Human colorectal cancer tissues

A total of 27 cases of colorectal cancer tissues and matching adjacent non-tumor colorectal tissues were collected from the First Affiliated Hospital of Jinzhou Medical University, China (No. 201923). And, all patients enrolled in the current study provided written informed consent. All sample collection and processing were undertaken according to the Declaration of Helsinki and ethical approval was obtained from the Ethics Committee of the First Affiliated Hospital of Jinzhou Medical University.

Cell culture

The human colorectal carcinoma cell lines (HCT116, HCT8, SW480) and HEK293 were cultured in the medium with 10% FBS (Biological Industries, Kibbutz Beit Haemek, Israel), penicillin (100 unit/mL), and streptomycin (100 μg/mL) in a 37 °C humidified atmosphere of 5% CO2.

Antibodies and reagents

The antibodies against MICALL2, TRIM21, Lamin B1, β-catenin, E-cadherin, Vimentin, c-Myc, Cyclin D1, Flag tag, His tag, Ubiquitin, and GAPDH were purchased from Proteintech (Proteintech, Chicago, IL, USA). The Lipofectamine 3000 and TRIzol™ reagent were purchased from Invitrogen (Invitrogen, Carlsbad, CA, USA). Nuclear and cytoplasmic extraction kit was purchased from Thermo Fisher Scientific (Waltham, MA, USA). The PAGE Gel Silver Staining kit (Cat. No. G7210) and MG132 (IM0310) were supplied by Beijing Solarbio Science & Technology Co., Ltd. (Beijing, China). Cycloheximide (CHX) (Cat. No.A49960) was purchased from Shanghai Acmec Biochemical Co., Ltd. (Shanghai, China).

Plasmids construction

The plasmids of pcDNA3.1-His-MICALL2, p3xFlag-CMV-TRIM21 and other truncated mutants were constructed based on human cDNAs of MICALL2 and TRIM21 were purchased from Miaolingbio Bioscience & Technology Co., Ltd. (Wuhan, China). Two MICALL2 specific targeting shRNAs with the following target sites were cloned in the lentiretroviral vector pLKO.1‐puro (Addgene, Cambridge, MA, USA): shMICALL2#1, 5’- CCGGTCTTGCACACGAGCAGAACTTCTCGAGAAGTTCTGCTCGTGTGCAAGATTTTTG-3’ and 5’-AATTCAAAAATCTTGCACACGAGCAGAACTTCTCGAGAAGTTCTGCTCGTGTGCAAGA-3’; shMICALL2#2, 5’-CCGGTGTCGTCCTTGTAGTACACTTC TCGAGAAGTGTACTACAAGGACGACATTTTTG-3’ and 5’-AATTCAAAAATGTCGTCCTTGTAGTACACTTCTCGAGAAGTGTACTACAAGGACGACA-3’; and shCtr, 5’-CCGGACGTGACACGTTCGGAGAATTCTCGAGAATTCTCCGAACGTGTCACGTTTTTTG-3’ and 5’-AATTCAAAA AACGTGACACGTTCGGAGAATTCTCGAGAAT TCTCCGAACGTGTCACGT-3’.

Construction of colorectal cancer cell lines with stable overexpression or knockdown of MICALL2

The constructed plasmid with pcDNA3.1-His-MICALL2 or control vector were transfected into HCT116 cells using Lipofectamine® 3000 reagent according to the manufacturer's instruction. After 48 h post-transfection, cells were selected in Geneticin (Invitrogen, Waltham, MA, USA) for 4 weeks, and cell colonies were selected and amplified for further studies. For stable silenced cell lines, HEK293T cells were cotransfected with shMICALL2‐pLKO.1 with packing plasmids by using Lipofectamine® 3000. After 48 h post-transfection, the harvested supernatants containing packaged lentivirus were used to infect HCT8 cells. Puromycin was then added into the culture to screen for stable cell lines.

Quantitative real-time polymerase chain reaction (qRT-PCR)

Total RNA was extracted from frozen tissue using TRIzol reagent and reverse transcribed into cDNA using a cDNA synthesis kit (Abm, Milton, ON, Canada). Real-time PCR analysis was performed with ABI 7500 real-time PCR system to specifically assess the relative abundances of MICALL2 mRNAs. The MICALL2 and 18S rRNA primers were as follows: MICALL2, 5’-AGTGACATCGTGGACTCGCT-3’ and 5’- TGGAGGCCCAGCTTCTCAATC-3’; 18S rRNA, 5’- GAAACGGCTACCACATCC-3’ and 5’-ACCAGACTTGCCCTCCA-3’. The experiment was repeated three times, and the relative expression level of the target gene was normalized to the mean of 18S rRNA. The data were analyzed using the comparative threshold cycle (2−ΔΔCT) method.

Tissue microarray (TMA) and immunohistochemical (IHC) analyses

Human colorectal cancer TMA (Shanghai Superchip Biotechnology Co., Ltd., Shanghai, China) consisting of 75 colorectal cancer samples and paired adjacent tissue samples were used to perform IHC by the streptavidin-peroxidase method (ZSGB-BIO, Beijing, China). To quantify the expression in the colorectal cancer tissues and matched adjacent non-tumor colorectal tissues, at least five random fields at 200 × magnification in each section were selected. For each field, integrated optical density (IOD) of protein was assessed using Image J software (NIH, Bethesda, MD, USA). Average optical density (AOD = IOD/Area) was used in this study for statistical analysis. Finally, the correlation between the expression levels of protein (AOD) and clinicopathological features in colorectal cancer patients was assessed. Besides, IHC staining scores were obtained to assess the correlation of MICALL2 with TRIM21 expression in serial colorectal cancer tissues by multiplying scores representing the staining intensity and positive cells percentage as described previously [17].

Immunoprecipitation (IP) and immunoblot analysis

Cell extracts were prepared with cell lysate (50 mM NaF, 1 mM Na3VO4, 1 mM DTT, 1 mM PMSF, protease inhibitors cocktail). After centrifugation, equal amounts of lysates were used to be immunoprecipitated with antibodies overnight at 4 °C and Protein A/G-Agarose. Subsequently, the immune complexe was subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes and probed with the corresponding primary and HRP- conjugated secondary antibodies, as well as chemiluminescence visualization.

Mass spectrometry analysis

Similar to the IP assay, cellular protein extracts were incubated with MICALL2 antibody followed by protein A/G agarose beads. The recovered protein in the immune complexes associated with MICALL2 or IgG were seperated by gel electrophoresis. The bands that specifically bind to MICALL2 were excised, and liquid chromatography tandem mass spectrometry (LC–MS/MS) was performed at PTM Biolab Hangzhou (Hangzhou, China).

Nuclear and cytoplasmic fractionation

The cells were harvested after centrifugation and washed twice with PBS. Subsequently, the nuclear and cytoplasmic extracts were prepared in accordance with the NE-PER Nuclear and Cytoplasmic Extraction Kit instruction (Thermo Scientific, Waltham, MA, USA). The purities of the nuclear and cytoplasmic extracts were assessed by Western blot with Lamin B1 and GAPDH antibody, respectively.

Wound healing assay

Colorectal cancer cells were cultured to confluence on 12-well plates. A wound area was scraped carefully with a 20 µL sterile pipette tip and detached cells were removed. At least five images were taken under an inverted microscope at 0 and 24 h following the wound formation. The wound area was measured using Image-J software (NIH, Bethesda, MD, USA). The cell migration rate (% wound closure) was calculated as follows: cell migration rate = [(wound area at 0 h)—(wound area at 24 h)] / (wound area at 0 h) × 100%.

Soft agar colony formation assay

Cells were suspended in 0.7% agarose and placed in 12-well plates. Then, the cells were cultured for two weeks, and then colonies were fixed with 4% paraformaldehyde and stained with 1% crystal violet. The number of colonies was counted with Image J software.

Immunofluorescence (IF) staining

Briefly, cells were cultivated on glassbottom culture dishes and fixed with 4% paraformaldehyde before being rinsed three times with PBS and treated with blocking solution. Then, cells were incubated sequentially with primary antibodies overnight at 4 °C, Alexa Fluor 488 and 594-conjugated secondary antibodies (1:500, Thermo Fisher) for 1 h at 37 °C, and 4,6-diamidino-2-phenylindole (DAPI) nuclear dye for 10 min at room temperature. The cells were imaged using a Leica confocal laser-scanning microscope.

RNA-Seq and bioinformatics

Total RNA was isolated from MICALL2-overexpressed HCT116 cells and control cells using Trizol Reagent (Thermo Fisher Scientific, Carlsbad, CA, USA), and subsequently purified using the Qiagen MinElute column kit (Germantown, MD, USA). Then, the RNA quality, library construction, and paired-end mRNA next-generation sequencing were sequentially performed on Illumina platform by Novogene Corporation Inc (Beijing, China). Next, the differentially expressed genes (DEGs) were statistically assessed by R/Bioconductor package DESeq2 (version 1.30.1) with the following criteria: |log2 (FoldChange)|> 1 and P-adj. < 0.05. Gene Ontology (GO) analysis was performed using the National Institutes of Health Database for Annotation, Visualization, and Integrated Discovery (DAVID, https://david.ncifcrf.gov/home.jsp) public online tool and visualized with R/Bioconductor package Goplot (version 1.0.2). UALCAN datasets was assessed for MICALL2 expression (http://ualcan.path.uab.edu/cgi-bin/ ualcan-res.pl). The GEPIA Database (http://gepia.cancer- pku.cn/ index.html) was used to analyze colorectal cancer patient survival and correlation of different genes expressions.

Animal studies

The animal experiment was carried out in accordance with a procedure approved by Jinzhou Medical University's Animal Experimentation Ethics Committee (Registration No.2021011501). Briefly, 5 × 106 of MICALL2-overexpressed HCT116 cells and control cells were subcutaneously injected into the right flank of 6 week old female BALB/c athymic nude mice (Charles River, Beijing, China). Tumor size was monitored over a 3-week period using calipers. Tumor volume was estimated according to the following formula: volume = (length × width2)/2. Tumors were removed and weighed when the mice were euthanized 21 days following treatment.

Statistical analysis

GraphPad Prism 8.0 was used for statistical analysis. The Student’s t-test or one way analysis of variance (ANOVA) was performed to evaluate the differences between two groups or more than two groups. The chi-square test was performed to analyze the correlation between MICALL2 expression and clinicopathological features. The Pearson’s correlation analysis was performed to analyze the relationship between MICALL2 and TRIM21 expression in the colorectal cancer tissues. The value of P < 0.05 was considered statistically significant. The data was presented as mean ± SEM. The following asterisks represent statistical significance: *, P < 0.05; **, P < 0.01; ***, P < 0.001.