Cell culture

Human CRC cell lines, including DLD-1 (TCHu 134), HCT116 (TCHu 99), LoVo (TCHu 82), SW480 (SCSP-5033), and SW620 (TCHu 101), were purchased from the Cell Bank of the Chinese Academy of Science (Shanghai, China). In addition, MMP14 knockout (MMP14KO) cells were purchased from Guangzhou Ubigene Biosciences Co., Ltd. (Guangzhou, China). The cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (Bio-Channel, cat. no.: BC-M-005), supplemented with 1% antibiotics (Vicmed, cat. no.: VC2003) and 10% fetal bovine serum (FBS) (ExCell Bio, cat. no.: FSP500). All cultures were maintained in a humidified incubator at 37 ℃ with 5% CO2.

Transfection and lentivirus transduction

Plasmids and small interfering RNAs (siRNAs) were purchased from GenePharma (Shanghai, China), Hebio (Shanghai, China), GENEWIZ, Inc. (Suzhou, China), and YouBio (Hunan, China). The MMP14-ATGmut plasmid was designed to simultaneously mutate all 11 putative open reading frame (ORF) initiation codons within the MMP14 mRNA sequence, thereby restricting expression to MMP14 RNA without allowing translation. Transient transfection of plasmids and siRNAs was conducted using the liposomal transfection reagent (YEASEN, cat. no.: 40802ES08) and Lipofectamine® 2000 (Invitrogen, cat. no.: 1168019), respectively, in accordance with the manufacturer’s protocols. The lentivirus containing the MMP14-ATGmut plasmid was purchased from OBiO Technology Co., Ltd. (Shanghai, China). HCT116 cells were infected with the lentivirus to establish a stable MMP14 RNA overexpression cell line, and successfully transfected cells were selected using puromycin (Beyotime, cat. no.: ST551-10). The sequences of the siRNAs are detailed in Table S1.

Single-cell RNA sequencing (scRNA-seq) analysis

The Seurat package was employed to analyze single-cell transcriptomes. The functions FindIntegrationAnchors and IntegrateData were utilized to eliminate batch effects. The FindAllMarkers function was utilized to identify markers for each cell type, with cell types determined using the CellMarker 2.0 database. Gene set enrichment analysis (GSEA) of specific cell types was conducted using the fgsea package. The R code for GSE221575, GSE110009, and GSE225857 is available in the supplementary material of our previous study [17]. In addition, the R code for GSE178318 is provided in the Supplementary Material.

Transwell assay

Approximately 50 μl of diluted Matrigel (Corning, cat. no.: 356234) was pre-plated in each well of the chamber to determine the invasion levels, while Matrigel was omitted for migration level evaluation. HCT116 cells (6 × 104 cells/well) and LoVo cells (1 × 105 cells/well) underwent a 12 h starvation period in FBS-free DMEM before being seeded in the upper chamber (Corning, cat. no.: 3422) of the Transwell plate. DMEM supplemented with 20% FBS was added to the lower chamber. After a 24 h incubation, invading cells were fixed with 4% paraformaldehyde for 30 min and subsequently stained with crystal violet for an additional 30 min. Cell observations were conducted using an Olympus microscope, and data were analyzed using ImageJ software.

Western blot analysis

Total cellular proteins were extracted using RIPA lysis buffer (Beyotime, cat. no.: P0013B). Protein concentrations were determined using a BCA kit (Abbkine, cat. no.: KTD3001). The proteins were then separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was blocked with 5% skim milk for 2 h at room temperature, followed by overnight incubation with primary antibodies at 4 ℃. Afterward, the membrane was incubated with secondary antibodies at room temperature for 2 h. Finally, protein bands were visualized with an ECL luminescent solution (Abbkine, BMU102-CN). Western blot analysis was performed using the following antibodies: anti-E-cadherin (E-Cad) (ABclonal, A20798, 1:2000), anti-N-cadherin (N-Cad) (Abclonal, A3045, 1:1000), anti-Vimentin (Proteintech, 10366-1-AP, 1:2000), anti-GAPDH (Proteintech, 60004-1-Ig, 1:10,000), anti-Snail1 (Proteintech, 13099-1-AP, 1:1000), anti-SIRT3 (Santa Cruz, sc-365,175, 1:200), anti-H3K27cr (PTM BIO, PTM-545RM, 1: 1000), anti-MMP14 (Affinit, AF0212,1:1000), anti-STARD13 (Proteintech, 21325-1-AP,1:1000), and anti-H3 (Proteintech, 17168-1-AP, 1:10,000).

Reverse transcription-polymerase chain reaction (RT-PCR) assay

Total DNA was extracted utilizing the DNA extraction kit (TIANGEN, cat. no.: DP304-02). The RT-PCR assay was conducted using the 2× PCR Master Mix (Beyotime, cat. no.: D7228). The PCR products were then separated using 1% agarose gel (Servicebio. GC205013-100 g). The gel containing the PCR products was excised for sequencing.

Quantitative real-time PCR assay

Total RNA was isolated from tissues or cells using TRIzol reagent (Vazyme, cat. no.: R401-01-AA). The extracted RNA was reverse-transcribed into cDNA using the SweScript RT I First Strand cDNA Synthesis Kit (Servicebio, cat. no.: G3330-50). qRT-PCR was performed with the 2× SYBR Green qPCR Master Mix (High ROX) (Servicebio, cat. no.: G3322-05). Gene expression levels were normalized to GAPDH and calculated using the 2−ΔΔCt method. The sequences of primers used are provided in Table S2.

Cell fractionation assay

CRC cells were separated using the nuclear and cytoplasmic extraction kit (Beyotime, cat. no.: P0027) following the manufacturer’s instructions. Briefly, cells were lysed on ice for 15 min with pre-cooled Reagents A and B. The cell lysate was then centrifuged at 14,000 rpm for 10 min at 4 ℃, and the supernatant was collected for cytoplasmic RNA extraction using TRIzol reagent. The sediment was then resuspended in reagents A and B and incubated on ice for 45 min. Subsequently, the sediment was washed thrice with cold phosphate-buffered saline (PBS) before the extraction of nuclear RNA using a TRIzol reagent.

Luciferase activity assay

A pGL3-enhancer plasmid containing 2 kb fragment of the STARD13 promoter was obtained from GENEWIZ, Inc. (Suzhou, China). CRC cells were co-transfected with this plasmid along with either MMP14-ATGmut or siMMP14 for 48 h. The firefly luciferase activity was determined using the Mono-Lumi firefly luciferase reporter gene assay kit (Servicebio, cat. no.: G1702). The fluorescence intensity was determined using a microplate reader.

RNA fluorescent in situ hybridization (RNA-FISH) and immunofluorescence microscopy

A cyanine 5-labeled MMP14 RNA probe was procured from Integrated Biotech Solutions (Shanghai, China). Cells were seeded onto coverslips and incubated overnight. Subsequently, the cells were washed thrice with cold PBS and then permeabilized on ice for 5 min using CSK buffer [100 mM NaCl, 300 mM sucrose, 3 mM MgCl2, 10 mM PIPES (pH 6.8)]. Following permeabilization, the cells were fixed on ice with 4% paraformaldehyde for 10 min. The cells were then dehydrated sequentially in ethanol at concentrations of 70%, 80%, 95%, and 100%, with each step lasting 3 min.

The MMP14 probe was diluted using 2× hybridization buffer [4× SSC, 40% (w/v) dextran sulfate, 2 mg/ml bovine serum albumin (BSA), 400 mM RVC], and incubated in a humid chamber at 37 °C overnight in the dark. The following day, the coverslips were washed thrice with freshly prepared 50% formamide and 2× SSC buffer at 42 °C for 5 min each. Subsequently, cells were blocked with 3% BSA for 45 min and then incubated with an anti-SIRT3 antibody (Bioss, Bs-6105R, 1:25) at room temperature for 1.5 h. The secondary antibody (Bioss, bs-0295D-AF488, 1:50) was then incubated at room temperature for 30 min. Afterward, the cells were stained with DAPI (Servicebio, cat. no.: G1012) at room temperature for 10 min. Finally, the coverslips were sealed onto the slide with anti-fluorescence quenching (Beyotime, cat. no.: P0126).

Chromatin isolation by RNA purification (ChIRP) assay

Biotin-labeled MMP14 probes were designed and synthesized by RiboBio Co., Ltd. (Guangzhou, China), with the LacZ probe serving as a negative control. The ChIRP protocol was applied as previously described [18]. Briefly, cells were cross-linked with 1% glutaraldehyde, after which the cross-linking reaction was terminated with 1.25 M glycine solution. The cells were then sonicated with a 30 s pulse and 30 s pause for a total of 20 cycles. Next, the lysate was centrifuged, and the supernatant was collected. Subsequently, hybridization buffer was added to the supernatant in a volume equivalent to twice that of the supernatant, along with the probes. Subsequently, 120 μl of BeyoMag™ streptavidin magnetic beads (Beyotime, cat. no.: P2151) was added to the mixture and incubated at 37 °C for 5 h with shaking. The beads were subsequently washed five times and resuspended using lysis buffer. The samples were divided into two portions: 100 μl for RNA extraction and 900 μl for DNA extraction. RNA and DNA were isolated using phenol–chloroform–isoamyl alcohol extraction, and their enrichment was quantified using the qRT-PCR assay.

Chromatin immunoprecipitation (ChIP-seq and ChIP-qPCR) assay

Chromatin immunoprecipitation was performed as previously described [19]. In brief, cells were cross-linked with 1% formaldehyde, and the cross-linking reaction was subsequently terminated with 1.25 M glycine solution. The cells were then harvested and incubated in 200 μl of ChIP lysis buffer. The samples were then sonicated with a 10 s pulse and 20 s pause for a total of ten cycles. After sonication, the lysates were centrifuged, and the supernatant was collected. To this supernatant, 1 μg of primary antibody was added and incubated at 4 °C for 2 h. Following this, 25 μl of protein A/G magnetic beads (MCE, cat. no.: HY-K0202) were added and incubated at 4 °C for an additional 2 h. The beads were washed three times with TE buffer. Finally, DNA was collected, and its enrichment was assessed using the qRT-PCR assay or sequenced by Shanghai Orizymes Biotech. Co., Ltd. (Shanghai, China).

RNA immunoprecipitation (RIP) assay

The RIP assay was conducted as previously described [17]. In brief, cells were harvested using polysome lysis buffer. After cell lysis, the lysate was centrifuged, and approximately 2 μg of primary antibody was added to the supernatant, followed by incubation at 4 °C overnight. The following day, 80 μl of protein A/G magnetic beads was added to the mixture and incubated at 4 °C for 4 h. The beads were then washed eight times. Afterward, the beads were resuspended in 100 μl of polysome lysis buffer with 0.1% SDS and 30 μg of Proteinase K, and incubated at 50 °C for 30 min. The supernatant was collected, and 10 μl of phenol–chloroform–isoamyl alcohol was added. The samples were then centrifuged and the supernatant was collected. RNA was subsequently precipitated using 5 μl of linear polyacrylamide (10 μg/ml), 12 μl of 3 M sodium acetate, and 250 μl of pre-chilled ethanol. Finally, RNA enrichment was determined by qRT-PCR.

The following antibodies were used: H3K27cr, SIRT3, H3K4me3 (Abcam, ab213224), H3K27ac (Abcam, ab177178), H3K27me3 (Abcam, ab192985), SIRT5 (Santa Cruz, SC-271635), SIRT1 (Proteintech, 13,161–1-AP), SIRT2 (Proteintech, 19,655–1-AP), SIRT6 (Proteintech, 13,572–1-AP), HDAC2 (Santa Cruz, SC-9959), EP300 (Santa Cruz, SC-48343), GCN5 (Affinity, DF3383), EED (Abcam, ab240650), SUZ12 (Abcam, ab175187), and EZH2 (Abcam, ab191250).

Immunohistochemistry (IHC)

Tissue samples were baked at 65 °C for 4 h. The tissue sections were subsequently deparaffinized by incubating them twice in xylene and anhydrous ethanol, followed by sequential incubation in graded alcohol solutions of 95%, 85%, 75%, and 50%. Antigen retrieval was then conducted using 0.01 M citrate buffer. Peroxidase activity was terminated using 3% hydrogen peroxide. The tissue sections were blocked with 10% BSA for 30 min and then incubated with primary antibodies at 4 °C overnight. Afterward, they were incubated at room temperature for 30 min with horseradish peroxidase (HRP)-conjugated secondary antibodies. The samples were then incubated with the DAB staining solution in a humidified chamber for 3 min, followed by counterstaining with hematoxylin for 1 min. Finally, the tissue sections underwent a graded alcohol series (75%, 80%, and 95%) for 5 min each, followed by dehydration in absolute ethanol and xylene for 10 min.

Hematoxylin and eosin (HE) staining

Tissue sections were stained with hematoxylin for 2 min, followed by successive incubation in 1% aqueous ammonia solution and eosin for 5 s each. After staining, the sections underwent treatment in a graded series of alcohol solutions: 85%, 95%, and 100% for 2 min each, followed by treatment with 100% alcohol for 4 min and xylene I and II for 10 min each. Finally, the tissue sections were sealed with neutral resin.

Animal assay

Five-week-old BALB/c nude mice were procured from GemPharmatech (Nanjing, China) and randomly divided into two groups, each consisting of six mice. HCT116 cells, either with or without stable overexpression of MMP14 RNA, were injected via the tail vein at a dose of 2 × 106 cells per injection. The mice were euthanized 5 weeks post-injection. Subsequently, the lungs were collected, photographed, and analyzed. This study was approved by the Ethical Committee for Animal Experimentation and the Ethics Committee of the Affiliated Hospital of Xuzhou Medical University (202306T019).

Statistical analysis

Data were presented as means ± SD. Differences between the two groups were analyzed using an unpaired two-tailed Student’s t-test, with a significance threshold set at P < 0.05. GraphPad Prism (version 8.0) was employed for graph generation and data analyses.