lncRNA SNHG6 regulates EZH2 expression by sponging miR-26a/b and miR-214 in colorectal cancer

The human colorectal cancer cell lines (HCT-116, HCT-8, SW-480, SW-620, DLD-1, and HT-29) and the human normal colorectal epithelial cell FHC were obtained from American Type Culture Collection. SW-480, SW-620, DLD-1, HT-29, and FHC cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum. HCT-116 and HCT-8 cells were cultured in RPMI-1640 medium with 10% fetal bovine serum. Cells were maintained in a humidified atmosphere of 5% CO₂ at 37 °C. All cells were authenticated via Short Tandem Repeat DNA profiling and routinely tested and found negative for mycoplasma infection.

A total of 120 CRC tissue and 80 adjacent nontumor tissue samples were obtained from patients during operations at the Affiliated Nanjing First Hospital of Nanjing Medical University (Nanjing, China). All collected tissue samples were immediately snap frozen in liquid nitrogen and stored at − 80 °C until used. The patient characteristics are listed in Table 1. This study was approved by the ethics committee on Human Research of the Nanjing First Hospital and written informed consents were obtained from all patients.

Total RNA was isolated from the tissue samples and cells by TRIzol (Invitrogen, USA) according to the manufacturer’s protocol. lncRNA and mRNA detection was performed using a SYBR Green PCR Kit (Takara, Japan) with an ABI 7500 System. GAPDH expression was employed as a control for normalization. MicroRNA detection was performed using a miDETECT A Track Kit (RiboBio, China). The expression of the small nuclear RNA U6 was used as a control for normalization. For SNHG6 copy number detection, genomic DNA was isolated using a Genomic DNA Purification Kit (Promega, USA). QRT-PCR was performed using SYBR Premix Ex Taq (Takara, Japan) with an ABI 7300 System. POLR2A, RPP14, andTBX15 expression levels were employed for normalization. These three loci are housekeeping genes, and their copy numbers are stable across the population. A sample with a mean expression of SNHG6 relative to these reference genes greater than 1.5 is defined as copy number gain. A sample with a mean expression of SNHG6 relative to these reference genes less than 0.6 is defined as copy number loss. Each experiment was repeated at least three times. Primers are listed in Additional file 1: Table S1.

The full-length complementary cDNAs of human SP1 was synthesized and cloned into the expression vector pcDNA3.1 (Invitrogen, China). The full-length complementary cDNA of SNHG6 (or cDNA containing mutations) was synthesized and cloned into the expression vector pLenti-Glll-GMV-GFP-2A-Puro (Applied Biological Materials, Canada). The small hairpin RNA (shRNA) of SNHG6 was synthesized and cloned into the piLenti-shRNA-GFP vector (Applied Biological Materials, Canada). siRNAs were synthesized by GenePharma (China). MicroRNA mimics and inhibitors were synthesized by RiboBio (China). The plasmid vectors and siRNAs were transfected into CRC cells using Lipofectamine 2000 (Invitrogen, USA) according to the manufacturer’s instructions. All siRNA and shRNA sequences are listed in Additional file 1: Table S2.

For the SNHG6 promoter luciferase reporter assay, different fragment sequences containing predicted SP1 binding sites were synthesized and then cloned into the pGL3-basic firefly luciferase reporter (GeneCreat, China). The pRL-TK vector was employed as a control. For the microRNA target gene luciferase reporter assay, target sequences containing the predicted microRNA binding sites (or containing mutations in the predicted microRNA binding sites) were synthesized and inserted into the psiCHECK-2 vector (Promega, USA). Luciferase activity was measured with a Dual Luciferase Assay system (Promega, USA).

ChIP assay was performed using the ChIP Assay Kit (Beyotime, China) according to the manual instructions with slight modifications. Briefly, HCT-116 and HCT-8 cells were cross-linked with 1% formaldehyde solution for 10 min at room temperature and quenched with 125 mM glycine. DNA fragments ranging from 200 to 500 bp were obtained via sonication. Then, the lysate was immunoprecipitated with anti-SP1, anti-H3K27me3, or IgG antibody. Immunoprecipitated DNA fragments were analyzed by qRT-PCR. ChIP primers are listed in Additional file 1: Table S1. The antibodies used in ChIP assay are listed in Additional file 1: Table S3.

Five-week-old male BALB/c nude mice were maintained under specific pathogen-free conditions and manipulated according to the protocols approved by the Animal Care Committee of Nanjing Medical College. Stably transfected HCT-116 cells (5 × 10⁶/0.2 ml PBS) were implanted into two sides of the same nude mouse in the armpit. Xenografts were examined every 4 days with digital calipers, and tumor volumes were calculated using the following equation: volume = 1/2 (length × width²). Twenty-three days later, the mice were sacrificed, and volumes of tumors were measured. For the in vivo tumor metastasis assay, the indicated cells (3 × 10⁶/0.2 ml PBS) were injected via the tail vein into nude mice; 60 days later, all mice were euthanized; and the lungs were surgically dissected. The samples were embedded in paraffin for hematoxylin and eosin (HE) staining and immunohistochemistry staining.

FISH assays were performed using a Fluorescent In Situ Hybridization Kit (RiboBio, China) according to the protocol. Cy3-labeled SNHG6 probes were designed and synthesized by RiboBio (China).

The nuclear and cytosolic fractions were separated using a PARIS Kit (Invitrogen, USA) according to the manufacturer’s instructions.

SNHG6 template DNA was transcribed in vitro with Biotin RNA Labeling Mix and T7 RNA polymerase (Roche, Switzerland) and purified with an RNeasy Mini Kit (Qiagen, USA) according to the manufacturer’s instructions. RNA-bound beads were incubated with total cell lysates of HCT-116, and eluted RNA was purified and assessed by qRT-PCR.

An EZ Magna RNA immunoprecipitation Kit (Millipore, USA) was used according to the manufacturer’s guidelines. Briefly, HCT-116 cells were lysed in RIP lysis buffer. Magnetic beads were preincubated with anti-AGO2 or IgG antibody for 30 min at room temperature, and the cell lysates were immunoprecipitated with beads for 6 h at 4 °C. Then, the immunoprecipitated RNA was purified and detected by qRT-PCR. The antibody information is listed in Additional file 1: Table S3.

Gene set enrichment analysis (GSEA) was used to explore the pathways and gene sets associated with SNHG6 in colorectal cancer. Gene expression profiles of 481 colorectal cancer samples were downloaded from TCGA dataset. According to the SNHG6 expression level, the top 25 % and the bottom 25 % of samples were grouped as the high SNHG6 group and low SNHG6 group respectively. GSEA v3.0 was used to determine whether the members of the gene sets from the MSigDB database were randomly distributed at the top or bottom of the ranking [21]. The number of permutation was 1000, and the threshold for the nominal P value was set to 0.05. If most members of a gene set were positively or negatively related to SNHG6, the set was considered to be associated with SNHG6.

All statistical analyses were performed using SPSS 18.0 (SPSS, USA) and GraphPad Prism 6 (GraphPad, USA) software. Chi-square test was used to analyze the different distribution of clinical variables. Differences in the level of gene expression were analyzed using Student’s t test. Univariate and multivariate Cox proportional hazards regression models were used to analyze potential factors associated with prognosis. Overall survival was estimated with the Kaplan–Meier method, and the log-rank test was employed to evaluate differences. For in vitro and in vivo experiments, a t test or analysis of variance was used to evaluate the differences between different groups. All P values were two-tailed, and P < 0.05 was considered statistically significant. All data are presented as the mean ± standard deviation (SD) from at least three independent replicates.

A complete description of the methods, including cell growth and colony formation assays, 5-Ethynyl-20-deoxyuridine (EdU), flow cytometry, protein extraction and western blot, Immunohistochemistry (IHC) and immunofluorescence (IF), wound healing assay, transwell migration and matrigel invasion assays and TUNEL assay are available in Additional file 2: Supplemental materials and methods.

We first analyzed the lncRNA expression profiles of CRC tissue and the adjacent normal tissue samples in The Cancer Genome Atlas (TCGA) [22]. Many abnormally expressed lncRNAs were identified. SNHG6 ranked as one of the top remarkably upregulated lncRNAs with relatively high abundance (Fig. 1a). We then analyzed microarray datasets of two independent CRC cohorts and found that SNHG6 expression was also upregulated (Fig. 1b). Besides, we detected SNHG6 expression in 80 paired colorectal cancer tissue and adjacent normal tissue samples by qRT-PCR, and SNHG6 expression was significantly upregulated in 88.8% (71 of 80 paired) of the colorectal cancer tissue samples (Fig. 1c). In addition, we analyzed SNHG6 expression in human colorectal cancer cell lines. As shown in Fig. 1d, SNHG6 expression was upregulated in all six colorectal cancer cell lines (SW-620, HCT-116, DLD-1, HCT-8, HCT-29, and SW-480) compared with the human colorectal epithelial cell line FHC (Fig. 1d). To explore the clinical relevance of SNHG6 in CRC, we divided the enrolled patients into two groups according to SNHG6 expression. As shown in Table 1, statistical analysis demonstrated that SNHG6 expression was correlated with tumor invasion depth (P = 0.010), distant metastasis (P = 0.001), lymph node metastasis (P = 0.013), and TNM stage (P = 0.008). We also analyzed SNHG6 expression in different CRC stages using TCGA data, and advanced stage tumors had higher SNHG6 expression (Additional file 3: Figure S1a). GSEA also revealed significant relations between the expression of dysregulated genes in CRC and SNHG6 (Fig. 1e). In addition, Kaplan–Meier survival analysis showed that high SNHG6 expression was significantly correlated with poor overall survival (P = 0.002) and disease-free survival (P = 0.036, Fig. 1h, i). The same result was observed in two independent CRC cohorts from the R2 database (http://​hgserver1.​amc.​nl/​cgi-bin/​r2/​main.​cgi) (Additional file 3: Figure S1b). To further evaluate the prognostic value of SNHG6, both univariate and multivariate analyses were performed. The univariate analysis results indicated that SNHG6 expression (HR = 3.24, 95% CI = 1.84–5.86, P < 0.001), TNM stage (HR = 2.72, 95% CI = 1.48–4.88, P < 0.001), and distant metastasis status (HR = 4.82, 95% CI = 2.52–8.96, P < 0.001) were prognostic factors. In addition, the multivariate analysis of the three prognosis factors revealed that SNHG6 expression was an independent prognostic biomarker (HR = 2.48, 95% CI = 1.60–5.86, P = 0.002) for colorectal cancer (Table 2). These results reveal that SNHG6 upregulation may play a critical role in the development and progression of colorectal cancer.

We next explored which factors induced high SNHG6 expression in CRC. SNHG6 is located on chromosome 8q13, a region with frequent copy number amplification in CRC. We speculated DNA copy number gains might be responsible for its upregulation. GSEA results also indicated that SNHG6 expression was positively correlated with the expression of genes in adjacent chromosomal regions (Fig. 2a). We then detected the genomic copy number levels of SNHG6 in 30 CRC tissue samples, and copy number gains was identified in 30% (9 of 30) colorectal cancer tissues (Fig. 2b). We also detected the SNHG6 genomic copy number levels in CRC cell lines, and SNHG6 copy number gains were observed in HCT-8 and HT-29 cells (Fig. 2c). Besides, we explored transcription factors that could potentially upregulate SNHG6 in CRC. We used the JASPAR CORE database to search for transcription factor binding sites in the SNHG6 promoter [23]. We found two putative SP1 binding sites: E1 (CCTCCGCCCCC, − 125 bp to − 115 bp) and E2 (ACTCCGCCTCA, − 901 bp to − 891 bp), got relatively high scores. We then analyzed SP1 ChIP-Seq data of HCT-116 downloaded from the Encyclopedia of DNA Elements (ENCODE) database. As shown in Fig. 2d, SP1 was highly enriched in the SNHG6 promoter region. We then silenced SP1 in HCT-116 and HCT-8 cells, and SNHG6 expression was decreased. In contrast, SP1 overexpression increased SNHG6 expression (Additional file 3: Figure S2a and Fig. 2e). In addition, we found SNHG6 expression was elevated in samples with high SP1 expression and that SNHG6 expression was positively correlated with SP1 expression in CRC tissues (Fig. 2f and Additional file 3: Figure S2b). Besides, luciferase reporter assays showed SP1 mainly bound to the E1 site of SNHG6 promoter (Fig. 2g). Furthermore, ChIP assays performed on E1 region of the SNHG6 promoter indicated SP1 interacted with the SNHG6 promoter region directly (Fig. 2h). Overall, the above results indicate that the upregulation of SNHG6 in CRC is partly due to SNHG6 genomic copy number gains and SP1 activation.

To further elucidate the role of SNHG6 in CRC, we first designed two independent small interfering RNAs (siRNAs) targeting SNHG6. As shown in Additional file 3: Figure S3a and b, these siRNAs could silence its expression and SNHG6 overexpression vectors could increase its expression efficiently. Then, CCK-8 assays demonstrated that SNHG6 knockdown inhibited HCT-116 and HCT-8 cell growth and SNHG6 overexpression promoted HCT-116 and HCT-8 cell growth significantly (Fig. 3a and Additional file 3: Figure S3c). Similarly, clone formation assays showed that SNHG6 knockdown decreased the clone-forming ability of CRC cells and SNHG6 overexpression increased the clone-forming ability of CRC cells (Fig. 3b and Additional file 3: Figure S3d). GSEA results revealed significant relations between the expression of cell cycle-related genes and SNHG6 in CRC (Fig. 3c). We then employed flow cytometry cell cycle assays and Ethynyl deoxy Uridine (EdU) dye assays to detect cell cycle progression and proliferation rates of SNHG6-silenced CRC cells. As we speculated, SNHG6 knockdown could induce cell cycle arrest and decrease the cell proliferation rate (Fig. 3d, e). In addition, western blotting results showed that the expression levels of cell cycle-related proteins cyclin D1, CDK4, and CDK6 were all decreased in si-SNHG6 transfected CRC cells (Fig. 3f). Moreover, GSEA results also indicated that SNHG6 expression exhibited significant relations with the expression of DNA repair and apoptosis-related genes in CRC (Fig. 3g). Flow cytometry cell apoptosis analysis showed SNHG6 knockdown significantly increased the proportion of apoptotic cells (Fig. 3h). Besides, SNHG6 silencing increased the expression of apoptosis-related proteins cleaved caspase-3, cleaved PARP, and Bax (Fig. 3i). In addition, we found that caspase-3 inhibitor Z-DEVD-FMK treatment (50 μM, 24 h) could partially abolish apoptosis of SNHG6 knockdown CRC cells (Additional file 3: Figure S3e). Taken together, these results demonstrate that SNHG6 can affect CRC cell growth by facilitating cell cycle progression and inhibiting cell apoptosis.

Subsequently, we explored the effects of SNHG6 on CRC metastasis. GSEA results indicated that SNHG6 expression was significantly correlated with the expression of metastasis-related genes (Fig. 4a). Transwell assays demonstrated that SNHG6 knockdown inhibited the migration and invasion abilities of HCT-116 and HCT-8 cells, and SNHG6 overexpression increased the migration and invasion abilities of HCT-116 and HCT-8 cells (Fig. 4b and Additional file 3: Figure S3f). The wound-healing assays showed that CRC cells transfected with SNHG6 siRNAs underwent slower scratch wound closure than the negative control (NC) cells (Fig. 4c). Besides, western blotting results showed that the protein level of the epithelial marker E-cadherin was markedly increased in SNHG6-silenced CRC cells. Conversely, the expression of the mesenchymal markers vimentin and MMP-9 was decreased (Fig. 4d). The same results were observed in immunofluorescence assays (Fig. 4e).

To evaluate the biological functions of SNHG6 in vivo, HCT-116 cells stably transfected with sh-SNHG6#1, SNHG6, or the corresponding empty vectors were subcutaneously or intravenously injected into nude mice. We found that tumor lumps in the sh-SNHG6#1 group were significantly smaller than those in the empty vector group. Conversely, the tumor volumes in the SNHG6 overexpressing group were larger than those in the empty vector group (Fig. 5a). At the end of this experiment, the mice were sacrificed, and we measured SNHG6 expression in each group. As expected, tumors formed from SNHG6 knockdown cells had lower SNHG6 expression and formed from SNHG6 overexpressing cells had higher SNHG6 expression (Fig. 5b). For lung metastasis, the number of metastatic nodules in the sh-SNHG6#1 group was lower than that in the vector control group. Accordingly, the number of metastatic nodules was increased in the SNHG6 overexpressing group (Fig. 5c). Besides, tumor tissues collected from the sh-SNHG6#1 group had lower Ki67-positive rates, whereas tissues collected from the SNHG6 overexpressing group had higher Ki67-positive rates than those from the corresponding control group. We also detected E-cadherin and vimentin expression in xenograft tumor tissues by immunohistochemistry. We found the E-cadherin expression was upregulated in the sh-SNHG6#1 group and downregulated in the SNHG6 overexpressing group. Vimentin expression exhibited the opposite trend (Fig. 5d). In addition, the terminal transferase dUTP nick end labeling (TUNEL) assays demonstrated that the tissues form the sh-SNHG6#1 group had higher cell apoptotic rates, whereas the tissues from the SNHG6 overexpressing group had lower cell apoptotic rates (Additional file 3: Figure S4a). Taken together, these results indicate that SNHG6 promotes tumor growth and metastasis in vivo, which is consistent with its functions in vitro.

To elucidate the potential molecular mechanisms through which SNHG6 contributes to the progression of CRC, we first examined its localization in CRC cells, because the functions of a lncRNA depended on its subcellular distribution [24]. Through FISH and subcellular fractionation assays, we identified that SNHG6 was mostly expressed in the cytoplasm (Fig. 6a, b). Many cytoplasmic lncRNAs have been reported to act as ceRNAs by competitively binding microRNAs. Thus, to demonstrate whether SNHG6 acts as a ceRNA, we first used miRcode software to explore miRNAs that could potentially target SNHG6 [25]. We found that a set of microRNAs (miR-214-3p, miR-26a-5p, miR-26b-5p, miR-1297, miR-17-5p, miR-21-5p, miR-139-5p, miR-181a-5p, miR-20a-5p, and miR-20b-5p) were predicted to have a high probability of combining to SNHG6. We next conducted RNA pull-down experiments with biotin-labeled SNHG6 in HCT-116 cells. As shown in Fig. 6c, miR-214-3p, miR-26a-5p, miR-26b-5p, miR-17-5p, and miR-139-5p could be pulled down by SNHG6. To further identify which microRNAs could interact with SNHG6 directly, we constructed corresponding dual luciferase reporter vectors for these five microRNAs. Dual luciferase reporter assays in HCT-116 cells indicated that only overexpression of miR-214-3p, miR-26a-5p, and miR-26b-5p could significantly decrease the luciferase activity (Additional file 3: Figure S5b). The efficiencies of mimics and inhibitors of the microRNAs are shown in Additional file 3: Figure S5a. In addition, to examine whether miR-214-3p, miR-26a-5p, and miR-26b-5p could bind to the predicted target sites in SNHG6, we constructed wild-type and mutant-type (putative binding sites for the microRNAs were mutated) luciferase reporter vectors of SNHG6. As expected, co-transfection of the wild-type SNHG6 luciferase vector (Luc-SNHG6-wt) with the miR-214-3p, miR-26a-5p, or miR-26b-5p mimics, but not the mutant SNHG6 vector (Luc-SNHG6-mt#1, Luc-SNHG6-mt#2), significantly decreased the luciferase activity (Fig. 6d). We also found overexpressing SNHG6 could reduce miR-214-3p, miR-26a-5p, and miR-26b-5p expression and knockdown of SNHG6 could increase these microRNA expressions significantly (Additional file 3: Figure S5c). Besides, correlation analysis revealed that there were negative correlations between the expression levels of SNHG6 and these microRNAs in 30 CRC tissues (Additional file 3: Figure S5d). Moreover, RIP assay results showed SNHG6, miR-214-3p, miR-26a-5p, and miR-26b-5p were all significantly enriched in AGO2-containing micro-ribonucleoprotein complexes, suggesting that the AGO2 protein bound directly to SNHG6, miR-214-3p, miR-26a-5p, and miR-26b-5p in CRC cells (Fig. 6e). The above results demonstrate that SNHG6 acts as a molecular sponge for miR-26a/b and miR-214 in CRC cells.

We next explored whether oncogenic functions of SNHG6 were dependent on sponging miR-214-3p, miR-26a-5p, and miR-26b-5p. The results of CCK-8 assays showed that miR-214-3p, miR-26a-5p, or miR-26b-5p downregulation could rescue the growth inhibition of HCT-116 cells caused by SNHG6 knockdown (Additional file 3: Figure S6a). In addition, by performing EdU assays, we found that SNHG6 overexpression could significantly increase HCT-116 cell proliferation rates compared with cells containing empty vectors and this increase could be eliminated when miR-214-3p, miR-26a-5p, or miR-26b-5p were transfected (Fig. 6f and Additional file 3: Figure S6b). Similarly, transwell assays showed that SNHG6 overexpression could significantly increase the migration and invasion abilities of HCT-116 cells and these increases could again be partially abolished when miR-214-3p, miR-26a-5p, or miR-26b-5p were transfected (Fig. 6g, h, Additional file 3: Figure S6c and d).

Taken together, these results indicate that SNHG6 promotes CRC progression by serving as a ceRNA for miR-214-3p, miR-26a-5p, and miR-26b-5p.

The functions of microRNAs rely on their downstream targets. Through literature review, we found that the oncogene EZH2 was reported to be a common target of these microRNAs. Our results also demonstrated that EZH2 expression was elevated in CRC tissues and that the overexpression of miR-214-3p, miR-26a-5p, or miR-26b-5p could decrease EZH2 expression in HCT-116 cells (Additional file 3: Figure S7a and b). Thus, we speculated that EZH2 might be involved in tumor-promoting functions of SNHG6. By expression analysis in HCT-116 cells, we found that when SNHG6 was silenced, EZH2 was downregulated. Besides, the inhibition of miR-214-3p, miR-26a-5p, or miR-26b-5p in SNHG6-silenced cells reversed the decrease of EZH2. Western blotting assays also showed that the inhibition of miR-214-3p, miR-26a-5p, or miR-26b-5p could rescue the EZH2 protein level decrease induced by SNHG6 knockdown (Fig. 7a). In addition, the overexpression of SNHG6 could upregulate EZH2, and transfection of the miR-214-3p, miR-26a-5p, or miR-26b-5p mimics abolished EZH2 increase in SNHG6-upregulated cells. To further verify that SNHG6 could regulate EZH2 expression by sponging these microRNAs, we constructed a SNHG6-mt vector, which contains mutations in the putative miR-214-3p, miR-26a-5p, and miR-26b-5p binding sites (Additional file 3: Figure S7c). As expected, transfecting the mutant SNHG6 plasmids into cells did not increase EZH2 expression. Meanwhile, the western blotting results were in agreement with the qPCR results (Fig. 7b). We then constructed luciferase reporter vectors Luc-EZH2 containing the 3′-untranslated region (3’-UTR) of EZH2. Dual luciferase reporter assays in HCT-116 cells showed that SNHG6 knockdown reduced Luc-EZH2 luciferase activity significantly, and the transfection of the miR-214-3p, miR-26a-5p, or miR-26b-5p inhibitors antagonized this decrease. We also found that the transfection of wild-type SNHG6 plasmids but not the SNHG6-mt plasmids could increase Luc-EZH2 luciferase activity, and the overexpression of miR-214-3p, miR-26a-5p, or miR-26b-5p could weaken the luciferase activity increase induced by ectopic SNHG6 expression (Fig. 7c). Besides, RIP assay results in SNHG6-silenced HCT-116 cells showed that the enrichment of Ago2 on SNHG6 was decreased and the enrichment of Ago2 on EZH2 was increased. Opposite changes were observed in SNHG6 overexpressed HCT-116 cells (Fig. 7d). Moreover, we observed a positive correlation between the expression levels of SNHG6 and EZH2 in 30 CRC tissues. The correlation between the expression levels of SNHG6 and EZH2 was confirmed in five independent CRC cohorts from the GEO database (Additional file 3: Figure S7d). The GSEA results also indicated a significant negative correlation between the expression of SNHG6 and the targets of EZH2 (Fig. 7e). In addition, immunohistochemistry assays showed that the EZH2 expression was significantly decreased in SNHG6 knockdown cells formed xenograft tumors and significantly increased in SNHG6 overexpression cells formed xenograft tumors (Additional file 3: Figure S7e). IHC assays also demonstrated that EZH2 protein levels were higher in high SNHG6 expression CRC tissues (Additional file 3: Figure S7f). Collectively, these results suggest that SNHG6 can release EZH2 by sequestering endogenous miR-214-3p, miR-26a-5p, and miR-26b-5p in CRC cells.

To investigate whether SNHG6 exerted oncogene functions by modulating EZH2, we first examined the effect of EZH2 on SNHG6-induced promotion of cell growth. The CCK-8 assay results showed that silencing EZH2 could abolish the growth acceleration of CRC cells induced by SNHG6 overexpression, and the EdU assay results revealed that silence of EZH2 could impair the increase of cell proliferative rates induced by SNHG6 upregulation (Fig. 8a, b). In addition, the increased cell migration and invasion abilities in SNHG6 overexpressing CRC cells were reversed by EZH2 knockdown (Fig. 8c). Many studies have shown that EZH2 can transcriptionally repress the INK4B-ARF-INK4A tumor suppressor locus to drive cell cycle progression and promote epithelial-mesenchymal transition (EMT) by suppressing the epithelial marker E-cadherin. Therefore, we next explored whether SNHG6 influenced the expression of these EZH2 targets. As shown in Fig. 8d, SNHG6 knockdown increased P14^ARF, P15^INK4b, P16^INK4a, and E-cadherin expression. Besides, ChIP assays revealed enrichment of the H3K27me3 modification induced by PRC2 was reduced in their promoter region after SNHG6 knockdown (Fig. 8e). Western blotting results showed that SNHG6 overexpression decreased the protein levels of P14^ARF, P15^INK4b, P16^INK4a, and E-cadherin, and these decreases could be rescued by EZH2 silencing. In comparison, SNHG6 knockdown inhibited P14^ARF, P15^INK4b, P16^INK4a, and E-cadherin expression in CRC cells (Fig. 8f). Moreover, SNHG6 overexpression increased the protein levels of cell cycle-related proteins and EMT related proteins, while SNHG6 silencing decreased them. Similarly, their increases induced by SNHG6 were impaired by EZH2 knockdown (Fig. 8g). Together, these data suggest that SNHG6 contributes to CRC progression through regulating EZH2 and its downstream targets.