The data of breast cancer patients were downloaded from the TCGA database (https://portal.gdc.cancer.gov). RNA-seq data from the TCGA-BRCA project was subjected to STAR workflow and extracted in transcripts per million (TPM) format to obtain gene expression and clinical data. Data processing and visualization were performed using R software (version 4.2.1). The prognostic value of FAM134B in breast cancer was analyzed through Kaplan-Meier Plotter database (https://kmplot.com/analysis/), including overall survival (OS) and disease-free survival (DFS).

The human normal breast epithelial cell line MCF-10A and human breast cancer cell lines MCF-7 and MDA-MB-231 were bought from Cobioer (Nanjing, China). MCF-10A cells were seeded in BEGM medium (BeNa Culture Collection, China), MCF-7 cells were cultured in RPMI-1640 medium (GIBCO, USA) and MDA-MB-231 cells were cultured using L15 medium (GIBCO). The conditioned medium contains 10% fetal bovine serum (FBS) and 100 U/mL penicillin-streptomycin (Sigma-Aldrich, USA). All of the cell lines used in current study have been tested to confirm the absence of mycoplasma contamination.

Recombinant lentiviral vectors specifically designed for FAM134B (shFAM134B) and control non-targeting vector (shNC) were constructed by GenePharma (Shanghai, China). MCF-7 and MDA-MB-231 cells were transfected with the vectors at multiplicities of infection (MOI) ranging from 10 and 100, in the presence of 5 mg/mL polybrene. After transfection, the supernatant containing the viral particles was replaced with fresh DMEM. Following 72 h of incubation, the cells were selected with 2 μg/mL puromycin to establish stably transfected shFAM134B cell line.

For qRT-PCR, total RNA was extracted from cells with TRIzol reagent (Invitrogen, USA) according to the manufacturer’s protocol. Complementary DNA (cDNA) was synthesized from RNA with Reverse Transcription Kit (Takara, Japan). Then qRT-PCR assay was performed by SYBR^® Premix Ex TaqTM (Takara) according to the instruction. The GAPDH was used as an endogenous control gene for normalizing the expression of FAM134B. The primer sequences are shown in Table 1.

The proteins were extracted by radio immunoprecipitation assay (RIPA) buffer (Sigma-Aldrich, USA) and quantified by BCA Assay Kit (Sigma-Aldrich). A total of 20 μg protein was treated with 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene fluoride (PVDF) membranes (Invitrogen). The membranes were blocked by 3% bovine serum albumin (BSA, Sigma-Aldrich), then primary antibodies including FAM134B, LC3B, IRE1α, p-IRE1α, PERK, p-PERK, CHOP and GAPDH were added and incubated overnight at 4°C. After that, secondary antibody horseradish peroxidase (HRP)-conjugated IgG (Abcam) was appended and incubated for 1 h at 37°C. The intensity of immunoreactive bands was normalized to that of GAPDH by ImageJ 1.4 software.

Cell viability was detected by cell counting kit-8 (CCK-8, Yeasen, China). Breast cancer cells were seeded into a 96-well plate at a density of 3 × 10³ cells/ml after transfection. The culture medium was added to 10 μl CCK-8 solution and cultured for 1.5 h, and the optical density (OD) value was measured at 450 nm at 0, 24, 48 and 72 h.

The breast cancer cells were cultured in a 6‐well plate (1×10³ cells per well) and incubated for 1 week at 37°C. Cells were then washed twice with PBS, fixed with 4% formaldehyde for 15 min, and stained for 30 min with Giemsa staining solution (Beyotime Biotechnology, China). Colonies with a diameter ≥ 100 μm were counted, and the assay was performed in triplicate.

The apoptotic rate of cells was determined under the guidelines of Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN Biotech, China). MCF-7 and MDA-MB-231 cells were sequentially stained with Annexin V-FITC and propidium iodide (PI), followed by incubation in a dark environment for 15 min. Subsequently, the status of cell apoptosis was calculated using a MoFlow flow cytometer (Beckman Coulter, USA).

The cells were cultured in 6-well plates, gently detached by 0.25% trypsin, and fixed with 4% glutaraldehyde at 4°C for 2 h. Cells were then fixed with 1% osmium tetroxide for another two hours, stained with filtered uranyl acetate, and dehydrated using ethanol and acetone. Subsequently, the cells were encapsulated in Epon resin, and both semi-thin (0.5 μm) and ultra-thin (60 nm) sections were meticulously prepared. Sections were stained with lead citrate and uranyl acetate, then observed under a Hitachi H-7500 transmission electron microscope (Hitachi, Japan), and images were captured using a sophisticated Gatan780 system.

Cells were rinsed thrice with PBS and fixed with freshly prepared methyl alcohol at 20°C for 20 min. Cells were exposed to primary antibody (Abcam) overnight at 4°C, and subsequent to washing by PBS, stained with fluorescent secondary antibody (Abcam) for 1 h at room temperature. Post rinsing with PBS, cells were stained with 4,6-diamidino-2-phenylindole (DAPI) for 15 min, and washed thrice with PBS. Cell images were acquired using a confocal microscope (Nikon, Japan).

The study was approved by the Institutional Animal Care and Use Committee of Shanghai General Hospital, School of Medicine, Shanghai Jiao Tong University (Approval No. 202501001). All animals received humane care according to the criteria outlined in the Guide for the Care and Use of Laboratory Animals. The mice were euthanized if they showed any adverse signs or symptoms of diseases which were beyond our intention in the experimental protocol. A total of 18 BALB/c nude mice (female, 5–6 weeks old, 18–22 g weight, achieved from the Laboratory Animal Center of Shanghai Jiao Tong University) were randomly divided into 6 groups (3 mice each group). Mice were kept in the animal house facility under a 12-h reversed light/dark cycle, 22°C with free access to water and food. Next, 0.2 mL of above cell suspension that contained 2 × 10⁴ cells was injected into the back of each mouse. The experiment lasted three weeks, and the mice in each group were monitored twice a week to the end of the experiment. After 3 weeks, mice were sacrificed by inhaling CO₂. The tumor tissues were excised, then the volume and weight of the tumor tissues were tested. Western blot was performed to detected the expression of FAM134B and autophagy-related proteins in the tumor tissues.

All experiments were performed in triplicate. Statistical analysis was executed with GraphPad Prism 8.0 (GraphPad Software, USA). Data were presented as the mean ± standard deviation (SD). To ascertain group differences, Student’s t test was employed. For multifaceted comparison studies, One-Way Analysis of Variance (ANOVA) methodology was implemented. Correlation between FAM134B expression and clinicopathologic characteristics of patients was analyzed using Chi-square test. Statistically significant results were determined when P value < 0.05.

In previous study, we have already discovered that FAM134B can promote the progression of hepatocellular carcinoma, in order to explore whether FAM134B also plays a role in breast cancer, a comprehensive analysis of its correlation with survival rates among breast cancer patients was initially conducted using the Kaplan-Meier Plotter database. Based on the TCGA dataset, breast cancer patients were divided into FAM134B high-expression and low-expression groups using the median expression level of FAM134B as the cutoff.The results indicated that patients with elevated levels of FAM134B exhibited reduced OS (P = 0.024, Figure 1A) and DFS (P = 3.2e^-07, Figure 1B) compared to those expressing lower levels of FAM134B. Correlation analysis between FAM134B expression and clinicopathological characteristics was performed. Results demonstrated that FAM134B expression was significantly associated with patient age, Pathologic T stage, progesterone receptor (PR) status, estrogen receptor status, and human epidermal growth factor receptor 2 (HER2) status (Table 2). We also conducted an analysis of the differentially expressed genes between these two groups. For differential expression analysis, criteria of adjusted p.adjust)<0.05 and log₂ fold change (log2FC)≥1 or ≤-1 were applied. A total of 1,824 differentially expressed mRNAs were identified, including 419 significantly upregulated and 1,405 significantly downregulated in the FAM134B high-expression group (Figure 1C). The differential expression results were visualized by a volcano plot (figure omitted). Subsequently, an intersection analysis was conducted between the 1,824 differentially expressed genes and autophagy-related genes (Human Autophagy Database, HADb, https://www.autophagy.lu/), and Venn diagram revealed 10 common genes (Figure 1D, Table 3). These 10 autophagy related genes showed significant differences between the FAM134B high-expression group and the FAM134B low-expression group, suggesting that FAM134B may be associated with autophagy in breast cancer. Moreover, investigations employing qRT-PCR on human normal breast epithelial cells (MCF-10A) and breast cancer cells (MCF-7 and MDA-MB-231), detected higher expression of FAM134B mRNA in breast cancer cells (P<0.001, Figure 1E). Western blotting further indicated an enhanced level of FAM134B protein expressed in breast cancer cells compared to normal breast epithelium cells (P<0.001, Figure 1F). These findings suggested that FAM134B might contribute to the progression of breast cancer.

qRT-PCR and western blotting were used to verify transfection efficiency. The qRT-PCR outcome indicated a reduction in FAM134B expression levels within the cells post transfection with shFAM134B (P<0.01, Figure 2A), and the results of western blot showed similar results on the level of FAM134B protein (P<0.001, Figure 2B), demonstrating successful transfection. The results of the CCK-8 assay indicated that the proliferation ability of breast cancer cells decreased after silencing FAM134B (P<0.05, Figure 2C). Colony formation assays further demonstrated that the number of colonies in the shFAM134B group was significantly lower than that in the shNC group (Figure 2D). These results indicated that FAM134B knockdown inhibited breast cancer cell proliferation.

Subsequently, we examined the apoptosis rate of breast cancer cells post transfection by flow cytometry assay. The results indicated a significant increase in the apoptosis rate of breast cancer cells following silence of FAM134B (P<0.001, Figure 3A). Transmission electron microscopy revealed an increase in the number and size of autophagosomes in shFAM134B-transfected cells compared to shNC-transfected cells (Figure 3B). Furthermore, the experimental findings from immunofluorescence demonstrated a higher content of LC3 protein in the shFAM134B group compared to the control group (Figure 3C). These results suggest that downregulation of FAM134B induced apoptosis and autophagy in breast cancer cells.

To further investigate the mechanism of the influence of FAM134B on breast cancer, we examined the expression levels of LC3I, LC3II, IRE1α, p-IRE1α, PERK, p-PERK, and CHOP as markers for autophagy and ER stress in breast cancer cells through western blot. The results revealed a significant increase in the expression of LC3II protein after downregulating FAM134B (P<0.01). Furthermore, the expression levels of p-IRE1α, p-PERK, and CHOP proteins in the shFAM134B group were significantly elevated compared to the control group (P<0.01, Figure 4). The findings suggested that downregulation of FAM134B stimulated autophagy and induce ER stress in breast cancer cells.

Tumor xenograft experiment was performed to verify the effect of FAM134B in vivo. The mice were euthanized 21 days post-injected with tumor cells, and the tumor tissues were isolated (Figure 5A). The experimental results indicated that the volume (P<0.001, Figure 5B) and weight (P<0.001, Figure 5C) of the tumor tissues were lower in shFAM134B than those in shNC group. In addition, the expression levels of LC3I, LC3II, IRE1α, p-IRE1α, PERK, p-PERK, and CHOP in tumor tissues were detected through western blot. The results revealed an increase in the expression of LC3II protein after downregulating FAM134B (P<0.01). Furthermore, the levels of p-IRE1α, p-PERK, and CHOP proteins in the shFAM134B group were significantly risen compared to the shNC group (P<0.01, Figure 6). These results indicated that FAM134B knockdown suppressed the growth of breast cancer not only in vitro but also in vivo.