Cell culture and treatment

Human embryonic kidney cells HEK293T (RRID: CVCL_0063), human luminal A breast cancer cells MCF-7 (RRID: CVCL_0031) and T47D (RRID: CVCL_0553), human TNBC cells BT-549 (RRID: CVCL_1092), MDA-MB-231 (RRID: CVCL_0062) and MDA-MB-468 (RRID: CVCL_0419), and mouse breast cancer cells 4T1 (RRID: CVCL_0125) were purchased from Servicebio company. These cells were meticulously nurtured in a conducive environment, specifically in Dulbecco’s modified Eagle medium (DMEM, Thermo, MA, USA), fortified with 10% fetal bovine serum (FBS, Thermo, MA, USA), 100 µg/ml streptomycin, and 100 units/ml penicillin, all within the confines of a controlled incubation chamber maintained at a temperature of 37 °C, and replete with a humidified atmosphere infused with 5% CO2. The siRNA oligonucleotides targeting SENP2, miR-145-5p mimic and inhibitor were purchased from GenePharma (Supplementary Table S1). Cells were transiently transfected with Lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. Briefly, the DNA and Lipofectamine 2000 was diluted in Opti-MEM medium (Thermo, MA, USA) and incubated for 5 min. The DNA and Lipofectamine 2000 were mixed and allowed to form complex for 20 min at room temperature and then added to the cells. The transfected cells were cultured in a humidified incubator for indicated experiments. All human cell lines have been authenticated using STR profiling within the last three years, and all experiments were performed with mycoplasma-free cells.

Xenograft mouse models

Five-week-old female BALB/c SCID mice were purchased from Beijing Vital River Laboratory Animal Technology and used for experiments. All mice were housed at room temperature (22 °C), with a 12-hour light-dark cycle and ad libitum access to food and water. sh-SENP2 or control MDA-MB-231 cells (4 × 106) were subcutaneously implanted into the left flank of SCID mice. The growth of tumors was determined by measuring of the dimensions of the xenograft every 4 days before reaching a mean volume of 100 mm3. The body weight of the mice was recorded every 4 days, and the mice were monitored each day. Tumor volumes were calculated with the formula: volume (mm3) = 0.5 × longest tumor diameter × (shortest tumor diameter)2. At the endpoint, the mice were euthanized, and the tumors were collected for weight and Western blotting analysis. The animals were housed in 2 to 5 mice per cage. All animal studies were performed according to the protocol “Guide for the Care and Use of Laboratory Animals”, which was approved by the esteemed Institutional Animal Care and Use Committee of Shaanxi Normal University, and all manipulations were carried out according to established guidelines.

Breast cancer specimens and immunohistochemistry (IHC) staining

Breast cancer patients were diagnosed at the Affiliated Hospital of Southwest Medical University, and the detailed clinical data were listed in Supplemental Table S7. For our investigations, tissue microarray chips comprising a total of 48 pairs of tumors and their corresponding adjacent tissues were procured from Biotech Well (Supplemental Table S8). It is essential to underscore that these tumor and adjacent non-cancerous tissue specimens were originally collected during surgical procedures and were obtained primarily for diagnostic and therapeutic purposes. Paraffin-embedded sections of 10 randomly selected breast cancer specimens were performed by IHC staining of SENP2 and ERK2 expression by an IHC kit (Vector Laboratories, CA, USA) according to the manufacturer. The staining results were scored by multiplying the percentage classification by the intensity [28]. All human specimens were analyzed for the current study with appropriate IRB approved by the Affiliated Hospital of Southwest Medical University, and the studies abide by the Declaration of Helsinki principles. The GEPIA2 database (http://gepia.cancer-pku.cn) was applied to perform differential expression analysis of target genes between tumor and normal samples, as well as survival analysis to investigate the prognostic value of gene expression levels in breast cancer patients. The last date of access to the platform was October 30, 2023 with email address at qiyitao@snnu.edu.cn.

Construction of plasmid

The eukaryotic expression plasmids harboring wild-type RGS-His-SENP2, the catalytic mutant RGS-His-SENP2, HA-SUMO1, and HA-SUMO2 have been comprehensively elucidated in prior literature [9, 12]. The FLAG-ERK2 plasmid was kindly provided by Dr. Qiao Wu from Xiamen University, and the SUMO site mutation of ERK2 was generated using the Quick-change site-directed mutagenesis kit (Vazyme, Nanjing, China) with the indicated primers (Supplementary Table S2). All plasmids were confirmed by DNA sequencing (Supplementary Table S3).

Generation of lentivirus and transduction

The sh1-SENP2 and sh2-SENP2 knockdown cell lines were generated using the lentiviral system (System Biosciences, CA, USA). Lentiviral expression plasmids, including pCDH-SENP2, wild-type and SUMOylation site mutant pCDH-ERK2, were generated via a standard PCR-based strategy with the indicated primers (Supplementary Table S2). sh-NC was purchased from System Biosciences, and sh-SENP2 plasmids were kindly provided by Dr. Jinke Cheng in Shanghai Jiao Tong University. The lentivirus was generated by HEK293TN cells by co-transfecting the plasmids along with package (psPAX2) and envelope (pMD2.G) plasmids. Cell culture medium was harvested 2 days after transfection and transduced into target cells. The successfully transduced cells were selected by puromycin incubation for 2 days. Real-time PCR and immunoblotting (IB) were performed to confirm the efficiency of gene knockdown or overexpression.

Dual-luciferase reporter assay

The potential interacting sites of miR-145-5p with the 3′-untranslated region (UTR) of SENP2 mRNA were constructed and cloned between the BamHI and XbaI sites of the pGL3-Promoter luciferase expression vector (Promega, WI, USA). Wild-type or mutant pGL3-SENP2 vectors were constructed. Subsequently, HEK293T cells were co-transfected with wild-type or mutant pGL3-SENP2 and then treated with miR-145-5p mimic or negative control. After 2 days of co-transfection, luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Beyotime, Beijing, China). Renilla luciferase activity was used for normalization.

Immunocytochemistry staining

Cells transfected with the specified plasmids were cultured on coverslips. Subsequently, the cells underwent a series of procedures, including washing with PBS, fixation using 4% paraformaldehyde (PFA, Thermo, MA, USA), permeabilization with Triton X-100, and incubation with the designated antibodies. Mitigation of non-specific antibody binding was achieved through treatment with 10% goat serum (Thermo, MA, USA) under ambient conditions. The primary antibody was diluted within Triton X-100 and subjected to incubation at 37 °C for 1 h. After this, the cells underwent triple PBS washes, followed by incubation for 1 h at ambient temperature with secondary antibodies labeled with Alexa Fluor 488 or 546 fluorophores (Thermo, MA, USA). The cells underwent a tripartite process involving a series of PBS washes, incubation with DAPI for 15 min, and subsequent mounting utilizing the anti-fade mounting solution (Maokangbio, Shanghai, China). Next, they were subjected to examination using confocal laser scanning microscopy (Leica, Wetzlar, Germany).

Fluorescence in situ hybridization

Briefly, paraffin sections of breast cancer and adjacent non-cancerous tissues were deparaffinized with xylene and rehydrated with ethanol. The sections were then treated with antigen retrieval buffer for 10 min and protease K for 15 min. The sections were incubated with pre-hybridization buffer for 1 h, followed by incubation with the miR-145-5p probe at 37 °C overnight. Subsequently, the sections were washed with ice-cold PBS, incubated with DAPI for 10 min and then detected by confocal scanning microscopy (Nikon, Tokyo, Japan).

Cell viability assay

Cellular seeding took place at a density of 2000 cells per well within 96-well plates, followed by an incubation period in a suitable culture medium. Following an incubation period of 1, 2, 3, and 4 days, cell viability of control and stably transfected cells was determined employing 3-(4,5)-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Sigma, MO, USA) in accordance with the manufacturer’s protocol. Concisely, a solution consisting of 10 µL of 4 mg/mL MTT was introduced into 100 µL of culture media. Following a 3-hour incubation at 37 °C, 100 µL of dimethyl sulfoxide (DMSO, Thermo, MA, USA) was administered to each well. Absorbance measurements were conducted employing a multimode microplate reader (Thermo, MA, USA).

Colony formation ability assay

Cells were seeded in a 24-well plate at approximately 300 cells per well. After incubation overnight, the cells were treated with the indicated agents and incubated at 37 °C with 5% humidified CO2 for two weeks. The medium was changed every three days. The formed colonies were washed twice with cold PBS, fixed with methanol for 15 min, stained with crystal violet, and then detected by confocal laser scanning microscopy (Nikon, Tokyo, Japan).

Wound healing assay

Cells were seeded in 6-well plates at 6 × 105 cells per well. When the cells reached 70% confluence, the cell monolayer was gently scratched with a sterile 100 µl pipette tip to generate a mechanical wound. The cells were cultured in serum-free media at different times. The images were captured with an inverted microscope, and the scratch area was measured with ImageJ software. Average scratch width = scratch area/length. Relative width = 48-hour scratch width/0-hour scratch width.

Transwell invasion assay

The assay for cell migration was conducted employing a 24-well Boyden chamber transwell insert (BD Biosciences, CA, USA). The Matrigel matrix (Corning, NY, USA) at a concentration of 200 µg/mL was introduced into the upper chamber. A total of 1 × 105 cells, suspended in 150 µL of serum-free DMEM, were introduced into the upper chamber. The lower chamber of each well was filled with 400 µL of DMEM supplemented with 10% FBS. Following an incubation period of 24 h in a controlled environment at 37 °C with 5% CO2, cells that migrated to the lower surface of the upper chamber membrane were subsequently stained with 0.1% crystal violet and subsequently visualized using confocal laser scanning microscopy (Leica, Wetzlar, Germany).

mRNA isolation and real-time PCR

To conduct a quantitative analysis of gene expression, we employed the RNeasy kit (Qiagen) to extract total mRNA from either cultured or transfected cells. Following extraction, the RNA underwent DNase treatment (Promega, WI, USA), and its concentration was quantified through absorbance measurement at 260 nm. An equivalent quantity of RNA (1 mg) served as the substrate for the synthesis of complementary DNA, a process facilitated by the high-capacity cDNA reverse transcription kit (Thermo, MA, USA). Quantitative real-time PCR was conducted employing reaction mixtures comprising cDNA, primers as specified (Supplementary Table S4), and SYBR Green reagent (Thermo, MA, USA). The ABI StepOne system (Perkin-Elmer, MA, USA) served as the platform for this analysis. The thermal protocol consisted of an initial denaturation step at 95 °C for 10 min, followed by 38 cycles of denaturation at 95 °C for 15 s, annealing and amplifying at a temperature specific to the primer sets used 60 °C for 30 s. Upon completion of the cycles, a final elongation step at 72 °C for 5 min ensured full extension of all amplified products. Real-time PCR was conducted in triplicate, and standard deviations, indicative of experimental errors, were subsequently calculated. Data analysis was executed employing ABI PRISM software (Perkin-Elmer, MA, USA). This software facilitates the determination of the threshold cycle, signifying the cycle number at which the fluorescence intensity significantly exceeds that of the background fluorescence.

Western blotting and immunoprecipitation

The transfected cells were harvested and subsequently homogenized under chilled conditions utilizing lysis buffer containing a protease inhibitor (Targetmol, Shanghai, China). Total protein content was quantified through the utilization of the BCA assay (Servicebio, Beijing, China). Equimolar quantities of protein (20 µg in each lane) were subjected to electrophoretic separation and subsequently transferred onto PVDF membranes by electroblotting. The PVDF membrane was blocked with 5% nonfat dried milk, followed by overnight incubation with primary antibodies diluted with BSA (Supplementary Table S5). Subsequent washing was carried out, followed by incubation with peroxidase-conjugated secondary antibodies. Protein levels were subsequently determined with a chemiluminescence system (Qinxiang, Shanghai, China) after additional TBST washing steps. In the context of immunoprecipitation (IP) experiments, whole cell lysates were subjected to overnight incubation with antibodies as specified. All incubations were rigorously carried out at a temperature of 4 °C with continuous agitation. The protein complex, bound by antibodies, was effectively captured through the introduction of protein A/G agarose (refer to Supplemental Table S6), followed by an additional 2-hour incubation. After centrifugation, the protein A agarose pellet was obtained, and the IP protein complex was subsequently eluted with SDS-PAGE sample buffer, followed by Western blotting analysis utilizing the specified antibody.

Statistical analysis

GraphPad Prism (USA) was used to evaluate the data. All data are presented as the means ± S.E.M., and experimental procedures were independently conducted in a minimum of three separate investigations. Group comparisons were conducted utilizing Student’s t test for pairwise comparisons, one-way ANOVA followed by Tukey’s or Dunnett’s test for multiple-group comparisons, or two-way ANOVA followed by Bonferroni test for comparisons involving more than two groups. Statistical comparisons were made to assess variances between groups, with statistical significance defined as p < 0.05 (* p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001).