Long non-coding RNA LINC01234 regulates proliferation, migration and invasion via HIF-2α pathways in clear cell renal cell carcinoma cells

Introduction

In 2018, it was predicted that 403,262 new cases would be diagnosed with kidney cancer in 185 countries and 175,098 cases would be dead (Bray et al., 2018). In 36 kinds of cancers, the morbidity and mortality of kidney cancer were 2.2% and 1.8% respectively (Bray et al., 2018). Clear cell renal cell carcinoma (ccRCC) is the most common subtype of RCC and it accounts for 75% (Song et al., 2018). Although surgery is still the preferred therapeutic option for the localized and locally advanced ccRCC, the long-term prognosis remains unsatisfactory and unpredictable. Current existed evaluation approach for prognosis of ccRCC is mainly based on clinicopathologic data, such as TNM staging. However, it does not reflect the biological heterogeneity of cancer (Cheng, 2018). Therefore, there is an urgent need for discovering a new prognostic model and biomarkers for ccRCC. Moreover, it is necessary to understand the molecular mechanisms of the prognostic biomarkers underlying ccRCC development.

Long non-coding RNA (lncRNA) is a kind of RNA transcripts with a length of >200 nucleotides. Unlike mRNA, it does not encode proteins. Currently, it is reported that lncRNA is engaged in numerous important biological processes and the development and progression of numerous human diseases, including ccRCC (Esteller, 2011; Gupta et al., 2010; Jin et al., 2017; Martens-Uzunova et al., 2014; Ponting, Oliver & Reik, 2009; Quinn & Chang, 2016; Rinn & Chang, 2012). The overexpression, deficiency or mutation of lncRNA are associated with the tumor formation, progression, metastasis and prognosis in many human malignancies including ccRCC (Esteller, 2011; Ghaffar et al., 2018; Gupta et al., 2010; He et al., 2018; Jin et al., 2017; Martens-Uzunova et al., 2014). However, the functions of the majority of lncRNA are not well understood. In our study, we constructed a lncRNA-based prognostic model and identified six lncRNAs as independent prognostic biomarkers in ccRCC, including LINC01234.

Several studies showed that LINC01234 was upregulated and had oncogenic potentials in several cancers, such as gastric cancer (Chen et al., 2018), esophageal cancer (Ghaffar et al., 2018), and colorectal adenocarcinoma (He et al., 2018). Most ccRCC are associated with loss of von Hippel-Lindau tumor suppressor (pVHL) function and deregulation of hypoxia pathways (Schödel et al., 2016). Adaptation to hypoxia plays an important role in the progression of ccRCC (Garje et al., 2018). Hypoxia is mediated via hypoxia-inducible factors (HIFs) HIF-1α and HIF-2α (Semenza, 2012). Recently, studies showed that HIF-2α, rather than HIF-1α, was a predominant driver in renal cancer progression (Keith, Johnson & Simon, 2011). Although HIF-1α can act as a ccRCC tumor suppressor, HIF-1α activity is commonly diminished by chromosomal deletion in ccRCC (Schödel et al., 2016). Conversely, HIF-2α has emerged as the key HIF isoform acting as an oncogene that is essential for ccRCC tumor progression (Meléndez-Rodríguez et al., 2018; Schödel et al., 2016). The polymorphisms at the HIF-2α gene locus predispose to the development of ccRCC, and HIF-2α promotes tumor growth (Schödel et al., 2016). Indeed, preclinical and clinical data have shown that pharmacological inhibitors of HIF-2α can efficiently inhibit ccRCC growth (Meléndez-Rodríguez et al., 2018). However, the role of LINC01234, as well as the relationship between LINC01234 and HIF-2α in ccRCC remains unclear. In the present study, we showed that LINC01234 was likely to regulate the progression of ccRCC by HIF-2α pathways. Therefore, LINC01234 might serve as a promising prognostic biomarker and a potential therapeutic target for patients with ccRCC.

Materials and Methods

Results

Identification of differentially expressed lncRNAs and key prognostic lncRNAs in patients with ccRCC

A total of 70 normal tissue samples and 541 cancer tissue samples from patients with ccRCC were collected. A total of 11,368 lncRNAs were extracted from the transcriptome profiling. Compared with the normal tissues, a total of 1541 lncRNAs were identified as differentially expressed lncRNAs in tumor tissues (FDR < 0.05 and |log₂FC| > 2), including 1075 upregulated (log₂FC > 2) and 466 downregulated (log₂FC < −2) lncRNAs (Fig. 1A) (Table S2). Preliminarily, a total of 323 statistically significant lncRNAs were considered to be related to the prognosis by the univariate cox regression (Table S3). Next, through the LASSO regression, 13 lncRNAs were identified as key prognostic lncRNAs (Figs. 1B and 1C), which were used for the further establishment of the risk score model by multivariate cox regression.

Establishment and evaluation of the prognostic model in ccRCC

The median cutoff point of the risk scores calculated by multivariate cox regression was 0.842. All patients were divided into the high-risk group and low-risk group. It was revealed that the patients in the high-risk group had a significantly worse overall survival (OS) rate than those in the low-risk group (p < 0.001) (Fig. 2A). The AUC was 0.753 (3-year ROC curve) and 0.784 (5-year ROC curve) respectively, and the C-index was 0.753 (Fig. 2B). In addition, it also presented the relationship between the survival time and the risk score for patients (the death and the alive) (Fig. 2C). Moreover, a heatmap was plotted to illustrate the expression levels of the key prognostic lncRNAs in the high-risk group and low-risk group (Fig. 2D).

Identification of independent prognostic biomarkers

The multivariate cox regression revealed HR and 95% CI for the 13 key prognostic lncRNAs with a forest plot (Fig. 3). It indicated six statistically significant lncRNAs as the independent prognostic biomarkers, including lncRNAs AC009654.1, AC012615.3, AC092490.2, AL357507.1, LINC01234 and LINC01956. Moreover, K–M survival analysis was performed for the six lncRNAs. It revealed that all the six lncRNAs with high expression levels predicted a significantly worse OS rate than the low expressed one (Figs. 4A–4F). Therefore, they could serve as the adverse independent prognostic factors. Furthermore, we validated the expression levels and prognostic significance of the six lncRNAs in patients with ccRCC via GEPIA server. It suggested that AL357507.1, LINC01234, and LINC01956 were highly expressed at higher pathological stage of the disease, while LINC01234 exhibited the most significance in terms of expression at different pathological stage of the disease (Figs. 5A–5F). Moreover, GEPIA server revealed the significance of LINC01234 in terms of survival time. The high expression level of LINC01234 predicted a significantly worse disease-free survival rare or OS rate than the low expressed one (Figs. 5G and 5H). Unfortunately, GEPIA server could not provide the prognostic significance of the other five lncRNAs because the server showed the sample size was insufficient.

LINC01234 knockdown suppressed the proliferation and clone formation of ccRCC cells

Knockdown of LINC01234 was performed in Caki-2 and A498 cells by the lentivirus-mediated shRNA transfection. It suggested that the expression of LINC01234 was reduced in Caki-2 and A498 cells, which was validated by qPCR (Fig. 6A). Next, the CCK-8 assay revealed the proliferations of Caki-2 and A498 cells were significantly suppressed (Fig. 6B and 6C). Moreover, cell colony formation assay was performed to analyze the role of LINC01234 in the colony formation of Caki-2 and A498 cells. As shown in Figs. 6D–6I, the clonogenic capacities of Caki-2 and A498 cells were dramatically inhibited. Obviously, it indicated that LINC01234 played an important role in the proliferation and colony formation of Caki-2 and A498 cells.

LINC01234 depletion inhibited the migration and invasion of ccRCC cells

The migration capabilities of Caki-2 and A498 cells were assessed by Transwell migration assay, while the invasion capabilities of these cells were assessed by Transwell Matrigel invasion assay. The results of the Transwell assay indicated that LINC01234 knockdown significantly inhibited the migration capabilities of Caki-2 and A498 cells (Figs. 7A–7F). Similarly, the invasion capabilities of Caki-2 and A498 cells were also suppressed following LINC01234 depletion (Figs. 7G–7L). These findings demonstrated that LINC01234 played an important role in the migration and invasion capacities of ccRCC cells.

LINC01234 knockdown suppressed EMT process in ccRCC cells

EMT process was closely related to the migration and invasion of cancer cells. Therefore, the mRNA levels of EMT-associated genes and the levels of EMT-associated proteins were detected by RT-PCR and western blots respectively. As shown in Figs. 8A and 8B, we found that the mRNA level of epithelial marker E-cadherin was increased, while the mRNA level of mesenchymal marker N-cadherin was decreased in Caki-2 and A498 cells following LINC01234 knockdown. Similarly, as shown in Fig. 8C, the protein expression levels of mesenchymal markers Vimentin and N-cadherin were significantly decreased in Caki-2 and A498 cells with LINC01234 knockdown, while the protein expression level of epithelial marker E-cadherin was upregulated. Moreover, the protein expression level of the transcription factor Snail was decreased in Caki-2 and A498 cells with LINC01234 knockdown. In addition, the protein expression level of β-catenin was also inhibited in Caki-2 and A498 cells following LINC01234 depletion.

Discussion

LncRNA was a kind of long RNA transcripts (>200 nucleotides) and it had no apparent protein-coding potentials (Quinn & Chang, 2016). Even so, lncRNA possessed a wide range of biological functions involved in multiple vital cellular activities (Shen et al., 2015). Generally, lncRNA achieved its function by regulating gene expression in the levels of epigenetics, transcription and post-transcription (Lee, 2012; Wang & Chang, 2011). It could serve as a molecular signal, a molecular decoy, a molecular guide, or a molecular scaffold to achieve its functions (Wang & Chang, 2011). The function of lncRNA was associated with its subcellular localization (Wang & Chang, 2011). More specifically, lncRNA might be involved in chromatin regulation, gene transcription and alternative splicing of transcripts when it was in nucleus, while if it was in cytoplasm, it might serve as a competing endogenous RNA (ceRNA), and regulated the stability or translation of mRNA (Yang et al., 2019).

Recently, more and more evidences indicated that aberrations of lncRNA, such as overexpression, deficiency or mutation, played an important role in malignant phenotypes of cancers (Schmitt & Chang, 2016), including tumor formation, progression, metastasis and poor prognosis (Esteller, 2011; Gupta et al., 2010; Martens-Uzunova et al., 2014; Yu et al., 2017; Yue et al., 2016). Some aberrant lncRNAs were also associated with lots of malignant biological behaviors of cancer cells, such as proliferation, apoptosis, migration and invasion (Ellinger et al., 2016; Huang et al., 2017; Yue et al., 2016). It was also reported that some aberrant lncRNAs could serve as prognostic indicators in ccRCC, such as lncRNA Fer1L4 (Cox et al., 2020). With the development of molecular biological techniques and bioinformatics, more and more lncRNAs were marked as novel biomarkers and prognostic signatures for ccRCC utilizing TCGA database. For example, lncRNA Fer1L4 was overexpressed in ccRCC tissues, and its high expression levels were found in higher grade, higher stage, and metastatic tumors (Cox et al., 2020). LncRNA Fer1L4 overexpression was also an independent prognostic factor for patients with ccRCC (Cox et al., 2020). It was also reported an 11-lncRNA signature (AC245100.1, AP002761.1, LINC00488, AC017033.1, LINC-PINT, COL5A1-AS1, AC026471.4, AL009181.1, LINC00524, HOTTIP, AL078590.3) and a six-lncRNA signature (CTA‑384D8.35, CTD‑2263F21.1, LINC01510, RP11‑352G9.1, RP11‑395B7.2, RP11‑426C22.4) were clearly linked to the OS rate of ccRCC patients based on TCGA database (Zeng et al., 2019; Zhang et al., 2019).

In the present study, utilizing the TCGA database, we identified 1,541 differentially expressed lncRNAs. More importantly, we not only constructed a 13-lncRNA-based risk score model with moderate accuracy, but also identified six independent adverse prognostic lncRNAs for patients with ccRCC, including lncRNA AC009654.1, AC012615.3, AC092490.2, AL357507.1, LINC01234 and LINC01956. It was similar to a recent study which suggested that lncRNA AC009654.1, AC092490.2, LINC00524, LINC01234 and LINC01885 were significantly associated with ccRCC prognosis (Zhang et al., 2020). The expression levels of the six lncRNAs above were upregulated in ccRCC tissues and the high expression levels of them predicted a worse OS in ccRCC patients. Furtherly, we investigated their expression levels at different pathological stages and validated their prognostic significance in ccRCC patients via GEPIA server. It revealed AL357507.1, LINC01234, and LINC01956 were highly expressed at higher pathological stages of the disease, while LINC01234 exhibited the highest significance in terms of expression at different pathological stages of the disease. It was a very interesting finding, because the pathological stage was closely associated with the prognosis of ccRCC patients. Moreover, GEPIA server revealed the significance of LINC01234 in terms of survival time. Unfortunately, GEPIA server could not provide the prognostic significance of the other five lncRNAs because the server showed the sample size was insufficient. Besides, I also referred to the recent studies and references about these six lncRNAs. Nevertheless, except limited researches for LINC01234, there are no investigations for them currently and it deserves to further researches. Therefore, we mainly focused on lncRNA LINC01234 for the subsequent experiments.

Recently, partial functions and mechanisms of LINC01234 (also known as LCAL84) were reported in cancers, such as gastric cancer (Chen et al., 2018), esophageal cancer (Ghaffar et al., 2018), and colorectal adenocarcinoma (He et al., 2018). LINC01234 was upregulated and had oncogenic potentials in esophageal carcinoma cells in vitro (Ghaffar et al., 2018; He et al., 2018). LINC01234 was significantly associated with the prognosis of colorectal adenocarcinoma and the malignant biological behaviors of esophageal carcinoma cells including proliferation, migration, invasion and apoptosis (Ghaffar et al., 2018; He et al., 2018). Besides, LINC01234 expression was significantly upregulated in gastric cancer tissues and was associated with larger tumor size, advanced TNM stage, lymph node metastasis, and shorter survival time (Chen et al., 2018). Moreover, LINC01234 could serve as ceRNA to regulate core-binding factor β (CBFB) expression by sponging miR-204-5p to regulate the apoptosis, growth arrest and tumorigenesis in gastric cancer (Chen et al., 2018). In our study, we also explored the role of LINC01234 in ccRCC. It indicated that LINC01234 expression was upregulated in ccRCC tissues. LINC01234 was expressed increasingly as the stage increased. The high expression level of LINC01234 predicted a significantly worse disease-free survival rate or OS rate than the low expressed one for the patients with ccRCC. Besides, LINC01234 knockdown inhibited proliferation, migration, invasion and EMT process of ccRCC cells. More importantly, LINC01234 knockdown impaired the expression of HIF-1a, HIF-2a, VEGFA, EGFR, c-Myc, Cyclin D1 and MET in Caki-2 and A498 cells following LINC01234 depletion.

EMT was considered as an essential process during development whereby epithelial cells acquired mesenchymal, fibroblast-like characteristics and displayed reduced intracellular adhesion and increased motility (Aigner et al., 2007; Moreno-Bueno, Portillo & Cano, 2008). EMT played a critical role in in the progression of primary tumors towards spread and metastasis, as well as the migration and invasion of malignant tumor cells (Gloushankova, Zhitnyak & Rubtsova, 2018; Peinado, Olmeda & Cano, 2007; Yang et al., 2018). Recently, an increasing number of studies supported the role of lncRNAs in the regulation of tumor progression and metastasis through the regulation of EMT (Gugnoni & Ciarrocchi, 2019). In the carcinogenic progression, downregulation of cell-adhesion molecules like epithelial cadherins, occludins, claudins, certain cytokeratins, and ZO-1 together with the coordinated upregulation of mesenchymal cadherins, vimentin, fibronectin and β1 and β3 integrins, promoted loss of cell-cell adhesion and apico-basal polarity and acquisition of invasive and migratory capacity (Gugnoni & Ciarrocchi, 2019; Lu & Kang, 2019). A group of transcription factors including Snail, Slug, Twist, zinc finger E-box-binding homeobox 1 and 2 (ZEB1, ZEB2) were well known to regulated EMT process partially or completely (Gugnoni & Ciarrocchi, 2019; Yang et al., 2018). Therefore, we detected the mRNA levels of EMT-associated genes and the expression levels of EMT-associated proteins in ccRCC cells by qPCR and western blots respectively following LINC01234 knockdown. It revealed that the mRNA level of epithelial marker E-cadherin was increased, while the mRNA level of mesenchymal marker N-cadherin was decreased in Caki-2 and A498 cells following LINC01234 knockdown. The protein expression levels of the transcription factor Snail and the epithelial markers N-cadherin and Vimentin were reduced, while the protein expression level of the mesenchymal marker E-cadherin was up-regulated in A498 and Caki-2 cells with LINC01234 knockdown. These findings indicated that the function of LINC01234 was associated with EMT process. EMT was impaired after LINC01234 knockdown. In addition, we also found the inhibition of β-catenin pathway contributed to the EMT impairment after LINC01234 depletion. All these evidences suggested that LINC01234 knockdown could inhibit the cell proliferation, migration and invasion, as well as EMT process in ccRCC. During EMT process, LINC01234 knockdown might suppress the expression of transcription factor Snail, and further stimulate the expression of E-cadherin, and inhibit the expressions of Vimentin and N-cadherin, which might result in a inhibition of malignant biological behaviors of ccRCC cells, such as cell proliferation, migration and invasion.

Hypoxia could induce ccRCC cells to undergo EMT, angiogenesis and metastasis (Meléndez-Rodríguez et al., 2018; Zhang et al., 2017). Adaptation to a hypoxic environment played an important role in the progression of ccRCC (Garje et al., 2018). Hypoxia was mediated via HIFs (Semenza, 2012). Previously, HIF-1α was supposed to be a key oncogenic factor, but recent evidence showed HIF-2α was a predominant driver in renal cancer progression (Keith, Johnson & Simon, 2011). Currently, HIF-1α is supposed to be a ccRCC tumor suppressor, but the activity of HIF-1α is commonly diminished by chromosomal deletion in ccRCC (Schödel et al., 2016). Conversely, HIF-2α has emerged as an oncogene that is essential for ccRCC tumor progression (Meléndez-Rodríguez et al., 2018; Schödel et al., 2016). The polymorphisms at the HIF-2α gene locus predispose to the development of ccRCC, and HIF-2α can promote tumor growth (Schödel et al., 2016). Indeed, preclinical and clinical data have shown that pharmacological inhibitors of HIF-2α can efficiently inhibit ccRCC growth (Meléndez-Rodríguez et al., 2018). HIF-2α was found to be more sensitive to moderate hypoxia and showed more enduring expression in hypoxic conditions (Zhang et al., 2017). HIF-2α could translocate to the nucleus and bind to the hypoxia response elements (Garje et al., 2018). This binding resulted in the expression of several target genes involved in angiogenesis, proliferation, migration and invasion of cancer cells, such as VEGFA, EGFR, c-Myc, Cyclin D1 and MET (Garje et al., 2018). VEGFA played an important role in the formation of blood vessels, which was closely associated with carcinogenesis (Shi et al., 2019). In ccRCC, as a well-known target of HIF-2α, VEGFA also played a vital role in angiogenesis and was a key target of anti-cancer therapeutic agents (Garje et al., 2018; Meléndez-Rodríguez et al., 2018). Besides, EGFR, c-Myc and Cyclin D1 were associated with ccRCC cell cycle and proliferation (Meléndez-Rodríguez et al., 2018). EGFR signaling could also influence ccRCC patient survival (Meléndez-Rodríguez et al., 2018). Moreover, MET was related to ccRCC metastasis (Meléndez-Rodríguez et al., 2018). Based on this above, we detected HIF-2α pathways after LINC01234 depletion. It revealed that the mRNA levels of HIF-2α and VEGFA were decreased in A498 and Caki-2 cells with LINC01234 knockdown. Similarly, the expression levels of proteins HIF-2α, VEGFA, EGFR, c-Myc, Cyclin D1 and MET were reduced in A498 and Caki-2 cells with LINC01234 knockdown. In our study, LINC01234 was expressed increasingly as the stage increased and its high expression level predicted a significantly worse disease-free survival rate or OS rate for the patients with ccRCC. LINC01234 knockdown suppressed cell proliferation, migration and invasion of ccRCC cells. Combined with all these findings above, it suggested that LINC01234 knockdown might suppress the expression of HIF-2α, and then inhibit the expression of VEGFA, EGFR, c-Myc, Cyclin D1 and MET, which might further inhibit the proliferation, metastasis and then influence the survival of ccRCC patient.

Unfortunately, there was several limitations in our study. Firstly, the function of lncRNA was associated with its subcellular localization, but we did not identify the subcellular localization of LINC01234 in ccRCC cell lines. Secondly, although LINC01234 functioned as ceRNA to regulate CBFB expression by sponging miR-204-5p in gastric cancer, we did not identify any miRNAs as direct targets of LINC01234 to investigate whether LINC01234 was a ceRNA for miRNAs in ccRCC. It deserves to more investigations.

Conclusions

In summary, we constructed a lncRNA-based prognostic model with moderate accuracy and identified LINC01234 as an independent prognostic biomarker in ccRCC. Moreover, LINC01234 knockdown might inhibit the proliferation and metastasis of ccRCC cells by suppressing HIF-2α pathways. Therefore, LINC01234 might serve as a promising prognostic biomarker and a potential therapeutic target for patients with ccRCC.

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