LARP6 suppresses colorectal cancer progression through ZNF267/SGMS2-mediated imbalance of sphingomyelin synthesis

Cell culture

Normal human colon mucosal epithelial cell (NCM460) and human CRC cell lines (SW480, DLD1, RKO, LOVO, CACO2, HCT116, and SW620) were obtained from the cell bank at the Chinese Academy of Sciences (Shanghai, China). Cells were authenticated by short tandem repeat (STR) profiling after receipt and were propagated for less than 6 months after resuscitation. Maintained in a humidified chamber containing 5% CO2 at 37 °C, cells were routinely cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS; Gibco, USA) and 1% antibiotics (Gibco, USA).

Patient samples

The Institute Research Medical Ethics Committee of Nanfang Hospital (Guangzhou, China) granted approval for this study. Informed consent was obtained from all patients. 61 pairs of fresh CRC tissues and matched adjacent normal tissues were collected randomly from primary CRC patients without any treatment before surgery in Nanfang Hospital from 2018 to 2020, and 49 pairs of them were analyzed by RT-PCR, and 12 pairs were analyzed by WB. Paraffin-embedded CRC tissues and matched adjacent normal tissues of 165 primary CRC patients were gathered in Nanfang Hospital from 2017 to 2021, and none of the patients received any preoperative chemotherapy or radiotherapy before surgery. Serial sections of paraffin-embedded CRC tissues used for IHC were obtained from the above 165 patients. Three cohorts of tissue samples for RT-PCR, WB and IHC are obtained from different primary CRC patients. The details of clinical information is provided in the Additional files [see Additional file 1].

Mice

BALB/c nude mice (male, 4 weeks old) were provided by the animal center of Guangdong Province. All mice experiments were approved by the Committee on the Ethics of Animal Experiments of Southern Medical University. All animal studies were strictly complied with regulations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health.

RNA extraction and quantitative real-time PCR (qPCR, RT-PCR)

According to the manufacturer’s instruction, total RNA in cells or tissues were extracted with TRIzol reagent (TaKaRa, Japan). cDNAs were generated with PrimeScript RT-PCR Kit (TaKaRa, Japan). RT-PCR was conducted using SYBR Premix Ex Taq (TaKaRa, Japan) on ABI7500 Real-time PCR system (Applied Biosystems, Foster City, USA). Relative mRNA expression was calculated according to the work by Pfaffl [21].

Protein extraction and western blotting (WB)

With RIPA buffer containing a protease inhibitor and a phosphatase inhibitor (FDbio, China), cells and tissues were lysed, then proteins were harvested. Proteins were separated by SDS-polyacrylamide gel electrophoresis (PAGE) and electrotransferred onto a polyvinylidene difluoride (PVDF) membrane (Merck millipore, USA). Having been incubated with primary and secondary antibodies, protein expression was visualized with an enhanced chemiluminescence system. Primary antibodies used are listed as follows: anti-LARP6 antibody (1:500; Abcam, Britain), anti-GAPDH antibody (1:1000; Proteintech, USA), anti-ZNF267 antibody (1:1000; Novus, USA), anti-SGMS2 antibody (1:1000; Novus, USA), anti-P62 antibody (1:1000; Proteintech, USA), anti-LC3B antibody (1:500; ABclonal, USA).

Immunohistochemistry (IHC)

IHC staining of paraffin-embedded human or mice tissues was carried out according to protocols. With dewaxing, hydration, antigen repair and block successively, sections were incubated in primary antibodies overnight at 4 °C. Next day, sections were put in room temperature for 1 h to rewarm, followed by secondary antibody incubation for 1 h in room temperature. Having been stained with 3,3-diaminobenzidine (DAB, ZSGB-BIO, China), the slides were counterstained with hematoxylin, dehydrated and mounted. Primary antibodies used are listed below: anti-LARP6 antibody (1:50; Abcam, Britain), anti-ZNF267 (1:100; Novus, USA), anti-SGMS2 (1:300; Novus, USA).

Performed by two independent pathologists blinded to the clinical data, IHC staining was scored as the product of staining intensity (0–3) and percentage of positive area (0–100) (overall score range = 0–300).

Lentivirus and plasmids transfection

Lentiviral vectors were constructed by GENECHEM Biotech at Shanghai, China (http://genechem.bioon.com.cn/). Luciferase-tagged LARP6-overexpressed vectors and control vectors were transfected into SW480 and DLD1 cells, while LARP6 shRNA and control short hairpin RNA were transfected into CACO2 and SW620 cells to generate cells with stable knockdown of LARP6. Transfection was carried out in accordance with protocol. Briefly, cells (1 × 105 per well) grown in 6-well plates were transducted with lentivirus for 24 h, followed by selection with puromycin (4 μg/ml) for 48 h post transduction for 4–5 days. Effect of overexpression or knockdown was analyzed by qRT-PCR and western blotting.

Overexpression and knockdown of ZNF267 and SGMS2 in CRC cells were achieved by plasmids and siRNA (small interfering RNA) transfection using Lipofectamine 3000 (Life Technologies, USA). Transfection protocol was resemble to Lentivirus.

CCK8 and colony formation assay

For cell proliferation assay, cells (1 × 103 cells per well) were seeded on 96-well plates and cell proliferation were determined for 7 days with cell counting kit-8 (DOJINDO Laboratories, Japan) according to instructions. For colony formation assay, cells (500 cells per well) were seeded on 6-well plates and cultured for 2 weeks. Colonies were fixed in methanol and stained with 1% Giemsa. Colonies containing more than 50 cells for each well were counted. All observations were reproduced at least three times in independent experiments.

Migration and invasion assay

For cell migration analysis, 2 × 105 cells suspended in serum-free media were seeded into the 8-μm-pore upper chambers and incubated in RPMI1640 with 10% FBS of the lower chamber of 24-well plates (Corning, USA). After regular culture for 20–48 h, cells were fixed with methanol and then stained with Giemsa. Migratory cells were calculated in five randomly chosen fields (magnification, 200×). For invasion assay, the upper chamber was coated with 200 μg/ml Matrigel (Corning, USA) and 2 × 105 cells were seeded into the upper chamber. Other procedures were resemble to migration assay. All experiments were repeated at least three times.

Orthotopic colorectal cancer mice model

Luciferase-tagged LARP6 shRNA and control shRNA were transfected into CACO2 cells. 2 × 106 CACO2 cells with or without LARP6 knockdown were then injected into the subserous layer of the cecum in nude mice (BALB/c-nu/nu, male, 4 weeks old, 8 mice per group). To assess metastasis of CRC in mice model, at 60 days after surgery, mice were injected intraperitoneally with luciferase substrate (Promega), and luciferase activity using an instrument (FX Pro, USA) was non-invasively detected. The mice were then killed, with intestine and liver tissues separated for further assays.

RNA immunoprecipitation (RIP)

Cells were gathered for RNA immunoprecipitation assay as kit instructed (Abcam, Britain). Cell lysate was incubated in anti-LARP6 antibody overnight at 4 °C, followed by conjugating to protein dynabeads, with serum (IgG) as a control group. RNA was extracted using TRIzol following manufacturer’s instructions (TaKaRa, Japan). cDNAs generation and RT-qPCR were performed as described earlier. The fold enrichment for each target was measured by comparing the Ct values of LARP6-immunoprecipitated fraction to the IgG isotype fraction and normalized using the ΔCt formula. GAPDH was used as a negative control.

Biotinylated RNA pull-down assay

Biotin-labeled ZNF267 RNA probe was designed, synthesized and purchased from Sangoon Biotech (Shanghai, China). With continuous rotation, purified biotinylated ZNF267 RNA probe was incubated with total cell lysates for 1 h at room temperature. Complexes were isolated with streptavidin-conjugated Dynabeads (Invitrogen, USA), followed by boiling with SDS-PAGE loading buffer for 5 min. The pull-down materials were subsequently analyzed by western blotting with LARP6-specific antibody.

mRNA stability analysis

Cells were treated with actinomycin D (2 μg/ml; FDbio, China) for 0, 0.5, 1, 3, 5 and 7 h, followed by RNA extraction at every time points. cDNA generation and RT-PCR were the same as described earlier. ZNF267 mRNA abundance was analyzed, with GAPDH as the endogenous control. ZNF267 mRNA level was normalized to the 0 h time point.

Polysome analysis

On being equally plated on cell culture dish at the concentration of approximately 20–25%, cells were maintained for 2–3 days until became 90% confluent. As it was mentioned [22], cells were treated with 100 μg/ml cycloheximide (CHX, Selleckchem, China) for 10 min, prior to lysing in 300 μL of lysis buffer. Nuclei and membrane debris were then removed by centrifuging at 12000 g, 10 min. The lysate was loaded onto a sucrose gradient (10–50% sucrose(w/v)) and centrifuged in a SW41Ti rotor (Beckman, USA) for 1.5 h at 39000 rpm at 4 °C. Fractions were collected by density gradient fractionation system Piston Gradient Fractionator™ (BIOCOMP, Canada). cDNA generation and RT-PCR were the same as described earlier.

Chromatin immunoprecipitation (ChIP)

ChIP was carried out as Chromatin Immunoprecipitation kit (Abcam, Britain) described. Immunoprecipitation reactions were performed with anti-ZNF267 antibody (5 μg; Novus, USA), and IgG was as a control. Purified DNA was used for RT-PCR, and primers were designed specific to SGMS2 promoter. The fold enrichment was measured by comparing the Ct values of ZNF267-immunoprecipitated fraction to the IgG isotype fraction and normalized using the ΔCt formula.

Dual-luciferase reporter gene assay

All experiments were performed according to the kit instructions (Yeasen, China). Cells were inoculated in a 96-well plate and cell lysis buffer was transferred to the black microplate, then firefly luciferase reaction solution was added and the firefly luciferase activity was determined. After inocubating with Renilla luciferase reaction solution, the activity was measured.

Total ceramide and sphingomyelin level detection

With quantitative cell or tissue lysate collected, the total ceramide level was measured using a human ceramide ELISA Kit (Enzyme-linked Biotechnology, Shanghai, China) according to the manufacturer’s instructions, and the total sphingomyelin level was measured using a human sphingomyelin Kit (Abcam, Britain).

Transmission electron microscopy (TEM)

With cell pellets about the size of a grain of rice collected, sample was fixed with 2% glutaraldehyde at 4 °C overnight. Samples were then postfixed with 1% OsO4 dissolved in 0.1 M PBS for 2 h and dehydrated using an ascending gradual series (50–100%) of ethanol and infiltrated with propylene oxide. After sectioning and staining with uranyl acetate and lead citrate, samples were viewed via TEM (HITACHI, HT7700, Japan).

Autophagy flux detection

Transfected with RFP-GFP-LC3B lentivirus, cells with LARP6 overexpression or interference and control cells were fixed using 4% paraformaldehyde and counterstained with DAPI. With confocal microscope, autophagy activity was assessed by quantitation of the number of red and yellow puncta in cells, counting at least 10 cells per group.

Statistical analysis

Each assay was performed in at least three independent experiments. Statistical analysis were finished using SPSS software (version 23.0, IBM Corp, Armonk, NY, USA). A two-tailed, unpaired, or paired Student t test was used to compare the variables of two groups, and one-way or two-way ANOVA were performed for multi-group comparisons. P < 0.05 was considered statistically significant (*P < 0.05, **P < 0.01, ***P < 0.001, ns means no statistic difference). The error bars represent Mean ± SD.

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