Identification of lncRNA, miRNA and mRNA expression profiles and ceRNA Networks in small cell lung cancer
BMC Genomics volume 24, Article number: 217 (2023)
Small cell lung cancer (SCLC) is a highly lethal malignant tumor. It accounts for approximately 15% of newly diagnosed lung cancers. Long non-coding RNAs (lncRNAs) can regulate gene expression and contribute to tumorigenesis through interactions with microRNAs (miRNAs). However, there are only a few studies reporting the expression profiles of lncRNAs, miRNAs, and mRNAs in SCLC. Also, the role of differentially expressed lncRNAs, miRNAs, and mRNAs in relation to competitive endogenous RNAs (ceRNA) network in SCLC remain unclear.
In the present study, we first performed next generation sequencing (NGS) with six pairs of SCLC tumors and adjacent non-cancerous tissues obtained from SCLC patients. Overall, 29 lncRNAs, 48 miRNAs, and 510 mRNAs were found to be differentially expressed in SCLC samples (|log2[fold change] |> 1; P < 0.05). Bioinformatics analysis was performed to predict and construct a lncRNA-miRNA-mRNA ceRNA network, which included 9 lncRNAs, 11 miRNAs, and 392 mRNAs. Four up-regulated lncRNAs and related mRNAs in the ceRNA regulatory pathways were selected and validated by quantitative PCR. In addition, we examined the role of the most upregulated lncRNA, TCONS_00020615, in SCLC cells. We found that TCONS_00020615 may regulate SCLC tumorigenesis through the TCONS_00020615–hsa-miR-26b-5p–TPD52 pathway.
Our study provided the comprehensive analysis of the expression profiles of lncRNAs, miRNAs, and mRNAs of SCLC tumors and adjacent non-cancerous tissues. We constructed the ceRNA networks which may provide new evidence for the underlying regulatory mechanism of SCLC. We also found that the lncRNA TCONS_00020615 may regulate the carcinogenesis of SCLC.
Small-cell lung cancer (SCLC) is one of the most aggressive malignant neuroendocrine (NE) tumors , accounting for 15% of newly diagnosed lung cancers. Compared with NSCLC, SCLC has strong invasiveness, rapid progress, early development of widespread metastases, and many gene mutations. However, there is no major driver gene mutation in SCLC, and no targeted drugs can be used ; chemotherapy and radiotherapy are still the main treatment methods for SCLC. Despite initial chemosensitivity, nearly all patients relapse with resistant disease within only a few months, resulting in a 5-year survival rate of 5–10% . The treatment for SCLC has not been significantly improved for more than 30 years . Part of the reason for the lack of progress is that SCLC is rarely treated by surgical resection, and, accordingly, scientific research has been hindered by the lack of available tissue samples. Further research for the molecular mechanisms underlying the occurrence and development of SCLC is urgently needed.
Tough incapable of encoding proteins, lncRNAs hold an indispensable role in epigenetics and gene expression regulation . Emerging studies  have established that lncRNAs are crucial during tumor progression as oncogenes or tumor suppressor genes. They participate in biological processes including cell growth, anti-apoptosis, migration, and invasion. Hence, research on lncRNAs may hold significant value in understanding tumor development and progression.
With the rapid development of next-generation sequencing (NGS) technology, long non-coding RNAs (lncRNAs), originally considered as junk molecules, have been identified and demonstrated to play pivotal roles in epigenetics and gene expression regulation. Increasing number of studies indicate that many types of cancer are associated with abnormal expression of lncRNAs, which participate in biological processes of tumor initiation and progression, such as cell proliferation, apoptosis, invasion, and chemosensitivity [5, 6]. As a classic molecular mechanism of lncRNA, the lncRNA-miRNA-mRNA competitive endogenous RNAs (ceRNA) regulatory network is implicated in the development of colorectal cancer , pancreatic carcinoma , prostate cancer , lung cancer , and others.
The lncRNA MALAT1 is upregulated in NSCLC and has been reported to modulate miR-204/SLUG and miR-124/STAT3 through two different axes to regulate the progression of lung cancer [11, 12]. Another lncRNA, LOC285194, is downregulated in NSCLC and suppresses NSCLC through targeting p53 . In SCLC, the lncRNA TUG1 promotes cell growth and chemoresistance through regulating the expression of LIMK2b via EZH2 . Knockdown of the lncRNA HIF1A-AS2 increases doxorubicin sensitivity of SCLC cells and decreases autophagy . These newly identified lncRNAs and miRNAs with aberrant expression levels can serve as therapeutic targets and potential diagnostic markers for lung cancer. However, except for these findings, little is known about the role of lncRNAs in SCLC.
In the present study, we identified the global expression profiles of lncRNAs, miRNAs, and mRNAs in SCLC tissues and paired adjacent non-cancerous tissues from six SCLC patients. To the best of our knowledge, this is the first study reporting the RNA expression patterns in SCLC tissues via NGS technology. We predicted a ceRNA network with the differentially expressed lncRNAs, miRNAs, and mRNAs based on miRanda and TargetScan databases. Furthermore, we found that the lncRNA TCONS_00020615, which was the most upregulated gene in SCLC tissues, regulated the proliferation and migration of SCLC cells. The TCONS_00020615–hsa-miR-26b-5p–TPD52 axis may play a pivotal role in SCLC tumorigenesis. Taken together, our study may facilitate the description of the underlying molecular mechanisms of SCLC. Our study may also help in identifying more diagnostic markers and developing novel therapeutic strategies for SCLC.
Human SCLC samples and expression profile dataset
Information of patients and specimens have been provided in our previous report . Briefly, the study included 16 SCLC patients (patient 1–16) who underwent surgery and four SCLC patients (patient 17–20) who underwent fibro-bronchoscopy evaluation without chemotherapy or radiotherapy at our hospital between September 2014 and August 2019. For NGS analysis, six paired SCLC and corresponding adjacent normal tissues were randomly selected, numbered as patient1-6. All the 16 pairs (patient 1–16) of SCLC and adjacent noncancerous tissues, and four SCLC samples (patient 17–20) without matched normal tissue, were used for qRT- PCR validation experiments.
RNA isolation & RNA-Seq library preparation
Total RNA isolation and RNA-Seq library preparation were performed as previously described . Briefly, total RNA was extracted from human SCLC tissues using TRIzol reagent (Life Technologies, Carlsbad, CA). RNA purity, RNA concentration, and RNA integrity were examined with Agilent 2100 bioanalyzer before RNA-Seq library construction. The library construction was performed using the VAHTS Total RNA-seq (H/M/R) Library Prep Kit for Illumina R (Vazyme Biotech, Nanjing, China) according to the kit’s instructions. The samples were then sequenced (150-bp paired-end RNA-Seq reads) on the Illumina Hiseq X10 platform (Illumina, CA, USA).
Differentially expression analysis
The raw RNA sequencing (lncRNA, miRNA, and mRNA) reads were post-processed and normalized using the trimmed mean of M-values (TMM) method. EdgeR package in R (version 3.4.1) was used to identify the differentially expressed mRNAs, lncRNAs, and miRNAs between each pair of SCLC sample and adjacent normal sample. The cut-off criteria were set as p ≤ 0.05 and |log2(fold change) |> 1. The heat map was plotted using the pheatmap function of pheatmap package version 1.0.8.
Interactions between lncRNAs and miRNAs were predicted using the miRcode database . The MiRanda and TargetScan databases were used to retrieve miRNA-targeted mRNAs. Only the mRNAs targeted by miRNAs present in the two databases were used to construct a ceRNA network. A lncRNA-miRNA-mRNA regulatory network was constructed to visualize the interactions using Cytoscape v3.6.1 .
Functional enrichment analysis
Kyoto Encyclopedia of Genes & Genomes (KEGG) enrichment [19,20,21] and Gene ontology (GO) [22, 23] analyses were employed to elucidate the potential roles of differentially expressed mRNAs in the ceRNA network. The top 20 enriched KEGG pathways and the top 10 GO terms were plotted.
Cell lines, cell culture and establishment of stable knockdown cell lines
Human SCLC cells (NCI-H1688) were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). Cells were grown in RPMI 1640 medium (Gibco, USA) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin (Gibco, USA). DNA sequences of shRNAs targeting TCONS_00020615 were subcloned into the pLKO.1-TRC lentivirus vector. The sequences of shRNAs against TCONS_00020615 were 5'-CAGCCCACAAATAAACTGGTA-3'; 5′-GTGCAGGTTGCATTTACTTAT-3'; 5′-AACCAACAGCTTTCAAAGTAA-3'; shRNA directed against GFP (target sequence: 5'-GCAAGCTGACCCTGAAGTTCAT-3') was used as the control scrambled shRNA (shCtrl). Cells were infected with lentivirus carrying specific shRNA in the presence of polybrene (8 μg/mL, sigma) for 24 h. The medium was replaced with fresh medium and the cells were cultured for 48 h. Then the cells were screened with selection medium containing 2.5 μg/mL puromycin until there was no live cell left in the mock group.
Quantitative real-time PCR (qRT-PCR)
Total RNA (1 μg) from each sample was reverse-transcribed to cDNA using HiScript® II Q RT SuperMix for qRT-PCR (Vazyme). Quantitative PCR was performed on a ROCHE LightCycler® 480 instrument (ROCHE, Basel, Switzerland) using AceQ qRT-PCR SYBR Green Master Mix (without ROX) (Vazyme). All the primers were designed with Primer Premier 5.0 (PREMIER Biosoft, Palo Alto, CA). The qRT-PCR primers used in this study are listed in supplementary Table S1. The relative standard curve method (2 − ΔΔCt) was used to determine the relative lncRNAs and mRNAs expression, β-actin was used as the reference.
Cell proliferation assay
Cell proliferation was monitored using Cell Counting Kit-8 (CCK8, Vazyme) according to the manufacturer’s instructions. For colony formation assay, cells were seeded on 6-well plates at a density of 2,000 cells per well and cultured in RPMI 1640 medium containing 10% FBS for 10–14 days. Cells were rinsed with PBS and fixed with 4% formaldehyde for 15 min, then stained with 0.5% crystal violet for 30 min. Cell colonies were photographed using a digital camera at 5 × magnification and counted.
Costar Transwell chambers with 8-μm aperture (Corning, USA) were used in migration assays. The cells (1 × 105) were suspended in 100 μl RPMI 1640 medium without FBS and transferred to the upper Transwell chambers (Becton, Dickinson and Company, USA), and 600 μl RPMI 1640 medium containing 10% FBS was added into the lower chambers. After 24 h of incubation, cells that passed through the membrane were fixed with 4% formaldehyde and stained with 0.5% crystal violet. Stained cells were imaged and counted using a microscope at 100 × magnification.
Wound healing assay
Cells were seeded on 6-well plates and grown to 90–100% confluency. Then cells were scratched with a 10 μl sterile pipette tip, washed twice with phosphate buffer saline (PBS), and cultured in RPMI 1640 supplemented with 1% FBS. The wound width was imaged at 0 and 48 h after wounding using a microscope (Leica, Wetzlar, Germany) at 100 × magnification.
Data were analyzed and displayed using GraphPad Prism 5. Data are expressed as means ± SEM. Unpaired two tailed Student’s t-test was used for statistical calculations. Statistically significance was defined as P < 0.05. Leave-one-out cross-validation was performed with the qRT-PCR results of sixteen paired (case1 to case16) SCLC and normal tissues in Fig. 5. Generalize linear model was used. Receiver operating characteristic (ROC) curve was drawn by the ggplot2 package on the R platform. Area under Curve (AUC) were calculated by the pROC R package .
Identification of differentially expressed lncRNAs, miRNAs and mRNAs in SCLC tissues
The NGS technology was utilized to profile lncRNA, miRNA, and mRNA expression in SCLC tumors and adjacent noncancerous tissues from six SCLC patients. In total, 44,191 lncRNAs, 2,358 miRNAs, and 50,869 mRNAs were identified. Compared with previously published studies, 10,425 (23.59%) detected lncRNAs were newly found in this study (Fig. 1a). Most of these detected lncRNAs were 400–1,000 nt in length (Fig. 1b), and most miRNAs consisted of 21–23 nucletides (Fig. 1c). Distributions of lncRNAs and mRNAs in human chromosomes were determined as shown in Fig. 1d; lncRNAs and mRNAs were generally distributed on many chromosomes.
We compared the expression profiles of lncRNAs, miRNAs, and mRNAs between each pair of SCLC and the corresponding adjacent non-cancerous samples. We selected RNAs with differential expression in all groups using the following cut-off criteria: |log2(Fold Change)|> 1 and p < 0.05. The analysis strategy and procedure of the current study are shown in Fig. 2 Overall, 29 lncRNAs (14 upregulated and 15 downregulated), 48 miRNAs (25 upregulated and 23 downregulated), and 510 mRNAs (225 upregulated and 285 downregulated) were dysregulated in the SCLC tissues compared with the non-cancerous tissues. Hierarchical clustering of the identified differentially expressed lncRNAs, miRNAs, and mRNAs were visualized in heatmaps, which exhibited distinguishable characteristic between SCLC tissues and adjacent non-cancerous tissues (Fig. 3).
The top 10 upregulated and downregulated lncRNAs, miRNAs, and mRNAs are listed in Supplementary Table S2. As shown in Table 1, the lncRNA, miRNA, and mRNA with the highest levels of upregulation were TCONS_00223926 (log25.87), hsa-miR-182-3p (log25.12), and CPLX2 (log27.70), respectively; those exhibiting the largest amounts of downregulation were TCONS_00160619 (-log24.826066667), hsa-miR-451a (-log24.99), and SFTPA1 (-log27.36), respectively.
Establishment of a ceRNA regulatory network
One of the most well-characterized functions of lncRNAs is to act as ceRNAs [25, 26]. To clarify the interaction between these differentially expressed lncRNAs, miRNAs, and mRNAs, we analyzed the lncRNA-miRNA-mRNA regulatory network. The differentially expressed lncRNAs, miRNAs, and mRNAs included in the ceRNA network were filtered as follows. First, we used the miRcode database to predict interactions between lncRNAs with miRNAs. We used miRanda and TargetScan databases to retrieve miRNA-targeted mRNAs. Only miRNA-targeted mRNAs present in both two databases were selected. Second, lncRNAs and mRNAs had to exhibit the same trends of expression, while miRNAs were required to show a trend in the opposite direction. Third, the Pearson correlation coefficient between the differently expressed lncRNAs and mRNAs was required to be greater than 0.8. There were at least three microRNA response elements (MREs) between lncRNAs and miRNAs as well as between miRNAs and mRNAs. As a result, 9 lncRNAs, 11 miRNAs, and 240 mRNAs were selected and used to generate the ceRNA network (Fig. 4a).
Functional enrichment analysis of differentially expressed mRNAs
To figure out the biological significance of altered levels of RNAs, we further analyzed the 240 ceRNA network-relevant mRNAs through GO and KEGG pathway analyses. GO analysis showed that single-organism cellular process, single-multicellular organism process, and multicellular organism development were the top three enriched biological processes. In the analysis of molecular function, the top three terms were binding, protein binding, and molecular transducer activity. According to the cellular component analysis, proteins encoded by these RNAs were mainly located in cell periphery, plasma membrane, and extracellular region (Fig. 4b). KEGG pathway analysis revealed that these mRNAs were mostly enriched in cytokine-cytokine receptor interaction, pathways in cancer, MAPK signaling pathway, cell adhesion molecules (CAMs), and focal adhesion, which may be related to the progression of SCLC (Fig. 4c).
Validation of the differently expressed lncRNAs and mRNAs
To verify the reliability of the RNA-seq data, we firstly examined the expression levels the top three upregulated and downregulated lnRNAs (TCONS_00223926, TCONS_00097709, TCONS_00100327, TCONS_00160619, TCONS_00093045, TCONS_00160600) and mRNAs (CPLX2, XKR7, STXBP5L, SFTPA1, ITLN1, MCEMP1) with qRT-PCR (Fig. 5a- l). qRT-PCR results showed that mRNA levels of all the twelve transcripts had significant changes in SCLC tissues, which were consistent with NGS data. Especially, the upregulated STXBP5L showed 165-fold change and downregulated ITLN1 showed 0.0007-fold change in SCLC tissues (Fig. 5m).
To identify potential therapeutic targets and molecular mechanisms for SCLC, we also examined the expression levels of the lncRNAs and mRNAs involved in the ceRNA network. Four up-regulated lnRNAs and mRNAs related to them were selected for further research. qRT-PCR results showed that the expression levels of the four lncRNAs, including TCONS_00020615, TCONS_00055555, TCONS_00106091 (also known as LINC00511), and TCONS_00130858, were quiet low in non-cancerous tissues (controls). However, they were significantly upregulated in the corresponding SCLC tissues, especially TCONS_00020615 and TCONS_00055555, which were up-regulated 346-fold and 311-fold in the SCLC tissues, respectively (Fig. 6a–d, k). We selected eight cancer-related mRNAs, including CDCA3 , DIAPH3 , MCM7 , NCAPG2 [30, 31], TPD52 , PRC1 , SETD6  and KIF22 , for qRT-PCR examination. Consistent with the RNA-seq results, mRNA levels of all the eight genes showed significant increases in SCLC tissues (Fig. 6e–l). Fold changes of qRT-PCR validated lncRNAs and mRNAs expression are shown in Fig. 6m. PRC1 exhibited the highest level of upregulation (405-fold; Fig. 6m). Such notable changes in the expression levels of these RNAs in SCLC tissues indicated they may play essential roles in SCLC progression.
To confirm the diagnostic value of these RNAs for SCLC, we performed leave-one-out cross-validation with the qRT-PCR results of the four lncRNAs and eight mRNAs in the sixteen paired (patient 1 to patient 16) SCLC and normal tissues. ROC curves were drawn to evaluate diagnostic efficiency. Results showed that all the twelve RNAs had high accuracy in distinguishing SCLC from the normal tissues (Fig. 7).
Knock down of TCONS_00020615 hampered the proliferation and migration of SCLC cells
TCONS_00020615 was the lncRNA exhibiting the highest levels of upregulation in SCLC tissues; therefore, we further investigated its potential role in SCLC tumorigenesis. TCONS_00020615 is a 2627-bp-long novel transcript with a sequence complementary to that of PROX1. It is mapped to Chromosome 1: 213,983,793–213,986,419 reverse strand (Supplementary Fig. S1).
To investigate the role of TCONS_00020615 in SCLC tumorigenesis, first, we examined the expression of TCONS_00020615 in human bronchial epithelial cell lines HBE and BEAS-2B as well as in SCLC cell lines H446 and H1688. TCONS_00020615 expression levels in H1688 cells were significantly higher than those in HBE and BEAS-2B cells (Supplementary Fig. S2), so H1688 cells were used for the sequencing studies. Lentivirus-mediated shRNA expression was used to generate a cell line with stable TCONS_00020615 knockdown. Expression of TCONS_00020615 in the knockdown cells were markedly suppressed compared with the control cells as determined by qRT-PCR (Fig. 8a). MTT and colony formation assays were performed in H1688 cells to assess SCLC cell proliferation. TCONS_00020615 knockdown significantly suppressed the proliferation (Fig. 8b) and number of clones (Fig. 8c) in H1688 cells. Wound healing and transwell assays performed to detect cell migratory activity of H1688 cells revealed that TCONS_00020615 knockdown remarkably suppressed the migration and invasion of H1688 cells (Fig. 8d and e). These results suggest that TCONS_00020615 may participate in the SCLC tumorigenesis.
TCONS_00020615–hsa-miR-26b-5p–TPD52 axis is considered as a potential pathway linked to SCLC
According to the ceRNA hypothesis, lncRNAs indirectly regulate the expression of mRNAs with a positive correlation. We analyzed the relation between the expression levels of TCONS_00020615 and those of related mRNAs in the constructed ceRNA network. The results indicated that the expression of TCONS_00020615 was positively correlated with that of TPD52 and NCAPG2. However, the p-value for NCAPG2 was not significant (Fig. 9a-c). The miRNA miR-26b-5p was suggested as a target of TCONS_00020615 and an upstream miRNA of TPD52 in the predicted ceRNA network. Results from miRcode database showed that miR-26b-5p had a latent binding site complementary to TCONS_00020615 (Fig. 9d). TargetScan analysis also revealed that TPD52 was considered as a putative target of miR-26b-5p (Fig. 9e). Moreover, the potential binding site of miR-26b-5p and TPD52 is broadly conserved among vertebrates (Fig. 9f). These results indicated that there may be a regulatory relationship between TCONS_00020615 and TPD52.
To the best of our knowledge, this is the first report providing expression profiles of lncRNAs, miRNAs, and mRNAs in human SCLC tumors and paired adjacent non-cancerous tissues. Comprehensive identification and analyses of the differentially expressed lncRNA-miRNA-mRNA ceRNA regulatory network further indicate potential mechanisms of SCLC progression. The lncRNA with the highest levels of upregulation, TCONS_00020615, may have a role in SCLC tumorigenesis. We anticipate that our work will serve as a valuable resource for future research, which may include diagnosis, mechanistic analyses, and therapy of SCLC.
SCLC is the most malignant subtype of lung cancer . 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 . 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 . 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 [10,11,12]. 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 qRT-PCR. 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 , gastric cancer  and cervical cancer . In NSCLC, two potential LINC00511 targets, enhancer zeste homolog 2 (EZH2) and lysine-specific demethylase 1 (LSD1), have been verified [42, 43]. 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 , renal cell carcinoma , gastric cancer  and ovarian cancer .. 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 , hepatocellular carcinoma , breast cancer , and non-small cell lung cancer (NSCLC) [51, 52]. In NSCLC tissues, miR-26b-5p expression was down-regulated compared with the adjacent non-tumor tissues . Functionally, overexpression of miR-26b-5p suppressed cell proliferation and induces apoptosis in NSCLC by targeting EZH2 . 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 .
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 . PRC1 also regulates the tumorigenesis of other types of cancer, including breast cancer , uveal melanoma , ovarian cancer , and childhood cancers, such as Ewing sarcoma (EwS) . DIAPH3 is the secondary upregulated gene (about 100-fold). Previous studies reported that DIAPH3 is also significantly upregulated in lung adenocarcinoma , pancreatic cancer , and hepatocellular carcinoma . 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 . Zhang et al. reported that CDCA3 is regulated by miR-4677-3p during lung cancer development . Four other genes involved in the ceRNA network, including MCM7 , NCAPG2 , SETD6 , and KIF22  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 can be summarized as follows: First, due to the heterogeneity of SCLC, we adopted relaxed screening conditions and used p-values instead of q-values (corrected p-values) to identify differentially expressed genes. However, it is worth noting that all 24 transcripts selected for qRT-PCR validation exhibited significant changes in our study. Second, the molecular mechanism of TCONS_00020615 in SCLC still needs to be further explored and verified. Third, 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.
Availability of data and materials
Raw sequencing reads of transcriptome Seq of the six pairs of SCLC and corresponding adjacent normal tissues can be obtained from NCBI Sequence Read Archive (SRA) (SRA accession: PRJNA553289). https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA553289
Small cell lung cancer
Long non-coding RNA
Competing endogenous RNA
Kyoto encyclopedia of genes and genomes
Quantitative real-time PCR
Fetal bovine serum
Phosphate bufered saline
Cell Counting Kit-8
Haddadin S, Perry MC. History of small-cell lung cancer. Clin Lung Cancer. 2011;12(2):87–93.
Hann CL, Rudin CM. Management of small-cell lung cancer: incremental changes but hope for the future. Oncology. 2008;22(13):1486–92.
van Meerbeeck JP, Fennell DA, De Ruysscher DK. Small-cell lung cancer. Lancet. 2011;378(9804):1741–55.
Koinis F, Kotsakis A, Georgoulias V. Small cell lung cancer (SCLC): no treatment advances in recent years. Transl Lung Cancer Res. 2016;5(1):39–50.
Sheng J, Lv E, Xia L, Huang W. Emerging roles and potential clinical applications of long non-coding RNAs in hepatocellular carcinoma. Biomed Pharmacother. 2022;153:113327.
Mirzaei S, Paskeh MDA, Okina E, Gholami MH, Hushmandi K, Hashemi M, Kalu A, Zarrabi A, Nabavi N, Rabiee N, et al. Molecular Landscape of LncRNAs in Prostate Cancer: A focus on pathways and therapeutic targets for intervention. J Exp Clin Cancer Res. 2022;41(1):214.
Weng X, Liu H, Ruan J, Du M, Wang L, Mao J, Cai Y, Lu X, Chen W, Huang Y, et al. HOTAIR/miR-1277-5p/ZEB1 axis mediates hypoxia-induced oxaliplatin resistance via regulating epithelial-mesenchymal transition in colorectal cancer. Cell Death Discov. 2022;8(1):310.
Xu J, Xu J, Liu X, Jiang J. The role of lncRNA-mediated ceRNA regulatory networks in pancreatic cancer. Cell Death Discov. 2022;8(1):287.
Liu M, Shen A, Zheng Y, Chen X, Wang L, Li T, Ouyang X, Yu X, Sun H, Wu X. Long non-coding RNA lncHUPC1 induced by FOXA1 promotes tumor progression by inhibiting apoptosis via miR-133b/SDCCAG3 in prostate cancer. Am J Cancer Res. 2022;12(6):2465–91.
Wang J, Yuan Y, Tang L, Zhai H, Zhang D, Duan L, Jiang X, Li C. Long non-coding RNA-TMPO-AS1 as ceRNA binding to let-7c-5p upregulates STRIP2 expression and predicts poor prognosis in lung adenocarcinoma. Front Oncol. 2022;12:921200.
Li J, Wang J, Chen Y, Li S, Jin M, Wang H, Chen Z, Yu W. LncRNA MALAT1 exerts oncogenic functions in lung adenocarcinoma by targeting miR-204. Am J Cancer Res. 2016;6(5):1099–107.
Li S, Mei Z, Hu HB, Zhang X. The lncRNA MALAT1 contributes to non-small cell lung cancer development via modulating miR-124/STAT3 axis. J Cell Physiol. 2018;233(9):6679–88.
Zhou H, Chen A, Shen J, Zhang X, Hou M, Li J, Chen J, Zou H, Zhang Y, Deng Q, et al. Long non-coding RNA LOC285194 functions as a tumor suppressor by targeting p53 in non-small cell lung cancer. Oncol Rep. 2019;41(1):15–26.
Niu Y, Ma F, Huang W, Fang S, Li M, Wei T, Guo L. Long non-coding RNA TUG1 is involved in cell growth and chemoresistance of small cell lung cancer by regulating LIMK2b via EZH2. Mol Cancer. 2017;16(1):5.
Guclu E, ErogluGunes C, Kurar E, Vural H. Knockdown of lncRNA HIF1A-AS2 increases drug sensitivity of SCLC cells in association with autophagy. Med Oncol. 2021;38(9):113.
Zhang C, Zhang B, Yuan B, Chen C, Zhou Y, Zhang Y, Sheng Z, Sun N, Wu X. RNA-Seq profiling of circular RNAs in human small cell lung cancer. Epigenomics. 2020;12(8):685–700.
Jeggari A, Marks DS, Larsson E. miRcode: a map of putative microRNA target sites in the long non-coding transcriptome. Bioinformatics. 2012;28(15):2062–3.
Kohl M, Wiese S, Warscheid B. Cytoscape: software for visualization and analysis of biological networks. Methods Mol Biol. 2011;696:291–303.
Kanehisa M, Goto S. KEGG: kyoto encyclopedia of genes and genomes. Nucleic Acids Res. 2000;28(1):27–30.
Kanehisa M, Furumichi M, Sato Y, Kawashima M, Ishiguro-Watanabe M. KEGG for taxonomy-based analysis of pathways and genomes. Nucleic Acids Res. 2023;51(D1):D587–92.
Kanehisa M. Toward understanding the origin and evolution of cellular organisms. Protein Sci. 2019;28(11):1947–51.
Ashburner M, Ball CA, Blake JA, Botstein D, Butler H, Cherry JM, Davis AP, Dolinski K, Dwight SS, Eppig JT, et al. Gene ontology: tool for the unification of biology. Gene Ontol Consortium Nat Genet. 2000;25(1):25–9.
Gene Ontology C. The Gene Ontology resource: enriching a GOld mine. Nucleic Acids Res. 2021;49(D1):D325–34.
Robin X, Turck N, Hainard A, Tiberti N, Lisacek F, Sanchez JC, Muller M. pROC: an open-source package for R and S+ to analyze and compare ROC curves. BMC Bioinformatics. 2011;12:77.
Salmena L, Poliseno L, Tay Y, Kats L, Pandolfi PP. A ceRNA hypothesis: the Rosetta stone of a hidden RNA language? Cell. 2011;146(3):353–8.
Poliseno L, Salmena L, Zhang J, Carver B, Haveman WJ, Pandolfi PP. A coding-independent function of gene and pseudogene mRNAs regulates tumour biology. Nature. 2010;465(7301):1033–8.
Adams MN, Burgess JT, He Y, Gately K, Snell C, Zhang SD, Hooper JD, Richard DJ, O’Byrne KJ. Expression of CDCA3 Is a prognostic biomarker and potential therapeutic target in non-small cell lung cancer. J Thorac Oncol. 2017;12(7):1071–84.
Xiang G, Weiwei H, Erji G, Haitao M. DIAPH3 promotes the tumorigenesis of lung adenocarcinoma. Exp Cell Res. 2019;385(1):111662.
Wang J, Amin A, Cheung MH, Shi L, Liang C. Targeted inhibition of the expression of both MCM5 and MCM7 by miRNA-214 impedes DNA replication and tumorigenesis in hepatocellular carcinoma cells. Cancer Lett. 2022;539:215677.
Chen X, Guo J, Ren W, Zhou F, Niu X, Jiang X. LncRNA-AL035458.2/hsa-miR-181a-5p axis-mediated high expression of NCAPG2 correlates with tumor immune infiltration and non-small cell lung cancer progression. Front Oncol. 2022;12:910437.
Zhan P, Xi GM, Zhang B, Wu Y, Liu HB, Liu YF, Xu WJ, Zhu Q, Cai F, Zhou ZJ, et al. NCAPG2 promotes tumour proliferation by regulating G2/M phase and associates with poor prognosis in lung adenocarcinoma. J Cell Mol Med. 2017;21(4):665–76.
Kumamoto T, Seki N, Mataki H, Mizuno K, Kamikawaji K, Samukawa T, Koshizuka K, Goto Y, Inoue H. Regulation of TPD52 by antitumor microRNA-218 suppresses cancer cell migration and invasion in lung squamous cell carcinoma. Int J Oncol. 2016;49(5):1870–80.
Zhan P, Zhang B, Xi GM, Wu Y, Liu HB, Liu YF, Xu WJ, Zhu QQ, Cai F, Zhou ZJ, et al. PRC1 contributes to tumorigenesis of lung adenocarcinoma in association with the Wnt/beta-catenin signaling pathway. Mol Cancer. 2017;16(1):108.
Admoni-Elisha L, Elbaz T, Chopra A, Shapira G, Bedford MT, Fry CJ, Shomron N, Biggar K, Feldman M, Levy D. TWIST1 methylation by SETD6 selectively antagonizes LINC-PINT expression in glioma. Nucleic Acids Res. 2022;50:6903.
Pike R, Ortiz-Zapater E, Lumicisi B, Santis G, Parsons M. KIF22 coordinates CAR and EGFR dynamics to promote cancer cell proliferation. Sci Signal. 2018;11(515):eaaq1060.
Nakanishi K, Mizuno T, Sakakura N, Kuroda H, Shimizu J, Hida T, Yatabe Y, Sakao Y. Salvage surgery for small cell lung cancer after chemoradiotherapy. Jpn J Clin Oncol. 2019;49(4):389–92.
Oronsky B, Reid TR, Oronsky A, Carter CA. What’s new in SCLC? Rev Neoplasia. 2017;19(10):842–7.
Liu X, Xing H, Liu B. Current status and future perspectives of immune checkpoint inhibitors in extensive-stage small cell lung cancer. Am J Cancer Res. 2022;12(6):2447–64.
Lu G, Li Y, Ma Y, Lu J, Chen Y, Jiang Q, Qin Q, Zhao L, Huang Q, Luo Z, et al. Long noncoding RNA LINC00511 contributes to breast cancer tumourigenesis and stemness by inducing the miR-185-3p/E2F1/Nanog axis. J Exp Clin Cancer Res. 2018;37(1):289.
Sun CB, Wang HY, Han XQ, Liu YN, Wang MC, Zhang HX, Gu YF, Leng XG. LINC00511 promotes gastric cancer cell growth by acting as a ceRNA. World J Gastrointest Oncol. 2020;12(4):394–404.
Yu CL, Xu XL, Yuan F. LINC00511 is associated with the malignant status and promotes cell proliferation and motility in cervical cancer. Biosci Rep. 2019;39(9):BSR20190903.
Sun CC, Li SJ, Li G, Hua RX, Zhou XH, Li DJ. Long intergenic noncoding RNA 00511 acts as an oncogene in non-small-cell lung cancer by binding to EZH2 and suppressing p57. Mol Ther Nucleic Acids. 2016;5(11):e385.
Zhu FY, Zhang SR, Wang LH, Wu WD, Zhao H. LINC00511 promotes the progression of non-small cell lung cancer through downregulating LATS2 and KLF2 by binding to EZH2 and LSD1. Eur Rev Med Pharmacol Sci. 2019;23(19):8377–90.
Zhao Q, Zhang B, Li Z, Tang W, Du L, Sang H. Effects of IncRNA PROX1-AS1 on proliferation, migration, invasion and apoptosis of lung cancer cells by regulating MiR-1305. J Healthc Eng. 2022;2022:9570900.
Rudzinska M, Czarnecka-Chrebelska KH, Kuznetsova EB, Maryanchik SV, Parodi A, Korolev DO, Potoldykova N, Svetikova Y, Vinarov AZ, Nemtsova MV, et al. Long non-coding PROX1-AS1 expression correlates with renal cell carcinoma metastasis and aggressiveness. Noncoding RNA. 2021;7(2):25.
Guo T, Wang W, Ji Y, Zhang M, Xu G, Lin S. LncRNA PROX1-AS1 facilitates gastric cancer progression via miR-877-5p/PD-L1 Axis. Cancer Manag Res. 2021;13:2669–80.
Zhao L, Li JF, Tong XJ. Long noncoding RNA PROX1-AS1 promoted ovarian cancer cell proliferation and metastasis by suppressing KLF6. Eur Rev Med Pharmacol Sci. 2020;24(12):6561–8.
Marquez ME, Sernbo S, Payque E, Uria R, Tosar JP, Querol J, Berca C, Uriepero A, Prieto D, Alvarez-Saravia D, et al. TGF-beta/SMAD pathway is modulated by miR-26b-5p: another piece in the puzzle of chronic lymphocytic leukemia progression. Cancers (Basel). 2022;14(7):1676.
Tang G, Zhao H, Xie Z, Wei S, Chen G. Long non-coding RNA HAGLROS facilitates tumorigenesis and progression in hepatocellular carcinoma by sponging miR-26b-5p to up-regulate karyopherin alpha2 (KPNA2) and inactivate p53 signaling. Bioengineered. 2022;13(3):7829–46.
Ma S, Wei H, Wang C, Han J, Chen X, Li Y. MiR-26b-5p inhibits cell proliferation and EMT by targeting MYCBP in triple-negative breast cancer. Cell Mol Biol Lett. 2021;26(1):52.
Liu Y, Zhang G, Chen H, Wang H. Silencing lncRNA DUXAP8 inhibits lung adenocarcinoma progression by targeting miR-26b-5p. Biosci Rep. 2021;41(1):BSR20200884.
Wu T, Chen W, Liu S, Lu H, Wang H, Kong D, Huang X, Kong Q, Ning Y, Lu Z. Huaier suppresses proliferation and induces apoptosis in human pulmonary cancer cells via upregulation of miR-26b-5p. FEBS Lett. 2014;588(12):2107–14.
Byrne JA, Frost S, Chen Y, Bright RK. Tumor protein D52 (TPD52) and cancer-oncogene understudy or understudied oncogene? Tumour Biol. 2014;35(8):7369–82.
Li X, Li J, Xu L, Wei W, Cheng A, Zhang L, Zhang M, Wu G, Cai C. CDK16 promotes the progression and metastasis of triple-negative breast cancer by phosphorylating PRC1. J Exp Clin Cancer Res. 2022;41(1):149.
Bakhoum MF, Francis JH, Agustinus A, Earlie EM, Di Bona M, Abramson DH, Duran M, Masilionis I, Molina E, Shoushtari AN, et al. Loss of polycomb repressive complex 1 activity and chromosomal instability drive uveal melanoma progression. Nat Commun. 2021;12(1):5402.
Wang L, He M, Fu L, Jin Y. Role of lncRNAHCP5/microRNA-525-5p/PRC1 crosstalk in the malignant behaviors of ovarian cancer cells. Exp Cell Res. 2020;394(1):112129.
Li J, Ohmura S, Marchetto A, Orth MF, Imle R, Dallmayer M, Musa J, Knott MML, Holting TLB, Stein S, et al. Therapeutic targeting of the PLK1-PRC1-axis triggers cell death in genomically silent childhood cancer. Nat Commun. 2021;12(1):5356.
Rong Y, Gao J, Kuang T, Chen J, Li JA, Huang Y, Xin H, Fang Y, Han X, Sun LQ, et al. DIAPH3 promotes pancreatic cancer progression by activating selenoprotein TrxR1-mediated antioxidant effects. J Cell Mol Med. 2021;25(4):2163–75.
Dong L, Li Z, Xue L, Li G, Zhang C, Cai Z, Li H, Guo R. DIAPH3 promoted the growth, migration and metastasis of hepatocellular carcinoma cells by activating beta-catenin/TCF signaling. Mol Cell Biochem. 2018;438(1–2):183–90.
Zhang K, Hu H, Xu J, Qiu L, Chen H, Jiang X, Jiang Y. Circ_0001421 facilitates glycolysis and lung cancer development by regulating miR-4677-3p/CDCA3. Diagn Pathol. 2020;15(1):133.
This work was supported by Nanjing Medical Science and Technique Development Foundation (JQX19010) to Chenxi Zhang; Nanjing Health Science and Technology Development Major Project (ZDX22001).
Ethics approval and consent to participate
All the tissue specimens for this study were obtained with informed patient consent from the Nanjing Chest Hospital Biobank. All methods were performed in accordance with the Declaration of Helsinki, and the research protocol was reviewed and approved by the Medical Ethics Commission of Nanjing Chest Hospital.
Consent for publication
The authors declare no competing interests.
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
The RT-qPCR primer used in this study. Supplementary Table S2. The top 10 upregulated and downregulated mRNAs, miRNAs, and lncRNAs. Supplementary fig S1. Characteristics of TCONS_00020615. Schematic representation of TCONS_00020615 and PROX1. Supplementary fig S2. Relative expression levels of TCONS_00020615 in HBE, BEAS-2B, H1688, and H446 cells.
About this article
Cite this article
Zhang, C., Zhou, Y., Zhang, B. et al. Identification of lncRNA, miRNA and mRNA expression profiles and ceRNA Networks in small cell lung cancer. BMC Genomics 24, 217 (2023). https://doi.org/10.1186/s12864-023-09306-4