The CRC datasets GSE32323 [26] and GSE26571 [27] in Gene Expression Omnibus (GEO) database, and colon adenocarcinoma (COAD) and rectum adenocarcinoma (READ) datasets in The Cancer Genome Atlas (TCGA) database were downloaded to analyze the expression levels of differentially expressed genes (DEG) were in normal tissues and tumor tissues. Based on the condition of log₂fold-change > 1 and p < 0.05, the upregulated DEG were obtained and shown as a Venn diagram.

The Database for Annotation, Visualization and Integrated Discovery online tool was used for gene ontology (GO) analysis. The top 10 terms in biological process, cellular compartment, and molecular function with minimal significance were plotted.

The CRC samples and adjacent non-tumor tissues (n = 40) were collected from CRC patients, and stored at −80°C after freezing in liquid nitrogen for the subsequent experiments. This study has been approved by the Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University (approval number: KYLL202375). All patients provided informed consents for taking part in this study.

HCT-116, RKO, SW480, DLD-1 and NCM460 cells were purchased from iCell Bioscience Inc. in Shanghai, China. HCT-116 cells were cultured in McCOY’s 5A medium (Catalog Number [No.] PM150710) supplied by Procell Life Science & Technology Co., Ltd. (Procell) in Wuhan, China. RKO cells were cultured in minimum essential medium (No. 41500, Solarbio, Beijing, China). The RPMI-1640 medium (No. 31800, Solarbio) was used for culturing DLD-1 and NCM460. The above cells were lived in the medium with 1% penicillin (No. C8251, Solarbio), 1% streptomycin (No. S8290, Solarbio) and 10% fetal bovine serum (No. 11011-8611, Tianhang Biotechnology, Huzhou, China) in an incubator with 5% CO₂ at 37°C. L15 medium (No. PM151010, Procell) containing 100 kU/L penicillin, 100 mg/L streptomycin was used for SW480 cells under 100% air conditions. The sequences of short hairpin RNAs (shRNAs) targeting RCN1 were as follows: shRCN1-1: 5′-GGATGAGAAGCTAACTAAA-3′, shRCN1-2: 5′-GGACGGGAAGTTAGACAAA-3′. The sequence targeting shRNA targeting IP3R1 (shIP3R1) was 5′-GATAGAGATTGTCAGATTA-3′. RKO and SW480 were transfected with shRNAs using Lipofectamine 3000 (No. L3000015) purchased from Invitrogen (Carlsbad, CA, USA). At 24 h after transfection, apoptosis of CRC cells was induced by 1 µM TG (No. T863962, Macklin, Shanghai, China).

TRIpure lysis (No. RP1001, BioTeke, Beijing, China) was used to isolate RNA from cells and tissues. These RNA samples were reversely transcribed to cDNA using BeyoRT II M-MLV reverse transcriptase (No. D7160L) purchased from Beyotime (Shanghai, China). Real-time PCR was performed using 2 × Taq PCR MasterMix (No. PC1150, Solarbio) and SYBR Green (No. SY1020, Solarbio) according to the manufacturer’s instructions. Sequences of primers used for relative RCN1 mRNA expression detection were listed in Suppl. Table S1. The thermocycling protocol was shown in Suppl. Table S2.

The harvested cells were mixed with the RIPA lysis (No. R0010, Solarbio) and 1 mM PMSF (No. P0100, Solarbio) for extracting proteins. Protein samples were separated through SDS-PAGE and transferred onto a polyvinylidene fluoride (No. IPVH00010, Millipore, Bedford, MA, USA) membrane. The membrane was incubated at 4°C overnight with primary antibodies after blocking for 1 h. Subsequently, the membrane was incubated with secondary antibodies at 37°C for 1 h. The details of antibodies were listed in Suppl. Table S3. Thereafter, the protein bands on the membrane were visualized by employing enhanced chemiluminescence (No. PE0010, Solarbio).

Cells were collected and seeded in a 96-well plate (5 × 10³ cells per well). After treatment with TG for 24 h, cells in each well were incubated with CCK-8 solution (10 μL) (No. KGA317, KeyGEN BioTECH, Nanjing, China) at 37°C for 2 h. The optical density value of cells was assessed at 450 nm using a microplate reader (BioTek, Winooski, VT, USA).

Cell apoptosis was determined using an Annexin V-FITC/PI Apoptosis Detection Kit (No. KGA1102, KeyGEN BioTECH). After centrifugation at 150×g for 5 min, cells from each group were harvested and mixed in binding buffer (500 µL). Cells were then stained with Annexin V-FITC (5 μL) followed by mixing with PI (5 μL) for 15 min in the dark. NovoCyte flow cytometer (Agilent, Santa Clara, CA, USA) was used for measurement of apoptosis rate.

Apoptotic cells were stained by the mixture of enzyme solution and label solution in In Situ Cell Death Detection Kit (Red) (No. 12156792910, Roche, Basel, Switzerland) at 37°C for 1 h in the dark. Following the rinsing by phosphate buffer saline, cell sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (No. D106471-5mg, Aladdin, Shanghai, China) for 5 min. Next, the cells were observed and captured using a microscope (Olympus, Tokyo, Japan).

The intracellular Ca²⁺ concentration was detected by a Fluo-4 AM fluorescent probe (No. MX4504, Shanghai Maokang Biotechnology Co., Ltd. (Maokang), Shanghai, China). Cells in each group were centrifugated at 150×g for 5 min. Then, they were incubated in serum-free medium containing Fluo-4 AM (4 μM) at 37°C for 30 min. Subsequently, cells were photographed using a BX53 fluorescence microscope (Olympus). For the quantification of Ca²⁺ concentration, harvested cells was evaluated by flow cytometry.

RhoD-2AM (Red) and Mito-Tracker Green fluorescent probes (No. MX4507, Maokang) were used to label mitochondrial Ca²⁺ in CRC cells. Cells were incubated in serum-free medium with Rhod-2 AM (4 μM) and Mito-Tracker Green (100 nM, No. MX4309, Maokang) at 37°C for 30 min. Thereafter, cells were observed and captured using the aforementioned fluorescence microscope.

The mitochondrial membrane potential (MMP) in RKO and SW480 cells was detected using the JC-1 staining kit (No. C2006, Beyotime) according to the instructions provided by the manufacturers. Briefly, cells in each group were stained by JC-1 solution (0.5 mL) for 20 min. Cells were collected and resuspended by JC-1 staining buffer (1×) (200 μL). Finally, the MMP was examined by flow cytometry.

Cells were fixed with 4% polyformaldehyde for 15 min. After rinsing by phosphate buffer saline for three times, cells were covered by 0.1% tritonX-100 (No. ST795, Beyotime) for 30 min and then incubated by 1% bull serum albumin (No. A602440-0050, Sangon Biotech, Shanghai, China) for 15 min. Next, cells were incubated with primary antibodies at 4˚C overnight, including rabbit anti-RCN1 antibody (dilution 1:500, No. ab210404, Abcam) and mouse anti-IP3R1 antibody (dilution 1:50, No. sc-271197, Santa Cruz). Cells were then incubated with the same dilution ratio (1:1000) of Cy3-conjugated goat anti-rabbit IgG antibody (No. ab6939, Abcam) and FITC-conjugated goat anti-mouse IgG antibody (No. ab6785, Abcam). Next, nuclei of RKO and SW480 cells were counterstained with DAPI (No. D106471-5mg, Aladdin). After treatment with mounting medium (No. S2100, Solarbio), fluorescence in cells was recorded using the aforementioned fluorescence microscope.

Statistical analysis was performed using GraphPad Prism software 8.0 (GraphPad, San Diego, CA, USA). Student’s t-test was used for analyzing differences between two groups. One-way analysis of variances was performed to evaluate differences among three or more groups. The p-values < 0.05 indicated statistically significant differences. All data were displayed as the mean ± SD.

To identify genes that potentially regulate the phonotypes of CRC tissues, we downloaded the GSE32323, GSE26571, COAD and READ datasets from the GEO and TCGA databases. The data from these datasets showed that 35 genes were upregulated in CRC tissues (Fig. 1A). The top 10 GO pathways in biological process, cellular compartment, and molecular function significantly enriched for these genes are presented in Fig. 1B. Previous studies have reported that the CREC protein family participates in the process of multiple diseases, and is associated with the activity and transport of Ca²⁺ in the cytosol [16]. The expression levels of RCN1, RCN2, RCN3, SDF4 and CALU in GSE32323 are presented as a heatmap. RCN1 showed high expression in CRC tissues (Fig. 1C), while its effect on the apoptosis of CRC cells remains unclear. The location of the CREC family genes on the chromosome is displayed in Fig. 1D. Additionally, the RCN1 expression in tumor tissue was significantly upregulated in GSE32323 and GSE26571 datasets (Fig. 1E). Relative RCN1 mRNA level was assessed in 40 pairs of tumors and non-tumor tissues. RCN1 expression was significantly increased in CRC tumor tissues (Figs. 1F and 1G). Therefore, we confirmed that RCN1 was highly expressed in RCR tissues.

Compared with NCM460 cells, RCN1 expression was obviously increased in CRC cell lines at mRNA and protein levels (Fig. 2A). RKO and SW480 cells were transfected with two pieces of shRCN1-1 and shRCN1-2. In the two types of CRC cells, relative mRNA and protein levels of RCN1 were reduced significantly (Figs. 2B and 2C). Additionally, previous reports demonstrated that the apoptosis of RKO and SW480 was promoted by TG-induced ER stress [28,29]. The viability of RKO and SW480 cells was suppressed by the absence of RCN1 (Fig. 2D).

Moreover, as displayed in Fig. 3A, knockdown of RCN1 obviously promoted the apoptosis of RKO and SW480 cell lines. B-cell lymphoma-2 (Bcl-2) protein expression was markedly suppressed by silencing RCN1. In contrast, absence of RCN1 increased the protein expression of Bcl-2-associated X (Bax) (Fig. 3B). Furthermore, after transfected with shRCN1s, TUNEL-positive cell rate upregulated remarkably (Fig. 3C). Collectively, these findings revealed that loss of RCN1 function suppressed cell viability and further promoted ER stress‑induced apoptosis of CRC cells.

When defective proteins accumulate abnormally, the PERK, IRE1 and ATF6 signaling pathways are activated to induce further ER stress and cell apoptosis. PERK, IRE1 and ATF6 are crucial factors in the three pathways. These ER stress pathways are activated to mediate the UPR and subsequent cellular stress [30]. Depletion of RCN1 markedly induced the phosphorylation of PERK and IRE1 (Figs. 4A and 4B). Moreover, ATF6 was activated in RKO and SW480 cells. Knockdown of RCN1 also increased the levels of CHOP (Figs. 4C and 4D). The above findings suggested that absence of RCN1 activated the PERK, IRE1 and ATF6 pathways and facilitated UPR in CRC cells.

Concentration of Ca²⁺ has been considered an important factor in cell apoptosis [31]. Intracellular Ca²⁺ homeostasis is the basis for maintaining normal cellular structure and function [32]. As depicted in Figs. 5A and 5B, intracellular Ca²⁺ was labeled with Fluo-4 AM fluorescent probes in RKO and SW480 cells. The intracellular Ca²⁺ concentration was significantly upregulated by the absence of RCN1, suggesting that a decrease in RCN1 expression disrupted the intracellular Ca²⁺ homeostasis in CRC cells. Additionally, the mitochondrial Ca²⁺ was stained with Rhod-2AM, and the staining results demonstrated that mitochondrial Ca²⁺ was obviously increased in RCN1-silenced RKO and SW480 cells (Figs. 5C and 5D). Moreover, a reduction in MMP was observed after knockdown of RCN1 in CRC cells. Notably, downregulation of shRCN1s significantly increased the MMP in cells with low MMP (Fig. 5E). The expression of mitochondrial cytochrome c was reduced in RKO and SW480 cells by knocking down RCN1. Meanwhile, cytochrome c in cytoplasm was significantly upregulated by depletion of RCN1 (Fig. 5F). Therefore, the intracellular Ca²⁺ homeostasis was disrupted by the downregulation of RCN1 in CRC cells.

Previous research has demonstrated that IP3R1 is involved in the release of ER Ca²⁺ through interaction with RCN1 [22]. Therefore, we investigated whether the RCN1 regulates ER stress-induced apoptosis by affecting IP3R1. The results of fluorescence co-localization analysis revealed that both RCN1 and IP3R1 were abundant in the cytoplasm of untreated RKO and SW480 cells (Fig. 6A). As displayed in Fig. 6B, RCN1 and IP3R1 were significantly downregulated by knocking down RCN1, and IP3R1 expression was obviously reduced by silencing IP3R1 in RKO cells. The depletion of IP3R1 further downregulated the protein expression of RCN1 and IP3R1 in the RCN1-reduced RKO cells. Cell apoptosis induced by knocking down RCN1 was suppressed in CRC cells after reduction of IP3R1 (Fig. 6C). RCN1 silencing-induced the damage to intracellular Ca²⁺ homeostasis induced by shRCN1 was reversed by the silencing of IP3R1 (Fig. 6D). In conclusion, the findings demonstrated that IP3R1 mediates ER stress-induced apoptosis promoted by RCN1 reduction.