Patients’ clinical information and tissue sample

CRC patients (n = 41) with primary CRC tissues and matched adjacent normal tissues were obtained from the biological sample bank of the Sixth Affiliated Hospital, Sun Yat-sen University (SYSU).

We also acquired primary CRC and paired adjacent normal tissues from 233 patients from the Six Affiliated Hospital of SYSU and conducted tissue microarrays. Before surgical resection of the tumor, none of the patients received chemotherapy, radiotherapy, or other related treatments. This study was approved by the Institutional Ethics Committee of the Six Affiliated Hospital, Sun Yat-sen University, and performed in accordance with the Declaration of Helsinki.

Immunohistochemistry (IHC) assay

Tissue microarray and related clinicopathological information were collected from the Sixth Affiliated Hospital of SYSU. IHC for MCCC2 was performed on the CRC tissue microarray slides. The slides were first incubated at 60 ℃ for 4–6 h, next deparaffinized with xylene, rehydrated with decreasing ethanol concentrations, and then heated in citrate buffer for 25 min. When dropped to room temperature, an IHC kit (cat. no. SP9000; ZSGB-Bio) was used to block endogenous peroxidase activity. Slides were blocked with 5% goat serum for 1 h and incubated with anti-MCCC2 antibody (1:500, cat. no. HPA038300, RRID:AB_2675941, Sigma) overnight at 4 ℃. The next day, after three washes with PBST, the slides were incubated with secondary antibody at room temperature for 1–2 h and then stained with a DAB kit (cat. no. ZLT-9017, ZSGB-Bio). After the experiments, slides were observed under a microscope. The IHC scores were independently assessed by two pathologists. In accordance with the ROC curve analysis, an IHC score of 10.3 was selected as the cutoff value that divided the cohort in to two groups: MCCC2 low expression (n = 98) and MCCC2 high expression (n = 112).

Cell lines and plasmids

All the cell lines used in this study were obtained from the American Type Culture Collection (ATCC Manassas, VA, USA). The cells were maintained in DMEM (Gibco) supplemented with 10% Fetal Bovine Serum (Gibco) and typically cultured at 37 ℃ in a humidified incubator with 5% CO2. HCT116 cells were transfected with lentiviruses containing the relevant shRNA or FLAG-MCCC2 plasmid. Puromycin dihydrochloride (2 μg/mL) was used to treat and screen cells. Wild-type MCCC2 (NM_022132.4, amino acids 1–564) gene, mutant MCCC2 (amino acids ∆2–22, 543aa; amino acids ∆343–372, 534aa) were cloned into the pEZ-Lv242 vector (1 × FLAG-tagged). The shRNA sequences targeting MCCC2 were cloned into the psi-LVRU6GP vector (EGFP). To generate MCCC2 KO HCT116 cells, three sgRNAs targeting exon 1 of MCCC2 were cloned into the pCRISPR-CG041 vector. Empty vectors were purchased from GeneCopoeia (FulenGen Co., Ltd., Guangzhou, China).

shMCCC2 sequences: 5′-GCAGGTTACCAGTTATATGAC-3′.

siMCCC2-1: 5′-GGGCCCAAGAAATTGCCAT-3′.

siMCCC2-2: 5′-GGATCTTGGAGGTGCTGAT-3′.

Generation of MCCC2 Knockout cells

HCT116 cells were transfected with the three pCRISPR-CG041-a/b/c vectors at a ratio 1:1:1 using Lipofectamine® 3000 reagent. The cells were cultured with neothramycin (800 μg/mL) for 5 days. The positive cells were planted into 96-well plates to isolate single-cell clones, whose DNA was extracted and subjected to T7 endonuclease survey assay and subsequent Sanger sequencing.

MCCC2 sgRNA-a: 5′- GCCGGGCCGCGCGCCTATCA-3′.

MCCC2 sgRNA-b: 5′-CCGCCATGTGGGCCGTCCTG-3′.

MCCC2 sgRNA-c: 5′-CTTGGGCTCTGCCCTCTACC-3′.

Western blotting

Cellular proteins were extracted using routine cell lysis solution and boiled in 1 × SDS loading buffer for 10 min. Proteins were separated by SDS-PAGE (8% or 10% separation gel) and transferred to PVDF membranes. The membranes were probed with antibodies against MCCC2 (1:1000 dilution, cat. no. HPA038300, RRID:AB_2675941, Sigma), MFN1 (1:1000 dilution, cat. no. AP16037c, Abcepta), MFN2 (1:1000 dilution, cat. no. AP8840c, Abcepta), OPA1 (1:1000 dilution, cat. no. AP20727c, Abcepta), FLAG (1:1000 dilution, cat. no. 390002, ZEN-BIOSCIENCE), and TERF2 (1:1000 dilution, cat. no. 22020-1-AP, Proteintech). GAPDH (1:2000 dilution, cat. no. 390035, ZEN-BIOSCIENCE) and α-tubulin (1:20,000 dilution, cat. no. 66031–1-Ig, Proteintech) antibodies were used as loading controls.

Cell proliferation assay

The cells were counted and seeded at a density of 5000 cells/well in 96-well plates. A real-time cell analyzer (RTCA, xCELLigence system, ACEA Biosciences, USA) and IncuCyte (Zoom, Essen Bioscience, USA) were used to monitor the cell growth. The live cells were recorded automatically every 120 min, and fresh culture medium was exchanged every 3 days. The experiments were conducted in triplicates.

Colony formation assay

Five hundred cells were counted and seeded in six-well plates for 2 weeks in a humidified incubator with 5% CO2, and the medium was exchanged every 3 days. The cells were washed with PBS and fixed with 4% paraformaldehyde for 30 min. The colonies were stained with 0.1% crystal violet for 15 min and washed with PBS. The experiments were conducted in triplicates.

Wound-healing assay

Cells were seeded into 24-well plates, which were placed in a 4-well silicone insert (Ibidi, Martinsried, Germany) per well, and incubated until 100% confluence in each well. After removing the silicone insert, the cells were cultured in an FBS-free culture medium. Live cell images were collected in an IncuCyte Essens Bioscience incubator every 120 min.

Transwell invasion assays

Cell invasion assays were performed in Transwell chambers containing 8-μm Transwell filters (Corning, NY, USA) placed in 24-well plates. The upper chambers were covered with Matrigel (Corning, NY, USA) and placed in at 37 ℃ incubator for 1 h. Then, 1 × 105 cells were counted, seeded into upper chamber, and incubated with FBS culture medium at 37 ℃ for 36–48 h. The upper chamber was wiped with cotton swabs, and the invading cells were attached to the lower surface of the membrane. The cells were fixed with 4% paraformaldehyde and stained with crystal violet for 5 min. Photographs were obtained using a brightfield microscope (Olympus, Japan).

Co-immunoprecipitation (Co-IP)

Whole-cell protein lysates were prepared using a cell lysis solution (NP-40, phosphatase inhibitors, and protease inhibitors). Nuclear proteins were extracted using a nuclear and cytoplasmic extraction kit, according to the manufacturer’s instructions (cat. no. P0027, Beyotime). Fifty microliters of Protein A/G magnetic beads (cat. no. HY-K0202, MCE) were incubated with ~ 2 μg antibody or IgG at 4 ℃ for 2–3 h, then incubated with cell lysates at 4 ℃ overnight. The complexes were washed with lysis buffer for eight times before preparation for immunoblotting.

Mitochondrial staining for quantity measurement, JC-1 assay, and intracellular ROS assay by flow cytometry

Cells were incubated with 100 nM MitoTracker Red CMXRos (cat. no. C1035, Beyotime) in HBSS (Ca+ and Mg+ free) at 37 ℃ for 30 min. The cells were subjected to a CytoFLEX flow cytometer (Beckman Coulter, USA) to determine the mitochondria-targeted red fluorescent probe for quantification at an excitation wavelength of 549 nm and analyzed using CytExpert software (Beckman Coulter, USA).

Cells were detected using an enhanced mitochondrial membrane potential assay kit with JC-1 (cat. no. C2003S, Beyotime), according to the manufacturer’s instructions. Briefly, the precipitated cells (~ 5 × 105 cells) were harvested and resuspended in 500 µL of cell culture medium. The cell suspension was incubated with 500 µl JC-1 at 37 ℃ for 20 min in the dark. The cell pellet was washed with JC-1 dyeing washing buffer for four times, then resuspended in 1 mL dyeing washing buffer. The prepared cellular samples were immediately loaded onto a CytoFLEX flow cytometer (Beckman Coulter, USA) and analyzed using the CytExpert software (Beckman Coulter, USA).

Intracellular ROS was measured using an oxidation-sensitive fluorescent probe (DCFH-DA) and a Reactive Oxygen Species Assay Kit (cat. no. S0033S, Beyotime). The non-fluorescent molecule (DCFH-DA), which is deacetylated intracellularly by nonspecific esterase, is robustly oxidized to fluorescent DCF in the presence of intracellular ROS. The fluorescence intensity reflects the level of oxidative stress. The cell precipitate (~ 5 × 105 cells) was harvested and resuspended in 10 µM DCFH-DA dissolved in cell-free medium at 37 ℃ for 30 min and then washed three times with PBS. The prepared cellular samples were immediately loaded onto a CytoFLEX flow cytometer (Beckman Coulter, USA) at an excitation wavelength of 488 nm and analyzed using CytExpert software (Beckman Coulter, USA).

Measurement of cellular ATP levels

Cellular ATP levels were assayed using an ATP bioluminescence assay kit (cat. no. S0026, Beyotime) according to the manufacturer’s instructions. Briefly, cells were lysed and centrifuged at 12,000 rpm for 5 min at 4 ℃. The supernatants were mixed with the working dilution of ATP detection in a white 96-well plate. The luminance (RLU) was measured using a Thermo Scientific Varioskan Flash instrument.

Telomeric restriction fragment (TRF) analysis

Genomic DNA was isolated using the AxyPrep Genomic DNA Miniprep Kit (cat. no. AP-MN-MS-GDNA-4, Axygen). Four micrograms of genomic DNA was digested with RsaI and HinfI before being resolved by electrophoresis (0.7% agarose gel, 14 h, 1.5 V/cm), The agarose gel was dried for 30 min at 50 ℃ and hybridized with radiolabeled 32P and telomere probe (TTAGGG)4. The signal intensity was quantified using a phosphor screen (Typhoon, GE). The average telomere length was analyzed and calculated using ImageJ software (RRID:SCR_003070) and ImageQuant TL.

Average TRF length was calculated from the scanned image from phosphorImager. Briefly, a grid of 150 boxes was divided over each lane, and the signal intensity for each box were calculated. The signal intensity could reflect the number of repeats in the certain molecular weight. Average TRF length could be obtained by weighted calculation [27].

qRT-PCR, Q-TRAP

Total RNA was extracted using the RNA-Quick Purification Kit (cat. no. ES-RN001, esunbio) and quantified using Nanodrop 100 (Thermo Fisher Scientific, USA). A total of 500 ng RNA was reverse-transcribed using the ReverTra Ace® qPCR RT Master Mix with a gDNA remover kit (cat. no. FSQ-301, TOYOBO). Quantitative polymerase chain reaction (qPCR) was performed using GoTaq® qPCR Master Mix (Promega) on an ABI Quantistudio 7 Flex real-time PCR system. Quantification was performed using comparative CT (delta-delta CT). The primers used were:

MCCC2 FP: 5′-GCCATGGCTGATGAAAACAT-3′

MCCC2 RP: 5′-TCAGCACCTCCAAGATCCTC-3′

MFN1 FP: 5′-CAAGGTGAATGAGCGGCTTTCCAA-3′

MFN1 RP: 5′-ATGCAGGCATCTTTCCATGTGCTG-3′

MFN2 FP: 5′-TGTCTGGGACCTTTGCTCATCTGT-3′

MFN2 RP: 5′-TTCCTGAGCAGCTTTGCTTTGCTC-3′

OPA1 FP: 5′-GCATGCTAAAGGCACACCAAGTGA-3′

OPA1 RP: 5′-TTCCCGCAGGCGAGGATAGTTATT-3′

β-Actin FP: 5′-TTGTTACAGGAAGTCCCTTGCC-3′

β-Actin RP: 5′-ATGCTATCACCTCCCCTGTGTG-3′

A qPCR-based telomerase repeated amplification protocol (Q-TRAP) was used to detect telomerase activity. Cells (1 × 106) were harvested and lysed in NP-40 lysis buffer (1 mM DTT, protease inhibitors, and RNase inhibitor) and diluted to 1 × 103 cells/μL, then centrifuged at 12,000 rpm for 10 min at 4 ℃. The supernatant was collected and the protein concentration of each sample was measured. Typically, 1–3 μg of the protein lysate was detected by PCR. Each 20 μL Q-TRAP reaction contained 2 μL of the eluted proteins, primers, 1 mM EGTA, and GoTaq® qPCR Master Mix (Promega). The reaction mixtures were incubated at 30 ℃ for 30 min and then PCR-amplified using an ABI QuantiStudio 7 Flex real-time PCR system (USA). The amplification program is 90 s at 94 ℃; 40 cycles of 30 s at 94 ℃ and 60 ℃ each [28].

TS primer: 5′-AATCCGTCGAGCAGAGTT-3′.

ACX primer: 5′-GCGCGG(CTTACC)3CTAACC-3′.

Transmission electron microscopy (TEM)

Cells were suspended in the fixation solution overnight at 4 ℃ and processed by the electron microscopy service center at core facilities for medical science of SYSU following a conventional protocol. The samples were observed using electron microscopy (JEM-1400, JEOL, Japan) at 15,000× magnification.

In vivo subcutaneous tumor growth assay

BALB/c nude mice were subcutaneously injected with 1 × 106 stable MCCC2 KD HCT116 cells and control cells; each group contained six mice. After 11 days of monitoring, the mice were sacrificed and the tumors were harvested. All the specimens were fixed in formalin and embedded in paraffin for slide preparation. The slides were stained with H&E, anti-Ki67, and anti-MCCC2 antibodies.

Statistical analysis

Statistical analyses were performed using GraphPad Prism 9.3.0 (RRID:SCR_002798) (Chicago, IL., USA) or SPSS 25.0 (RRID:SCR_002865) (California, USA). Associations between MCCC2 expression and clinical variables were analyzed using chi-squared test. Overall survival was analyzed using Kaplan–Meier analysis, and the p value was calculated using the log-rank test. A Cox regression analysis model was used to evaluate univariate and multivariate survival analyses. Differences between groups were analyzed using two-tailed Student’s t-test. Two categorical independent variables in multiple comparisons were analyzed by two-way ANOVA. Significance was set at p < 0.05.