SNORD93 suppresses colorectal cancer progression by inducing G0/G1 arrest and apoptosis

Introduction

Colorectal cancer (CRC) is the third most common cancer and the second leading cause of cancer-related death, accounting for approximately 10% of all cancer cases (1). Progress in early detection protocols, systematic surveillance, and optimized therapeutic interventions has driven measurable reductions in CRC burden, evidenced by declining incidence and mortality trends (2,3). Despite therapeutic advances, CRC prognosis remains suboptimal, with 5-year recurrence rates reaching 25% in unresectable cases and 35–45% among stage II/III patients following curative surgery (4). Alarmingly, early-onset CRC incidence escalates annually, evidenced by 1.2% year-over-year mortality increases in under-50 cohorts [2005–2019] (5). This substantial disease burden underscores the critical demand for innovative molecular signatures and druggable targets in CRC management.

Small nucleolar RNAs (snoRNAs), a subclass of noncoding RNAs, are typically distributed in the nucleolus (6). snoRNAs have been identified as housekeeping genes due to their unique function in the posttranscriptional modification of RNAs, including ribosomal RNA (rRNA) and small nuclear RNA (snRNA), via base pairing with target RNAs (7,8). In 2002, Chang et al. discovered that h5sn2, a H/ACA box snoRNA, exhibits lower expression in patients with meningioma and contributes to a poorer prognosis, establishing a groundbreaking connection between snoRNA and cancer development (9). Since then, numerous studies have revealed that snoRNAs act dually in both promoting and suppressing malignancy (10,11). For instance, SNORD88B, as an oncogene, initiates hepatocarcinogenesis in liver cancer. After SNORD88B anchors WRN in the nucleolus, Hippo signaling is inactivated as a result of X-ray repair cross complementing 5 (XRCC5) interacting with the serine/threonine kinase 4 (STK4) promoter to suppress its transcription (12). Conversely, SNORA21 was found to function as a tumor inhibitor, inducing cells to stagnate in the G0/G1 phase (13). Consequently, examining the regulatory role of snoRNAs in the pathogenesis and progression of cancer may be helpful in identifying novel markers for the diagnosis, treatment, and prognosis of patients with cancer.

SNORD93, a subclass of C/D box snoRNA, has been found to play a significant role in tumor development. Although there is currently no widely documented evidence or consensus in the scientific literature regarding recurrent genomic amplifications or mutations in SNORD93, several studies have reported its altered expression in certain types of neoplasms. For instance, SNORD93 is aberrantly expressed in renal clear-cell carcinoma and is associated with poor patient prognosis (14). Moreover, SNORD93 can be upregulated to promote the invasion of breast cancer and can be converted into the brief fragment snoRNA-derived small RNA (sdRNA93), resembling microRNA (miRNA), to inhibit the production of the pipecolic acid and sarcosine oxidase (PIPOX) (15). However, the expression of SNORD93 and its biological significance in CRC have not been clarified. This research delineates SNORD93’s functional impact and associated molecular pathways in colorectal carcinogenesis. Analyses revealed significant SNORD93 underexpression in CRC specimens, indicating tumor-suppressive roles during malignant progression. We present this article in accordance with the MDAR reporting checklist (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1236/rc).

Methods

Tissue samples

This study examined 13 paired CRC tissue samples and their adjacent nontumorous tissues obtained from 13 patients who had undergone surgical excision at the Quanzhou First Hospital Affiliated to Fujian Medical University. The CRC diagnosis was confirmed via a histological analysis of each tissue sample collected. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by institutional ethics board of Quanzhou First Hospital Affiliated to Fujian Medical University (2023, No. 218), and informed consent was taken from all the patients.

Cell culture

The human CRC cell lines HT-29 (HTB-38), SW480 (CCL-228), and HCT-15 (CCL-225), and the normal human intestinal epithelial crypt cell (HIEC) line (CRL-3266) were sourced from the American Type Culture Collection (ATCC; Manassas, VI, USA). All cell lines were then cultured in RPMI-1640 complete medium (Gibco, Thermo Fisher Scientific, Waltham, MA, USA) containing 10% fetal bovine serum (FBS; Gibco), and 1% penicillin-streptomycin (Invitrogen, Thermo Fisher Scientific) in a 5% CO₂ humidified atmosphere incubator at 37 ℃.

Plasmids and transfection

The full-length SNORD93 sequence was cloned into the pGPU6-GEP-Neo vector (Invitrogen) to establish SNORD93 overexpression. Cells were transfected with the pGPU6-SNORD93 plasmid via Lipofectamine 3000 (Invitrogen) according to the manufacturer’s protocol. The expression of SNORD93 was further confirmed via quantitative real time polymerase chain reaction (qRT-PCR).

RNA extraction and qRT-PCR

Total RNA was extracted with a column extraction kit, and RT-PCR was performed with HiScript III RT SuperMix according to the manufacturer’s protocol (Nanjing Vazyme Biotech Co., Ltd., Nanjing, China). RNA was then reverse transcribed into complementary DNA (cDNA). Subsequently, SNORD93 messenger RNA (mRNA) expression was measured with qRT-PCR and amplification via the SYBR Green Master Mix Kit (Nanjing Vazyme Biotech Co., Ltd.). The expression was normalized to 18S rRNA, and the relative expression was evaluated with the 2^−ΔΔCt method.

Cell Counting Kit-8 (CCK-8) assay

CCK-8 was used to assess cell viability following the transfection of CRC cells with pGPU6-SNORD93 or the control. The cells were seeded into the 96-well plates with 5,000 cells in each well and then cultivated for 0, 24, 48, and 72 hours, after which 10 µL of CCK-8 reagent was added. Following this, the plate was incubated for 1.5 hours at 37 ℃, which was followed by the measurement of the optical density values at 450 nm.

Colony formation assay

Colony formation assay was employed to evaluate the cell capacity of colony formation after the CRC cells were transfected with pGPU6-SNORD93 or the control. The cells were cultured in six-well plates with 1,000 cells in each well, and the medium was refreshed every 2 days. Fourteen days later, the cell colonies became visible and were fixed with formaldehyde for 30 minutes and stained with crystal violet for 30 minutes. Cell colonies were then counted manually after the background color cleared.

Cell cycle assay

Cell cycle assay was applied to analyze the cell cycle profile of CRC cells that were respectively treated with pGPU6-SNORD93 or the control. After 48 hours, the CRC cells were harvested and then washed twice in cold phosphate-buffered saline (PBS), resuspended in 70% ethanol, and fixed overnight at 4 ℃. Subsequently, they were treated with propidium iodide (PI) for 30 minutes at 37 ℃. For the cell apoptosis assay, the percentages of cells in different phases of the cell cycle were determined via flow cytometry, with the analysis being conducted with FlowJo v. 10.6.2 software (BD, Franklin Lakes, NY, USA).

Cell apoptosis assay

Cell apoptosis was detected in CRC cells transfected with the control or pGPU6-SNORD93. After 48 hours, the CRC cells were digested with trypsin without EDTA and harvested following treatment with the Annexin-V-FITC binding buffer, Annexin-V-FITC, and PI (cat. No. C1067S; Beyotime Biotechnology, Shanghai, USA). Subsequently, the CRC cells were incubated for 20 minutes at room temperature and shielded from light and subjected to an ice bath, after which flow cytometry was performed. PI produced red fluorescence, whereas Annexin V-FITC produced green fluorescence.

Wound healing assay

Wound healing assay was used to measure the migration capacity of CRC cells after transfection with the control or pGPU6-SNORD93. A total of 5×10⁵ CRC cells were cultured in six-well plates, and then a vertical wound was marked in a Petri dish with the tip of a 200-µL pipette. The outcomes of scratch migration to the central zone were evaluated at 0, 24, and 48 hours, respectively.

Western blotting (WB)

Cellular proteins were extracted with RIPA lysis buffer, and protein lysates were collected following centrifugation. Subsequently, a bicinchoninic acid (BCA) assay was used to quantify the proteins. Following denaturation, protein samples were separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and the proteins were subsequently transferred to polyvinylidene fluoride (PVDF) membranes. Following this step, the membranes were blocked with 5% nonfat milk in Tris-buffered saline containing Tween-20 (TBST) for 2 hours at room temperature, which was followed by overnight incubation at 4 ℃ with the following primary antibodies: p21 [cat. No. 2947T; Cell Signaling Technology (CST), Danvers, MA, USA], cyclin D3 (cat. No. 2936T; CST), cyclin E2 (cat. No. 4132P; CST), CDK9 (cat. No. 2316P; CST), caspase 3 (cat. No. 14220T; CST), caspase 7 (cat. No. 12827T; CST), caspase 8 (cat. No. 4790T; CST), Bim (cat. No. 2933P; CST), receptor-interacting protein (RIP) (cat. No. 3493T; CST), phosphorylated RIP (p-RIP) (cat. No. 65746T; CST), mixed lineage kinase domain-like protein (MLKL) (cat. No. 14993T; CST), phosphorylated MLKL (p-MLKL) (cat. No. 91689T; CST), E-cadherin (cat. No. 3195T; CST), and β-catenin (cat. No. 8480T; CST). β-actin (cat. No. 3700; CST) and GAPDH (cat. No. 60004-1-Ig; Proteintech, Rosemont, IL, USA) were used as the reference proteins. The membrane was incubated with secondary antibodies, either goat anti-mouse IgG (cat. No. 21005148; Proteintech) or goat anti-rabbit IgG (cat. No. 7074P2; CST), following a triple wash with PBS for 2 hours at room temperature. After three washes, proteins were detected on the membranes with enhanced chemiluminescence (ECL) reagent.

Statistical analysis

For statistical analysis, we used GraphPad Prism 8 software (Dotmatics, Boston, MA, USA). Data normality was verified using Shapiro-Wilk tests (α=0.05). Non-normally distributed data were analyzed with Mann-Whitney U tests, and normally distributed data were compared using Student’s t-test for two-group comparisons. One-way analysis of variance (ANOVA) was applied to compare data between multiple groups. When ≥2 hypotheses were tested concurrently on a single dataset, appropriate multiple testing corrections were applied. All experiments included ≥3 biological replicates (independently cultured cells). P values less than or equal to 0.05 were considered statistically significant.

Results

SNORD93 was downregulated and correlated with poor prognosis in CRC

The abundance of SNORD93 in patients with CRC was detected via qRT-PCR to clarify the relationship between SNORD93 and CRC. The expression of SNORD93 was significantly downregulated in CRC tissue as compared with adjacent normal tissues (P=0.03; Figure 1A and Table 1). Furthermore, we analyzed the SNORD93 expression patterns in a subset of CRC cell lines (SW480, HT-29, and HCT-15) and the normal HIEC line. As expected, SNORD93 exhibited lower expression in the CRC cell lines (HIEC: SW480, P<0.001; HIEC: HT-29, P<0.001; HIEC: HCT-15, P=0.002; Figure 1B). Pilot data from 13 patients suggested SNORD93 dysregulation may contribute to CRC progression in preliminary observations; large-scale validation is required in the future. Therefore, we further screened SNORD93 in the SNORic databases (16). The patients were divided into two groups based on the median expression level of SNORD93, and the association between SNORD93 expression and overall survival was determined. The results demonstrated that a lower SNORD93 expression was associated with a significantly worse prognosis (P=0.001; Figure 1C). Preliminary clinical data supported by public cohorts and the cellular-level evidence consistently suggest SNORD93 as a potential tumor suppressor.

Figure 1 Reduced SNORD93 expression in CRC. (A) Expression levels of the SNORD93 mRNA in 13 CRC tissues and paired adjacent tissues were determined via qRT-PCR analysis (P=0.03), normalized to 18S rRNA. (B) Expression levels of the SNORD93 mRNA in CRC cell lines (SW480, HT-29, and HCT-15) relative to normal HIEC controls, quantified by qRT-PCR analysis (HIEC: SW480, P<0.001; HIEC: HT-29, P<0.001; HIEC: HCT-15, P=0.002). (C) Prognostic relevance of SNORD93 abundance in colorectal cancer survival outcomes. Green color indicates patients with CRC and low SNORD93 expression, while red color indicates patients with CRC and high SNORD93 expression (P=0.001). Data are presented as mean ± SD (n=3). *, P<0.05; **, P<0.01; ***, P<0.001. CRC, colorectal cancer; HIEC, human intestinal epithelial crypt cell; mRNA, messenger RNA; NT, normal tissue samples; qRT-PCR, quantitative real-time polymerase chain reaction; rRNA, ribosomal RNA; SD, standard deviation; T, tissue samples.

SNORD93 inhibited the proliferation of CRC cells

Tumor cell proliferation is one of the most significant indicators of a malignant phenotype. To determine the biological effects of SNORD93 on CRC tumor progression, we upregulated the expression of SNORD93 in the HCT-15 and HT-29 cells via transfection with pGPU6-SNORD93. The transfection of pGPU6-SNORD93 markedly enhanced the expression of SNORD93 in both the HCT-15 and HT-29 cell lines (HCT-15: P=0.004; HT-29: P=0.01; Figure 2A). As expected, the CCK-8 assay confirmed that the proliferation was inhibited when the SNORD93 expression in CRC cell lines was increased (HCT-15: P<0.001; HT-29: P=0.03; Figure 2B). In line with this, the colony formation assay indicated that overexpression of SNORD93 resulted in fewer and smaller colonies of CRC cells (HCT-15: P=0.002; HT-29: P=0.02; Figure 2C). Taken together, these investigations suggest that the overexpression of SNORD93 significantly constrains the proliferation ability of CRC cells.

Figure 2 SNORD93 constrained the proliferation of CRC cells. (A) Ectopic SNORD93 expression in HCT-15 and HT-29 cells via overexpression vectors significantly elevated transcript levels compared to pGPU6-NC controls (HCT-15: P=0.004; HT-29: P=0.01). (B) CCK-8 was used to compare the effects of SNORD93-overexpression vector and control pGPU6-NC vector on the proliferation of the HCT-15 and HT-29 cell lines (HCT-15: P<0.001; HT-29: P=0.03). (C) Clonogenic potential of HCT-15 and HT-29 lines was assessed following SNORD93 overexpression vector, with pGPU6-NC vector transfected cells serving as controls, stained with crystal violet (HCT-15: P=0.002; HT-29: P=0.02). Data are presented as mean ± SD (n=3). *, P<0.05; **, P<0.01; ****, P<0.0001. CCK-8, Cell Counting Kit-8; CRC, colorectal cancer; OD, optical density; SD, standard deviation.

SNORD93 limited CRC growth by regulating the cell cycle and promoting apoptosis

The proliferation of normal cells depends on appropriate cell cycle signals, which restrict unfettered proliferation (17). We thus sought to determine whether the function of SNORD93 in cell proliferation was associated with the cell cycle. The SNORD93-overexpressing and negative-control CRC cells were stained with PI. Data from flow cytometry revealed that SNORD93 enhanced the proportion of CRC cells in the G0/G1 phase (Figure 3A,3B). Subsequently, the expression of cell cycle-related proteins was detected. As shown in Figure 3C, SNORD93 enhanced G1 block-related protein p21 Waf1/Cip1 while simultaneously decreasing the levels of cyclin D3, cyclin E2, and CDK9.

Figure 3 SNORD93 inhibited the proliferation of CRC. (A,B) Comparison of the effect of SNORD93-overexpression vector and control pGPU6-NC vector on the cell cycle of CRC cell lines via flow cytometry (HCT-15: P=0.001; HT-29: P=0.048). (C) Western blot profiling of cell cycle regulators in SNORD93-overexpressing CRC cells (HCT-15, p21 Waf1/Cip1: P=0.004; cyclin E2: P=0.047; cyclin D3: P=0.04; CDK9: P=0.03) (HT-29, p21 Waf1/Cip1: P=0.007; cyclin E2: P=0.008; cyclin D3: P=0.03; CDK9: P=0.01). (D) Comparison of the effect of the SNORD93-overexpression vector and the control pGPU6-NC vector on apoptosis in CRC cell lines via flow cytometry. (E) Western blot profiling of apoptosis markers in SNORD93-overexpressing CRC cells (HCT-15, caspase 8: P=0.044; caspase 3: P=0.044; caspase 7: P=0.006; Bim: P=0.01) (HT-29, caspase 8: P=0.01; caspase 3: P=0.02; caspase 7: P=0.008; Bim: P=0.002). (F) Western blot profiling of necroptosis markers in SNORD93-overexpressing CRC cells (HCT-15, p-RIP: P=0.02; RIP: P=0.97; p-MLKL: P=0.04; MLKL: P=0.27) (HT-29, p-RIP: P=0.04; RIP: P=0.14; p-MLKL: P=0.01; MLKL: P=0.16). Data are presented as mean ± SD (n=3). ns, P>0.05; *, P<0.05; **, P<0.01. CRC, colorectal cancer; MLKL, mixed lineage kinase domain-like protein; NC, negative control; p-MLKL, phosphorylated MLKL; p-RIP, phosphorylated RIP; PI, propidium iodide; RIP, receptor-interacting protein; SD, standard deviation.

Evasion from apoptosis is one of the hallmarks of cancer, and deregulated apoptotic signaling induces continuous proliferation, ultimately leading to the formation of cancer (18). To further elucidate the mechanism by which SNORD93 affects CRC cell proliferation, we conducted Annexin V/PI staining on CRC cells after they were transfected with pGPU6-NC or pGPU6-SNORD93 to analyze cell death and apoptosis. The upregulation of SNORD93 increased the proportion of apoptotic cells (Figure 3D and Table 2). Subsequently, we measured the expression of apoptotic proteins and found that SNORD93 stimulated the expression of caspase 3, caspase 7, caspase 8, and Bim (Figure 3E). In addition, the phosphorylation of both RIP and mixed lineage kinase domain-like protein (MLKL), which are essential cellular elements during necroptosis, increased in pGPU6-SNORD93-treated CRC cells (Figure 3F). SNORD93 dysregulation coincides with elevated necroptosis marker, the p-RIP/p-MLKL, suggesting potential engagement of necroptosis. Our in vitro findings demonstrated that SNORD93 overexpression hinders the proliferation of CRC by arresting the cell cycle and inducing apoptosis.

SNORD93 failed to influence CRC cell migration

Tumor metastasis is a characteristic trait of the most aggressive tumors and a major contributor to cancer-related death. Epithelial-mesenchymal transition (EMT) is an essential cellular process in which epithelial cells exhibit mesenchymal characteristics. Generally, EMT has been associated with various tumor functions, including tumor cell migration, metastasis (19), and chemotherapy resistance (20). We thus sought to clarify SNORD93’s biological role in metastasis and the expression of EMT-related genes. As shown in Figure 4A,4B, the migration capacity of CRC cells was not altered in response to SNORD93 overexpression. Additionally, the expression of E-cadherin and β-catenin showed no significant difference between the control and SNORD93-overexpressing groups (Figure 4C). Therefore, the overexpression of SNORD93 exerted a nonsignificant effect on the migratory capacity of CRC cells.

Figure 4 SNORD93 overexpression did not modulate CRC cell migration. (A,B) The effects of increased expression of SNORD93 on the migration of HCT-15 and HT-29 cell lines were detected via a wound healing assay and compared with the pGPU6-NC control vector (HCT-15: P=0.78; HT-29: P=0.85). (C) Western blot profiling of migratory markers in SNORD93-overexpressing CRC cells (HCT-15, E-cadherin: P=0.94; β-catenin: P=0.46) (HT-29, E-cadherin: P=0.19; β-catenin: P=0.47). Data are presented as mean ± SD (n=3). ns, P>0.05. CRC, colorectal cancer; NC, negative control; SD, standard deviation.

Discussion

snoRNAs, a subclass of noncoding RNAs, are typically distributed in the nucleolus, range in length from 60 to 300 nt, and have no protein-coding capacity (21). Evidence suggests that the dysregulation of snoRNA is strongly connected with the occurrence and progression of tumors, including CRC. Gómez-Matas et al. analyzed the whole snoRNome in CRC to identify potentially noninvasive snoRNA-based biomarkers in fecal samples (22). SNORA28 overexpression promotes the development and radioresistance of CRC cells. By recruiting bromodomain-containing protein 4 (BRD4), SNORA28 functions as a molecular decoy, raising the level of H3K9 acetylation at the LIFR promoter region. In turn, this increases the proliferation and radioresistance of CRC cells by inducing LIFR transcription, which then activates the JAK1/STAT3 pathway (23). Moreover, SNORD126 promotes the proliferation of CRC through increasing the expression of fibroblast growth factor receptor 2 (FGFR2) and activating the phosphoinositide 3-kinase-Akt (PI3K/Akt) pathway (24). It has also been found that the upregulation of SNORD44, when combined with GAS5, leads to caspase-dependent apoptosis, which inhibits CRC cells from proliferating (25). Moreover, SNORD1C is highly expressed in CRC and enhances the stem cell properties of CRC cells through activating the Wnt/β-catenin pathway (26). Therefore, clarifying the expression pattern of snoRNA in CRC and its regulatory role in the pathogenesis and progression of CRC may be beneficial in identifying novel markers for the diagnosis, treatment, and prognosis of patients with CRC.

Given the potential relevance of the dysregulated expression of snoRNAs in CRC and the fact that SNORD93 exerts functions in multiple cancers, we sought to characterize the expression pattern of SNORD93 and the possible molecular mechanisms underlying its role in CRC development. Indeed, we found that SNORD93 was significantly downregulated in CRC. Notably, patients with CRC with a lower expression of SNORD93 experienced poor prognosis. However, Zhao et al. reported the upregulation of SNORD93 in renal clear-cell carcinoma tissues (14). Thus, the expression patterns of SNORD93 may differ across types. Similarly, SNORD44 has been reported to be weakly expressed in prostate cancer but overexpressed in breast cancer (27,28).

We further clarified how SNORD93 is related to the malignant features of CRC. Initially, our findings indicated that SNORD93 blocked cell proliferation but failed to impact cell migration in CRC. As shown in Table 1, despite the small sample size, no statistically significant associations were observed—including in the analysis of metastatic lymph node status. Additional evidence indicated that exogenous SNORD93 did not influence the expression level of E-cadherin or β-catenin, which are respectively associated with EMT. These data led us to conclude that SNORD93 regulates cell proliferation rather than cell migration in CRC. Similarly, in another study, SNORD113-1 was found to significantly suppress cancer growth in hepatocellular carcinoma cells but to not affect their migration or invasion (29). However, this observation appears to contradict Patterson et al.’s finding that MDA-MB-231 metastatic breast cancer cells exhibit elevated levels of SNORD93 expression and that the migration of MDA-MB-231 cells can be inhibited via anti-sdRNA93 (15). Overall, snoRNAs function as either oncogenes or tumor-suppressor genes and regulate the biological behavior of malignancies in a tumor- and tissue-specific manner. Our results demonstrate that SNORD93 functions as a tumor suppressor in CRC. However, our data do not support the involvement of SNORD93 in metastatic processes in breast cancer.

All cancers depend on continuous cell division, and controlled cell cycle progression typically prevents the excessive proliferation of cancer cells (30). According to previous research, SNORD78 inhibits the proliferation of non-small cell lung cancer by inducing G0/G1 phase arrest, while SNORD76 delays cell entry into the S phase in glioblastoma (31,32). Meanwhile, SNORD52 promotes the G2/M cell cycle transition in hepatocellular carcinoma (33). In line with this, our results suggested that upregulation of SNORD93 limits CRC cell proliferation by arresting tumor cells in the G0/G1 phase. As expected, its upregulation was accompanied by a higher expression of p21 protein, G0/G1 phase arrest, and a decreased expression of the proteins cyclin E2, cyclin D3, and CDK9, which are associated with the transition from the G1 to the S phase. In contrast, Shen et al. observed that SNORA24 promoted the G1/S phase transition, contributing to colony formation, cell proliferation, and the growth of xenograft tumors in CRC (34).

Induction of cell apoptosis is inexorably linked with the development of cancer, with the activation of caspases resulting in the programmed execution of cell apoptosis. To further clarify the possible mechanism underlying SNORD93’s tumor-suppressive function, we examined caspase-dependent apoptosis. Our findings indicated that the upregulation of SNORD93 effectively promoted cell apoptosis and the production of caspase 8, Bim, caspase 3, and caspase 7, among which caspase 8 is well-known to activate the death receptor signaling cascade (35). Bim translocates and attaches to Bcl-2 after receiving apoptosis signaling, triggering the release of cytochrome C and inducing the mitochondrial apoptosis pathway, with apoptosis being achieved via the respective activation of the effector caspase 3 and caspase 7. Similarly, the overexpression of SNORD44 with its host GAS5 stimulates cystatin-dependent apoptosis, which inhibits CRC cell proliferation (22).

Caspase 8 is critical to initiating both exogenous apoptosis and necroptosis (36). Necroptosis is a newly identified form of programmed necrotic cell death. The ripoptosome is a protein complex that contains FADD, RIPK1, caspase 8, and cFLIP, and the formation of the ripoptosome is mediated via the recruitment of RIPK1 and the phosphorylation of RIPK3, after which it phosphorylates MLKL to build the necrosome. Large pores generated by MLKL oligomers in phosphatidylinositol phosphate-rich patches in the plasma membrane result in necroptotic cell death (37,38). Notably, necroptosis involves a dual regulation of tumor occurrence and development (39,40). For instance, Feng et al. found that necroptosis inhibited the proliferation, migration, and invasion of CRC (41). However, in the enteritis mouse model experiments of Liu et al., necroptosis was observed to participate in the growth of early-stage CRC (42). Surprisingly, our data show that SNORD93-mediated cell death exhibits features consistent with necroptosis, suggesting SNORD93 prevents the proliferation of CRC cells by triggering necroptosis-like cell death. Although elevated p-RIP and p-MLKL indicate engagement of necroptosis pathways, the absence of functional inhibitor data precludes definitive causal attribution. Future studies should validate these observations using necroptosis-specific inhibitors (e.g., necrostatin-1).

Considered cumulatively, our findings indicate a suppressive function of SNORD93 in CRC tumorigenesis, which may facilitate the development of the snoRNA-based diagnosis and treatment of cancers. However, the mechanisms underlying SNORD93’s oncogenic function need to be further clarified in subsequent research. Moreover, the clinical translation potential of SNORD93 is particularly noteworthy, given the emerging utility of snoRNAs in liquid biopsy applications. snoRNAs can be detected in liquid biopsies, such as blood plasma, serum, and other body fluids, through use of advanced technologies such as next-generation sequencing (NGS), qRT-PCR, and microarrays. These methods enable the quantification of snoRNAs that are present within extracellular vesicles, such as exosomes, or that exist as cell-free RNAs. Liquid biopsy analyses have revealed tissue-specific snoRNA expression profiles (e.g., CRC, breast cancer, and lung cancer), positioning them as promising noninvasive biomarkers for the early diagnosis, prognosis, and monitoring of recurrence. Furthermore, longitudinal changes in snoRNA levels may serve as indicators of treatment efficacy or resistance, supporting personalized therapeutic approaches. Therefore, our future research will prioritize clinical validation of SNORD93 in liquid biopsies to translate these findings into clinical practice.

Conclusions

Our findings demonstrated that the inhibition of SNORD93 is closely related to the poor outcome of patients with CRC. Notably, SNORD93 inhibited proliferation by delaying cells’ entry into the S phase, inducing apoptosis and necroptosis in CRC. Further, additional prospective studies in larger series of patients are required to confirm the clinical utility of SNORD93 and to determine if it may serve as a novel biomarker and therapeutic target for CRC.

Acknowledgments

None.

Footnote

Reporting Checklist: The authors have completed the MDAR reporting checklist. Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1236/rc

Data Sharing Statement: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1236/dss

Peer Review File: Available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1236/prf

Funding: This study was supported by the Natural Science Foundation of Fujian Province (No. 2020J011286) and the Quanzhou Science and Technology Plan Project (No. 2025QZNG019).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tcr.amegroups.com/article/view/10.21037/tcr-2025-1236/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved. The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by institutional ethics board of Quanzhou First Hospital Affiliated to Fujian Medical University (2023, No. 218) and informed consent was taken from all the patients.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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