue for future works in this research area. In general, the findings indicated the tumorigenic role of LINK-A in these cell lines.

Conclusion
Our observations obtained by the down-regulation of LINK-A in two cell lines as a representative model for lung cancer suggest an oncogenic function for LINK-A. Reduced expression of LINK-A significantly decreased cell proliferation, viability, migration, and invasion abilities. Also, we detected the occurrence of apoptosis following by LINK-A down-regulation. License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.