Biological functions and clinical significance of long noncoding RNAs in bladder cancer

Bladder cancer (BCa) is one of the 10 most common cancers with high morbidity and mortality worldwide. Long noncoding RNAs (lncRNAs), a large class of noncoding RNA transcripts, consist of more than 200 nucleotides and play a significant role in the regulation of molecular interactions and cellular pathways during the occurrence and development of various cancers. In recent years, with the rapid advancement of high-throughput gene sequencing technology, several differentially expressed lncRNAs have been discovered in BCa, and their functions have been proven to have an impact on BCa development, such as cell growth and proliferation, metastasis, epithelial-mesenchymal transition (EMT), angiogenesis, and drug-resistance. Furthermore, evidence suggests that lncRNAs are significantly associated with BCa patients’ clinicopathological characteristics, especially tumor grade, TNM stage, and clinical progression stage. In addition, lncRNAs have the potential to more accurately predict BCa patient prognosis, suggesting their potential as diagnostic and prognostic biomarkers for BCa patients in the future. In this review, we briefly summarize and discuss recent research progress on BCa-associated lncRNAs, while focusing on their biological functions and mechanisms, clinical significance, and targeted therapy in BCa oncogenesis and malignant progression.

• LncRNAs are a large class of noncoding RNA transcripts longer than 200 nucleotides that play important roles in biological processes, especially in cancer progression.
• LncRNAs can regulate the progression of bladder cancer.
• LncRNAs have the potential to accurately predict BCa patient prognosis and associated with clinicopathologic characteristics.

OPEN QUESTIONS
• Are lncRNAs involved in the posttranscriptional regulation of bladder cancer genes?
• How can we target lncRNAs to modulate the mechanism of bladder cancer progression?
• Are more multicenter cohort studies needed to verify the clinical value of lncRNAs in bladder cancer?
BACKGROUND As one of the most common urinary malignancies, bladder cancer (BCa) ranks within the top 10 cancers associated with high morbidity and mortality globally [1]. As a highly heterogeneous cancer, non-muscle-invasive BCa accounts for more than 75% of all BCa cases, while muscle-invasive BCa accounts for the remainders [2]. In current clinical practice, pathological biopsy with cystoscopy is considered to be the most reliable method for detecting BCa [3]. A major achievement in BCa therapies has been obtained. There is a wide range of BCa treatment plans, including surgical resection, chemotherapy, radiotherapy, and immunotherapy [4]. Despite recent progress in various cystoscopy and treatment options, the outcome of BCa patients is still not optimistic. The main reason for the low 5-year survival rate of advanced BCa patients is postoperative recurrence and uncontrollable distant metastasis [5]. Therefore, elucidating the molecular mechanisms and identifying potential therapeutic targets in BCa patients are of great significance.
The Cancer Genome Atlas (TCGA) has identified molecular aberrations at the DNA, RNA, protein, and epigenetic levels via massive numbers of human tumors analyzed. These sequencing results have confirmed that only 1-2% of human DNA is proteincoding genes, while more than 90% of the human gene (called noncoding RNAs) is transcribed to a universal team of RNA transcripts except protein-coding functions [6][7][8]. Long noncoding RNAs (lncRNAs), a large class of noncoding RNA transcripts, consist of more than 200 nucleotides [9]. With the rapid development of high-throughput genome sequencing technologies, lncRNAs are reported to play important roles in biological processes, especially in cancer progression, cell proliferation, differentiation, and metastasis. Several lncRNAs such as HOTAIR, PVT1, and H19, have been found to influence carcinogenesis and progression in colon cancer [9]. Recent studies have demonstrated that lncRNAs play important roles in tumor development and progression and aberrant expression of lncRNAs has been reported in BCa [10]. However, there are no studies that have systematically analyzed the role and mechanism played by lncRNAs in BCa. This review summarizes the functions and mechanisms, and clinical significance of lncRNAs in the oncogenesis and malignancy of human BCa within the last 10 years. Gibb et al. suggested that the importance of lncRNAs is rising, as they play roles in the cancer paradigm demonstrating potential functions in both oncogenic and tumor-suppressive pathways [11]. The study of lncRNAs in cancer progression has gradually developed. Studies have demonstrated that the expression of lncRNAs is related to the development and progression of BCa. It has been reported that lncRNAs are engaged in the regulation of cell growth and proliferation, tumor progression, and drug chemoresistance in BCa cells (Table 1).

CELL PROLIFERATION
Aberrant tumor cell proliferation can sustain active proliferative states, playing an important role in tumor growth [12]. UCA1 was the first reported oncogenic lncRNA and is overexpressed enormously in BCa and promotes BCa progression by regulating several targets and pathways [13]. First, UCA1 interferes with the chromatin redesigning activity of BRG1 and binds to the P21 promoter, thereby proliferating tumor cells [14]. The transcriptional activation of UCA1 through C/EBPα additionally contributes to elevated viability and reduced apoptosis of BCa cells [15]. Second, UCA1 regulates miR-16/GLS2 expression and suppresses ROS formation [16]. Via the mTOR/STAT3cascade and the miR143/ HK2 axis, UCA1 also enhances cancer cell glucose metabolism [17]. Third, UCA1 was also reported to influence AKT expression and activity, and its alteration parallels the expression and phosphorylation of CREB to promote the proliferation and regulation of the cell cycle [18]. BMP9 upregulates AKT phosphorylation levels and increases UCA1 expression to promote the proliferation and metastasis of BCa cells [19].
The PI3K/AKT signaling pathway is the most generally activated pathway in human malignant tumors, and its activation increases the activity of nutrient transporters and metabolic enzymes to reprogram cellular metabolism inflicting tumor cell proliferation [20]. HULC promotes BCa cell proliferation via regulation of the PI3K/AKT signaling pathway and ZIC2 [21]. ATB, an oncogene, is overexpressed to promote cell proliferation and migration by regulating miR-126/KRAS via the PI3K/AKT signaling pathway [22]. FOXD2-AS1 negatively regulates the expression of TRIB3 (a negative regulator of AKT) and promotes cell proliferation, and migration [23]. However, the expression of LINC00641 is significantly decreased in BCa, and its upregulation markedly inhibits the proliferation, and metastasis of BCa cells via the PTEN/ PI3K/AKT axis [24]. LOC572558 inhibits BCa cell proliferation by regulating the AKT/MDM2/P53 axis [25]. For other pathways, HCG22 negatively regulates the PTBP1-mediated Warburg effect by destabilizing human antigen R (HuR) to suppress cell proliferation, and progression [26]. IGFBP4-1 promotes cell proliferation and cell cycle progression and inhibits cell apoptosis by activating the JAK/STAT signaling pathway [27]. E2F4 is reported to be critical for MIR497HG silencing. MIR497HG suppresses cell proliferation, and metastasis by inhibiting YAP, SMAD3, BIRC5, and CCND1 expression (key genes of Hippo/Yap and TGF-b/Smad signaling) [28].
In addition to their binding to miRNAs, some newly reported lncRNAs directly bind proteins and participate in proliferation processes. The knockdown of ZFAS1 represses BCa cell proliferation by upregulating KLF2 and NKD2 expression [59]. SNHG5 promotes BCa cell proliferation by targeting P27 [60]. GClnc1 has been shown to significantly promote cell proliferation, metastasis, and invasiveness in BCa via the LIN28B/let-7a/MYC axis [61]. Upregulation of CASC9 is induced by STAT3 to promote BCa cell proliferation, migration, and invasion by interacting with EZH2 and affecting the expression of PTEN [62]. As a tumor suppressor, GAS5 has been reported to inhibit BCa proliferation by regulating CDK6 and CCL1 expression [63,64]. BRE-AS1 inhibits cell proliferation and accelerates cell apoptosis by mediating STAT3 expression [65]. ZNF503-AS1 can recruit transcription factor GATA6 to upregulate SLC8A1 expression, thereby increasing the intracellular Ca 2+ concentration, repressing proliferation, and enhancing the apoptosis of BCa cells [66].
In addition, downregulation of LINC00346 inhibits BCa cell proliferation and migration, and induces cell apoptosis [67].     Fig. 1 The overview of the ceRNA hypothesis.
Y. Zhang et al.
CRNDE strengthens cell migration and proliferation and inhibits cell apoptosis in BCa [68]. CCAT1 promotes BCa cell proliferation, migration, and invasion [69]. AFAP1-AS1 promotes the proliferation ability and invasiveness of BCa cells [70]. Overexpression of DGCR5 markedly inhibits proliferation and its ectopic expression leads to decreased BCa cell migration, invasion, and EMT, and promotes apoptosis [71].

CELL APOPTOSIS
Regulated cell death (RCD), also named cell suicide pathways, is of great importance in organismal development, homeostasis, and cancer pathogenesis [72]. Autophagy is an evolutionarily conserved process, in which dysfunctional cellular components are sequestered into lysosomes and degraded [73]. This process maintains cellular energy levels and promotes cellular survival. LncRNAs are reported to modulate autophagy [74]. ADAMTS9-AS2 inhibits BCa progression by affecting several key autophagy and apoptotic proteins [75]. Similarly, a study by Ying et al. demonstrated that insufficient expression of MEG3 could activate autophagy and promote cell proliferation [76]. Another study by Liu et al. showed that lowexpression of MEG3 inhibits apoptosis of BCa by regulating miR-96 along with TPM1 [77]. In contrast, UCA1 targets miR-582-5p and promotes BCa invasion, migration, growth, and drug resistance through ATG7-mediated autophagy inhibition [78].
Numerous studies indicate that the activity of Wnt/β-catenin signaling can either foster or restrain the processes of apoptosis based on specific cellular environmental stimuli [79,80]. Low TUG1 expression inhibits BCa cell proliferation and induces apoptosis by promoting ZEB2 mediated miR-142 suppression via inactivation of the Wnt/β-catenin pathway [81]. LINC00511 knockdown suppresses the proliferation and promotes apoptosis of BCa cells by suppressing the activity of the Wnt/β-catenin signaling pathway [82]. Cao et al. showed that SNHG16 is overexpressed in BCa tissues and cell lines and can notably promote proliferation by suppressing apoptosis of BCa cells by targeting P21 expression and regulating the miR-98/STAT3/Wnt/ β-catenin axis [83,84].

ANGIOGENESIS
Angiogenesis plays a critical role in tumorigenesis and the diffusion of malignant lesions by enhancing nutrient and oxygen supplies as well as providing a conduit for distant metastasis [157]. FAM83H-AS1 binds to c-Myc-mediated ULK3 to activate the Hedgehog signaling pathway, and FAM83H-AS1 knockdown inhibits the expression of CD31 and VEGFA (indicators of angiogenesis), suggesting that FAM83HAS1 promotes growth, metastasis, and angiogenesis of BCa cells through ULK3 upregulation and hedgehog activation [158]. In contrast, downregulation of RP11-79H23.3 led to higher CD31 and S100A4 expression and more microvessels. Moreover, RP11-79H23.3 can regulate the expression of the miR-107/PTEN axis and activate the PI3K/AKT signaling pathway to contribute to the proliferation, migration, apoptosis, and angiogenesis of BCa cells [159].

CHEMORESISTANCE AND RADIO-RESISTANCE
As a first-line treatment for BCa in clinical practice, chemotherapy reduces tumor masses in most patients. However, most patients gradually become unresponsive after multiple treatment cycles and eventually suffer tumor recurrence [160]. Several lncRNAs have been shown to modify the chemotherapy response in BCa.
Cisplatin, a basic drug of first-line treatment for chemotherapy, is shown to significantly improve the prognosis in sensitive patients [161]. As an oncogene, TUG1 induces the expression of EZH2 and directly sponges miR-194-5p. Low levels of miR-194-5p result in increased expression of CCND2, which promotes the chemoresistance of BCa cells to cisplatin [162]. Moreover, TUG1 knockdown enhances the sensitivity of BCa cells to adriamycin [163]. LINC00857 knockdown sensitizes BCa cells to cisplatin, by negatively regulating the target gene LMAN1, indicating that LINC00857 can regulate sensitive patient responses to platinumbased chemotherapy [164]. In cisplatin-resistant BCa cells, a high level of HIF1A-AS2 enhances the expression of HMGA1 to constrain the transcriptional activity of p53 family proteins, which affects cisplatin-induced apoptosis [165]. A previous study reported that DLEU1 enhances cisplatin resistance by competitively regulating miR-99b and restoring the expression of the target gene HS3ST3B1 [166]. Downregulated MALAT1 enhances the cisplatin sensitivity of BCa cells via the miR-101-3p/VEGFC axis [167]. MST1P2 has been found to regulate the miR-133b/SIRT1 axis and suppress the sensitivity of BCa cells to cisplatin [168]. UCA1 decreases the cisplatin sensitivity of BCa cells by enhancing the expression of Wnt6 [169]. lncRNAs can also inhibit drug resistance and promote the chemosensitivity of BCa cells to cisplatin. For example, overexpression of MEG3 sensitizes BCa cells to the chemotherapy drug cisplatin [170].
Gemcitabine is another cytotoxic chemotherapeutic agent of BCa cells, but the majority of patients, similar to those treated with cisplatin, ultimately experience tumor recurrence [171]. The upregulation of LET hinders BCa recurrence when treating with gemcitabine. However, the proinflammatory cytokine TGFβ1 can directly decrease LET expression levels in gemcitabine-resistant patients [172]. However, FOXD2-AS1 positively regulates ABCC3 protein via miR-143 targeting, and its knockdown suppresses the 50% inhibitory concentration of gemcitabine, the expression of drug resistance-related genes (MDR1, MRP2, LRP1), invasion, and ABCC3 protein expression in gemcitabine-resistant BCa cells [173]. High-expression levels of CDKN2B-AS are related to low gemcitabine sensitivity, and downregulated CDKN2B-AS gene levels inactivate the Wnt signaling pathway and ultimately affect the sensitivity of BCa cells to gemcitabine [174]. Similarly, the high expression of GHET1 is associated with low gemcitabine sensitivity in BCa patients, and knockdown of GHET1 advances gemcitabineinduced cytotoxicity [175]. In addition, UCA1 activates the transcription factor CREB, by binding with its promoter and leading to miR-196a-5p expression, while knockdown of UCA1 decreases chemosensitivity to cisplatin/gemcitabine by inhibiting BCa cell growth [176].
More investigations have revealed that lncRNAs also play an important role in chemosensitivity to doxorubicin in BCa. HOTAIR overexpression promotes cell proliferation and inhibits chemosensitivity to doxorubicin, while cell apoptosis is induced by doxorubicin, and GAS5 enhancement reduces chemotherapy resistance to doxorubicin [177,178].
For the radioresistance of BCa, the miR-145/ZEB2 axis mediates TUG1 function in EMT and radioresistance, and TUG1 downregulating increases radiosensitivity in BCa by inhibiting the targeting gene HMGB1 [179,180].

BCA STEM CELLS
Although both cancer stem cells (CSCs) and normal tissue stem cells possess the abilities to undergo self-renewal and differentiation, self-renewal is typically deregulated in CSCs [181]. LncRNAs have been reported to regulate cellular identity and differentiation in cancer. Depletion of ASAP1-IT1 in T24 cells reduces the CD44 population, whereas forced overexpression of ASAP1-IT1 in J82 cells enhances cancer cell stemness, suggesting that ASAP1-IT1 is sufficient and necessary for the maintenance of stemness [182]. Overexpression of NCK1-AS1 reduces miR-143 expression and promotes proliferation and increases CD133 expression [183]. HOXA-AS2 is upregulated in BCa cells and Wang et al. reported that it is positively correlated with the expression of OCT4. In addition, HOXA-AS2 promotes the migration, invasion, and stemness of BCa cells [184]. SOX2OT is highly expressed in BCa, upregulates SOX2 expression by sponging miR-200c, and downregulates SOX2OT to inhibit BCSC self-renewal, cell migration, invasion, and EMT [185]. LBCS can inhibit BCSC self-renewal and chemoresistance by suppressing SOX2 expression [186].

LNCRNAS ARE ASSOCIATED WITH CLINICOPATHOLOGICAL CHARACTERISTICS
Numerous reports show that lncRNAs have two main functions in promoting or inhibiting tumor development. Further analysis has shown that lncRNAs are closely related to many clinicopathological characteristics, such as stage, tumor size, and grade ( Table 2). The risk of tumor development in BCa varies according to the patient's age and sex [187]. Interestingly, lncRNAs have no relationship with patient sex, while two studies have reported that CASC9 and PlncRNA-1 are associated with patient age. CASC9 upregulation is significantly positively correlated with BCa tumor invasion depth, histological grade, and age; however, sex and tumor volume were not related to CASC9 expression levels [62,102,148].
For BCa tumor size, several lncRNAs are related. The high expression level of ZNFX1-AS1 is related to advanced clinical stages and tumor size [47]. High expression of ZEB2-AS1 and SNHG5 is significantly correlated with tumor size, lymph node metastasis, and clinical stage [60,93]. Patients with advanced-stage disease have higher levels of OIP5-AS1 expression than those with early-stage disease. High OIP5-AS1 expression is also observed in muscular invasion or large tumors [94]. Similarly, increased SNHG1 expression is closely correlated with tumor size, stage, invasion, and metastasis [188]. CCAT1 is positively related to clinical stage, tumor grade, and tumor size [69]. Increased ARSR expression is positively correlated with higher histological grade and larger tumor size [146]. SNHG3 [137], RMRP [132], PCAT6 [189], and LSINCT5 [149] expression positively correlated with tumor size and TNM stage, while high expression of MAFG-AS1 [118,153], SNHG7 [90,103], and TMPO-AS1 [33,34] was closely related to histological grade, tumor size, TNM stage, and clinical stage of BCa patients. However, many lncRNAs are important for tumor size suppression. CASC2a is highly negatively correlated with pathological T and N stages, and tumor size [190]. HCG22 expression correlates with pathological stage, metastasis, and a large tumor range [26]. The MBNL1-AS1 expression level correlates with the clinical stage, tumor size, and focal classification [55]. In addition, downregulated MAGI2-AS3 correlates with the number of tumors, stage, grade, and stage [125,150].
Accumulating evidence has revealed that the TNM stage, grade, and clinical/pathological stage of BCa can reflect the status of tumor development. High expression of HOTAIR and CDKN2B-AS is associated with a worse tumor grade. In addition, high expression of 5 lncRNAs positively correlates with tumor stage [21,23,37,53,54], while higher expression of 11 other lncRNAs is related to worse TNM stage [52,61,68,70,98,110,128,156,162,182]. The expression of 4 lncRNAs is positively associated with an advanced disease stage and poor tumor grade [85,154,191]. Higher expression levels of 6 lncRNAs are associated with high tumor grade and advanced TNM stage Y. Zhang et al. [42,108,134,185,192,193], while some other 4 lncRNAs are significantly correlated with T stage or metastasis, in addition to tumor grade [27,36,119,175]. The expression level of 4 lncRNAs positively correlates with tumor progression stage and TNM stage [59,88,184,194]. The expression of SNHG16 [83], BCAR4 [32], and SLCO4A1-AS1 [45] is related to metastasis and pathological stage. In addition, the high expression levels of LINC01296 [155], Carlo-7 [75], and ROR1-AS1 [54] are correlated with advanced tumor stage, higher tumor grade, and metastasis. In contrast, the expression of MIR143HG and MIR503HG is negatively correlated with tumor grade, advanced stage, and lymph node metastasis [111,151]. Decreased expression of GAS5 [195] and DBCCR1-003 [196] is observed in BCa patients with higher grades, while LINC00675 [112] expression is decreased in lymph node-metastatic MIBC tissues compared to those without lymph node metastasis. Decreased expression of other lncRNAs, such as LBCS [186], MEG3 [77], and TP73-AS1 [152], is strongly associated with tumor stage, grade, and/or TNM stage.

CONCLUSION
Researchers have already found that more than hundreds of lncRNAs could affect the initiation and progression of BCa. In the past 10 years, several biological functions of lncRNAs have been reported, especially in the past two years. As described in this review, more than 100 lncRNAs influence the proliferation, apoptosis, invasion, migration, metastasis, drug resistance, and even CSCs in BCa. Other BCa-related lncRNAs can act as ceRNA regulatory mechanisms to regulate various processes in tumors (Fig. 2). The studies reviewed here also indicate that lncRNAs are strongly associated with BCa patients' clinicopathological characteristics and prognosis, demonstrating that lncRNAs may be potential diagnostic and prognostic biomarkers for BCa patients. Several questions remain regarding the role of lncRNAs in BCa. Evidence indicates that one lncRNA can regulate more than one gene. The relationship between such genes should be further investigated. Apart from acting as miRNA sponges and via ceRNA mechanisms, other important mechanisms, such as ubiquitination and other posttranscriptional modifications, should be studied. Moreover, clinical studies with a large sample should be designed to explore the roles of lncRNAs in BCa from the perspectives of epigenetics and posttranscription. In addition, multicenter cohort studies are necessary to validate the diagnostic, prognostic and therapeutic value of lncRNAs in BCa.

DATA AVAILABILITY
All data generated or analyzed during this study are included in this published article.