Long intergenic non‐coding RNA Linc00485 promotes lung cancer progression by modulating miR‐298/c‐Myc axis

Long non‐coding RNAs (lncRNAs), which are non‐protein‐coding transcripts, are emerging as novel biomarkers for cancer diagnosis. Their dysregulation is increasingly recognized to contribute to the development and progression of human cancers, including lung cancer. Linc00485 is a newly discovered cancer‐related lncRNA; however, little is known about its role in lung cancer progression. In this study, we found that the expression of Linc00485 was significantly increased in human lung cancer tissue and associated with malignant phenotypes, including tumour‐node‐metastasis (TNM) stage, metastasis and relapse. Furthermore, the proliferative, migratory and invasive abilities of lung cancer cells in vitro were significantly enhanced by overexpression of Linc00485 but inhibited by its silencing. Mechanistically, Linc00485 regulated the expression of c‐Myc by directly binding to miR‐298; the effects of Linc00485 overexpression could be significantly reversed by a c‐Myc inhibitor or small interfering RNA. Xenotransplantation experiments showed that Linc00485 silencing significantly weakened the proliferation potential of A549 cells in vivo. Overall, these findings indicate that Linc00485 overexpression down‐regulates miR‐298, resulting in the up‐regulation of c‐Myc and thereby promoting the development of lung cancer.

Non-coding RNAs, which include microRNAs, long non-coding RNAs (lncRNAs), P-Element induced wimpy testis-interacting RNAs, and circular RNAs (circRNAs), have traditionally been considered to be non-functional transcriptional by-products. 4 However, recent studies have shown that non-coding RNAs have important biological functions in cells. 5,6 MicroRNAs and other small RNAs modulate gene expression by binding to untranslated regions (UTRs) of mRNA. 4,7 LncRNAs 8,9 and circRNAs 10 indirectly affect gene expression by functioning as sponges for microRNAs. Previous studies have suggested that lncRNAs have important roles in various types of cancer. [11][12][13][14] LincRNAs, a type of lncRNA, have been implicated in the initiation and progression of ovarian, pancreatic, and lung cancer. [14][15][16][17] Linc00152 was shown to promote the proliferation of colorectal cancer by regulating the expression of miR-139-5p, which is associated with metastasis and chemotherapy resistance. 18 Linc00284 knockdown up-regulated MEST expression by recruiting NF-κB1, reducing angiogenesis in ovarian cancer and restraining cancer cell migration and invasion. 15 We recently showed that Linc00485 was significantly up-regulated in lung cancer tissues compared with adjacent normal tissues, and that it had a significant positive correlation with the expression of the c-Myc oncogene.
The aim of the present study was to investigate the biological function of Linc00485 in lung cancer development, thus identifying a potential target for the diagnosis and treatment of lung cancer.

| Human lung cancer samples
Forty-nine paired lung cancer tissues and adjacent normal tissues were collected from Fujian Medical University Union Hospital. None of the patients who provided samples had undergone preoperative chemoradiotherapy, and their lung cancer was not accompanied by other disease. Twenty-nine patients were male and 20 were female, with an average age of 56.40 ± 7.10 years. There were 26 cases of lung adenocarcinoma and 23 cases of lung squamous cell carcinoma.
Regarding TNM staging, 14 cases were stage I, 18 cases were stage II, and 17 cases were stage III. Further details are given in Table 1.
The pathology of tumour tissues was strictly diagnosed by two independent expert pathologists. Tissues were immediately stored in liquid nitrogen at −196°C after surgery. All patients provided informed consent. The study was approved by the Ethics Committee of Fujian Medical University Union Hospital.

| Cell culture
Non-small-cell lung cancer (NSCLC) cell lines (A549 and H460 cells), human lung adenocarcinoma H1975 cells, lung epithelial BEAS-2B cells, and 293T cells were purchased from the Cell Bank of the Chinese Academy of Sciences and maintained in our laboratory.

| Scratch wound assay
As described previously, 19 cells were plated into six-well dishes with a density of 2 × 10 6 cells per well. When the cells reached 90% confluence, a gap in the confluent monolayer was created using a 200 μL sterile pipette tip. The detached cells were removed using PBS. The wound healing process was recorded and photographed under a microscope every 12 hours. The experiment was repeated independently at least three times.

| Transwell migration and invasion assays
Cells were collected, resuspended using serum-free medium, and diluted to a final concentration of 1 × 10 5 cells/mL.  20 The experiment was repeated independently at least three times.

| Colony formation assay
Cells in logarithmic growth phase were collected and seeded into six-well plates (1 × 10 3 cells per well) and cultured for 10 consecutive days. Culture medium was refreshed every 48 hours.
Subsequently, the cells were fixed with 75% ethanol for 30 minutes at room temperature and stained with 0.1% crystal violet for 20 minutes, followed by washing three times with PBS. Cells were counted for colonies containing at least 50 cells. Ten fields of view per group were observed and photographed under a microscope. The experiments were repeated independently at least three times.

| RNA immunoprecipitation assay
The RNA immunoprecipitation (RIP) assay was performed using a Magnetic RIP kit (Merck KGaA) according to the manufacturer's instructions. Briefly, the cells were collected, washed twice with cold PBS, and resuspended in an equal volume of RIP lysis buffer. Then, they were placed on ice for 5 minutes, centrifuged at 12 000× g and 4℃ for 10 minutes. Next, 100 μL of supernatant was added to 900 μL of RIP Immunoprecipitation Buffer following incubation overnight at 4°C. Magnetic beads containing the immunoprecipitated RNAprotein complex were treated with proteinase K. The total RNA in the precipitate was extracted according to the instructions and prepared for subsequent qPCR analysis.

| Western blotting
Whole-cell lysates were prepared using pre-cooled RIPA lysis buffer

| Statistical analysis
Data were analysed using SPSS 21.0 statistical software and expressed as mean ± SD. Differences between two groups were analysed by Student's t test. Comparisons among multiple groups with uniform variance were performed using one-way ANOVA with post hoc Fisher's least significant difference (LSD) test. Pvalues less than 0.05 were considered to indicate statistical significance. Each experiment was repeated independently at least three times.

Gene Forward sequence Reverse sequence
Linc00485

| Linc00485 is up-regulated in lung cancer tissues and predicts poor outcomes in patients with lung cancer
In this study, we found that the expression of Linc00485 in lung cancer tissues was significantly higher than that in adjacent normal tissues ( Figure 1A). Linc00485 levels were associated with TNM stage in patients with lung cancer; the expression of Linc00485 was strikingly elevated in the tumour tissues of patients with stage III/ IV lung cancer in comparison with those of patients with stage I/II lung cancer ( Figure 1B). Moreover, Linc0048 levels were markedly

| Linc00485 silencing inhibits and Linc00485 overexpression promotes proliferation of lung cancer cells
To understand the role of Linc00485 in lung cancer progression, cells'

| Silencing of Linc00485 suppresses and overexpression of Linc00485 facilitates migration and invasion of lung cancer cells
Through in vitro scratch assays and transwell migration and invasion assays, we further confirmed that Linc00485 silencing sig- Linc00485-silenced A549 cells ( Figure 3J).

| Linc00485 exerts its biological function by targeting miR-298
By searching https://omict ools.com/diana-lncba se-tool, we identified the sites of binding between miR-298 and Linc00485 ( Figure 4A). Expression of miR-298 was found to be significantly

| Linc00485/miR-298 axis regulates proliferation, migration, and invasion of lung cancer cells
To determine whether Linc00485 mediates the behaviours of lung cancer cells by sponging miR-298, a mimic/inhibitor of miR-298 or a negative control was transfected into A549 cells. We observed that  Linc00485 knockdown compared with the control group (n = 6 mice per group) ( Figure 6N).

| Linc00485/miR-298/c-Myc axis is involved in the proliferation, migration, and invasion of lung cancer cells
To elucidate the molecular mechanism by which the Linc00485/ miR-298/c-Myc axis regulates lung cancer progression, the c-Myc   Figure 7U).

| D ISCUSS I ON
Accumulating evidence indicates that smoking, environmental pollution, diet and genetic susceptibility are responsible for the initiation and progression of lung cancer. 23,24 However, the pathogenesis of lung cancer has not yet been fully elucidated. LincRNA, a long-chain non-coding RNA, has been confirmed to be involved in cancer biology. [25][26][27] In this study, we showed that the expression of Linc00485 was significantly up-regulated in lung cancer tissues, suggesting that Linc00485 may be an oncogene.
Previous studies have found that Linc00485 plays an important part in the development of uterine leiomyoma. 28 Moreover, in lung adenocarcinoma, Linc00485 up-regulated the expression of cell cycle checkpoint kinase 1, thereby enhancing the resistance of lung cancer cells to chemotherapy. 29 Here, our results confirmed that up-regulation of Linc00485 in lung cancer tumour tissues and cells facilitated lung cancer cell proliferation, migration, and invasion.

CO N FLI C T S O F I NTE R E S T
The authors confirm that there are no conflicts of interest.

E TH I C A L A PPROVA L
This study was approved by the ethics committee of Fujian Medical University Union Hospital, and written informed consents were obtained from patients enrolled in this study.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data supporting the findings of this study are included within the article or are available upon request from the corresponding author.