MALAT1 accelerates the development and progression of renal cell carcinoma by decreasing the expression of miR‐203 and promoting the expression of BIRC5

Abstract Objective We aimed to investigate the roles of the lncRNA MALAT1 in renal cell carcinoma (RCC) progression. Methods qRT‐PCR was used for the assessment of BIRC5, miRNA‐203 and MALAT1 expression. Furthermore, the targeted relationships between miR‐203 and BIRC5, as well as MALAT1 and miR‐203, were predicted by the miRanda/starBase database and verified by dual‐luciferase reporter gene assay. The effects of MALAT1, miRNA‐203 and BIRC5 on cell proliferation, cell cycle, cell apoptosis, cell invasion and cell migration were studied by using CCK‐8, flow cytometry, transwell and wound healing assays, respectively. In addition, the effects of MALAT1 on RCC tumorigenesis were evaluated in vivo by nude mouse tumorigenesis. Results The expression levels of BIRC5 and MALAT1 were higher in RCC tissues and cell lines than in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. In contrast, the expression of miRNA‐203 in RCC tissues and cell lines was higher than that in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line. BIRC5 and MALAT1 promoted cell proliferation yet decreased the percentage of RCC cells at G0/G1 phase. Conclusions Our study demonstrated that MALAT1 functions as a miR‐203 decoy to increase BIRC5 expression in RCC.


MicroRNAs (miRNAs) are 19-22 nucleotide-long non-coding
RNAs that function as negative regulators of translation and are involved in many cellular processes. Increased levels of specific miRNAs have been closely related to a variety of diseases, such as cancers, diabetes, obesity and cardiovascular disease. 7 miRNAs are non-coding RNAs, and it has been estimated that 30% of all genes in animals are regulated by translational and post-transcriptional repression, cleavage or destabilization. 8  is downregulated in multiple tumour types and causally linked to oncogenesis. 9 miRNAs are also aberrantly expressed in several eukaryotic organisms to regulate the stability and processing of target mRNA through directly binding to 3′UTRs. miRNAs have been reported to be involved in cell proliferation, cell differentiation and cell apoptosis, resulting in a reduction in the microRNA levels of hundreds of small target mRNAs. 10 miR-203 is a tumour suppressor in a variety of human cancers, such as hepatocellular carcinoma (HCC), 11 prostate cancer 12 and liver cancer. 13 Besides, miR-203 can directly suppress the expression of transcription factor p63 during epidermal differentiation, thus limiting the proliferation potential and inducing the withdrawal of the cell cycle to eventually promote epidermal differentiation. 14 In rhabdomyosarcoma cells, the overexpression of miR-203 suppressed cell growth and promoted myogenic differentiation. 15 Currently, lncRNAs are differentially expressed in various tissues and have essential functions in gene regulatory processes in normal cells and cancer cells. Furthermore, many lncRNAs are associated with chromatin modification complexes and act as miRNA sponges, which adjust gene expression. 16 LncRNAs are the largest class of non-coding RNAs. Noncoding RNAs, once thought to be part of the transcriptional noise, now constitute a regulatory layer of transcriptional and post-transcriptional regulation. The enhanced transcriptional noise and gene expression regulatory function of lncRNAs are fully supported by their functional roles observed in various important biological environments. 17 In previous reports, high expression levels and the acute hypoxic induction of MALAT1 in several mouse organs suggested a hitherto unrecognized role of this lncRNA in systemic adaptation hypoxia. 18 In addition, the upregulation of MALAT1 was correlated with cancer progression and poor prognosis in clear cell renal cell carcinoma. 19 However, the roles of MALAT1 in RCC progression need to be further understood.
BIRC5 (also known as survivin) is a critical anti-apoptotic protein that is been involved in many cancer types. BIRC5 inhibits apoptosis-related signalling pathways and promotes cell proliferation to affect cancer progression. 20 BIRC5, which encodes surviving, is upregulated in both adenocarcinoma and squamous cell carcinoma tissues, and the high expression of BIRC5 is related to poor survival in adenocarcinoma, but not squamous cell carcinoma. In addition, survivin was identified as a candidate marker of aggressiveness in clear cell renal cell carcinoma (ccRCC), and high expression levels of survivin protein predicted a poor outcome for ccRCC patients. 21 In addition, the ratio of the miR-195 level to the BIRC5 level was associated with both recurrence-free and overall survival in lung adenocarcinoma. 22 Previous researches showed that the miR-195/BIRC5 axis is a potential target for the specific treatment of lung adenocarcinoma, especially for NSCLC (non-small-cell lung carcinoma). 22 BIRC5 is a new member of inhibitor of IAP family, the proteins of which regulate the cell cycle and apoptosis. Besides, the expression of BIRC5 was induced by hypoxia, 23 and BIRC5 promoted angiogenesis and was strongly correlated with cell proliferation. 24 There is increasing evidence that indicated that BIRC5 is highly expressed in most human tumours and closely related to tumour progression, tumour recurrence, chemotherapy resistance and poor prognosis. 25,26 The aim of our study was to investigate the roles of MALAT1/ miR-203/BIRC5 in the development and progression of RCC, which might provide us with more diagnostic and therapeutic strategies for RCC in the future.

| Immunohistochemistry (IHC)
The tissue sections were dried at 60°C for 1 hour and then dewaxed by an automatic dyeing machine. The tissue sections were incubated after being washed with PBS with 3% hydrogen peroxide at room temperature for 6 minutes. The sections were then immersed in 0.01 M 3% citrate buffer. Afterwards, they were heated at 95°C for 10 minutes in a microwave and cooled to room temperature.
After 30 minutes, non-immune goat serum was added and the sections were incubated overnight with BIRC5 (1:1000 v/v) and Ki-67 (1:300 v/v) (Abcam) at 4°C. Afterwards, they were washed in PBS, labelled with HRP-labelled goat anti-rabbit IgG (Abcam) (1:1000 v/v) and incubated for 30 minutes at room temperature. Besides, the sections should be exposed to freshly prepared diaminobenzidine and stained for 4-6 minutes. The sections were also stained for 15 seconds with haematoxylin. Finally, the sections were rinsed with water.

| Dual-luciferase reporter gene assay
The targeted relationship between miR-203 and BIRC5 was pre- Similarly, the pGL3 luciferase reporter gene vector (Promega) loaded with either BIRC5-wt or BIRC5-mut was co-transfected with miR-203 mimics or control into HEK293T cells using Lipofectamine 2000 reagent (Invitrogen). The luciferase activities in cell lysates were measured with a Dual-Glo Luciferase Assay System (Promega) 48 hours after transfection (Promega) in accordance with the manufacturer's instructions. Each experiment was repeated at least three times.
Afterwards, the OD at 450 nm (OD450) was measured with a plate luminometer (Bio-Rad). The experiment was repeated at least three times.

| Flow cytometry analysis
For cell cycle experiments, different groups of cells were collected 72 hours after transfection and digested to obtain a cell suspension.
Cell suspension was centrifuged, and the supernatant was discarded.
Cells were washed twice with PBS to remove any residue and fixed in

| Wound healing assay
Cell motility was evaluated using a wound healing assay. RCC cells were plated in 24-well plates with serum-free medium until they reached 80% to 90% confluence. The cell monolayers were scratched across the centre of each well with a 10 μL micropipette tip. Wound healing was monitored at the indicated time points by phase-contrast microscopy with a 20 × objective and an inverted microscope. The experiment was performed at least three times.

| Transwell assay
Matrigel (BD Bioscience) and serum-free DMEM were thoroughly mixed and placed in a transwell chamber (Corning Incorporated). Cells at a concentration of 2.5 × 10 4 cells/mL in serum-free medium were placed into the upper chamber (500 μL for each chamber) of 24-well invasion chambers, while culture medium containing 20% FBS was added to the lower chamber. A 4% paraformaldehyde solution was utilized to immobilize the cells, and 0.1% crystal violet was used to stain RCC cells after 24-48 hours of incubation. Pictures were taken of each chamber, and the cells in 5-10 independent fields were counted.

| Western blot analysis
The concentration of each protein was determined using a Pierce BCA Protein Assay Kit (Pierce). After they were separated by 10% SDS-polyacrylamide gel electrophoresis (SDS-PAGE), the proteins were then transferred to polyvinylidene difluoride membrane (PVDF, Millipore, Billerica) for 120 minutes. Thereafter, membranes were blocked with TBST containing 5% skim milk and incubated with the primary antibody (anti-BIRC5, ab76424, 1:5000; anti-GAPDH, ab181602, 1:10 000). Subsequently, the membranes were incubated with a secondary antibody (anti-rabbit IgG H&L, ab6721, 1:10 000) for 1 hour at 37°C. All of the antibodies in this study were purchased from Abcam. Signal detection was conducted by the ECL system (Life Technology). The relative protein levels among the samples using the GAPDH density as an internal loading control were compared.

| Statistical analysis
Data are expressed as mean ± standard deviation (SD) from triplicate independent experiments. Student′s t test was used to compare differences in the two different groups with parametric variables.
For three or more groups, difference analysis was performed by one-way ANOVA. Statistical analysis was carried out with GraphPad Prism 6. P values <0.05 were considered as statistically significant.

| BIRC5 was overexpressed in RCC tissues and cells
The mRNA and protein expression of BIRC5 was higher in RCC tissues than in adjacent normal tissues as shown in Figure 1A Taken together, these data suggest that BIRC5 was overexpressed in RCC tissues and cells, and BIRC5 promoted the development and progression of RCC cells.
F I G U R E 1 BIRC5 expression was higher in RCC tissues and cell lines than in control tissues and cell lines. A, The expression of BIRC5 was higher in RCC tissues than in adjacent normal tissues, as shown by qRT-PCR. B, IHC results showed that the expression of BIRC5 was higher in RCC tissues than in adjacent normal tissues. C and D, Kaplan-Meier plot analysis illustrated that patients expressing high levels of BIRC5 presented a significantly poorer prognosis than those expressing low levels of BIRC5. E and F, The increased expression levels of BIRC5 were detected in four RCC cell lines (A498, 786-O, OS-RC-2 and CAKI-1) compared with its expression in a normal renal cortex proximal tubule epithelial cell line. Data were expressed as mean ± standard deviation of three independent experiments. **P < 0.01. ***P < 0.001

| MiR-203 directly targeted BIRC5 and suppressed the expression of BIRC5
The miRanda database predicted that that BIRC5 is a potential target of miR-203 ( Figure 2A). To verify the targeted relationship, a dual-luciferase reporter gene assay was employed. Dual-luciferase reporter gene assay showed that the luciferase activity of the group co-transfected with miR-203 mimics and BIRC5-wt was lower than that of the group co-transfected with miR-203 NC and BIRC5-wt

| Targeted relationship between miR-203 and MALAT1 was validated
Two potential binding sites between MALAT1 and miR-203 were identified based on starBase as shown in Figure 4A.

| Effects of MALAT1 on the proliferation, cell cycle, apoptosis and migration of RCC cells
The CCK-8 assay indicated that the upregulation of MALAT1 significantly increased cell viability, which was otherwise decreased with downregulation of MALAT1 in both A498 and OS-RC-2 cells ( Figure 6A

| MALAT1 promoted RCC tumorigenesis in vivo
As shown in Figure 7A

| D ISCUSS I ON
LncRNAs are important regulators of gene expression that interact with major signalling pathways for cell growth, proliferation, differentiation, apoptosis, migration, and invasion. 29 In some studies, MALAT1 acted as an miR-203 sponges to promote inflammation in myocardial ischaemia-reperfusion injury. 30 In our study, MALAT1 F I G U R E 6 Effects of MALAT1 on cell proliferation, cell cycle, apoptosis, invasion and migration of A498 and OS-RC-2 cells. A and B, The effects of MALAT1 on the proliferation of A498 cells and OS-RC-2 cells were examined by CCK-8 assay. C-F, The effects of miR-203 on the cell cycle (C and D) and apoptosis (E and F) in A498 cells and OS-RC-2 cells were measured by flow cytometry. G-K, The effects of miR-203 on the invasion and migration of A498 cells and OS-RC-2 cells were examined by transwell assay (G and H) and wound healing assay (I-K), respectively. Data were expressed as mean ± standard deviation of three independent experiments. *P < 0.05. **P < 0.01. ***P < 0.001 BIRC5 has been shown to inhibit cell apoptosis yet promote cell proliferation in human cancers. The overexpression of BIRC5 was observed in almost all human malignancies, and the increased expression of BIRC5 was correlated with poor clinical outcomes, tumour recurrence and drug resistance in cancer patients. 31 Recently, increasing numbers of studies have showed that BIRC5 is regulated by cytokines in lymphocytes and plays a vital role in the proliferation and survival of haematopoietic cells. 32 We verified that BIRC5 accelerated cell proliferation and the cell cycle, inhibited apoptotic pathways and promoted cell migration. Some articles also showed that BIRC5 inhibited caspase-dependent apoptotic pathways and caspase-independent apoptotic pathways as well as accelerated cell proliferation. 20 We investigated proposed targeted relationship between miR-203 and BIRC5. Our results showed that the overexpression of miR-203 inhibited BIRC5 expression, while the inhibition of an miR-203 inhibitor accelerated BIRC5 expression. Wang et al demonstrated that miR-203 suppressed the proliferation and migration of lung cancer cells and promoted their apoptosis by targeting SRC. 30 Zhang et al suggested that miR-203 inhibited tumour growth and invasion in oesophageal cancer by inhibiting Ran. 6 These studies are generally consistent with our results. In addition, we verified that miR-203 inhibited cell proliferation, cell cycle progression and cell migration but promoted apoptotic pathways by targeting BIRC5.
Moreover, MALAT1 was shown to be upregulated in RCC.
MALAT1 upregulated BIRC5 expression to promote cell proliferation, cell cycle progression and migration yet inhibited cell apoptosis in RCC through targeting miR-203. Besides, MALAT1 promoted OS tumour growth in vivo. 33,34 Furthermore, MALAT1 regulated ovarian cancer cell proliferation, migration and apoptosis through Wnt/βcatenin signalling pathway. 35 Our research showed that MALAT1 promoted RCC tumorigenesis in vitro. We validated that the expression level of MALAT1 was higher in RCC tissues and cell lines than in adjacent normal tissues and a normal renal cortex proximal tubule epithelial cell line.
RCC patients with high MALAT1 expression had more advanced clinical features and a shorter overall survival time than those with low MALAT1 expression. 36 MALAT1 promoted cancer cell proliferation and metastasis through activating the ERK/MAPK pathway and interacting with hnRNP during cell cycle regulation. 19 In the same way, our study explored the mechanism of action between them.
To conclude, we confirmed the direct targeted relationships be- lung cancer 39 and colorectal cancer. 40 The detection of MALAT1 has not yet been utilized for the diagnosis and treatment of RCC.
However, upgraded and integrated therapies could be developed based on the detection of MALAT1. More miRNAs specifically targeting MALAT1 in RCC needed to be uncovered for the development of integrated therapies. In addition, more specific mRNAs regulated by MALAT1 in RCC could be selected as potential therapeutic targets.
In conclusion, MALAT1 accelerates the development and progression of renal cell carcinoma by decreasing the expression of miR-203 and promoting the expression of BIRC5.

ACK N OWLED G EM ENTS
None.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

AUTH O R CO NTR I B UTI O N S
HMZ and WL designed the study; WYG and YY collected the data; HMZ and XDY analysed and interpreted the data; and JHZ involved in critical reviewing of the manuscript.

E TH I C S A PPROVA L
This study was approved by Shanghai General Hospital, The First People's Hospital Affiliated to Shanghai Jiaotong University.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.