Pan-cancer expression heterogeneity of TSPEAR-AS2
TSPEAR-AS2 is a recently identified disease-associated lncRNA. However, its role in human cancers is yet to be fully elucidated. To address this knowledge gap, we used clinical data from two tumor databases, GEPIA and ENCORI, to map the pan-cancer expression profile of TSPEAR-AS2. According to the GEPIA database, the expression of TSPEAR-AS2 was dysregulated in multiple cancers. For instance, its expression was decreased in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), acute myeloid leukemia (LAML), lung adenocarcinoma (LUAD), rectum adenocarcinoma (READ), testicular germ cell tumors (TGCT), thyroid carcinoma (THCA), and uterine corpus endometrial carcinoma (UCEC) (Fig. 1a, b), whereas it was increased in ESCA and stomach adenocarcinoma (STAD) (Fig. 1c). The expression of TSPEAR-AS2 was not significantly altered in other cancer types. Data from the ENCORI database showed that TSPEAR-AS2 expression was deregulated in seven cancer types. Its expression was down-regulated in THCA (Fig. 1d) and up-regulated in colon adenocarcinoma (COAD), ESCA, head and neck squamous cell carcinoma (HNSC), kidney renal clear cell carcinoma (KIRC), liver hepatocellular carcinoma (LIHC), and STAD (Fig. 1e). However, the expression of TSPEAR-AS2 was not significantly altered in other cancer types. These results suggest that TSPEAR-AS2 exhibits heterogeneous expression across cancers.
TSPEAR-AS2 shows heterogeneity in human pan-cancer. (a-c) The GEPIA database was used to explore the expression landscape of TSPEAR-AS2 in human pan-cancer. TSPEAR-AS2 was found to be dysregulated in nine cancer types, including CESC (306T vs 13N), LAML (173T vs 70N), LUAD (483T vs 347N), READ (92T vs 318N), TGCT (137T vs 165N), THCA (512T vs 337N), UCEC (174T vs 91N), ESCA (182T vs 286N), and STAD (408T vs 211N). (d-e) The ENCORI database was employed to characterize the expression profile of TSPEAR-AS2 in human pan-cancer. TSPEAR-AS2 was dysregulated in seven cancer types, including THCA (510T vs 58N), COAD (471T vs 41N), ESCA (162T vs 11N), HNSC (502T vs 44N), KIRC (535T vs 72N), LIHC (374T vs 50N), and STAD (375T vs 32N). T represents tumor, N represents Normal. *p < 0.05, ****p < 0.0001.
TSPEAR-AS2 is up-regulated in ESCA and serves as a potential clinical biomarker
Data from GEPIA and ENCORI showed that TSPEAR-AS2 expression was dysregulated in ESCA, STAD, and THCA (Fig. 2a). Therefore, we used the UALCAN database to analyze the expression of TSPEAR-AS2 in the subtypes of these three cancers. The results revealed significant differences in the expression of TSPEAR-AS2 among the subtypes. In ESCA, TSPEAR-AS2 expression was higher level in adenocarcinoma than in squamous cell carcinoma (Supplementary Fig. 1a). In STAD, TSPEAR-AS2 expression was higher in AdenoNOS, IntAdenoNOS, and IntAdenoPapillary subtypes but lower in AdenoDiffuse and IntAdenoMucinouse subtypes (Supplementary Fig. 1b). In THCA, TSPEAR-AS2 expression was higher in Classical and Follicular subtypes but lower in Tall subtypes (Supplementary Fig. 1c). The heterogeneity of TSPEAR-AS2 expression among these subtypes highlights its potential as a precise therapeutic target.
High TSPEAR-AS2 expression predicts worse patient prognosis in ESCA. (a) A comparative analysis of TSPEAR-AS2 expression from GEPIA and ENCORI. (b-d) GEPIA database was used to analyze the relationship between TSPEAR-AS2 and prognosis of ESCA, STAD, and THCA. (e) Validation of TSPEAR-AS2 expression in three independent ESCA datasets, TCGA, GSE53622, and GSE53624. (f–h) The diagnostic value of TSPEAR-AS2 in three ESCA cohorts (TCGA, GSE53622, and GSE53624) was evaluated by ROC. (i-j) KMP database was used to analyze the relationship between TSPEAR-AS2 and disease-free survival or overall survival of ESCA patients. (k) The expression levels of TSPEAR-AS2 in Het-1A, EC1, TE1, KYSE70, and KYSE150 cells were detected by qRT-PCR. AUC represents area under the curve. *p < 0.05, **p < 0.01.
The GEPIA database was used to analyze the relationship between TSPEAR-AS2 expression and patient survival in the three cancers. It was found that TSPEAR-AS2 was associated with the prognosis of ESCA but not with that of STAD or THCA (Fig. 2b–d), implying that TSPEAR-AS2 might play a crucial role in ESCA progression. The over-expression of TSPEAR-AS2 in ESCA was validated in three independent ESCA cohorts (TCGA, 11N vs. 161T; GSE53622, 60N vs. 60T; and GSE53624, 119N vs. 119T) (Fig. 2e). Subsequently, ROC analysis was used to evaluate the diagnostic value of TSPEAR-AS2 in ESCA. The results showed that TSPEAR-AS2 exhibited high diagnostic value in all three ESCA cohorts (Fig. 2f–h). Using the Kaplan–Meier Plotter database, we found that high expression of TSPEAR-AS2 was a risk factor for both disease-free survival (Fig. 2i) and overall survival (Fig. 2j) in patients with ESCA. We further confirmed that TSPEAR-AS2 was up-regulated in ESCA cell lines by qRT-PCR (Fig. 2k). These data collectively indicate that TSPEAR-AS2 is a promising diagnostic and prognostic biomarker for ESCA.
RNA m7G transferase METTL1 mediates the expression of TSPEAR-AS2
Post-transcriptional modifications, such as N6-nathyladenosine (m6A), 5-methylcytosine (m5C), and N7-methylguanosine (m7G), play a crucial role in regulating gene expression in eukaryotic cells. To explore the potential mechanism underlying the up-regulation of TSPEAR-AS2 in ESCA, we analyzed the reported epigenetic modification enzymes FTO (m6A)19, METTL3 (m6A)20, NSUN2 (m5C)21, NSUN6 (m5C)22, METTL1 (m7G)23, and WDR4 (m7G)24 in relation to TSPEAR-AS2 using the GEPIA and ENCORI databases. The results showed that the expression of METTL1 or NSUN6 was positively correlated with TSPEAR-AS2 in ESCA tumors, respectively (Fig. 3a, b). We then explored the expressions of METTL1 and NSUN6 in ESCA using GEPIA and ENCORI databases, and found that METTL1 (Fig. 3c), but not NSUN6 (Fig. 3d), was increased in ESCA tumors, suggesting that METTL1 was a key factor involved in the regulation of TSPEAR-AS2. Over-expression of METTL1 in ESCA cells (Supplementary Fig. 2a) led to significant up-regulation of TSPEAR-AS2 (Fig. 3e), suggesting that METTL1 induces the expression of TSPEAR-AS2.
Up-regulation of METTL1 increases TSPEAR-AS2 expression in ESCA. (a) GEPIA database was employed to analyze the correlation between TSPEAR-AS2 and FTO, METTL3, NSUN2, NSUN6, METTL1, or WDR4 expression in 182 ESCA samples, respectively. (b) ENCORI database was used to analyze the correlation between TSPEAR-AS2 and FTO, METTL3, NSUN2, NSUN6, METTL1, or WDR4 expression in 162 ESCA samples, respectively. (c-d) GEPIA and ENCORI databases were used to explore the expression status of METTL1 and NSUN6 in ESCA. (e) The effect of over-expression of METTL1 on TSPEAR-AS2 expression in ESCA cells was tested by qRT-PCR. ns represents no significance. *p < 0.05, ****p < 0.0001.
Loss of TSPEAR-AS2 suppresses ESCA cell proliferation and cell cycle progression
We then performed bioinformatics analysis to investigate the potential biological function of TSPEAR-AS2 in ESCA. We conducted a Reactome enrichment analysis based on co-expressed genes of TSPEAR-AS2 and found that TSPEAR-AS2 was involved in ESCA cell proliferation and stemness regulation (Supplementary Fig. 2b). In addition, LncACTdb 3.0 showed that TSPEAR-AS2 participated in cell growth and metastasis (Supplementary Fig. 2c). Subsequently, both gain-of-function and loss-of-function assays were performed to assess the biological role of TSPEAR-AS2 in ESCA. Transfection with OE-AS2 (a recombinant lentivirus used to over-express TSPEAR-AS2) up-regulated TSPEAR-AS2 expression in ESCA cells (Fig. 4a), whereas transfection with two siRNAs targeting TSPEAR-AS2 (si-AS2#1 and si-AS2#2) significantly decreased its expression in ESCA cells (Fig. 4b). Results from CCK-8 experiments showed that over-expression of TSPEAR-AS2 accelerated the proliferation of ESCA cells (Fig. 4c), whereas its knockdown inhibited the growth of ESCA cells (Fig. 4d, e). Similarly, over-expression of TSPEAR-AS2 promoted ESCA cell cycle progression (Fig. 4f), whereas its down-regulation arrested ESCA cell cycle at the G0/G1 phase (Fig. 4g).
TSPEAR-AS2 promotes ESCA cell proliferation and cell cycle progression. (a, b) The knockdown and over-expression effects of TSPEAR-AS2 were detected by qRT-PCR. (c-e) The effects of TSPEAR-AS2 over-expression and knockdown on ESCA cell proliferation were evaluated by CCK-8 assay. (f-g) The changes of ESCA cell cycle after over-expression and knockdown of TSPEAR-AS2 were analyzed by flow cytometry. *p < 0.05, **p < 0.01, ***p < 0.001.
TSPEAR-AS2 promotes cell metastasis, stemness, and tumor growth in ESCA
Transwell and tumor sphere formation assays were employed to observe the effects of TSPEAR-AS2 on ESCA cell migration and stemness, respectively. The results illustrated that knockdown of TSPEAR-AS2 inhibited the metastatic ability of ESCA cells, whereas its enforced expression promoted ESCA cell migration (Fig. 5a, b). Moreover, ablation of TSPEAR-AS2 suppressed the stem-like properties of ESCA cells, whereas its up-regulation enhanced cell stemness (Fig. 5c, d). To further investigated the role of TSPEAR-AS2 in ESCA tumorigenesis, we subcutaneously inoculated KYSE70 cells stably transfected with the sh-AS2 or sh-NC lentivirus into nude mice. The knockdown effect of sh-AS2 on TSPEAR-AS2 in KYSE70 cells was evaluated by qRT-PCR (Supplementary Fig. 2d). Compared with the control group, TSPEAR-AS2 knockdown significantly retards the growth of ESCA tumors in vivo, as evidenced by reduced tumor volume and weight (Fig. 5e, f). These findings suggest that TSPEAR-AS2 promotes metastasis, stemness, and tumor growth in ESCA.
TSPEAR-AS2 exacerbates tumor growth and as a potential therapeutic target. (a, b) Transwell assay was used to observe the effects of knockdown and over-expression of TSPEAR-AS2 on ESCA cell migration. (c, d) Tumor sphere formation assay was performed to investigate the effects of knockdown and over-expression of TSPEAR-AS2 on stemness maintenance of ESCA cells. (e, f) The effect of lentivirus-mediated TSPEAR-AS2 knockdown on ESCA tumor growth was determined using nude mouse tumor bearing experiment. (g, h) The effect of intratumoral injection of ASO-TSPEAR-AS2 on ESCA growth was evaluated by tumor bearing experiment in nude mice. *p < 0.05, **p < 0.01.
TSPEAR-AS2 is a potential therapeutic target for ESCA
Targeting key molecules via ASOs is considered a promising therapeutic strategy for cancer treatment. We synthesized an ASO targeting TSPEAR-AS2 and examined its effects on TSPEAR-AS2 in KYSE70 cells (Supplementary Fig. 2e). To preliminarily evaluate the effectiveness of targeting TSPEAR-AS2 for the treatment of ESCA, we established a subcutaneous tumor-bearing model using nude mice, and injected ASO-AS2 into the tumors twice a week for three weeks. The results showed that ASO-AS2 significantly inhibited tumor growth in vivo (Fig. 5g). Both tumor volume and tumor weight were lower in the ASO-AS2 group than in the control group (Fig. 5h), suggesting the potential of TSPEAR-AS2 as a therapeutic target for ESCA.
Knockdown of TSPEAR-AS2 inhibits interferon signaling in ESCA cells
To investigate the mechanism underlying the oncogenic role of TSPEAR-AS2 in ESCA, we performed quantitative proteomics analysis on KYSE70 cells transfected with sh-NC or sh-AS2. We found that TSPEAR-AS2 knockdown increased the expression of 106 proteins and decreased that of 124 proteins (Fig. 6a, b). These differentially expressed proteins are shown in a heat map and volcano plot (Fig. 6c, d). To identify pathways mediated by TSPEAR-AS2, we performed pathway enrichment analyses of these 124 down-regulated proteins.
TSPEAR-AS2 regulates interferon signaling in ESCA. (a–d) Quantitative proteomics was used to reveal the changes of protein levels in ESCA cells after TSPEAR-AS2 deficiency. (e, f) GO-BP and Reactome analyses were performed to explore enrichment pathways of 124 down-regulated proteins. (g, h) The effects of TSPEAR-AS2 ablation on the levels of 12 interferon signaling pathway-related proteins in ESCA cells. *p < 0.05, **p < 0.01, ***p < 0.001.
Gene Ontology analysis showed that TSPEAR-AS2 was involved in biological processes such as MDA-5 signaling pathway, type I interferon signaling pathway, response to type I interferon, interferon-gamma-mediated signaling pathway, and positive regulation of type I interferon production (Fig. 6e). Reactome analysis showed that TSPEAR-AS2 was implicated in interferon alpha–beta signaling, interferon gamma signaling, CTLA4 inhibitory signaling, PD-1 signaling, and interferon signaling (Fig. 6f). These results suggest that TSPEAR-AS2 primarily affects interferon signaling pathways. Quantitative proteomic analysis showed that down-regulation of TSPEAR-AS2 resulted in decreased expression of 12 interferon-signaling-related proteins (HLA-E, HLA-DRB5, IRF3, IFIT1, IFIT2, IFIT3, PARP9, PTPN11, STAT1, OAS2, OAS3, and OSAL) (Fig. 6g, h). Collectively, these findings indicate that TSPEAR-AS2 promotes ESCA development via interferon signaling.
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- Source: https://www.nature.com/articles/s41598-024-80439-6