LINC00958 promotes bladder cancer carcinogenesis by targeting miR-490-3p and AURKA

Background Bladder cancer is a prevalent malignancy of the urinary system, in which long non-coding RNAs (lncRNAs) are highly associated. We aimed to elucidate the role of LINC00958 in bladder cancer. Methods LINC00958 expression levels were measured using qRT-PCR. The interaction of LINC00958-miR-490-3p-AURKA was analyzed by luciferase, RIP, and RNA pull-down assays. The biological roles of LINC00958, miR-490-3p, and AURKA in bladder cancer cells were analyzed using CCK8, BrdU, and transwell assays. Results Increased expression of LINC00958 and AURKA was observed in bladder cancer tissues and cell lines. Decreased LINC00958 expression repressed bladder cancer progression and downregulation of miR-490-3p accelerated bladder cancer cell progression. Moreover, LINC00958 sponges miR-490-3p to upregulate AURKA expression, thereby promoting carcinogenesis in bladder cancer cells. Conclusions Our study revealed that LINC00958 facilitated cell proliferation and invasion, and suppressed cell apoptosis by sponging miR-490-3p and upregulating AURKA, thus inspiring a new treatment method for bladder cancer. Supplementary Information The online version contains supplementary material available at 10.1186/s12885-021-08882-6.

miRNAs are non-coding RNAs of approximately 20 nucleotides, which typically function by degrading the expression of their target genes [15]. Dysregulation of miRNAs has been regarded as a critical factor in cancer cell growth and apoptosis in cancers such as colon, ovarian, and lung cancer [16][17][18]. Both miR-490-3p and miR-490-5p are members of the miR-490 family, which participate in tumorigenesis [19,20]. Evidence has shown that miR-490-3p prevents cell growth and invasion, whereas it increases apoptosis in various cancers, such as prostate cancer [21], esophageal squamous cell carcinoma [22], and breast cancer [23]. Several studies have suggested the inhibitory action of miR-490-5p in bladder cancer [24][25][26]; however, no study has explored the molecular mechanism of miR-490-3p in the development of bladder cancer.
The aurora kinase A (AURKA) gene is located on chromosome 20q13.2 and consists of 12 exons. It encodes a cell cycle-regulated kinase that participates in microtubule formation [27,28]. High-throughput data analysis demonstrated that AURKA was identified as a tumor promoter in all types of cancers [29][30][31]. Upregulation of AURKA has been reported in bladder cancer progression, which might be a potential therapeutic target for bladder cancer [32][33][34]. For example, overexpression of AURKA accelerated cell proliferation and reduced cell apoptosis, and high AURKA expression predicted poor prognosis in bladder cancer [33]. Furthermore, miRNA-124-3p inhibited cell proliferation and migration, but enhanced cell apoptosis by reducing AURKA in bladder cancer [35]. Nonetheless, the effect of LINC00958 and miR-490-3p on AURKA remains unknown.
In this study, we sought to determine the role of the LINC00958-miR-490-3p-AURKA axis in bladder cancer. We hypothesized that LINC00958 aggravates bladder cancer progression via the miR-490-3p-AURKA axis.

Samples and cells
Cancer tissues and adjacent normal tissues from 34 patients with bladder cancer, who provided informed written consent, were obtained from our hospital. The study was approved by the ethics committee of our hospital. The clinicopathological characteristics of the patients are listed in Table 1. Human bladder cancer cell lines (5637, RT4, T24, and UMUC3) and a normal human ureteral epithelial cell line (SV-HUC-1) were purchased from ATCC (Manassas, VA, USA). 5637 and UMUC3 cells were cultured in RPMI-1640 medium (Gibco, Waltham, MA, USA), RT4 and T24 cells were cultured in McCoy's 5A medium (Gibco), and SV-HUC-1 cells were cultured in Ham's F-12 K medium (Gibco) with 10% FBS (Gibco) at 37°C with 5% CO 2 .

RT-qPCR
Trizol reagent (Cat#: 15596018, Thermo Fisher Scientific, Waltham, MA, USA) was used to extract mRNA and lncRNA. The miRcute miRNA extraction kit (DP501, Tiangen, Beijing, China) was used for miRNA extraction. The PrimeScript First Strand cDNA Synthesis kit (RR037A, Takara, China) was used to convert mRNA and lncRNA to cDNA. The All-in-One miRNA First Stand cDNA Synthesis Kit (AMRT-0060, GeneCopoeia, Rockville, MD, USA) was used to convert miRNA into cDNA. Finally, RT-qPCR was performed on the ABI 7500 Sequence Detection System with SYBR Premix Ex Taq (RR420A, Takara, China) for mRNA and lncRNA, and All-in-One miRNA qRT-PCR Detection Kit (AOMD-Q050, GeneCopoeia) for miRNA. The quantification of LINC00958 and AURKA relative to GAPDH, and miR-490-3p relative to U6 was performed using the formula 2 -ΔΔCt . All primers are listed in Table 2.

Subcellular localization
The PARIS Kit (AM1921, Life biosciences, Boston, MA, USA) was used to extract the subcellular fractionation for the LINC00958 localization experiment. After obtaining the nuclear and cytoplasmic fractions, we detected the LINC00958 level in the nucleus and cytoplasm by RT-qPCR, where GAPDH was used as the internal control of the cytoplasmic fraction and U6 was used as the internal control of the nuclear fraction.

CCK8 assay
The

RIP assay
The Magna RIP RNA-binding protein immunoprecipitation kit (Cat#: 17-700, Millipore, Burlington, MA, USA) was used to detect the interaction between LINC00958 and miR-490-3p. The prepared cell lysates of RT4 and T24 cells transfected with miR-490-3p mimic or NC mimic were immunoprecipitated with magnetic beads conjugated with Ago2 (Cat#: ab186733, Abcam) or the control IgG (Cat#: ab172730, Abcam) for 4 h at 4°C. Finally, the precipitates were collected, and the enrichment of LINC00958 was subjected to RT-qPCR.

Western blotting (WB) analysis
Whole-cell lysates of RT4 and T24 cells were treated with RIPA buffer (Cat#: #20-188, Sigma-Aldrich). Then, 30 μg of proteins were separated on 12% SDS-PAGE gels and transferred to PVDF membranes. After blocking with 5% BSA in TBST and washing twice with TBST, the membranes were incubated with anti- Bax

Statistical analysis
Student's t-test for two groups and one-way analysis of variance (ANOVA) for multiple group comparisons were performed using GraphPad Prism 8.0 (GraphPad Prism, USA). Pearson's correlation analysis was used to detect the association between LINC00958 and miR-490-3p or AURKA and miR-490-3p. P < 0.05 was considered statistically significant. Data are shown as mean ± standard deviation (SD).

Results
The selection of LINC00958-miR-490-3p-AURKA axis as our study object LINC00958 has been extensively studied in some cancers such as head and neck cancers [36][37][38], gastric cancer [13], lung cancer [12], and cervical cancer [11,39]. It is also a potential oncogenic factor and a candidate prognostic biomarker in bladder cancer [40,41] and is associated with lymphangiogenesis and lymphatic metastasis [42]. Only Cui et al. reported LINC00958 in a ceRNA network in bladder cancer [14]. Thus, LINC00958 has been rarely studied in bladder cancer. To expand the downstream network of LINC00958, we first analyzed the GSE40355 and GSE37815 data series to identify differentially expressed miRNAs (DE-miRs) and differentially expressed mRNAs (DE-mR) in bladder cancer samples. By intersecting the predicted target miRNAs of LINC00958 using starBase and the DE-miR list (adjusted P < 0.05, logFC ≤ − 1.5), we identified miR-490-3p (Fig. 1A). The cancersuppressive role of miR-490-3p has been demonstrated in various cancers [43][44][45][46], but has not been studied in bladder cancer. Thus, we speculated that LINC00958 promotes bladder cancer malignancy by suppressing miR-490-3p. We sought to identify the potential downstream network of LINC00958-miR-490-3p. By intersecting the DE-mR list of GSE37815 (P < 0.05, logFC ≥1.5) and the target mRNAs of miR-490-3p predicted by TargetScan (version 7.2 of human database), we identified AURKA (Fig. 1B). AURKA has also been reported to be a significant facilitator in bladder cancer [33,34] and has been studied in miRNA-mRNA interaction networks [35]. However, AURKA has not been previously studied in a ceRNA network involving an lncRNA in bladder cancer. Thus, we hypothesized that AURKA might be regulated by LINC00958-miR-490-3p, thereby affecting the malignant phenotypes of bladder cancer.

LINC00958 is upregulated in bladder cancer tissues and cells
To analyze the biological function of LINC00958 in bladder cancer, LINC00958 expression in bladder cancer tissues and cells was first determined, which was dramatically elevated 4-fold in bladder cancer tissues compared to normal tissues ( Fig. 2A). Furthermore, we also analyzed the correlation between LINC00958 expression and certain clinical parameters such as sex, age, smoking, tumor size, tumor grade, tumor number, and pathological T stage, and found that increased LINC00958 expression was correlated with tumor grade (P = 0.020) and pathological T stage (P = 0.039) in bladder cancer (Table 1). LINC00958 expression in bladder cancer cell lines (5637, RT4, T24, and UMUC3) was significantly elevated by more than 2-fold compared to that in the normal human ureteral epithelium cell line (SV-HUC-1). Notably, the RT4 and T24 cell lines showed the highest LINC00958 expression; thus, we chose them for subsequent experiments (Fig. 2B). Next, we observed that 60% of LINC00958 was localized in the cytoplasm, while 40% of LINC00958 was found in the nucleus, suggesting that LINC00958 mainly exists in the cytoplasm (Fig. 2C). Then, we transfected siRNA-LINC00958 (Si-lnc) and negative control (NC) into RT4 and T24 cells and confirmed the silencing efficiency. As displayed in Fig. 2D, LINC00958 expression was reduced by over 50% in the Si-lnc groups compared to that in the blank groups of RT4 and T24 cells. These results indicated that LINC00958 was upregulated in bladder cancer tissues and cells.

LINC00958 enhances the progression of bladder cancer cells
To investigate whether LINC00958 plays a key role in bladder cancer, we performed a series of experiments in cells transfected with Si-lnc. The CCK8 assay showed that the cell viability levels in the Si-lnc group were significantly lower than those in the blank group of both RT4 and T24 cells (Fig. 3A). Meanwhile, the BrdU assay showed that cell proliferation levels in the Si-lnc group were dramatically decreased by 50% compared to that in the blank groups of RT4 and T24 cells (Fig. 3B). Subsequently, we checked the invasion ability of the cells; cell invasion levels in Si-lnc group were significantly downregulated by 80% in the RT4 cells and by 50% in the T24 cells compared to that in the blank group (Fig. 3C). Moreover, the expression of the pro-apoptotic protein Bax was dramatically increased in the Si-lnc group compared to the blank group of RT4 and T24 cells, while the anti-apoptotic protein Bcl-2 showed the opposite tendency (Fig. 3D). Thus, these results indicate that LINC00958 enhances cell proliferation and invasion, but suppresses apoptosis of bladder cancer cells.
LINC00958 sponges miR-490-3p in bladder cancer cells Next, we determined the interaction between LINC00958 and miR-490-3p. The binding site sequences of LINC00958 and miR-490-3p were predicted using star-Base (Fig. 4A). Next, we detected the interaction by luciferase activity assay, where the miR-490-3p mimics or negative control (NC), and psiCHECK2 LINC00958-wide type (lnc-WT) or psiCHECK2 LINC00958 mutant (lnc-MUT) were co-transfected into RT4 and T24 cells. The results showed that only the co-transfection of miR-490-3p mimic and lnc-WT group showed a 50% decrease in luciferase activity, suggesting that miR-490-3p indeed interacts with LINC00958 (Fig. 4B). We also found elevated levels of LINC00958 in cells overexpressing miR-490-3p by interacting with AGO2, suggesting that LINC00958 had coordinated with miR-490-3p in both RT4 and T24 cells (Fig. 4C). Additionally, as shown in Fig. 4D, we observed that the miR-490-3p expression was 50% lower in bladder cancer tissues than in adjacent normal tissues, and there was a negative correlation between LINC00958 and miR-490-3p levels in bladder cancer tissues (Fig. 4E). Furthermore, the miR-490-3p levels in RT4 and T24 cells were downregulated by over 50% compared to that in SV-HUC-1 cells (Fig. 4F). We further transfected siRNA-LINC00958 (Si-lnc), negative control (NC), and miR-490-3p inhibitor into RT4 and T24 cells. We found that the miR-490-3p level in the inhibitor groups was 70% lower than that in the blank groups; silencing LINC00958 led to a 1.5-fold increase in miR-490-3p levels and the miR-490-3p levels in the Si-lnc + inhibitor groups were similar to those in the blank groups (Fig. 4G). Thus, miR-490-3p is a downstream gene of LINC00958 in bladder cancer cells.

LINC00958 sponging miR-490-3p facilitates progression of bladder cancer
We further assessed whether LINC00958 regulates miR-490-3p to affect the progression of bladder cancer. First, we found that the cell viability in the miR-490-3p inhibitor group was significantly increased, while the cell viability in the Si-lnc + inhibitor group was similar to the blank group of both RT4 and T24 cells (Fig. 5A). In addition, cell proliferation in the miR-490-3p inhibitor group was elevated by 1.5-fold, while the cell proliferation in the Si-lnc + inhibitor group was similar to that in the blank group (Fig.  5B). Furthermore, the results of the transwell invasion assay showed that the cell invasion level in the miR-490-3p inhibitor group was significantly enhanced in RT4 and T24 cells, while the Si-lnc + inhibitor group presented an invasion level similar to that of the blank group of both RT4 and T24 cells (Fig. 5C). Moreover, cell apoptosis presented by the expression of pro-apoptotic protein Bax was significantly reduced in the miR-490-3p inhibitor group, while the Bax expression level in the Si-lnc + inhibitor group was similar to that of the blank groups of RT4 and T24 cells (Fig. 5D). Collectively, LINC00958 sponging miR-490-3p facilitates bladder cancer cell progression.

MiR-490-3p targets AURKA in bladder cancer cells
Subsequently, we sought to elucidate the interaction between miR-490-3p and AURKA. The binding site sequences of AURKA and miR-490-3p were predicted by TargetScan 7.2 (Fig. 6A). We further confirmed the interaction by luciferase activity assay, which showed that the co-transfection of miR-490-3p mimics and psiCHECK-2 AURKA 3′UTR WT plasmid showed a 50% decrease in luciferase activity compared to the NC groups, suggesting that miR-490-3p had a direct interaction with AURKA (Fig. 6B). Next, an RNA pull-down assay confirmed that miR-490-3p could bind to AURKA (Fig. 6C). In addition, miR-490-3p levels were enhanced by 3-fold in the tumor tissues compared to the normal tissues (Fig. 6D), and were negatively correlated with AURKA in bladder cancer tissues (Fig. 6E). Moreover, miR-490-3p was upregulated by approximately 3-fold in RT4 and T24 cells compared to SV-HUC-1 cells (Fig.  6F). Next, we treated RT4 and T24 cells with siRNA-AURKA (Si-AURKA), miR-490-3p inhibitor (inhibitor), and negative control (NC) to study the role of AURKA in bladder cancer. The siRNA-AURKA groups showed a 30% decrease in AURKA protein expression, while the AURKA protein expression in the inhibitor group was increased by nearly 1.3-fold when compared to that in the blank group. The levels observed in the Si-AURKA+ inhibitor groups were similar to those in the blank groups (Fig. 6G). Taken together, it can be concluded that miR-490-3p directly targets AURKA in bladder cancer cells.

MiR-490-3p targeting AURKA hampers bladder cancer cells progression
We further confirmed the biological action of miR-490-3p on AURKA in bladder cancer. First, we found that cell viability in the siRNA-AURKA group was dramatically impaired, while the cell viability in the Si-AURKA+ inhibitor group was similar to that in the blank groups of both RT4 and T24 cells (Fig. 7A). Additionally, cell proliferation in the siRNA-AURKA group decreased by 30%, while no significant difference was observed between the Si-AURKA+inhibitor group and blank groups in both RT4 and T24 cells (Fig. 7B). Moreover, the transwell invasion assay showed that cell invasion levels in the siRNA-AURKA group were decreased by 80% in the RT4 and 50% in the T24 cells, while the Si-AURKA+ inhibitor groups displayed similar cell invasion levels to blank groups in both RT4 and T24 cells (Fig. 7C). Finally, cell apoptosis levels in the siRNA-AURKA groups, indicated by the expression of the pro-apoptotic protein Bax, were evidently upregulated, while the expression levels in the Si-AURKA+inhibitor groups and the blank groups in both RT4 and T24 cells were similar (Fig. 7D).

Discussion
Our study confirmed that the expression of LINC00958 and AURKA was upregulated in bladder cancer, while the expression of miR-490-3p was downregulated. Furthermore, LINC00958 enhanced cell growth and invasion, but suppressed apoptosis of bladder cancer cells by inhibiting miR-490-3p and upregulating AURKA expression. Thus, the LINC00958-miR-490-3p-AURKA axis may be an effective therapeutic strategy for bladder cancer.
Multiple studies have suggested that miR-490-3p acts as a suppressor in most cancers, including hepatocellular carcinoma, gastric cancer, and breast cancer [51][52][53]. In bladder cancer, evidence has shown that miR-490-5p inhibits proliferation via G1-phase arrest of bladder cancer by targeting c-Fos [24]. Another study also revealed that upregulation of miR-490-5p led to decreased EGFR expression, hampering cell invasion in bladder cancer cells [25]. miR-490-3p and miR-490-5p are members of the family of miR-490, but the role of miR-490-3p in bladder cancer is unknown. Our study investigated the role of miR-490-3p in bladder cancer and found an obvious downregulation of miR-490-3p expression in bladder cancer. Inhibition of miR-490-3p elevated cell growth and suppressed apoptosis in bladder cancer. Additionally, miR-490-3p, sponged by LINC00958, could regulate its target gene AURKA, thereby inhibiting the progression of bladder cancer. Our study is the first to elucidate the role of miR-490-3p in bladder cancer.
Dysregulation of AURKA has been found in numerous cancers, including lung, cervical, and renal cancers, showing that it can facilitate cell growth and invasion, but represses cell apoptosis [54][55][56]. As referred to bladder cancer, Guo et al. [33] reported that AURKA expression was significantly elevated in bladder cancer tissues, and the high expression of AURKA presented a poor prognosis. Another study indicated that AURKA targeted by miR-124-3p could enhance proliferation and migration and impair apoptosis of bladder cancer cells [35,53]. Consistent with previous reports, a similar molecular mechanism of AURKA in bladder cancer was revealed in our study; high level of AURKA was also observed in bladder cancer, and the downregulation of AURKA could hamper bladder cancer progression. However, to investigate the mechanism of miR-490-3p/ AURKA on the proliferation, migration, and apoptosis of cancer cells, Si-AURKA and miR-490-3p inhibitor were transfected into RT4 and T24 cells. The results showed that cell proliferation and migration were increased and apoptosis was decreased after transfection with the miR-490-3p inhibitor. In addition, Si-AURKA transfection inhibited cell proliferation and migration and promoted apoptosis. Therefore, we speculated that the effect of the inhibitor and Si-AURKA on the cancer cells was offset by each other. Hence, AURKA could be targeted by miR-490-3p to affect bladder cancer progression. Finally, we further elucidated that LINC00958 facilitated bladder cancer progression via the miR-490-3p/AURKA axis. Thus, this study is the first to report that AURKA could be regulated by both LINC00958 and miR-490-3p in bladder cancer progression.
As previously reported, evidence showed that lncRNA RP11-480I12.5 promotes the progression of breast cancer cells through the miR-490-3p-AURKA-Wnt/β-catenin axis [53]. Therefore, whether the Wnt/β-catenin pathway or other pathways participate in the LINC00958/miR-490-3p/AURKA axis in bladder cancer needs further exploration. Meanwhile, the role of the LINC00958/miR-490-3p/AURKA axis in bladder cancer also needs to be proven both in vivo and clinically. In addition, this study screened the differentially expressed miRNAs and mRNAs in bladder cancer using GEO DataSets, and the targets of LINC00958, miR-490-3p, and AURKA will be further explored in the future using multiple databases such as TCGA and GEPIA.

Conclusions
Taken together, our findings indicate that LINC00958 aggravates cell growth and invasion, but hampers apoptosis of bladder cancer cells via the miR-490-3p/AURKA axis. This may provide a new direction for the clinical treatment of bladder cancer patients.
analysis and interpretation of data. All authors read and approved the manuscript.

Funding
Funding information is not available.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.