microRNA-1258 suppresses breast cancer progression by targeting LARP4B

Abstract

Background

Breast cancer (BC) is the most common cancer in women. The majority of BC-related deaths result from metastasis and invasion. MicroRNA (miRNA) is a key regulatory factor that suppresses the expression of target genes. miR-1258 is considered one of the miRNAs significantly associated with BC, but its downstream target genes have not been fully defined. Our study aimed to elucidate the role of miR-1258 in breast cancer cell proliferation, migration, and invasion, identify its potential targets, and investigate its regulatory mechanisms.

Methods

miR-1258 expression in breast cancer was measured by Real‑time quantitative PCR. The effects of the Cell Counting Kit-8 method, the Transwell migration and invasion method on the behavior of tumor-related cells were studied. In addition, the interaction between miR-1258 and LARP4B in cancer was elucidated using dual-luciferase assays and Pearson correlation analysis.

Results

miR-1258 is significantly downregulated in breast cancer, whereas LARP4B is significantly upregulated; both are closely associated with patients’ TNM staging. miR-1258 is an independent prognostic factor; patients with low expression of miR-1258 exhibit shorter overall survival. In BC cells, overexpression of miR-1258 inhibits cell proliferation, migration, and invasion. LARP4B is a potential target gene of miR-1258, and silencing LARP4B inhibits cellular biological functions.

Conclusions

Low expression of miR-1258 may indicate poor prognosis in BC whilst its overexpression significantly inhibits proliferation, migration and invasion of BC cells. miR-1258 may inhibit BC progression by negatively regulating LARP4B.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-025-04374-2.

Introduction

Breast cancer (BC) is globally recognized as a cancer that mainly affects women, is the most prevalent malignant tumor, and is a primary cause of cancer deaths among Chinese women [1]. Although many diagnostic and therapeutic strategies have been developed for BC, clinical outcomes remain unsatisfactory [2]. Therefore, in-depth investigation into the mechanisms underlying BC development and the identification of potential therapeutic targets is of paramount importance for reducing breast cancer mortality.

MicroRNAs (miRNAs) are characterized as uncoded monostand RNAs encoded by endogenous genes, which are essential regulators of the expressed target genes [3, 4]. Several miRNAs significantly associated with BC were identified by bioinformatics methods [5–8]. miR-1258 has also been associated with the development of various cancers, including oral squamous cell carcinoma, liver cancer, gastric cancer, and so on [9–11]. Many investigations have shown that miR-1258 exhibits low expression in BC [12, 13]. Therefore, this study investigated the effects of miR-1258 overexpression on BC cells and predicted potential target genes of miR-1258, aiming to elucidate the mechanism by which miR-1258 regulates BC progression.

RNA-binding proteins (RPBs) regulate mRNA translation and stability by recognizing and binding to specific RNA sequences or structural elements [14]. La-Related Protein (LARP) is a typical RBP defined by the highly conserved structural domain La motif that binds RNA and promotes translation of mRNAs [15]. Research indicates that LAPR plays a pivotal role in cancer progression, whilst LARP4B promotes the advancement of hepatocellular carcinoma [16]. However, the role of LARP4B in BC development remained unclear.

In this study, we investigated the expression level of miR-1258 in BC cells and determined its biological effects on the proliferation, migration, and invasion of BC cells. In addition, by comprehensively analyzing information from miRNA target prediction databases, the immediate target LARP4B of miR-1258 and the relationship between the two in BC were explored. In conclusion, our study aimed to examine the regulatory mechanism of miR-1258/LARP4B and supply a speculative basis for future prognosis and restorative care in BC.

Methods and materials

Study subjects

This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Medical Ethics Committee of Weifang People’s Hospital. A total of 107 patients who attended the hospital between July 2017 and July 2019 were selected, with each participant signing an informed consent form. Inclusion criteria: (1) Female patients primarily diagnosed with breast cancer based on cytological or histopathological examination and imaging studies; (2) No history of radiotherapy or chemotherapy; (3) No severe complications. Exclusion criteria: Pregnant or lactating women. The follow-up period was from October 1, 2017, to October 31, 2024, with a median follow-up time of 36.2 months. Patient death is the terminal event.

Sample collection

Collect patient tissue samples obtained during surgery, including tumor and normal para-cancerous tissue that meet the requirements for testing. The collected tissues are ascertained and arranged by the two pathologists. The tissue is frozen in fluid nitrogen and kept at −80 °C after labeling.

Cell culture and transfection

Human-origin BC cell types (MCF-7, MDA-MB-231, HCC-1937 and MDA-MB-468) and mammary cells (MCF-10 A) were from ATCC. Cells were cultured in DMEM medium, maintained in DMEM medium, and incubated at 37 °C in a 5% CO₂ incubator.

miR-1258 mimic or miR-1258 inhibitor and miRNA negative control (miR NC), along with LARP4B small interfering RNA (si-LARP4B) and negative control (si-NC), were procured from RiboBio (Guangzhou, China). Transfection was performed according to the protocol of the Lipofectamine 3000 kit (Invitrogen, USA). Seed MDA-MB-231 cells at a density of 3.0 × 10⁵cells per well in a 6-well plate. Add miR-NC, miR-mimic (100 nM), si-LARP4B, and si-NC to the MDA-MB-231 cells. Thoroughly mix the culture medium and transfection system, then place them in the incubator for 6 h of transfection.

Real‑time quantitative PCR

According to the instructions, extract total RNA using the Fast Pure Viral DNA/RNA Mini Kit. Refer to the SYBR Green Realtime PCR Master Mix manual. Configure the system using GAPDH and U6 as internal reference genes. Place the system in the LightCycler^® 96 PCR instrument. After obtaining the data, analyze expression changes using the 2^−ΔΔCt method. The primer sequences used for qRT-PCR in this study are listed in Supplementary Table S1.

Cell proliferation

Cell multiplication was performed by using the Cell Counting Kit-8 (CCK-8; Dojindo, Japan) according to the manufacturer’s protocol. Absorbance at OD 450 was counted with an enzyme marker.

Migration and invasion assays

Detection of cell migration and invasion capabilities using the Transwell method. Inoculate a 5 × 10⁴ cell suspension into the upper chamber, fill the lower chamber with DMEM medium, and incubate at 37 °C for 48 h. After removing non-migrated cells, fix the sample, stain it with crystal violet, and then count the cells under a microscope. In the invasion assay, cells on the upper surface of the membrane were removed. The adherent cells on the lower surface were then fixed, stained, and counted under a microscope.

Dual luciferase reporter gene assay

Wild-type (WT) and mutant (MUT) 3’-UTRs of LARP4B containing the miR-1258 binding site were cloned into the pmirGLO vector (Promega, Madison, WI, USA). Lipofectamine 3000 (Invitrogen) was employed to co-transfect the vector with miR mimics and miR inhibitors into MDA-MB-231 cells. Fluorescence activity was measured at 48 h post-transfection using the DLR assay kit (Shanghai Bio-Tech Co., Ltd.).

Western blot

Collect MDA-MB-231 cells and add RIPA lysis buffer (Beyotime, Shanghai, China) to extract proteins. Total protein concentration was determined using the BCA assay kit (Beyotime, Shanghai, China). Mix 20 µg of total protein with 5× loading buffer and boil for 10 min. After 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis, the protein was transferred to a polyvinylidene fluoride membrane. Seal with 5% skim milk for 1 h; add primary antibody LARP4B (1:1000; ab197085, Abcam, UK) and GAPDH (1:5000; AG019, Beyotime, Shanghai, China) and incubate overnight at 4 ℃; After washing the TBST membrane, mouse secondary antibody (1:3000; ab205719, Abcam, UK) was added and incubated at room temperature for 1 h. ECL chemiluminescence staining (Bio Rad ChemiDoc XRS+) was performed, and the grayscale values of protein bands were quantitatively analyzed using ImageJ software. The relative protein levels of the target protein were evaluated using GAPDH as an internal control.

Public dataset collection

Potential target genes of miR-1258 were predicted using the public databases TargetScan, miRDB, and miRWalk. Furthermore, potential binding sites between miR-1258 and LARP4B were identified using the StarBase database.

Statistical analyses

All experiments were repeated at least three times to ensure reproducibility. Statistical analysis and visualization were performed using SPSS 26.0 and GraphPad Prism 8.0, with results presented as mean ± standard deviation (SD). The Shapiro-Wilk test is used to assess the normality of a data distribution. For data following a normal distribution, independent samples t-tests or one-way analysis of variance (ANOVA) were employed to compare differences between two or more groups, with Tukey’s post hoc test conducted for multiple comparisons. For non-normally distributed data, the Mann-Whitney U test or Kruskal-Wallis H test shall be employed. Use the chi-squared (χ²) test to compare categorical variables. Kaplan-Meier and Cox regression analysis for prognostic information. Pearson correlation analysis for correlation analysis. Data were considered statistically significant at P < 0.05.

Results

miR-1258 is poorly expressed in BC

miR-1258 expression was significantly lower in tumor tissues versus normal tissues (P < 0.001) (Fig. 1A). miR-1258 expression levels were also remarkably lower in BC cells relative to normal breast cells (P < 0.001) (Fig. 1B). During follow-up, 34 deaths occurred (incidence rate 31.8%), and 73 cases were lost to follow-up (loss rate 68.2%). The primary reason for loss to follow-up was survival at the end of the follow-up period (70 cases), with 3 cases lost due to non-endpoint events. The distribution of loss to follow-up was uniform. Patients were divided into a miR-1258 high-expression group and a miR-1258 low-expression group using the median miR-1258 expression level as the cut-off point. Low expression of miR-1258 is associated with poor prognosis and shorter overall survival in patients (Fig. 1C). Low expression of miR-1258 was significantly associated with TNM staging (P = 0.025) (Table 1). Univariate and multivariate Cox regression analyses demonstrated that miR-1258 (HR = 0.418, 95% CI = 0.186–0.941), along with TNM staging (HR = 2.199, 95% CI = 1.117–4.328), was an independent prognostic indicator (Table 2). In addition, the Schoenfeld residual test was used to test all variables included in the model. The results showed that the P-value of the Schoenfeld residual test for TNM staging was 0.405, and the P-values of other variables were all > 0.05, indicating that all variables met the proportional hazards hypothesis (Table S2).

Fig. 1.

miR-1258 is poorly expressed in BC. Expression of miR-1258 in tumor tissues (A) and BC cells (B). C Overall survival based on miR-1258 expression. ^***P < 0.001

Table 1.

The association of miR-1258 with patients’ clinicopathological features

Variant	Cases (n = 107)	miR-1258			P
		Low (n = 53)		High (n = 54)
Age					0.625
< 55	51	24	27
≥ 55	56	29	27
Tumor size					0.638
< 2	55	27	28
≥ 2	52	26	26
Histological grade					0.387
I-II	57	26	31
III	50	27	23
TNM stage					0.025
I-II	55	20	35
III	52	33	19
Menopausal					0.919
Pre	50	25	25
post	57	28	29

TNM: Tumor node metastasis

Table 2.

Univariate and multivariable Cox regression analysis of overall survival

Variables	Total sample size	Number of events	Univariate analysis					Multivariate analysis
			B	SE	P	HR	95% CI	B	SE	P	HR	95% CI
Age	107	34	0.330	0.342	0.336	1.390	0.711–2.719	——	——	——	——	——
Tumor size	107	34	0.221	0.334	0.509	1.247	0.648–2.399	——	——	——	——	——
Histological grade	107	34	0.082	0.334	0.805	1.086	0.565–2.087	——	——	——	——	——
TNM stage	107	34	0.993	0.336	0.003	2.669	1.398–5.214	0.788	0.345	0.023	2.199	1.117–4.328
Menopausal	107	34	0.136	0.335	0.686	1.145	0.593–2.210	——	——	——	——	——
miR-1258	107	34	−1.086	0.402	0.008	0.344	0.156–0.756	−0.871	0.414	0.035	0.418	0.186–0.941

TNM: Tumor node metastasis, I-II = 0, III = 1; HR: Hazard Ratio; CI: Confidence interval

Overexpression of miR-1258 inhibits proliferation, migration and invasion of BC cells

In the BC cell line, MDA-MB-231 had the lowest expression level and was selected for in vitro experiments. Compared with the control group, miR-1258 mimics resulted in a significant increase in miR-1258 expression in BC cells (Fig. 2A). miR-1258 mimics significantly inhibited cellular proliferation, migration, and invasive capacity (Fig. 2B–D).

Fig. 2.

Overexpression of miR-1258 inhibits BC cell proliferation, migration and invasion. A miR-1258 expression. B Cell viability. C Cell migration. D Cell invasion. ^***P < 0.001; ^**P < 0.01

LARP4B is overexpressed in BC

Using the TargetScan, miRDB, and miRWalk databases to predict miR-1258 downstream targets, a total of 180 common target genes were identified, revealing that LARP4B may be one of the potential target genes of miR-1258 (Fig. 3A). The expression of LARP4B was significantly higher in tumor tissues analogized to normal tissues (P < 0.001) (Fig. 3B); the level of LARP4B expression was also significantly increased in BC cells relative to normal breast cells (P < 0.001) (Fig. 3C). Patients were categorized into high and low expression groups using the median LARP4B expression level as the cut-off point. High LARP4B observation predicted a poor prognosis and shorter overall survival of patients (Fig. 3D). The high expression of LARP4B was significantly correlated with TNM staging (P = 0.035) (Table 3).

Fig. 3.

LARP4B is overexpressed in BC. A miR-1258 target gene prediction. B Expression of LARP4B in tumor tissues. C Expression of LARP4B in BC cells. D Overall survival based on LARP4B expression. ^***P < 0.001; ^**P < 0.01

Table 3.

The association of LARP4B with patients’ clinicopathological features

Variant	Cases (n = 107)	LARP4B		P
		Low (n = 53)	High (n = 54)
Age				0.775
< 55	51	26	25
≥ 55	56	27	29
Tumor size				0.497
< 2	55	29	26
≥ 2	52	24	28
Histological grade				0.381
I-II	56	30	26
III	51	23	28
TNM stage				0.035
I-II	52	18	34
III	55	35	20
Menopausal				0.490
Pre	48	22	26
post	59	31	28

TNM: Tumor node metastasis

Silencing of LARP4B inhibits BC cell proliferation, migration and invasion

Silencing of LARP4B resulted in a marked decrease in LARP4B expression in BC cells versus controls (Fig. 4A, Fig.S1). Knockdown of LARP4B caused a significant reduction in the proliferative capacity of BC cells (Fig. 4B), which significantly inhibited the migration (Fig. 4C) and invasion (Fig. 4D) of cancer cells.

Fig. 4.

Silencing LARP4B inhibits BC cell proliferation, migration and invasion. A LARP4B expression in BC cells. B Cell viability. C Cell migration. D Cell invasion. ^***P < 0.001; ^**P < 0.01

miR-1258 has a targeting relationship with LARP4B

We identified a potential binding site between miR-1258 and LARP4B using publicly available databases (Fig. 5A). Dual luciferase reporter assay results demonstrated that the miR-1258 mimic significantly inhibited LARP4B wild-type luciferase activity, whereas the miR-1258 inhibitor markedly enhanced luciferase activity (Fig. 5B). Western blot results showed that miR-1258 mimetics significantly inhibited LARP4B protein expression, while miR-1258 inhibitors significantly promoted LARP4B protein expression (Fig. S2). Pearson correlation analysis based on 107 clinical samples in this study showed a significant negative correlation between miR-1258 and LARP4B (Fig. 5C, r = − 0.75, P < 0.001). Moreover, after controlling for other confounding factors (Age, Tumor size, Histological grade, Menopausal and TNM stage), miR-1258 and LARP4B remained significantly negatively correlated (partial correlation coefficient r = − 0.460, P < 0.001). Further confirmed the targeting relationship between miR-1258 and LARP4B.

Fig. 5.

miR-1258 has a targeting relationship with LARP4B. A Binding site of miR-1258 with LARP4B. B Dual luciferase reporter assay. C Pearson correlation analysis. ^***P < 0.001

The interaction between miR-1258 and LARP4B

Based on the target relationship between miR-1258 and LARP4B, miR-1258 mimics were co-transfected with LARP4B vectors into MDA-MB-231 cells to further investigate the interaction between miR-1258 and LARP4B in breast cancer. Results demonstrated that the miR-1258 analogue significantly suppressed LARP4B expression, whereas co-transfection with LARP4B markedly increased its expression (Fig. 6A). Furthermore, the miR-1258 analogue significantly inhibited the proliferation, migration, and invasion of breast cancer cells, whereas co-transfection with LARP4B markedly reversed this inhibitory effect (Fig. 6B–D).

Fig. 6.

miR-1258 regulates BC cell processes through LARP4B. A LARP4B expression in BC cells. B Cell viability. C Cell migration D Cell invasion. ^***P < 0.001; ^**P < 0.01

Discussion

Breast cancer is the most commonly diagnosed cancer worldwide, with high incidence and mortality rates. However, its pathogenesis remains poorly understood, posing a serious threat to patients’ quality of life and survival [17].

Numerous studies have demonstrated that microRNAs play a crucial role in cancer progression by directly targeting messenger RNAs [18, 19]. has-miR-557 is abnormally expressed in osteosarcoma and inhibits tumor growth by regulating KRAS [20]. miR-26a/miR-26b exhibits downregulated expression in squamous cell carcinoma of the tongue and suppresses its progression by targeting PAK1 [21]. miR-1258 is involved in multiple cancer progression. For example, miR-1258 inhibits cancer cell proliferation and induces senescence and apoptosis through the GRB2/Ras/Erk pathway in non-small cell lung cancer [22]. In osteosarcoma, miR-1258 interferes with cell proliferation by targeting AKT3 [23]. Furthermore, it has been reported that miR-1258 is also involved in BC and inhibits BC metastasis by regulating the expression of heparinase [8]. miR-1258 inhibits breast cancer progression by targeting E2F1 to regulate cell proliferation and the cell cycle [9]. In this study, miR-1258 exhibited downregulated expression in breast cancer (BC), with patients displaying its low expression demonstrating shorter overall survival and indicating poor prognosis. Furthermore, overexpression of miR-1258 inhibited the proliferation, migration, and invasion of BC cells, consistent with findings from previous studies [12, 24, 25]. These findings indicate that miR-1258 is an independent prognostic factor in breast cancer, and overexpression of miR-1258 may improve patient prognosis.

As a member of the LARP family, LARP4B plays a significant role in cancer progression. For instance, LARP4B promotes hepatocellular carcinoma progression by activating the SPINK1-mediated EGFR pathway [26]. LARP4B promotes the growth and metastasis of sarcomas and cancers [27]. Knocking down LARP4B induced a reduction in leukemia stem cells, impaired their self-renewal capacity, and prolonged the lifespan of MLL-AF9 acute myeloid leukemia recipient mice [28]. However, the role of LARP4B in breast cancer remains unexplored. Therefore, this study employed relevant experiments to investigate and confirm that LARP4B is a potential target gene of miR-1258. LARP4B is highly expressed in pancreatic cancer in mice, and its overexpression promotes the proliferation and invasion of pancreatic cancer cells, consistent with the findings of this study [29]. Our research has identified that LARP4B is overexpressed in breast cancer (BC). Silencing LARP4B inhibits the proliferation, migration, and invasion of BC cells. Furthermore, LARP4B exhibits a negative correlation with miR-1258 expression; co-transfection of LARP4B attenuates the inhibitory effect of miR-1258 mimics on BC cells. This finding is consistent with the results of Yin et al., who demonstrated that miR-106b suppresses the viability and metastasis of prostate cancer cells by targeting LARP4B [30]. These findings suggest that miR-1258 may play a role in regulating breast cancer progression by targeting LARP4B.

However, this study also has certain limitations. Firstly, there is a lack of in vivo experiments and diverse sample data. Secondly, there is a lack of in-depth investigation into the interactions between miR-1258 downstream target genes and related signaling pathways. Future research should conduct additional in vivo model experiments to thoroughly investigate the interactions between the miR-1258/LARP4B axis and other signaling pathways. This approach will provide a more comprehensive theoretical foundation and identify more effective therapeutic targets for the treatment and prognosis of breast cancer.

Conclusion

Low expression of miR-1258 may indicate poor prognosis in breast cancer, whilst its overexpression significantly inhibits proliferation, migration and invasion of breast cancer cells. miR-1258 may suppress breast cancer progression by targeting LARP4B.

Author contributions

Jianping Lai and Liang An did conceptualization, data curation, formal analysis, validation, writing—review & editing. Yongqing Zhang did conceptualization, methodology, investigation, and writing-original draft. Xinbo Wang did conceptualization, methodology, supervision, and writing-review.

Funding

No funding was received for conducting this study.

Data availability

The data used to support the findings of this study are available from the corresponding author upon request.

Declarations

Ethics approval and consent to participate

This study was conducted in accordance with the principles of the Declaration of Helsinki and approved by the Medical Ethics Committee of Weifang People’s Hospital (Approval number: 2024XX-012, Date: 2024-07-28). Each participant signed an informed consent form.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.