1 Introduction

Renal cell carcinoma (RCC) ranks as the third most prevalent urinary tract neoplasm, constituting about 90% of renal malignancies and 4% of all malignant tumours in the adult population [1]. Clear cell RCC (ccRCC), the most common and aggressive RCC subtype, represents 70–75% of RCC cases [2]. Owing to its elusive early clinical manifestations, more than 30% of ccRCC patients manifest metastases at their initial diagnosis, thereby forgoing surgical options. Additionally, roughly 40% of patients who underwent primary ccRCC resection still experienced cancer recurrence and metastasis, resulting in an overall 5-year survival rate less than 10% [3, 4]. Consequently, exploring the molecular underpinnings of ccRCC development and identifying potential targets for ccRCC assume paramount importance in enhancing ccRCC treatment.

Micro RNAs (miRNAs) are a class of small non-coding RNAs, consisting of approximately 22 nucleotides, that are indispensable factors in the regulation of tumorigenesis and metastasis [5]. miRNAs function as either tumour suppressors or oncomiRNAs by binding to the 3′-untranslated region (3′-UTR) of target genes, thereby downregulating the transcription of associated genes [6]. Extensive reports indicate that miR-320 family members, including miR-320a, miR-320b, miR-320c, miR-320d, and miR-4429 are downregulated in various malignancies and have been linked to tumour aggressiveness and an unfavourable prognosis [7,8,9,10,2.9 Cell migration and invasion assays

We employed Transwell chambers (Corning Costar Corp., Cambridge, MA, USA) with 8 μm pores, placed in 24-well culture plates. For the invasion assay, matrix gel (BD Bioscience, USA) stored at − 20 °C was allowed to dissolve overnight at 4 °C. It was subsequently diluted in a 1:8 ratio with FBS-free cell-affiliated medium, and 100 µL of the gel mixture was used to coat the upper chamber evenly, followed by an incubation period at 37 °C for 2 h. 786-O and Caki-2 cells, transfected for 48 h, were diluted with RPMI 1640 and McCoy's 5A medium without FBS, to a concentration of 5 × 105 cells per 200 µL, and seeded into the upper chamber. The lower chamber was supplemented with RPMI 1640 and McCoy's 5A medium containing 10% FBS. After 48 h of incubation, cells were fixed with 4% PFA for 15 min at room temperature and then stained with 1% crystal violet for 20 min. Subsequently, five different field of view statistics were taken at random.

2.10 Western blot

To prepare cell lysates, cells were collected at 70–80% confluence 48 h post-transfection. Protein concentrations were determined, and a loading buffer was prepared according to the Solabank BCA Protein Concentration Assay Kit (Bei**g, China). The prepared loading buffer was applied to individual lanes on a 4–15% gradient polyacrylamide gel (Mini-PROTEAN TGX™, Bio-Rad). Blotting was executed using a gel transfer system (Invitrogen) to transfer proteins onto PVDF membranes, which were then immersed in TBST (TBS containing 5% skim milk powder and 0.1% Tween) for 2 h. Following this, the blotted membranes were incubated with primary antibodies at a concentration of 1:1000 overnight at 4 °C. After washing the membrane three times, it was incubated with rabbit-specific secondary antibody (1:3000) for 2 h at room temperature. To develop the blotted membrane, the membrane was washed thrice with the ELC kit (New Cell & Molecular Biotech Co., Ltd. SuZhou, China), and the grayscale values of the target bands were assessed utilising Image J. GAPDH expression level was employed to normalise the target proteins. All antibodies were sourced from Abcam, including CD274 (ab205921), PI3K (ab302958), p-PI3K (#4228), p-AKT (ab8805), AKT (ab8805), and GAPDH (ab8245).

2.11 Dual-luciferase reporter assay

We amplified the 3′-UTR of CD274, which carried the putative binding site for miR-4429, and then cloned it into the pmirGLO vector. To eliminate complementarity with miR-4429, the miR-4429 complementary site within the CD274 3′-UTR, with the sequence 5′-CAGCUUU-3′, was individually mutated. 293T cells were seeded into 96-well plates at a density of 4000 cells per well and transfected with luciferase reporter vectors containing either the wild-type (WT) or mutated (MUT) CD274 3′-UTR (constructed by Ribobio), along with miR-4429 mimics or a negative control (NC). After 48 h of transfection, cells were harvested, and the Dual-Luciferase® Reporter Assay System was utilised to measure luciferase activity. Normalised luciferase activity was determined as the ratio of luciferase activity to Renilla luciferase activity.

2.12 Immunohistochemical staining

For immunohistochemistry, the hydrated sections were treated with an antigen repair solution (Shanghai Shumba Biotech; Shanghai, China). Subsequently, the slides were soaked in 3% H2O2 for 20 min and then incubated with Primary antibody (CD274, 1:150) was incubated at 4 °C for 12 h. The following day, the slides were rinsed and incubated with rabbit secondary antibody at 15 °C for 90 min and then transferred to 37 °C for 30 min. Separate staining with 3,3′-diaminobenzidine (DAB) and hematoxylin was conducted. The slides were then assessed and captured using an Olympus BX53 fluorescence microscope (Tokyo, Japan).

2.13 Statistical analysis

We conducted statistical analyses utilising SPSS 19.0 (IBM SPSS, Chicago, IL) and GraphPad Prism 6.0 (IBM, Armonk, NY, USA). Paired and unpaired samples were analyzed for statistical significance using t-tests. For samples with more than two groups, analysis of variance (ANOVA) was used. Correlations between gene expression levels were analyzed using Spearman's correlation analysis. All experiments were repeated at least three times. Statistical significance was set at P values < 0.05.

3 Results

3.1 MiR-4429 is significantly down-regulated in ccRCC tissues

To investigate the potential association of miR-320 family members with the progression of ccRCC, we conducted a comprehensive analysis of their expression patterns utilising the TCGA database and bioinformatics algorithms. The analysis was based on three dimensions, including differential expression, prognosis, and metastasis (relative to normal tissue) in ccRCC. In terms of prognostic stratification, we segregated patients into two groups: those with less than one year of survival and those with over five years of survival. In the context of metastasis, ccRCC patients were categorised into metastatic (M1) and non-metastatic (M0) groups.

Differential expression analysis revealed up-regulation of miR-320 family members, including miR-320a, miR-320b, miR-320c, and miR-320d, as depicted in Fig. 1a. However, when considering prognosis (Fig. 1b), there was no significant differences. In contrast, miR-320b exhibited significant variation in the context of the metastatic group, as shown in Fig. 1c.

Fig. 1
figure 1

MiR-4429 was significantly downregulated in ccRCC tissues a–c MiR-320a, miR-320b, miR-320c, and miR-320d expression were up-regulated in the a differentially expressed group compared to normal tissues. miR-320 family members were not significantly different in the b prognosis group, and only miR-320b was significantly different in the c metastasis group, d The expression levels of miR-320 family members were detected in the tissues of 30 ccRCC patients, and miR-4429 expression was significantly down-regulated. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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To further validate the expression patterns of miR-320 family members in ccRCC patients, we conducted RT-qPCR to assess their levels in 30 ccRCC tumour samples and compared with control tissues. The outcomes revealed a noteworthy down-regulation of miR-4429 in ccRCC tissues. However, the expression levels of miR-320a, miR-320b, miR-320c, and miR-320d did not exhibit statistically significant differences, as illustrated in Fig. 1d. This finding suggests that miR-4429, a member of the miR-320 family, may serve as an independent biomarker in the context of ccRCC.

3.2 Overexpression of miR-4429 inhibits proliferation, migration, and invasion of 786-O and Caki-2 cells in vitro

In order to elucidate the involvement of miR-320 family members in the proliferation and metastasis of ccRCC, we employed miR-4429, a representative of this family, and utilised RT-qPCR to assess its expression levels in various cell lines including HK-2, ACHN, 786-O, Caki-1, and Caki-2. Subsequently, we transfected 786-O and Caki-2 cells with mimic, inhibitor, and negative control to gauge transfection efficiency. The biological function of miR-4429 was rigorously examined through a battery of assays, including cell viability assay performed with CCK-8, clone formation assay, wound healing assay, and Transwell assay. Predictive target analysis of miR-4429 was conducted using miRDB and TargetScanv7.1, and all putative targets underwent functional enrichment analysis employing the KEGG database.

Our findings unveil a substantial downregulation of miR-4429 in 786-O and Caki-2 cell lines (Fig. 2a). Furthermore, the expression level of miR-4429 experienced a significant upregulation following transfection with miR-4429-mimic (Fig. 2b). Importantly, this overexpression of miR-4429 exerted a notable inhibitory effect on cell proliferation (Fig. 2c, d), migration, and invasion (Fig. 2e–g). These compelling results posit miR-4429 as a promising candidate in the role of a tumour suppressor in ccRCC.

Fig. 2
figure 2

MiR-4429 overexpression inhibited proliferation, migration, and invasion of 786-O and Caki-2 cells in vitro a miR-4429 mRNA levels were significantly down-regulated in renal cancer cell lines compared to HK-2 in renal tubular epithelial cells. b Transfection of miR-4429-mimic in 786-O and Caki-2 cells significantly up-regulated miR-4429 mRNA levels. c–f Overexpression of miR-4429 significantly inhibited cell proliferation rate (c), clone formation (d), wound healing (e), migration (f), and invasion (g). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

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3.3 CD274, a potential target of miR-4429, has elevated expression in ccRCC and positively correlates with histologic grading

As widely acknowledged, immune cell infiltration within the tumor microenvironment (TME) can profoundly influence the effectiveness of immunotherapy and the subsequent prognosis of ccRCC patients. Extensive research has demonstrated that CD274 (PD-L1) is prominently overexpressed on the surface of malignant tumour cells [12, 29]. In our research, we definitively identified CD274 as a direct target of miR-4429 and established a negative correlation between miR-4429 and CD274 mRNA expression levels. Our in vitro experiments demonstrated that miR-4429 overexpression significantly curtails the proliferation, migration, and invasion of ccRCC cells. Additionally, our analysis of the UALCAN database and clinical specimens unveiled the upregulation of CD274 in ccRCC and its positive correlation with the Fuhrman grade and the infiltration of PD-1, Immune infiltrating cells. Hence, the combinatory evaluation of miR-4429 and CD274 bears great potential for assessing the clinical prognosis of ccRCC patients, suggesting a plausible regulatory mechanism for CF274 as an oncogene in ccRCC.

While multiple studies have illuminated the association between abnormal microRNA expression and various intrinsic tumour processes, showcasing the substantial impact of microRNA up- or down-regulation in suppressing tumour cell proliferation, migration, in vitro invasiveness, and subcutaneous tumour growth, the clinical application of miRNA regulation and its targeted therapies remains constrained by limitations. Significantly, ccRCC as an immune-responsive tumour, is likely intertwined with immune system orchestration throughout its development and progression. Hence, modulating the immune response emerges as a promising avenue for ccRCC therapy. Consequently, delving into the potential of the miR-4429/CD274 axis as a target for immunomodulation and immunotherapy in ccRCC is merited. However, further investigations are warranted to ascertain whether CD274 fosters ccRCC progression by sustaining the activation of the PI3K/AKT signalling pathway.

In summary, our study has elucidated that miR-4429 overexpression yields a significant impediment to ccRCC progress, chiefly mediated through the PI3K/AKT signalling pathway. We have pinpointed CD274 as a direct and functional target of miR-4429, with its expression levels correlating with higher histological grading in ccRCC. Therefore, the newfound miR-4429/CD274 axis holds promise as a therapeutic target and prognostic biomarker for ccRCC patients.