LINC00470 promotes tumour proliferation and invasion, and attenuates chemosensitivity through the LINC00470/miR‐134/Myc/ABCC1 axis in glioma

Abstract Glioma is the most common primary malignant tumour in the brain; temozolomide (TMZ) is the most prevalent chemotherapeutic drug currently used to combat this cancer. We reported previously that the long intergenic non‐protein coding RNA 470 (LINC00470) is a novel prognostic biomarker for glioma and promotes the tumour growth in an intracranial transplantation mouse model. However, the effects of LINC00470 on glioma cell proliferation, invasion and TMZ chemosensitivity, as well as its molecular mechanism, remain unclear. In this study, we found elevated expression levels of LINC00470 and MYC in glioma tissues and cells and decreased expression of microRNA‐134 (miR‐134). Functional studies have shown that LINC00470 promotes proliferation and invasion, and attenuates chemosensitivity of glioma cells, while miR‐134 exerts the opposite effect. In the rescue experiments, the tumorigenic effect of LINC00470 was offset by miR‐134. In the mechanism study, we found that LINC00470 was a competitive endogenous RNA (ceRNA) of miR‐134 and that miR‐134 can directly target MYC and negatively regulate its expression. Furthermore, MYC was positively correlated with ATP‐binding cassette subfamily C member 1 (ABCC1) expression in glioma cells and MYC up‐regulated ABCC1 expression. Further studies found that LINC00470 regulated MYC by sponging miR‐134 to regulate the expression of ABCC1. We concluded that LINC00470 promoted the expression of MYC and ABCC1 by suppressing miR‐134, thus promoting glioma cell proliferation and invasion, and attenuating TMZ chemosensitivity. Moreover, the LINC00470/miR‐134/MYC/ABCC1 axis constitutes a potential therapeutic target.


| INTRODUC TI ON
Glioma is the most common primary malignant tumour of the human central nervous system, accounting for more than half of the primary malignant tumours in the brain. 1 Low-grade gliomas have a better prognosis when surgery is combined with radiotherapy and chemotherapy; however, the 5-year survival rate remains less than 5% in high-grade gliomas such as glioblastoma multiforme (GBM). This is closely related to the highly invasive and rapid proliferation of GBM. 2 Therefore, it is particularly important to further explore the molecular mechanism of the malignant progression of glioma. Temozolomide (TMZ) is a DNA-alkylating agent that crosses the blood-brain barrier and is considered a first-line treatment for GBM. 3 Although TMZ has been shown to suppress tumour growth and prolong the survival of GBM patients, resistance to it is common, causing many treatment failures. 4,5 Multidrug resistance (MDR) is the main reason for this chemotherapy resistance. 6 Long non-coding RNAs (lncRNAs), comprising a set of non-coding RNAs greater than 200 nucleotides in length, have been shown to play an important role in the occurrence and development of a variety of tumours, including gliomas. 7,8 Long intergenic non-protein coding RNA 470 (LINC00470) is a lncRNA located on chromosome 18p11.32. 9 In our previous studies, we found that LINC00470 activated the AKT signalling pathway and promoted the tumour growth of intracranial transplantation mouse model with glioma. 10 However, the role and mechanism of LINC00470 in glioma must be further elucidated; in particular, it remains unclear whether LINC00470 affects the chemosensitivity of glioma.
Unlike lncRNAs, microRNAs (miRNAs) contain only 22 nucleotides and regulate gene expression post-transcriptionally by targeting the 3' untranslated region (3'UTR) of messenger RNA (mRNA). 11 miRNAs regulate the expression of approximately 30%-50% of human genes. 12,13 In addition, more than 50% of miRNAs are involved in the occurrence and development of human tumours by directly acting on oncogenes or tumour suppressor genes. 12 Recent studies have shown that microRNA-134 (miR-134) plays an important role in tumour cell proliferation, apoptosis, invasion and metastasis. For example, miR-134 inhibits the proliferation of lung cancer cells by down-regulating the expression of EGFR. 14 It is known that miR-134 is down-regulated in gliomas and is negatively correlated with World Health Organization (WHO) glioma grades; the mechanism may involve the promotion of glioma cell migration and invasion by targeting the KRAS gene and activating the ERK signalling pathway. 15,16 However, a deeper understanding of the miR-134 mechanism in glioma remains to be investigated.
Recent studies report that lncRNA can act as a special 'sponge' to adsorb miRNA, thereby reducing the regulatory effect of miRNA on mRNA. 17 In our present study, we confirmed the role of LINC00470 as an oncogene in gliomas and found that LINC00470 acts as competing endogenous RNA (ceRNA) to inhibit miR-134 to further regulate the expression of the oncogene MYC. We also found a positive correlation between the expression of MYC and the expression of ATP-binding cassette subfamily C member 1 (ABCC1, also known as multidrug resistance protein 1) in gliomas and that LINC00470 affected the chemosensitivity of glioma cells by regulating the expression of ABCC1 by interacting with miR-134. Our results provide a new understanding of the role and mechanism of the LINC00470/miR-134/MYC/ABCC1 axis in glioma, which may contribute to the development of novel therapeutic targets and improve the chemosensitivity of glioma.

| Public database, tissue samples and cell lines
The expression levels of MYC and ABCC1 in 541 GBMs were obtained using a gene expression array (Affymetrix U133A; Affymetrix/ Thermo Fisher Scientific, Santa Clara, CA, USA) referenced in The Cancer Genome Atlas (TCGA) database (https://portal.gdc.cancer. gov/explo ration). Thirty-two glioma tissues and seven normal brain tissues were collected at the Department of Neurosurgery, Xiangya Hospital, Central South University, Hunan, China. After surgical resection, fresh specimens were sent to the Department of Pathology for neuropathological evaluation and the remaining specimens were frozen in liquid nitrogen immediately. The glioma group comprised eight WHO II, eight WHO III and sixteen WHO IV tumours and the normal brain tissues were taken from patients with brain trauma. Clinical parameters of thirty-two glioma patients are listed in Table S1. This study was approved by the Ethics Committee of Xiangya Hospital, Central South University, Hunan, China, and written informed consent was provided by all of the patients.

| Quantitative reverse transcription-PCR (RT-qPCR) and Western blotting
RT-qPCR and Western blotting were performed as described previously. 18 The following RT-qPCR primers were used: GAPDH,

| Cell viability assay
Cell activity was measured with a CCK8 assay. The transfected cells were cultured for 24 hours, then seeded in 96-well plates (2000 cells per well). After the cells attached, they were cultured for different time periods (0, 1, 2 and 3 days) after different treatments (with or without TMZ). Ten microlitres of CCK8 reagent was added to each well, and the absorbance was measured at 450 nm after incubation at 37°C for 3 hours.

| Immunohistochemistry
Immunohistochemistry was carried out as described previously. 19 The following antibody was used: rabbit polyclonal antibody to MYC (Sangon Biotech).

| Statistical analysis
All experiments were repeated three times, and data were statistically analysed using GraphPad Prism 5 (La Jolla, CA, USA) and SPSS version 20.0 (IBM Corp., Armonk, NY, USA). Descriptive statistical analysis was used to ensure normality. The Levene test was used to test the homogeneity of variance. Differences between the different groups were assessed using Student's t test or one-way ANOVA.
When the variance was unequal, t' test or Welch's ANOVA was used and Kruskal-Wallis test was used for verification. The correlation between the expression levels of the two genes was analysed using Pearson's chi-squared test. A P-value of <0.05 was considered to be statistically significant.

| LINC00470 was up-regulated in glioma tissues and cell lines
In this study, we analysed the expression of LINC00470 in 32 glioma tissues and 7 normal brain tissues from the Department of Neurosurgery, Xiangya Hospital, China, by RT-qPCR. We found that the expression of LINC00470 in glioma tissues was significantly upregulated relative to normal brain tissues ( Figure 1A). Meanwhile, compared with normal brain tissue cell line HEB, the expression levels of LINC00470 in glioma cell lines U87 and U251 were also up-regulated ( Figure 1B). These results indicated that LINC00470 may play a biological role in the development and/or progression of glioma.

| LINC00470 promoted glioma cell proliferation and invasion, and attenuated TMZ chemosensitivity
In order to explore the biological role of LINC00470 in glioma cells, an LINC00470 overexpression plasmid was transfected into cell lines U87 and U251. A CCK8 assay showed that the cell viability was significantly increased when LINC00470 was overexpressed in U87 and U251 cells compared with controls ( Figure

| LINC00470 was a sponge of miR-134
RT-qPCR analysis showed that the relative expression of miR-134 was significantly down-regulated in glioma tissues, while the expression of miR-134 in U87 and U251 cells was also down-regulated relative to HEB cells (Figure 2A-B). In addition, Pearson's chi-squared test revealed a significant negative correlation between the expression levels of LINC00470 and miR-134 in thirty-two glioma tissues ( Figure 2C). Previous studies report that lncRNAs interact with miR-NAs as sponges to regulate their biological functions. 17 To determine F I G U R E 1 RT-qPCR showed that LINC00470 was up-regulated in glioma and promoted glioma cell proliferation, invasion and TMZ resistance. A, The expression of LINC00470 in glioma tissues (n = 32) was significantly up-regulated relative to normal brain tissues (n = 7). GAPDH was the internal control. B, The relative expression of LINC00470 in glioma cell lines U87 and U251 was higher than that in normal cell line HEB. GAPDH was the internal control. C,D, The cell viability was increased when LINC00470 was overexpressed in U87 and U251 cells compared with controls. E,F, Overexpression of LINC00470 enhanced the invasive ability of U87 and U251 cells. The photographs were taken at 200 × magnification. Scale bar: 100 μm. G,H, LINC00470 enhanced the chemoresistance of U87 and U251 cells to TMZ. The data shown represent the mean ± SD of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. TMZ, temozolomide; RT-qPCR, quantitative reverse transcription F I G U R E 2 LINC00470 was a sponge of miR-134. A,B, The relative expression of miR-134 was down-regulated in glioma tissues and cell lines. U6 was the internal control. C, Pearson's chi-squared test showed a significant negative correlation between the expression of LINC00470 and miR-134 in glioma tissues. D, miR-134 was predicted to be a target miRNA of LINC00470. E, The luciferase activity of 239T cells co-transfected with miR-134 mimics and LINC00470-Wt was significantly reduced, while the luciferase activity of 239T cells co-transfected with miR-134 mimics and LINC00470-Mut was scantly affected. F, The expression of miR-134 was down-regulated when we overexpressed LINC00470 in U87 and U251 cells. U6 was the internal control. G, The expression of LINC00470 was down-regulated when we overexpressed miR-134. GAPDH was the internal control. The data shown represent the mean ± SD of at least three independent experiments.*P < 0.05; **P < 0.01; ***P < 0.001. NC, negative control F I G U R E 3 miR-134 suppressed glioma cell proliferation and invasion and enhanced TMZ sensitivity. A,B, The cell viability was decreased when miR-134 was overexpressed in U87 and U251 cells compared with controls. C,D, miR-134 mimics decreased U87 and U251 cell invasion ability. The photographs were taken at 200 × magnification. Scale bar: 100 μm. E,F, Overexpressed miR-134 enhanced the sensitivity of U87 and U251 cells to TMZ. The data shown represent the mean ± SD of at least three independent experiments. *P < 0.05; **P < 0.01; ***P < 0.001. TMZ, temozolomide; NC, negative control whether LINC00470 and miR-134 also exhibit similar interactions in gliomas, we first predicted the binding site of LINC00470 and miR-134 using bioinformatics analysis ( Figure 2D). Furthermore, in the dual-luciferase reporter assay, we found that the luciferase activity of 239T cells co-transfected with miR-134 mimics and LINC00470-Wt was significantly reduced, while the luciferase activity of 239T cells co-transfected with miR-134 mimics and LINC00470-Mut was scantly affected ( Figure 2E). In addition, expression of miR-134 was down-regulated when we overexpressed LINC00470 in glioma cells ( Figure 2F), whereas expression of LINC00470 was down-regulated when we overexpressed miR-134 ( Figure 2G). These data indicated that miR-134 is a direct target miRNA of LINC00470 and confirmed the mutual regulatory relationship between LINC00470 and miR-134.

| miR-134 suppressed glioma cell proliferation and invasion and enhanced TMZ sensitivity
From our previous results (Figure 2A-B), we determined that miR-134 was down-regulated in glioma tissues and cell lines.

| miR-134 mediated the tumour-promoting effect of LINC00470 in glioma cells
To

| Overexpression of LINC00470 upregulated the expression of MYC by regulating miR-134
It is well known that MYC, as a proto-oncogene, exhibits elevated levels of expression in most human tumours-including glioma-and promotes glioma growth. 20 Through bioinformatics analysis, we found that a binding site exists between miR-134 and the MYC 3'UTR ( Figure 5A). To confirm that MYC was a direct target of miR-134, we performed a dual-luciferase reporter assay.
F I G U R E 6 MYC was up-regulated in glioma tissues. A, The mRNA expression level of MYC was elevated in 32 glioma tissues. GAPDH was the internal control. B, Immunohistochemical staining images of grade II, III and IV glioma tissues and non-tumour brain tissues (magnification × 200). The immunohistochemical score indicated that MYC protein expression was dependent on WHO grade. Data are represented as mean ± SEM.*P < 0.05; **P < 0.01 These results suggested that LINC00470 up-regulated the expression of MYC by regulating miR-134. Besides, in our study, we also confirmed that the mRNA expression level of MYC was elevated in glioma tissues ( Figure 6A). Meanwhile, we confirmed that the protein expression of MYC in glioma was significantly up-regulated and positively correlated with pathological grade ( Figure 6B).

| D ISCUSS I ON
Recently, with more and more evidence showing that lncRNAs function as oncogenes or tumour suppressors, the study of lncRNAs has become increasingly important. 7 Although the roles of a few lncR-

NAs in gliomas have been depicted, little is known about LNC00470.
Our previous studies have demonstrated that elevated expression levels of LINC00470 were associated with poor prognosis in glioma patients and promoted the glioma growth in an intracranial transplantation mouse model by activating the AKT signalling pathway. 10 In the present study, we further confirmed that LINC00470 was highly expressed in gliomas and promoted glioma cell proliferation and invasion, and attenuated TMZ chemosensitivity. Our study clarified the new mechanism by which LINC00470 acts as a ceRNA to sponge miR-134, thereby regulating downstream molecules such as MYC and ABCC1 to maintain the malignant phenotype of glioma.
Given that previous studies report that lncRNAs competitively bind to miRNA response elements in cells as miRNA sponges to regulate mRNA expression, 21 we speculated that LINC00470 may have a similar mechanism. Using bioinformatics methods, we predicted the binding site of LNC00470 and miR-134. As expected, we confirmed the direct binding and regulatory relationship between LINC00470 and miR-134 using a luciferase reporter assay. Unlike LINC00470, miR-134 is a microRNA that has attracted considerable attention recently; it is considered to be a suppressor in a variety of tumours. of cell self-renewal and tumorigenesis. [29][30][31] In addition, MYC is one of the most frequently deregulated oncogenes currently known, and its forced expression promotes tumorigenesis by providing sufficient energy and metabolic substrates for uncontrolled cell proliferation. 30,32,33 Previous studies have provided sufficient evidence that MYC is up-regulated in gliomas and its forced down-regulation has been shown to promote cell apoptosis and reduce the formation of xenograft tumours in vivo. 34,35 Our study further confirmed the up-regulation of MYC at both mRNA and protein levels in gliomas.
Some studies have shown that MYC can be regulated by miRNAs at the post-transcriptional level. 36 Here, we identified that MYC was a novel target of miR-134 by dual-luciferase assay, by which it was regulated. Furthermore, we determined that LINC00470 indirectly regulates the expression of MYC by sponging miR-134, thus regulating the malignant phenotype and TMZ sensitivity of glioma cells.
Although TMZ is the preferred chemotherapeutic drug for the treatment of GBM, chemotherapy failure is common due to both inherent resistance and acquired resistance. 37   identified the LINC00470/miR-134/MYC/ABCC1 axis and illuminated its biological role and mechanism in glioma. This axis may constitute potential therapeutic targets for gliomas.

CO N FLI C T O F I NTE R E S T
The authors declare no conflict of interest. Data curation (supporting); Investigation (supporting). Yimin Pan: Formal analysis (supporting); Investigation (supporting). Qing Liu: Conceptualization (equal); Funding acquisition (lead).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data sets used and analysed during the current study are available from the corresponding author on reasonable request.