LINC00330 as a potential new therapeutic target and biomarker for ESCC

To screen and identify TAM-associated lncRNAs in ESCC, transcriptional sequencing data of cancerous and adjacent normal esophageal tissue from 312 patients with ESCC were collected from the GEO (GSE53625, GSE130078, and GSE45670) and TCGA databases as of 1 November 2018. Differential expression analysis across datasets yielded 3102 lncRNAs, with 1077 downregulated and 2025 upregulated (Fig. 1A & Additional file 1: P1–4). Then, lncRNAs significantly differentially expressed in at least two databases (266 upregulated and 123 downregulated, Additional file 1: P5–6) were intersected with 932 immune-related lncRNAs (Additional file 1: P7) from the immune-related GSEA molecular signature database, resulting in 99 ESCC immune-related lncRNAs (64 upregulated lncRNAs and 35 downregulated lncRNAs; Fig. 1B & Additional file 1: P8–9). Next, using the ImmLnc web tool, we analyzed the correlation between these 99 lncRNAs and immune cell infiltration (mainly CD8+ T cells, dendritic cells, neutrophils, CD4+ T cells, B cells, and macrophages) in the ESCC TME (Additional file 1: P10–11), and 11 lncRNAs associated with TAM infiltration were identified (Additional file 1: P12). Among them, six lncRNAs (two upregulated, NR2F1-AS1 and ZFHX4-AS1; four downregulated, HAND2-AS1, LINC00330, RP11-834C11.4, and TTTY10) were defined as ESCC TAM-associated lncRNAs, which were only associated with TAM infiltration and had no correlation with infiltration of other immune cells (|r|> 0.3, P < 0.05; Additional file 3: Table S1). Finally, a series of literature investigations, bioinformatics reanalysis, and preexperimental detection were carried out, and LINC00330 was ultimately selected as the focus of this project.

Fig. 1

LINC00330 is expected to become a new target and biomarker for the treatment of ESCC. A LncRNAs with significantly upregulated and downregulated expression in the GSE53625, GSE130078, GSE45670, and TCGA datasets. B Intersection of immune-related lncRNAs with significantly upregulated or downregulated expression in the GSE53625, GSE130078, GSE45670, and TCGA datasets. C Screening flow chart of the ESCC TAM-associated lncRNA LINC00330. D The expression level of LINC00330 in different parts of the human body. Red represents tumor tissue and green represents normal tissue. E, F LINC00330 was significantly expressed at low levels in the GSE53625 and GSE130078 datasets. G The expression of LINC00330 in esophageal carcinoma tissues and paired paracancerous tissues in the TCGA database. H RT‒PCR detection of LINC00330 expression in 22 pairs of ESCC clinical samples. I Kaplan‒Meier analysis of prognostic survival in patients with high or low expression of LINC00330 in ESCC. The results are presented as the mean ± SD, * P < 0.05, ** P < 0.01, *** P < 0.001

Subsequently, the expression profile and clinical relevance of LINC00330 in ESCC were evaluated. Analysis of sequencing data from ESCC tissue samples in the TCGA and GEO databases indicated that LINC00330 was highly expressed in normal esophageal epithelial tissue (Fig. 1D) and significantly downregulated in ESCC tissue (Fig. 1E–G). Similarly, LINC00330 expression was assessed in the cancerous and adjacent normal tissues of 22 patients with ESCC who were pathologically diagnosed and underwent tumor resection surgery. The results demonstrated that LINC00330 was consistently underexpressed in the majority of ESCC tissues (17/22) compared with adjacent normal tissues (Fig. 1H). Moreover, clinical data from the TCGA were used to evaluate the associations between LINC00330 levels and clinical parameters in patients with ESCC. Kaplan–Meier analysis revealed a close association between low LINC00330 expression and poor prognosis in patients with ESCC. Patients with low LINC00330 expression had significantly worse overall survival and disease-free survival (Fig1I). However, no significant correlation was observed with other clinical parameters, such as age, sex, TNM stage, or tumor grade (Additional file 3: Table S2). In summary, our study indicated that the downregulation of LINC00330 is significant in ESCC and that its expression can predict patient outcomes.

Overexpression of LINC00330 inhibits ESCC progression in vivo and in vitro

To further clarify the function of LINC00330, we first analyzed the relative expression levels of LINC00330 in the normal esophageal epithelial cell line SHEE and five ESCC cell lines (Additional file 3: Figure S1A). We selected the Kyse450 and EC109 cell lines for subsequent tests owing to their moderate levels of LINC00330 expression. Next, we constructed LINC00330 overexpression (LINC00330) and knockdown (sh-LINC00330) plasmids. Overexpression (Additional file 3: Figure S1B) and knockdown (Additional file 3: Figure S1C) of LINC00330 in ESCC cell lines were achieved by transient transfection or lentiviral packaging and infection, followed by a series of gain-of-function and loss-of-function experiments.

The results showed that overexpression of LINC00330 significantly inhibited the proliferation (Fig. 2A, and Additional file 3: Figure S1D) and invasion (Fig. 2B, and Additional file 3: Figure S1E) of ESCC cells and promoted their apoptosis (Fig. 2C, and Additional file 3: Figure S1F). Conversely, knockdown of LINC00330 promoted the proliferation (Fig. 2D, and Additional file 3: Figure S1G) and invasion (Fig. 2E, and Additional file 3: Figure S1H) of ESCC cells and inhibited apoptosis (Fig. 2F and Additional file 3: Figure S1I).

Fig. 2

LINC00330 inhibits ESCC progression in vivo and in vitro. A, D CCK-8 assays were used to detect the effect of LINC00330 overexpression or knockdown on the proliferation of EC109 cells. B, E Transwell assays were used to detect the effect of LINC00330 overexpression or knockdown on the invasion of EC109 cells. Scale bar = 200 μm. C, F Flow cytometry was used to detect the effect of LINC00330 overexpression or knockdown on apoptosis in EC109 cells. G‒J RT‒PCR and WB were used to detect the effect of LINC00330 overexpression or knockdown on the EMT of EC109 cells. K‒M Overexpression of LINC00330 inhibited tumor growth in mice, and the tumors produced at the same time became increasingly smaller. N–P Knockdown of LINC00330 promoted subcutaneous tumor formation in nude mice, resulting in increased tumor size and increased tumor weight. The figure shows the mean ± SD of the statistical results from three independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001

Additionally, when culturing ESCC cells with LINC00330 knocked down, we unexpectedly observed that these cells acquired mesenchymal-like morphological characteristics (morphology changed from polygonal or pebble-like to elongated spindle or spindle-shaped), whereas ESCC cells overexpressing LINC00330 maintained the morphological characteristics of epithelial cells better (the cell morphology was mostly polygonal or pebble-like) (Additional file 3: Figure S1J&K). Therefore, we further assessed the changes in epithelial–mesenchymal transition (EMT) markers in ESCC cells by RT‒PCR and WB. The results indicated that upregulation of LINC00330 expression in ESCC cells promoted the expression of the epithelial marker E-cadherin and reduced the expression of mesenchymal markers (Fibronectin, β-catenin, Vimentin, ZEB1, Snail, and N-cadherin; Fig. 2G&H, and Additional file 3: Figure S1L). Conversely, in cells with downregulated LINC00330, the epithelial marker E-cadherin was decreased, while mesenchymal markers were upregulated to varying degrees (Fig. 2I, J, and Additional file 3: Figure S1M).

We also evaluated the function of LINC00330 in vivo using a subcutaneous xenograft model in nude mice. Subcutaneous injection of LINC00330-overexpressing ESCC cells suppressed tumor growth, resulting in decreased tumor volumes and decreased body weights (Fig. 2K–M). In contrast, mice injected with LINC00330-knockdown ESCC cells displayed enhanced tumor formation capacity, with larger tumor volumes and heavier body weights than those of the control group (Fig. 2N–P).

In conclusion, the study highlights the role of LINC00330 as a key inhibitor in the development of ESCC and demonstrates its ability to inhibit tumor progression both in vitro and in vivo, suggesting its potential as a therapeutic target and biomarker for ESCC treatment.

LINC00330 plays a significant role in the reprogramming of TAMs in ESCC

Next, we evaluated the correlation between LINC00330 and ESCC TAMs. High-throughput single-cell sequencing data from cancerous tissues (14 samples) and adjacent noncancerous tissues (13 samples) of patients with ESCC were collected (PRJNA777911), and a detailed analysis of the cellular composition within the ESCC tumor microenvironment (TME) was conducted. The results demonstrated a rich immune cell population in the ESCC TME, particularly groups of B cells, T cells, and macrophages (Fig. 3A), consistent with previous reports [12, 25]. Additionally, using CIBERSORTx (https://cibersortx.stanford.edu/), we quantified 22 types of immune cell infiltrates from RNA sequencing data of ESCC in The Cancer Genome Atlas (TCGA) database. Similar to the existing findings, the four predominant types of infiltrating immune cells in the ESCC TME were CD4 memory resting T cells, M0 macrophages, CD8 T cells, and M2 macrophages (Fig. 3B). Further analysis revealed that TAM infiltration was significantly higher in tumor tissues than in adjacent noncancerous tissues, with a predominance of M2 TAMs in ESCC tissues (Fig. 3C, D). The expression of LINC00330 was negatively correlated with the infiltration of M2 TAMs (Fig. 3E).

Fig. 3

LINC00330 plays an important role in ESCC TAM reprogramming. A Cluster analysis and type annotation of esophageal cancer tissue cells from single-cell high-throughput sequencing data (PRJNA777911). B Evaluation of immune infiltration of 22 immune cell types in ESCA patient tumor tissues in the TCGA cohort using CIBERSORTx. C Histogram showing the proportion of immune cells in tumor tissue for each patient. D The proportion of M2 macrophages in tumor tissues was significantly greater than that in nontumor tissues (Student’s t test, P = 0.00017). E CIBERSORTx analysis showed that LINC00330 expression was negatively correlated with M2 macrophage infiltration

In addition, using the GEPIA database (http://gepia.cancer-pku.cn/), we evaluated the correlation between the expression of LINC00330 and specific markers of these immune cells within the ESCC immune microenvironment. There was no significant correlation between the expression of LINC00330 and the expression of CD4 memory resting T cells, such as CCR7 (R = −0.059, P = 0.43), CD8 T cells, CD8 (R = −0.075, P = 0.31), or CD27 (R = −0.07, P = 0.35). However, there were significant negative correlations with the panmacrophage markers CD106 (R = −0.33, P = 6.50 × 10−9) and CD81 (R = −0.47, P = 6.20 × 10−40). Notably, LINC00330 expression was significantly positively correlated with markers for M1 macrophages, such as IL-12α (R = 0.56, P = 0) and IRF5 (R = 0.48, P = 0), and negatively correlated with several M2 macrophage markers, including CD209 (R = −0.27, P = 8.50 × 10−16), CD163 (R = −0.24, P = 2.10 × 10−10), CD206 (R = −0.27, P = 1.60 × 10−13), and IL-10 (R = −0.2, P = 7.90 × 10−08) (Additional file 3: Table S3).

We also utilized RT–PCR to analyze the relationship between LINC00330 expression and the infiltration of different types of TAMs in ESCC clinical samples and mouse tumor tissues (collected from Figs. 2L and O). The analysis of ESCC clinical samples indicated that in the majority of tumor tissues with low expression of LINC00330, there was a significant increase in CD68+ TAM infiltration (Additional file 3: Figure S2A). Notably, the interstitial infiltration of CD80+ M1 macrophages was significantly reduced (Additional file 3: Figure S2B), while the accumulation of CD163+ M2 macrophages was more pronounced (Additional file 3: Figure S2C). Similarly, in mouse tumor tissues, the expression of the panmacrophage marker CD68 was lower in the LINC00330 group than in the control group (Additional file 3: Figure S2D) but was significantly greater in the sh-LINC00330 group (Additional file 3: Figure S2E). Furthermore, compared with those in the control group, the expression of the M1 markers CD80 and CD86 was significantly increased in the tumor tissues of the LINC00330 group (Additional file 3: Figure S2F, G), while M2 TAM infiltration was reduced (as indicated by the significant reduction in the expression of the M2 surface markers CD163 and CD206) (Additional file 3: Figure S2H, I). Conversely, compared with that in the control group, M1 TAM infiltration was reduced in the sh-LINC00330 group (Additional file 3: Figure S2J, K), while M2 TAM infiltration was significantly increased (Additional file 3: Figure S2L, M).

In summary, our study suggested that LINC00330 plays a significant role in the reprogramming of TAMs in ESCC. Increasing the expression of LINC00330 may improve the state of the ESCC TME, promoting the polarization of TAMs (especially M2 TAMs) toward the M1 phenotype.

LINC00330-mediated TAM reprogramming inhibits ESCC proliferation and migration

To further clarify the role of LINC00330 in ESCC TAM reprogramming, we conducted “ESCC-macrophage coculture experiments.” Initially, we overexpressed or knocked down LINC00330 in ESCC cells (Fig. 4A, and Additional file 3: Figure S3A&E) and collected the conditioned medium. Subsequently, human mononuclear THP-1 cells were induced with PMA to differentiate into macrophages; IFN-γ and IL-4 were used to induce macrophage polarization into M1 and M2 TAMs, respectively. The success of the induction was confirmed by RT‒PCR detection of M1 TAM markers (IL-12α and IRF5) and M2 TAM markers (CD163 and CD206; Fig. 4B). Then, conditioned media from ESCC cells under different conditions were cocultured with various types of macrophages (M0, M1, M2) to assess the impact of LINC00330 on TAM reprogramming. The results demonstrated that conditioned media from ESCC cells overexpressing LINC00330 promoted polarization toward M1 TAMs, as characterized by increased expression of IL-12α and IRF5 and decreased expression of CD163 and CD206 (Fig. 4C–E, and Additional file 3: Figure S3B-D). Conversely, conditioned media from ESCC cells with LINC00330 knockdown promoted polarization toward M2 TAMs (Fig. 4F–H and Additional file 3: Figure S3F- H).

Fig. 4

LINC00330 facilitates ESCC TAM reprogramming. A Overexpression or knockdown of LINC00330 in EC109 cells. B RT‒PCR was used to detect the expression of markers of M1 (IL-12α and IRF-5) and M2 (CD163 and CD206) macrophages. C, F M0 macrophages were cocultured with conditioned medium generated by the overexpression or knockdown of LINC00330 in EC109 cells, and the expression of M1 and M2 markers was detected by RT‒PCR. D, G M1 macrophages were cocultured with conditioned medium generated by the overexpression or knockdown of LINC00330 in EC109 cells, and the expression of M1 and M2 markers was detected by RT‒PCR. E, H M2 macrophages were cocultured with conditioned medium generated by the overexpression or knockdown of LINC00330 in EC109 cells, and the expression of M1 and M2 markers was detected by RT‒PCR. The data are presented as means ± SDs; * P < 0.05, ** P < 0.01, *** P < 0.001

Next, we overexpressed and knocked down LINC00330 in M1 and M2 macrophages to determine whether LINC00330 could endogenously affect macrophage polarization. As shown in Fig. 5A–D, overexpression of LINC00330 enhanced the expression of M1 markers (IL-12α and IRF-5) in both M1 and M2 macrophages while reducing the expression levels of M2 markers (CD163 and CD206). In contrast, knocking down LINC00330 inhibited M1 polarization and enhanced M2 polarization. We also performed similar experiments using flow cytometry and obtained consistent results: there was an increase in the number of CD86+ M1 macrophages and a significant decrease in the number of CD163+ M2 macrophages after LINC00330 overexpression (Fig. 5E, G). Conversely, after knockdown of LINC00330, the number of CD86+ M1 macrophages significantly decreased, while the number of CD163+ M2 macrophages increased (Fig. 5F, H). In summary, these data suggest that LINC00330 can promote TAM reprogramming. Upregulation of LINC00330 expression activates M1 polarization and inhibits M2 polarization.

Fig. 5

LINC00330 can mediate reprogramming of different types of TAMs. A, E Overexpression of LINC00330 in M1 macrophages. RT‒PCR and flow cytometry were used to detect the expression of LINC00330 and M1/M2 markers. B, F After LINC00330 was knocked down in M1 macrophages, RT‒PCR and flow cytometry were used to detect the expression of the LINC00330 and M1/M2 markers. C, G Overexpression of LINC00330 in M2 macrophages. RT‒PCR and flow cytometry were used to detect the expression of the LINC00330 and M1/M2 markers. D, H After LINC00330 knockdown in M2 macrophages, RT‒PCR and flow cytometry were used to detect the expression of LINC00330 and M1/M2 markers. The figure shows the mean ± SD of three independent experiments, * P < 0.05, ** P < 0.01, *** P < 0.001

Finally, we evaluated the effects of LINC00330-mediated TAM reprogramming on the progression of ESCC. We overexpressed or knocked out LINC00330 in M1 macrophages and cocultured the cells with ESCC cell lines. As shown in Additional file 3: Figure S4, compared with the control group, the overexpression of LINC00330 in M1 macrophages restricted the growth of ESCC cells (Additional file 3: Figure S4A), promoted apoptosis (Additional file 3: Figure S4B), and inhibited the invasion of ESCC cells (Additional file 3: Figure S4C). In contrast, sh-LINC00330 in M1 cells significantly promoted ESCC cell growth (Additional file 3: Figure S4D), inhibited apoptosis (Additional file 3: Figure S4E), and enhanced tumor cell invasion (Additional file 3: Figure S4F). We performed similar experiments in M2 macrophages and obtained the same results; that is, M1 polarization mediated by LINC00330 overexpression inhibited the proliferation and migration of ESCC cells and promoted apoptosis (Additional file 3: Figure S4G–I). However, knockdown of LINC00330 led to suppressed M1 polarization, activated M2 polarization, and consequently promoted the proliferation and invasion of ESCC cells while inhibiting apoptosis in ESCC cells (Additional file 3: Figure S4J–L).

In summary, our study demonstrated that LINC00330 plays a significant role in TAM reprogramming and that LINC00330-mediated TAM reprogramming can inhibit the progression of ESCC.

LINC00330 inhibits the expression of CCL2 protein and the activation of its downstream signaling pathway

To elucidate the molecular mechanisms underlying the effects of LINC00330. We collected a control group (NC) and LINC00330-overexpressing Kyse450 cells for transcriptomic sequencing and bioinformatics analysis. KEGG analysis revealed that LINC00330 primarily affected “environment information processing” (Fig. 6A). GO enrichment analysis of the top 30 downregulated genes related to LINC00330 revealed that LINC00330 significantly affected the “chemokine activity” and “chemokine-mediated signaling pathway” in the immune system (Fig. 6B), with CCL2 being the gene with the most overlap (Fig. 6C).

Fig. 6

LINC00330 can inhibit the expression of CCL2 and the activation of its downstream signaling pathway. A KEGG analysis of the physiological pathways associated with the main regulation of LINC00330. B, C The top 30 genes downregulated by LINC00330 were “pathway” enriched according to the Reactome database. D The subcellular localization of CCL2 and LINC00330 was determined by RT‒PCR after cytoplasmic nucleus isolation. E Silver staining analysis after the LINC00330 RNA pulldown experiment. F The interaction between CCL2 and LINC00330 was detected by WB after the RNA pulldown experiment. G The interaction between CCL2 and LINC00330 was detected by RT‒PCR after RIP. H Pearson correlation analysis was used to analyze the correlation between LINC00330 and CCL2 expression. I WB was used to detect the effect of LINC00330 overexpression or knockdown on CCL2 protein expression in EC109 cells. J, K The effect of LINC00330 overexpression or knockdown in ESCC cells on the CCL2/CCR2 axis and its downstream signaling pathway was detected via RT‒PCR. L, M The effect of LINC00330 overexpression or knockdown on the CCL2/CCR2 axis and its downstream signaling pathway in ESCC cells was detected via WB. The figure shows the mean ± SD of the statistical results from three independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001

To further elucidate the relationship between LINC00330 and CCL2, we first determined the subcellular localization of these proteins. Nuclear-cytoplasmic fractionation experiments revealed that LINC00330 and CCL2 were colocalized in the cytoplasm of ESCC cells (Fig. 6D). According to previous studies, the subcellular localization of lncRNAs is closely related to their functions. Cytoplasmic localization of lncRNAs often involves signal transduction, posttranscriptional regulation, translation, and posttranslational modifications [16]. Therefore, we hypothesize that LINC00330 exerts diverse functions by forming RNA‒protein complexes with proteins. To further clarify the relationship between LINC00330 and CCL2 proteins, we constructed sense and antisense probes for LINC00330, biotin-labeled them, and performed RNA pulldown experiments, silver staining (Fig. 6E) and CCL2-specific WB (Fig. 6F). The results showed that only the sense probe for LINC00330 immunoprecipitated with the CCL2 protein, while the negative control group and the antisense probe group did not show this interaction (Fig. 6E, F). This indicates that there is direct targeted binding between LINC00330 and the CCL2 protein. In addition, through RIP experiments, we confirmed the interaction between LINC00330 and the CCL2 protein (Fig. 6G). Subsequently, we also analyzed the correlation between LINC00330 and CCL2 through clinical correlation analysis, Pearson correlation analysis, and WB detection. We found that CCL2 was more highly expressed in ESCC tissues (Additional file 3: Figure S5A) and that its high expression was closely correlated with a higher stage grade (Additional file 3: Figure S5B) and poor prognostic survival (Additional file 3: Figure S5C, D), as opposed to LINC00330 (Fig. 1H, I). Pearson correlation analysis revealed that LINC00330 expression was negatively correlated with CCL2 expression (Fig. 6H). In addition, LINC00330 overexpression inhibited CCL2 protein expression, whereas LINC00330 knockdown significantly upregulated CCL2 protein expression (Fig. 6I). These results suggest that CCL2 is a target of LINC00330. LINC00330 can directly bind to the CCL2 protein to form RNA‒protein complexes that inhibit its translation and function.

Additionally, CCL2 preferentially binds to CCR2 receptors and initiates a variety of signal transduction pathways (including more classical M1/M2 activation pathways, such as the PI3K/AKT, ERK, JAK-STAT and NF-κB signaling pathways [26]), stimulating cell migration and mediating tumor pathogenesis [27]. In our study, GSEA and KEGG mapping revealed that LINC00330 can negatively regulate “chemokine-mediated signaling pathways,” such as the “JAK–STAT signaling pathway,” “MAPK signaling pathway,” and “PI3K–AKT signaling pathway” (Additional file 3: Figure S5E, F). Therefore, we examined the effect of changes in LINC00330 expression on the expression of key genes in the CCL2/CCR2 axis and downstream signaling pathways (such as CCL2/CCR2, JAK/STAT3, ERK1/2, and AKT/mTORC1). We first evaluated the correlation between the expression of LINC00330 and CCR2, as well as between CCL2 and CCR2, in ESCC clinical samples. The results showed a weak negative correlation between CCR2 and LINC00330 expression (Additional file 3: Figure S5G) and a positive correlation with CCL2 expression (Additional file 3: Figure S5H). Furthermore, CCR2 was found to be highly expressed in ESCC tumor tissues (Additional file 3: Figure S5I), and its high expression was indicative of poorer survival (Additional file 3: Figure S5J), similar to that of CCL2 (Additional file 3: Figure S5C&D), but in contrast to that of LINC00330 (Fig. 1I). Next, we examined the effect of changes in LINC00330 expression on the expression of key genes in the CCL2/CCR2 axis and downstream signaling pathways (such as CCL2/CCR2, JAK/STAT3, ERK1/2, and AKT/mTORC1). The results showed that overexpression of LINC00330 suppressed the expression of CCL2/CCR2 and inhibited the activation of key genes in signaling pathways. Conversely, knockdown of LINC00330 activated the CCL2/CCR2 axis and related downstream signaling pathways (Fig. 6J–M & Additional file 3: Figure S5K–M).

In summary, our research demonstrated a negative regulatory relationship between LINC00330 and CCL2. LINC00330 binds to the CCL2 protein and inhibits the expression of CCL2 and downstream signaling pathways. This finding suggested that LINC00330 can suppress the progression of ESCC by inhibiting the expression of CCL2, thereby blocking the activation of the CCL2/CCR2 axis and its downstream signal transduction pathways, promoting TAM reprogramming, and inhibiting ESCC progression.

CCL2 is critical for LINC00330-mediated TAM reprogramming and ESCC progression

Finally, we performed “rescue” experiments to investigate the role of CCL2 in LINC00330-mediated TAM reprogramming. We overexpressed or knocked down LINC00330 in EC109 cells and subsequently overexpressed or knocked down CCL2 (Fig. 7A, and Figure S6A&E). We collected conditioned media under these various conditions. These conditioned media were then cocultured with M0 macrophages (Fig. 7B, and Additional file 3: Figure S6B&F), M1 macrophages (Fig. 7C and Additional file 3: Figure S6C&G), and M2 macrophages (Fig. 7D and Additional file 3: Figure S6D, H). We assessed the expression of M1 and M2 markers by RT‒PCR. The results indicated that overexpression of LINC00330 promoted the polarization of various types of macrophages toward the M1 phenotype, as evidenced by increased expression of M1 markers and decreased expression of M2 markers. Conversely, knocking down LINC00330 promoted M2 polarization and inhibited M1 macrophage accumulation. Overexpression of CCL2 reversed the LINC00330-mediated reprogramming of TAMs, which was characterized by inhibited M1 polarization and significantly increased M2 polarization. Knocking down CCL2 further promoted polarization toward the M1 phenotype and suppressed the expression of M2 markers; however, overexpression of CCL2 aggravated the M2 polarization of TAMs caused by knockdown of LINC00330, promoting TAM reprogramming. Knocking down CCL2 somewhat alleviated the M2 polarization caused by sh-LINC00330 and promoted TAM reprogramming toward M1 (Fig. 7 and Additional file 3: Figure S6).

Fig. 7

CCL2 plays an important role in LINC00330-mediated TAM reprogramming in ESCC. A RT‒PCR was used to detect the expression of LINC00330 and CCL2 in EC109 cells under different treatment conditions. B M0 macrophages successfully induced were cocultured with conditioned medium generated by different experimental groups, and the expression of M1 and M2 markers was detected by RT‒PCR. C Successfully induced M1 macrophages were cocultured with conditioned medium generated by different experimental groups, and the expression of M1 and M2 markers was detected by RT‒PCR. D Successfully induced M2 macrophages were cocultured with conditioned medium generated by different experimental groups, and the expression of M1 and M2 markers was detected by RT‒PCR. The figure shows the mean ± SD of three independent experiments; * P < 0.05, ** P < 0.01, *** P < 0.001

We also detected the critical role of CCL2 in LINC00330-mediated ESCC progression. Initially, we overexpressed and knocked down CCL2 on the basis of LINC00330 overexpression (Fig. 8A, B, and Additional file 3: Figure S7A&B) and evaluated proliferation, invasion, and EMT of ESCC cells. The results revealed that overexpression of CCL2 could reverse the inhibitory effects of LINC00330 on ESCC cell proliferation (Fig. 8C, and Additional file 3: Figure S7C), invasion (Fig. 8G, and Additional file 3: Figure S7G), and EMT (Fig. 8H, and Additional file 3: Figure S7H). Conversely, knockdown of CCL2 further enhanced the anticancer effects mediated by LINC00330.

Fig. 8

CCL2 plays an important role in LINC00330-mediated ESCC progression. A, D RT–PCR was used to detect the expression levels of LINC00330 and CCL2 mRNA in EC109 cells. B, E WB was used to detect the expression level of CCL2 protein in EC109 cells. C, F CCK-8 assay was used to detect the effect of different treatments on cell proliferation in EC109 cells. G, I Transwell assays were used to detect the effect of different treatments on the invasion of EC109 cells. Scale bar , 200 μm. H, J RT‒PCR was used to detect the effect of different treatments on the EMT ability of EC109 cells. * P < 0.05, ** P < 0.01, *** P < 0.001

Additionally, we overexpressed or knocked down CCL2 on the basis of LINC00330 knockdown (Fig. 8D&E, and Additional file 3: Figure S7D&E) and assessed their impact on ESCC cell proliferation, invasion, and EMT. The results indicated that overexpression of CCL2 significantly exacerbated the promoting effects of LINC00330 knockdown on ESCC cell proliferation (Fig. 8F, and Additional file 3: Figure S7F), invasion (Fig. 8I, and Additional file 3: Figure S7I), and EMT (Fig. 8J, and Additional file 3: Figure S7J). Conversely, knockdown of CCL2 partially reversed this phenomenon. In summary, our study demonstrates that CCL2 is critical for LINC00330-mediated TAM reprogramming and ESCC progression.

In conclusion, as shown in the “Graphical Abstract” (Fig. 9), our study showed that LINC00330 was expressed at significantly lower levels in ESCC. Low expression of LINC00330 can upregulate the expression of endogenous CCL2 and activate downstream signaling pathways, leading to the deterioration of ESCC. Deteriorated ESCCs secrete CCL2 and recruit CCR2+ mononuclear cells to reside in ESCC tissues to form TAMs [