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Comprehensive analysis of lncRNAs as biomarkers for diagnosis, prognosis, and treatment response in clear cell renal cell carcinoma

Clear cell renal cell carcinoma (ccRCC) is the most common histological type of renal carcinoma and has a high recurrence rate and poor outcome. Accurate patient risk stratification based on genetic markers can help to identify the high-risk patient for early and further treatments and would promote patient survival. Long non-coding RNAs (lncRNAs) have attracted widespread attention as biomarkers for early diagnosis, treatment, and prognosis because of their high specificity and sensitivity. Here, we performed a systematic search in NCBI PubMed and found 44 lncRNAs as oncogenes, 18 lncRNAs as tumor suppressors, 199 lncRNAs as diagnostic biomarkers, 62 lncRNAs as prognostic biomarkers, and 3 lncRNAs as predictive biomarkers for ccRCC. We also comprehensively discuss the biological functions and molecular regulatory mechanisms of lncRNAs in ccRCC. Overall, the present study is a systemic analysis to assess the expression and clinical value of lncRNAs in ccRCC, and lncRNAs hold promise to be diagnostic, prognostic, and predictive biomarkers.


INTRODUCTION
Kidney cancer ranks as the 16th most common cause of cancer death worldwide, and there were 179,368 kidney cancer-related deaths and 431,288 new cases in 2020. 1 Clear cell renal cell carcinoma (ccRCC) accounts for 70%-80% of all kidney cancers. 2,3 Recent advances in imaging technology and increased use of screening modalities contribute to the detection of ccRCC at an early stage and promote the prognosis through partial nephrectomy. However, 30% of patients with this disease present metastasis at the time of diagnosis because of prior insipidity or absence of symptoms. 4 In addition, for ccRCC patients with metastasis or recurrence, the sensitivity to chemotherapy and radiotherapy is generally low, and the effect of targeted therapy including sunitinib, sorafenib, pazopanib, aldesleukin, and temsirolimus varies from person to person. The 5-year survival rate of metastatic ccRCC patients is just 8%-11.7%. 5,6 Hence, it is urgent for us to investigate the molecular mechanisms and identify new, sensitive, and reliable biomarkers for diagnosis and prediction of treatment response and prognosis in ccRCC, which can enhance the survival probability of patients.
Long non-coding RNAs (lncRNAs) are a type of non-coding RNA (ncRNA) >200 nucleotides in length and pervasively transcribed in the human genome, having high tissue specificity. 7-10 There were 9,640 lncRNA loci in the human genome according to a report by the ENCODE Project Consortium in 2012, and the number continues to increase. 8,11 lncRNAs play essential functions in virtually every aspect of cell biology, including cellular differentiation, proliferation, DNA damage response, dosage compensation, chromosomal imprinting, transcriptional regulation in cis or trans, nuclear domain organization, and so on. 12,13 Moreover, lncRNAs are emerging as new players in cancer and have regulatory functions in tumorigenesis, metastasis, and drug resistance with frequently abnormal expression. 14 The important function of lncRNAs in regulating gene expression is related to their complicated structures, because lncRNAs have a complex secondary and tertiary structure, which endows the abilities to bind protein, RNA, and/or DNA partners. Consequently, they possess several regulatory capacities. 15 Accordingly, dysregulated lncRNAs can contribute to various pathological events, including cancer initiation and progression. 16 For instance, HOTAIR overexpression promoted breast cancer metastasis as seen in in vivo assay by changing the cell expression profile, favoring metastasis. 17 Several lncRNAs were also found abnormally expressed in ccRCC and were involved in initiation and progression of ccRCC. [18][19][20][21][22] Additionally, dysregulated lncRNAs may be used as novel diagnostic, prognostic, and predictive biomarkers in ccRCC. 23-25 Therefore, we systematically analyzed the potential roles of these dysregulated lncRNAs as diagnostic, treatment response predictive, and prognostic biomarkers, their functions, and their molecular mechanisms in ccRCC.

RESULTS
A flowchart showing the selection process of lncRNA identification, inclusion, and exclusion criteria is presented in Figure 1. In brief, the initial records were 1,243 studies in the NCBI PubMed database, and of these a total of 939 duplicate studies were excluded. Next, we excluded 154 records after reading the titles and abstracts according to the inclusion and exclusion criteria (Figure 1). Then, we read and checked carefully the full texts of the remaining 150 publications. Accordingly, 54 publications out of these were excluded on the basis of the exclusion criteria ( Figure 1). In total, 96 publications were finally included in this systematic review. Among the 96 publications, 21 publications were about lncRNAs as competing endogenous RNAs (ceRNAs), 59 publications about oncogenes, 19 publications about tumor suppressors, 93 publications about diagnostic biomarkers, 65 publications about prognostic biomarkers, and 3 publications about therapeutic predictive biomarkers.
Regulatory mechanisms of lncRNAs in cancer lncRNAs are subject to fine-tuned regulation, ranging from epigenetic to posttranscriptional regulation. 26,27 Genetic and epigenetic changes are mainly responsible for the abnormal expression of lncRNAs. 28,29 Studies have shown that small-and large-scale mutations affect non-coding regions of the genome, including chromosomal translocations, copy number alterations, nucleotide expansions, and single-nucleotide polymorphisms (SNPs). 30 Dozens of lncRNAs abnormally expressed in cancers can also be regulated by specific oncogenic and tumor suppressor-related signals and regulatory factors such as Sp1, p53, and linc-p21. 31 In addition, lncRNAs can regulate protein, RNA, and/or DNA partners by forming a complicated network responsible for cancer initiation and development. 16 Therefore, the mechanisms for lncRNA regulation in ccRCC are complicated.
Among all the mechanisms, the ceRNA network is attracting increasing attention. In the ceRNA network, lncRNAs act as ceRNAs or natural microRNA (miRNA) sponges; they can bind to miRNAs through their miRNA binding sites (also referred to as miRNA response elements [MRE]), thereby de-repressing the target genes' mRNAs of the respective miRNAs. This indicates that these lncRNAs serve as posttranscriptional regulators of gene expression. 32 Accordingly, the ceRNA network in ccRCC is systematically summarized in Among these 19 lncRNAs, MALAT1, HOTAIR, and PCAT1 were reported to bind to two relevant miRNAs, respectively. MALAT1 functions as a ceRNA by binding miR-200s to regulate ZEB2 expression and also sponges miR-194-5p9 to regulate ACVR2B expression. 33,34 Similarly, HOTAIR regulates ST8SIA4 expression by binding to miR-124 20 and regulates HIF-1a expression by binding to miR-217. 18 PCAT1 sponges miR-539 and miR-656 for YAP, the same target. 35 From the intersection of the ceRNA network, two lncRNAs, MALAT1 and PVT1, are found to bind the same miRNA, miR-200s, in our ceRNA network ( Figure 2). MALAT1 binds miR-200s to regulate ZEB2 expression, whereas PVT1 binds miR-200s to regulate ZEB1 and BMT1 besides ZEB2 ( Figure 2). 22,33 lncRNA functions in ccRCC Fourteen hallmarks of cancer have been reported: evading growth suppressors, avoiding immune destruction, inducing angiogenesis, enabling replicative immortality, sustained proliferative signaling, resisting cell death, deregulating cellular epigenetics, activating invasion and metastasis, genome instability and mutation, tumor promoting inflammation, dedifferentiation and transdifferentiation, epigenetic dysregulation, altered microbiome, and altered neuronal signaling. 36,37 More and more lncRNAs have been reported to play critical roles as tumor oncogenes or tumor suppressors, involved in the cancer hallmark process of initiation, growth, and metastasis in ccRCC ( Figure 4; Table  S1). The dysregulated lncRNAs involved in the above processes and their molecular mechanisms in ccRCC are summarized in Figure 3 and Figure 4.

lncRNAs as oncogenes in ccRCC
Forty-four lncRNAs with abnormal expression were reported as oncogenes in 59 publications and were related to cell cycle, proliferation, apoptosis, migration, invasion, metastasis or epithelial-mesenchymal transition (EMT) in ccRCC ( Figure 4). Among them, MALAT1, PVT1, HOTAIR, and LUCAT1 were explored in more than 2 studies, and the corresponding reporting frequency of each lncRNA was There was also a direct interaction between MALAT1 and Livin protein. MALAT1 regulated and increased Livin levels by enhancing the stability of the protein via MALAT1 protein interaction, which pro-moted cell proliferation and suppressed cell apoptosis. 38 In addition, other studies identified that MALAT1 plays important roles in promoting cell cycle, invasion, and EMT (Figures 3 and 4; Table  S1). 38,39,44,45 PVT1 was found upregulated in ccRCC, and its high expression was associated with a shorter overall survival time. 42 In addition, PVT1 expression was closely correlated with TNM stage, Fuhrman grade, lymph node metastasis, and tumor size. Ren et al. 21 reported that PVT1 promotes proliferation, invasion, and EMT of ccRCC through downregulation of miR-16-5p. Moreover, PVT1 can inhibit ccRCC cell apoptosis by upregulating Mcl-1. 41 Correspondingly, knockdown of PVT1 induced apoptosis and cell cycle arrest in ccRCC, which was associated with the epidermal growth factor receptor pathway (Figures 3 and 4; Table S1). 42 It has been shown that HOTAIR promotes the development and progression of ccRCC. Hu et al. 19 found that increased expression of HOTAIR predicts a poor prognosis of ccRCC after surgery and HOTAIR can promote RCC cell proliferation and growth in vitro and in vivo. Hong et al. 18 demonstrated that HOTAIR facilitates ccRCC proliferation, migration, and the EMT process and inhibits apoptosis and that HOTAIR knockdown suppresses tumor growth. Furthermore, the migration of ccRCC cell was promoted by increased HOTAIR, which upregulated insulin growth factor-binding protein   Table S1).
LUCAT1 expression was significantly upregulated in tumor tissues compared with matched adjacent non-tumor tissues, promoting renal cancer cell proliferation, and its expression level was also associated with cancer grade, pathological stage, and survival time. 24 Additionally, Wang et al. 23 reported that LUCAT1 promotes proliferation and invasion of ccRCC cells by negatively regulating miR-495-3p. Zheng et al. 25 showed that LUCAT1 promotes proliferation and invasion through the AKT/GSK-3b signaling pathway in ccRCC (Figures 3 and 4; Table S1).

lncRNAs as tumor suppressors in ccRCC
Apart from roles as oncogenic lncRNAs in ccRCC, some lncRNAs, as tumor suppressors, have been proven to have significant regulatory effects on inhibiting cell proliferation and invasion in ccRCC. There are 18 lncRNAs described as tumor suppressors in 19 publications (Figures 3 and 4). Among them, MEG3 was researched in more than 2 studies. MEG3 expression was significantly decreased in ccRCC tissues and cells, and upregulating MEG3 expression effectively inhibited proliferation, accelerated cell apoptosis, and induced the G0/G1 phase cell cycle by increasing the expression of RASL11B. 47 Similarly, BANCR, considered as a tumor inhibitor, was significantly decreased in ccRCC, and its low expression was associated with poor prognosis. Moreover, BANCR overexpression inhibited the proliferation, migration, and invasion capacity of ccRCC, meanwhile, apoptosis was increased and G1 cell cycle arrest was induced in vitro. 48 Other lncRNAs like GAS5, LOC389332, SARCC, and TCL6 were reported as tumor suppressors in ccRCC as well. However, they are not drawn or discussed in more detail here because of the lack of in-depth mechanisms in published articles. [49][50][51][52] Clinical applications of lncRNAs in ccRCC Characterization of lncRNAs in the initiation and progression of ccRCC would be definitely beneficial for cancer diagnosis and therapy. The discovery of sensitive and specific biomarkers for ccRCC will facilitate early detection and improve current clinical management of ccRCC.

Diagnostic biomarkers in ccRCC
The lncRNA expression in ccRCC was analyzed in 93 studies. A total of 199 differentially expressed lncRNAs between ccRCC and healthy controls are shown in Table S2.

Prognostic biomarkers in ccRCC
Sixty-five studies analyzed the correlation between lncRNAs and prognosis of ccRCC patients. A total of 62 lncRNAs were significantly correlated with the prognosis of ccRCC patients (Table S3).
Among them, PVT1, MALAT1, and LUCAT1 had significant changes in more than 2 studies (Table S3). Increased expression of PVT1, LUCAT1 and MALAT1 were all associated with poor prognosis. PVT1 was the lncRNA most frequently studied as prognostic biomarker, and 5 publications indicated that high expression of PVT1 was associated with poor overall survival (OS) and diseasefree survival (DFS). 21,22,41,42,53 LUCAT1 was also a poor prognostic factor, and higher expression of LUCAT1 was related to a poorer OS and DFS in 3 studies. 23-25 MALAT1 was an important prognostic biomarker in 3 studies. The expression of MALAT1 was significantly higher in ccRCC with poor prognosis. 34,39,45 The other 59 lncRNAswere also markedly correlated with the prognosis of ccRCC patients (Table S3).

Treatment response-predictive biomarkers in ccRCC
Three studies focused on the role of lncRNAs as biomarkers for predicting therapeutic response in ccRCC, and the results showed that 3 lncRNAs including ADAMTS9-AS2, GAS5 and SARCC, were predictive biomarkers for drug treatment. However, each lncRNA was only analyzed in one study, without replicated results (Table S4).
All 3 lncRNAs were found to be associated with a favorable response to therapy. They can serve as useful biomarkers for drug effectiveness. ADAMTS9-AS2 attenuated the susceptibility of ccRCC cells to chemotherapy drugs such as cisplatin. 58 GAS5 was found to sensitize renal cell carcinoma to sorafenib. 59 In addition, SARCC was a key mediator to influence sunitinib efficacy, and enhancing the expression of SARCC was a novel therapeutic approach to enhance sunitinib efficacy in ccRCC treatment. 60

DISCUSSION
With the development of high-throughput sequencing technology and bioinformatics, lncRNA dysregulation has been found to be related to the initiation and progression of various types of cancers including ccRCC and is involved in multiple biological behaviors of cancer, including cell proliferation, apoptosis, migration, and metastasis. 61 Previous reviews and meta-analyses have reported the prognostic values of lncRNAs in multiple cancers, such as colorectal cancer, ovarian cancer, lung cancer, etc. 62,63 However, no one investigated ccRCC specifically. Therefore, we conducted this systematic analysis to highlight the values of lncRNAs in ccRCC. It should be pointed out that we excluded the papers reporting plasma/serum ncRNAs in ccRCCs for the following reasons. First, the studies reporting lncRNAs in plasma/serum samples are limited, not enough for a systematic discussion and analysis. 64,65 In addition, we were concerned that the expression of ncRNAs is not consistent or comparable between blood and tissue samples. Therefore, we only included the studies of lncRNAs in tissue samples of clear cell carcinoma of the kidney. We comprehensively clarified the association between lncRNA expression and molecular mechanisms, functions, and clinical applications in ccRCC. The ceRNA hypothesis particularly provides a new perspective in terms of studying tumor formation mechanisms and the developing cancer treatments. Therefore, we summarized the ceRNA network participating in ccRCC. In clinical application, we generalized abnormally expressed lncRNAs in ccRCC tissues as diagnosis, prognosis, and treatment response biomarkers.
The prognosis for ccRCC patients is favorable when detected in the early stages, but is poor when diagnosed in the advanced stages. 4 To select promising prognosis biomarkers, 62 lncRNAs were identified from publications. Briefly, PVT1, 21,22,41,42,53 MALAT1,34,39,45 and LUCAT1 23-25 were related to the patients' prognosis in 3 or more studies with concordant results. This implies that PVT1, MA-LAT1 and LUCAT1 can serve as potential prognostic biomarkers for ccRCC patients.
To identify treatment lncRNAs, 3 lncRNAs (ADAMTS9-AS2, LncRNA-SARCC, and GAS5) were recognized in 3 studies. LncRNA-SARCC, GAS5, and SARCC were found to be associated with treatment sensitivity of cisplatin, sorafenib, and sunitinib, respectively. 58-60 ADAMTS9-AS2 attenuated the chemosensitivity of ccRCC cells to cisplatin via the ADAMTS9-AS2/miR-27a-3p/ FOXO1 axis. 58 GAS5 overexpression conferred RCC cell resistance to sorafenib via the GAS5/miR-21/SOX5 axis. 59 SARCC influenced sunitinib efficacy through binding and destabilizing androgen receptor (AR) protein, which led to transcriptional increase of miR-143-3p expression and thus inhibited ccRCC tumor progression. 60 It has been shown that MALAT1 and PVT1 can act as diagnostic and prognostic biomarkers in ccRCC. MALAT1 is one of the first identified cancer-associated lncRNAs and has been found with poor prognosis in various cancers, such as lung cancer, 66-68 bladder cancer, 69,70 breast cancer, 71,72 and others. [73][74][75] In lung cancer, for instance, MALAT1 was upregulated and transcriptionally activated by Oct4 via enhancer binding to promote cell proliferation and motility and led to lung tumorigenesis and poor prognosis. 76 Similarly, in gastric cancer, MALAT1 levels were significantly higher in cases with distant metastasis than in cases without distant metastasis and healthy controls. In addition, high levels of plasma MALAT1 independently correlated to a poor prognosis for gastric cancer patients. Functional studies revealed that knockdown of MALAT1 could inhibit cell proliferation, cell cycle progression, migration, and invasion and promote apoptosis in gastric cancer cells. 77 In this paper, MALAT1 was found to be upregulated in the cancer tissues in 7 independent studies of ccRCC and may be used as a potential diagnostic biomarker. 22,33,34,[38][39][40]45 MALAT1 was also an important prognostic biomarker in 3 studies. 34,39,45 ccRCC patients with high MALAT1 expression had significantly shorter OS, and MALAT1 upregulation was correlated with big tumor size, advanced tumor stage, and lymph node metastasis in ccRCC patients. 34, 39,45 As for PVT1, previous studies found that it is upregulated in bladder cancer 78 , cervical cancer 79 , gastric cancer, 80,81 and other cancers. 82 Notably, PVT1 was the most studied prognostic biomarker for ccRCC in 5 studies, 21,22,41,42,53 and PVT1 was reported to be upregulated in ccRCC tissues. 21,22,41,42,53 It was demonstrated that ccRCC patients with higher expression of PVT1 have worse OS and DFS. In addition, high expression of PVT1 was significantly associated with larger tumor size, advanced TNM stage, and lymph node metastasis. 21,22,41,42,53 PVT1 can promote ccRCC cell proliferation, migration and invasion, and EMT, induce ccRCC cell cycle arrest, and inhibit apoptosis. 21,41,42,53 In conclusion, this article summarized the potential carcinogenesis and ceRNA network of dysregulated lncRNAs in ccRCC, then reviewed their functions in tumorigenesis and progression, and finally discussed the potential roles of lncRNAs in diagnosis, prognosis, and treatment response in ccRCC.

Paper search strategy
Studies for lncRNAs as diagnostic, prognostic, or predictive biomarkers of ccRCC were searched in PubMed until June 26, 2020. The search terms used for paper retrieval were as follows: (("long non-coding RNA") OR ("lncRNA") OR ("lincRNA") OR ("long intergenic non-protein coding RNA") OR ("long non-protein-coding RNA")) AND ("clear cell renal cell carcinoma OR ccRCC OR renal clear cell carcinoma OR suprarenal epithelioma OR kidney renal clear cell carcinoma OR KIRC").

Eligibility criteria
Eligible studies should fulfill the following criteria: (1) a definitive diagnosis of ccRCC with the gold standard and (2) independent original studies that evaluated the expression of lncRNAs in ccRCC tumor tissue as diagnosis, prediction of treatment response, or prognosis biomarkers in patient populations. The exclusion criteria were as follows: (1) duplicate reports; (2) written in a language other than English; (3) non-human studies; (4) case reports, comments, letters, and reviews or meta-analyses; (5) other diseases; (6) not clear cell carcinoma of the kidney clearly; (7) not lncRNA studies; (8) studies did not assess the role of lncRNA in diagnosis, prognosis, and treatment response. All evaluations were independently performed by two individual researchers to ensure the accurate inclusion of studies. The discrepancies were resolved by discussion.

Data extraction
Two reviewers (X.C. and P.W.) independently extracted the data from the included studies by using a standardized table that included the following items: the names of differentially expressed lncRNAs, result of the abnormal expression (up or down), sample size, control group size, testing methods, survival outcome, regulatory mechanisms, functions in ccRCC, and the PMID number of the studies.

Cytoscape
Cytoscape is public software for integrating biomolecular interaction networks with high-throughput expression data and other molecular states into a unified conceptual framework, and is applicable to any system of molecular components and interactions. 83 We used Cytoscape 3.7.2 to construct the frameworks of lncRNA cellular functions in ccRCC.

DECLARATION OF INTERESTS
The authors declare no competing interests.