SCLC is the most malignant subtype of lung cancer [33]. It has two stages: limited stage (LS-SCLC) and extensive stage (ES-SCLC), both of which show the characteristics of fast growth, strong invasiveness, early metastasis, and poor prognosis. Currently, the first-line therapy for SCLC patients is still etoposide plus carboplatin/cisplatin; nearly all ES-SCLC patients relapse with resistant disease within one year, with a median OS of approximately 10–11 months. In recent years, the emergence of immune checkpoint inhibitors has brought new hope for the treatment of ES-SCLC patients [34]. The median OS of SCLC patients treated with anti-PD-L1 mAb is about 12–13 months. Although ICIs plus chemotherapy improved OS of ES-SCLC patients, the therapeutic effect of ICI has not been breakthrough so far [35]. Thus, finding more effective predictive biomarkers and exploring different combination therapy strategies are urgently needed.
Notably, recent studies showed that lung cancer is also associated with dysregulated expression of lncRNAs [13–16]. Unfortunately, because only few tumor specimens can be obtained, information on the genomic profile of SCLC is very limited. In the present study, we performed RNA-Seq to compare the expression profiles of SCLC tumor and adjacent non-cancerous tissues from six SCLC patients. We identified dysregulated RNAs, including 29 lncRNAs, 48 miRNAs, and 510 mRNAs. We also constructed a lncRNA-miRNA-mRNA ceRNA network and finally identified a total of 15 lncRNAs, 17 miRNAs, and 89 mRNAs as potential mediators of SCLC tumorigenesis. We verified the four up-regulated lncRNAs (TCONS_00020615, TCONS_00055555, TCONS_00106091, and TCONS_00130858) and eight cancer-related mRNAs (CDCA3, DIAPH3, MCM7, NCAPG2, TPD52, PRC1, SETD6, and KIF22) involved in the ceRNA network by RT-RT-qPCR. All these lncRNAs and mRNAs exhibited significant upregulation in SCLC tissues.
Among the four up-regulated lnRNAs, TCONS_00020615 and TCONS_00106091 are verified lncRNAs. TCONS_00055555 and TCONS_00130858 are novel lncNRAs that are identified the first time in our sequencing results. TCONS_00106091 (LINC00511) has been studied in a variety of tumors, such as breast cancer [36], gastric cancer [37] and cervical cancer [38]. In NSCLC, two potential LINC00511 targets, enhancer zeste homolog 2 (EZH2) and lysine-specific demethylase 1 (LSD1), have been verified [39, 40]. Expression of LINC00511 was significantly upregulated (57-fold change) in SCLC tissues compared with adjacent non-tumor tissues. However, whether LINC00511 plays an important role in SCLC remains unclear. The role of LINC00511 in SCLC tumorigenesis will be investigated in our future studies.
TCONS_00020615, exhibiting 346-fold higher expression levels in the SCLC tissues, is the most up-regulated lncRNA. TCONS_00020615 is a novel transcript that is antisense to PROX1. Previous studies indicated that lncRNA PROX1-AS1 is up-regulated and acts as a tumor promoter in a wide range of human tumor types, including lung cancer [41], renal cell carcinoma [42], gastric Cancer [43], ovarian cancer [44] and prostate cancer [45]. However, expression levels of TCONS_00020615 have not been investigated. In our study, knockdown of TCONS_00020615 reduced the proliferation and migration of HCT1688 cells. Bioinformatic analysis results show that TCONS_00020615 is involved in small cell lung cancer possibly through the TCONS_00020615–hsa-miR-26b-5p–TPD52 pathway.
The miRNA miR-26b-5p has been identified as an oncogenic driver in a variety of cancers, including lymphocytic leukemia [46], ovarian cancer [47], hepatocellular carcinoma [48], breast cancer [49], and non-small cell lung cancer (NSCLC) [50–52]. In NSCLC tissues, miR-26b-5p expression was down-regulated compared with the adjacent non-tumor tissues [52]. Functionally, overexpression of miR-26b-5p suppressed cell proliferation and induces apoptosis in NSCLC by targeting EZH2 [51]. We also observed decreased miR-26b-5p expression in SCLC tissues in our study. There is a putative binding site of miR-26b-5p in TCONS_00020615, suggesting that TCONS_00020615 may exert its tumor-promoting role in SCLC by sponging miR-26b-5p. TPD52 exhibits an increased copy number, and is upregulated in a variety of cancers to accelerate tumor formation and progression by affecting cellular survival, proliferation, migration, invasion, and DNA repair[53].
Eight ceRNA-relevant mRNAs exhibited strinking upregulation in the SCLC samples. Among them, PRC1 showed the highest levels of upregulation (405-fold). Previous studies reported that PRC1 is upregulated in NSCLC tissues and promotes the proliferation and metastasis of NSCLC via activating the Wnt/β-catenin pathway [30]. PRC1 also regulates the tumorigenesis of other types of cancer, including esophageal cancer, breast cancer [54], uveal melanoma [55], ovarian cancer [56], colon cancer, and childhood cancers, such as Ewing sarcoma (EwS) [57]. DIAPH3 is the secondary upregulated gene (about 100-fold). Previous studies reported that DIAPH3 is also significantly upregulated in lung adenocarcinoma [26], pancreatic cancer [58], and hepatocellular carcinoma [59]. Furthermore, CDCA3, which encodes cell division cycle associated protein-3, is also found to be increased in NSCLC tissues and associated with poor patient prognosis [25]. Zhang et al. reported that CDCA3 is regulated by miR-4677-3p during lung cancer development [60]. Four other genes involved in the ceRNA network, including MCM7 [27], NCAPG2 [28], SETD6 [31], and KIF22 [32] have also been reported to involved in lung cancer progression. These eight genes might be potential therapeutic targets in the small-cell lung cancer, which have not been studied for now.
The limitations of this study are as follows. First, the molecular mechanism of TCONS_00020615 in SCLC still needs to be further explored and verified. Second, small sample size and difficulties in obtaining SCLC tissues limit the scope of our study. Further larger scale investigations are needed to analysis the relationship among lncRNAs and clinical features. Nonetheless, to our knowledge, the present study is the first to profile the expression of lncRNA, miRNA, and mRNA, as well as the underlying lncRNA-miRNA-mRNA ceRNA regulatory networks in SCLC. This information could be helpful in improving our understanding of the molecular mechanisms of SCLC and in improving the diagnosis and management of SCLC.
In conclusion, we revealed the expression profiles of lncRNAs, miRNAs, and mRNAs in six pairs of SCLC tumor tissues and adjacent non-cancerous tissues, then we constructed a ceRNA network through the RNA sequencing data. TCONS_00020615 was identified as the most upregulated lncRNA in SCLC patients and might be a potential prognostic marker and therapy target of SCLC. Our findings of these differentially expressed lncRNAs, miRNAs, and mRNAs in the ceRNA network would improve the understanding of the pathogenesis of SCLC and help develop more treatment approaches.