miR-27b-3p directly targets PPARG to regulate pancreatic cancer progression

Abstract

Background

miR-27b-3p is dysregulated in many cancers, but its expression, clinical role, and mechanism in pancreatic cancer are not fully understood.

Objective

This study aimed to evaluate the clinical value of miR-27b-3p in PC and its role in pancreatic cancer (PC) progression by targeting PPARG.

Method

qRT-PCR was used to measure miR-27b-3p and PPARG levels in 106 paired PC tissues and adjacent non-tumor tissues, and in PC cell lines (PANC-1, SW1990, BxPC-3, AsPC-1) and HPDE cells. Kaplan-Meier and multivariate Cox analyses assessed miR-27b-3p prognostic value. CCK-8 and Transwell assays evaluated the impact of miR-27b-3p inhibition on PC cell function. Dual-luciferase reporter and Pearson correlation analyses confirmed the targeting of PPARG by miR-27b-3p. A PPARG knockdown rescue experiment verified that miR-27b-3p function depends on PPARG.

Results

miR-27b-3p was highly expressed in PC tissues and cell lines and linked to lymph node metastasis, distant metastasis, elevated CA19-9 levels, and poor overall survival. Multivariate Cox analysis showed high miR-27b-3p expression as an independent predictor of poor prognosis. In vitro, miR-27b-3p inhibition suppressed PANC-1 cell proliferation, migration, and invasion. PPARG was a direct target of miR-27b-3p and was downregulated at both mRNA and protein levels. A negative correlation was found between miR-27b-3p and PPARG mRNA in PC tissues. PPARG knockdown reversed the effects of miR-27b-3p inhibition, confirming functional dependence.

Conclusion

miR-27b-3p functions as an oncogenic miRNA in PC by targeting PPARG and may be a prognostic biomarker and therapeutic target.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12672-026-04509-z.

Introduction

Pancreatic cancer (PC) is a highly aggressive malignant tumor characterized by a poor prognosis and persistently high mortality, with a 5-year survival rate of less than 10%, posing a significant challenge to global public health [1, 2]. In clinical practice, early detection is crucial for improving patient prognosis [3]. However, timely detection remains a major challenge. Currently, imaging examinations are the primary tools for early diagnosis, but are limited by low sensitivity and often failing to detect subtle early-stage lesions [4]. Furthermore, the early symptoms of PC are typically nonspecific and may mimic common gastrointestinal disorders, frequently resulting in misdiagnosis or delayed diagnosis [5]. As a consequence, most patients are diagnosed with PC at an advanced stage, during which therapeutic choices are restricted and clinical outcomes remain unsatisfactory [6]. Therefore, the identification and development of novel biomarkers hold great promise for improving early diagnosis and prognostic assessment of PC and represent an urgent priority in current research.

Dysregulated miRNA expression regulation often leads to the aberrant activation of target oncogenes or the suppression of tumor suppressor genes, thereby promoting the development and progression of cancer [7]. In PC, miRNAs contribute to disease progression via multiple mechanisms [8–10]. miR-27b-3p exerts regulatory effects in various types of cancer, including colorectal cancer, prostate cancer, and gastric cancer [11–13]. Although previous studies have demonstrated that miR-27b-3p is markedly upregulated in both plasma and tissue specimens from patients with PC [14], its exact biological functions and the molecular mechanisms underlying PC progression remain incompletely understood.

Peroxisome proliferator-activated receptor gamma (PPARG) is a key member of the nuclear receptor superfamily that regulates gene expression by binding to specific DNA response elements [15]. Accumulating evidence indicates that PPARG participates in the development and progression of various tumor types, such as breast cancer and prostate cancer [16, 17]. Recent studies have shown that PPARG, as a central regulator in the apoptotic pathway, promotes apoptosis of pancreatic cancer cells and suppresses their proliferation [18]. Bioinformatic analysis using TargetScan 7.2 predicts potential binding sites between miR-27b-3p and PPARG. Given that miR-27b-3p is consistently upregulated in pancreatic cancer and PPARG has been established as a tumor suppressor in this disease, their functional interaction is biologically plausible and warrants further investigation. However, the interaction mechanism between PPARG and miRNAs in pancreatic cancer remains poorly understood, and the functional role and clinical significance of the miR-27b-3p–PPARG axis have not yet been reported. Given the above background, this study aims to address the research gap regarding the regulatory mechanism and clinical value of the miR-27b-3p/PPARG axis in pancreatic cancer. By integrating clinical sample validation, in vitro functional assays, and target confirmation, we provide a novel foundation for prognostic assessment and targeted therapy in pancreatic cancer.

Patients and methods

Gathering of clinical specimens

A total of 106 consecutive patients with pathologically confirmed pancreatic ductal adenocarcinoma (PDAC) at Shanghai Baoshan Luodian Hospital between January 2018 and May 2024 were included in this research.Patients in this study were classified into the PC group for analysis.

Inclusion criteria: (1) pathologically confirmed diagnosis of PDAC; (2) underwent radical surgical resection; (3) no prior anti-tumor treatments, including chemotherapy, radiotherapy, or targeted therapy, before surgery; (4) availability of complete clinical and pathological data; (5) postoperative follow-up duration of at least 6 months.

Exclusion criteria: (1) concomitant presence of other malignant tumors; (2) severe hepatic or renal dysfunction, coagulation disorders, or other surgical contraindications; (3) loss to follow-up or withdrawal from the study during the observation period.

Intraoperative tissue collection focused on two types of samples: cancerous and adjacent non-cancerous tissues, with the latter harvested at a distance of no less than 3 cm from the tumor edge to ensure they were free from tumor cell infiltration.

Comprehensive clinical and pathological information was collected in an organized manner for each patient, covering variables such as age, sex, tumor stage based on the TNM classification system, lymph node metastasis status, occurrence of distant metastasis, and CA19-9.

Cell culture and transfection

Human PC cell lines PANC-1, MIA PaCa-2, BxPC-3, AsPC-1, and normal pancreatic ductal epithelial cells HPDE were acquired from the National Center for the Preservation of Certified Cell Cultures. Cells were cultured in DMEM medium (12491015, Thermo Fisher) supplemented with 10% fetal bovine serum (FBS; A5256701, Gibco), 100 U/mL penicillin, and 100 µg/mL streptomycin (15140122, Gibco), and maintained in a humidified incubator at 37 °C with 5% CO₂. When cell confluence reached 80%–90%, cells were detached using 0.25% trypsin (25200072, Gibco) for subculturing. Twenty-four hours prior to transfection, cells were seeded into 6-well or 96-well plates, and transfection was carried out when confluence reached 50%–60%.miR-27b-3p mimics, miR-27b-3p inhibitors, inhibitor-NC and mimic-NC were produced by RiboBio. The transfection of these constructs into target cells was performed utilizing Lipofectamine™ 3000 transfection reagent (L3000150, Thermo Fisher) in strict accordance with the protocols provided by the manufacturer.

qRT-PCR

RNA isolation: Tissue and cell samples were used for the isolation of total RNA with TRIzol reagent (R0016, Beyotime), in line with the manufacturer’s recommended protocols. After RNA extraction, the A260/A280 ratio was measured using NanoDrop 2000 (range: 1.8–2.0) to ensure that RNA quality met the experimental requirements.

Reverse transcription reactions: For mRNA reverse transcription, GeneCreate RT OneStep Mix for qRCR with dsDNase (JKR23014, GENE CREATE) was used. For miRNA reverse transcription, the Hifair^® miRNA 1st Strand cDNA Synthesis Kit (11148ES50, Yeasen) was utilized. Both reverse transcription processes were conducted following the specific reaction systems and thermal cycling parameters provided by their respective manufacturers.

qPCR: The level of miR-27b-3p was determined using miRNA Unimodal SYBR qPCR Master Mix (MQ102, Vazyme), with U6 as the internal control. For analyzing PPARG mRNA expression, ChamQ Universal SYBR qPCR Master Mix (Q711, Vazyme) was applied, and GAPDH served as the internal reference.

Cell growth assay

Transfected PANC-1 cells were plated into 96-well plates. At 24,48, and 72 h post-seeding, 10 µL of CCK-8 solution was dispensed into each well. Subsequently, the plates were incubated at 37 °C for another 2 h; following this incubation period, the OD at a 450 nm wavelength was detected using a microplate reader. The proliferative capacity of the cells was assessed according to the variations in OD values over the three time points, and each experiment was conducted independently in triplicate.

Transwell-based migration and invasion assays

For the migration assay, 5 × 10⁴ cells in serum-free medium were added to the upper chamber of a Transwell insert, while the lower chamber contained 10% FBS as a chemoattractant. Following 24 h of incubation, migrated cells on the membrane were fixed, stained with crystal violet, and counted using an optical microscope.

For the invasion assay, the upper chamber of the transwell insert was pre-coated with Matrigel matrix gel (BD Biosciences) that had been diluted at a 1:8 ratio. The remaining procedures were identical to those used in the migration assay. Cell invasion capacity was assessed after 48 h of incubation. Each experiment was independently replicated three times.

Dual-luciferase reporter assay

Potential binding sites between miR-27b-3p and PPARG were predicted using the TargetScan 7.2. Luciferase reporter vectors containing either the wild-type (WT) or mutant (MUT) 3’ untranslated region (3’UTR) of the PPARG gene were constructed. These vectors were then co-transfected alongside miR-27b-3p mimics into PANC-1 cells. Forty-eight hours after transfection, luciferase activity was assessed to experimentally confirm the regulatory interaction between miR-27b-3p and PPARG. Each experiment was carried out independently in triplicate to ensure reproducibility.

Analysis of statistical data

Statistical analyses and graphical visualization were conducted using GraphPad Prism 9.0 software. Quantitative data are expressed as mean ± standard deviation (x̄ ± SD). Comparisons between two independent groups were performed using independent sample t-tests, while one-way or two-way ANOVA with Tukey’s post hoc test was applied for comparisons across multiple groups. Categorical variables were analyzed using chi-square tests. Survival curves were generated using the Kaplan-Meier method, and group differences were assessed via the log-rank test. Pearson’s correlation coefficient was used to evaluate correlations between continuous variables. All statistical tests were two-sided, and a P value less than 0.05 was considered statistically significant.

Results

miR-27b-3p expression in PC tissues and its correlation with clinicopathological features

To characterize the expression pattern of miR-27b-3p in PC, we measured its levels in 106 paired PC tissues and adjacent non-tumor tissues using qRT-PCR. The results revealed that miR-27b-3p expression was significantly upregulated in PC tissues compared to adjacent non-tumor tissues (P < 0.001, Fig. 1A). Subsequently, patients were stratified into high-expression and low-expression groups according to the median miR-27b-3p expression level. The comparative analysis of the clinical and pathological features (Table 1) shows that there are significant differences between the two groups in LNM, distant metastasis, and serum CA19-9 levels (P < 0.001). On the other hand, factors such as age, gender, and tumor stage appear to be similarly distributed between the groups, with no statistically significant differences observed (P > 0.05).

Fig. 1.

PC exhibits overexpression of miR-27b-3p, which correlates with poor patient prognosis. A Comparison of miR-27b-3p between cancerous and adjacent non-cancerous tissues, ***P < 0.001; B Relationship between miR-27b-3p and prognosis in PC patients; C High miR-27b-3p expression functions as an independent adverse prognostic factor for PC patients

Table 1.

Association of miR-27b-3p expression with clinicopathologic characteristics of pancreatic cancer

Parameters	Low-expression group	High-expression group	χ²	P-value
Age (years)
≤ 60	34	26	0.47	0.680
> 60	23	23
Gender
Male	32	25	0.28	0.600
Female	25	24
T stage
I-II	30	23	0.34	0.560
III-IV	27	26
Distance metastasis
No	52	19	32.78	< 0.001
Yes	5	30
Lymph node metastasis
No	48	15	31.40	< 0.001
Yes	9	34
CA19-9(U/ml)
< 37	40	12	22.01	< 0.001
≥ 37	17	37

Prognostic value of miR-27b-3p in PC patients

Kaplan-Meier survival analysis demonstrated that PC patients with high miR-27b-3p expression had significantly poorer prognoses (P < 0.001; Fig. 1B).

Multivariate Cox regression analysis further confirmed that elevated miR-27b-3p expression is an independent adverse prognostic factor in pancreatic cancer (HR = 3.695, 95% CI: 1.124–12.156, P = 0.03, Fig. 1C). Notably, the prognostic value of miR-27b-3p was comparable to that of established clinical markers, including distant metastasis (HR = 2.681, P = 0.04), lymph node metastasis (HR = 2.237, P = 0.03), and elevated CA19-9 levels (HR = 5.395, P = 0.04, Fig. 1C).

miR-27b-3p expression in PC cell lines and transfection efficiency validation

Next, miR-27b-3p expression levels were examined in human pancreatic ductal epithelial (HPDE) cells and four pancreatic cancer cell lines (PANC-1, SW1990, BxPC-3, AsPC-1). miR-27b-3p expression was significantly elevated in all pancreatic cancer cell lines compared to HPDE cells, with the highest expression observed in PANC-1 cells (P < 0.001, Fig. 2A). Therefore, PANC-1 cells were selected as the representative in vitro model for subsequent functional studies.

Fig. 2.

miR-27b-3p expression profiles in PC cell lines and validation of inhibitor transfection efficiency. A Relative miR-27b-3p expression in cells, *P < 0.05, ***P < 0.001; B Changes in miR-27b-3p expression after transfection with the miR-27b-3p inhibitor or inhibitor NC, inhibitor NC vs. miR-27b-3p inhibitor, ##P < 0.01

To modulate miR-27b-3p expression, PANC-1 cells were transfected with miR-27b-3p inhibitor or inhibitor NC. qRT-PCR analysis confirmed efficient knockdown, as miR-27b-3p levels in the inhibitor group were significantly lower than those in the NC group (P < 0.001, Fig. 2B).

Effect of miR-27b-3p inhibition on PC cell proliferation, migration, and invasion

After 72 h of culture, the OD value in the miR-27b-3p inhibitor group was significantly lower than that in the NC group (P < 0.01, Fig. 3A), indicating that inhibition of miR-27b-3p suppresses PC cell proliferation.

Fig. 3.

Effects of miR-27b-3p inhibition on the functional phenotypes of PC cells. A CCK-8 was employed to assess cell proliferation capacity, **P < 0.01; B The Transwell migration was utilized to assess migratory capacity, ***P < 0.001; C The Transwell migration was employed to evaluate invasive capacity, ***P < 0.001

Compared with the NC group, transfection with the miR-27b-3p inhibitor led to a significant reduction in both migration (P < 0.001, Fig. 3B) and invasion (P < 0.001, Fig. 3C) of PANC-1 cells.

miR-27b-3p directly targets PPARG and regulates its expression in PC cells

To elucidate the molecular mechanism of miR-27b-3p in PC, potential target genes were predicted using TargetScan 7.2. The analysis identified a conserved 8-nucleotide binding site (positions 82–89) in the 3′UTR of PPARG with a high Context + + score (percentile 99), indicating a strong likelihood of functional interaction (Fig. 4A).

Fig. 4.

Validation of PPARG as a direct target gene of miR-27b-3p. A Potential miR-27b-3p binding sites in PPARG predicted by TargetScan; B Dual-luciferase reporter gene assay results verifying the targeting interaction between miR-27b-3p and PPARG; comparisons include inhibitor NC vs. miR-27b-3p inhibitor (***P < 0.001) and mimic NC vs. miR-27b-3p mimic (##P < 0.01); C Pearson illustrating the relationship between miR-27b-3p and PPARG in PC tissue samples; P < 0.001; D qRT-PCR indicated that miR-27b-3p inhibitor significantly upregulated PPARG mRNA expression in PANC-1 cells, **P < 0.01; E Western blot (left) and quantitative analysis (right) indicated that miR-27b-3p inhibitor markedly increased PPARG protein level in PANC-1 cells, ***P < 0.001, GAPDH served as the loading control

Subsequently, the direct targeting relationship was validated through a dual-luciferase reporter assay. In PANC-1 cells, co-transfection of PPARG-WT with miR-27b-3p mimics significantly reduced luciferase activity (P < 0.001), whereas miR-27b-3p inhibitors significantly increased it (P < 0.01). In contrast, no significant change in luciferase activity was observed upon co-transfection with PPARG-MUT (P > 0.05, Fig. 4B), confirming the specific and direct binding of miR-27b-3p to the PPARG 3′UTR.

Pearson correlation analysis revealed a significant negative correlation between miR-27b-3p and PPARG mRNA expression (P < 0.001, Fig. 4C), consistent with the predicted targeting relationship.

To further validate its regulatory role, functional experiments were performed in PANC-1 cells. qRT-PCR analysis demonstrated that miR-27b-3p inhibitor treatment significantly upregulated PPARG mRNA expression (P < 0.01, Fig. 4D). Western blotting and quantitative analysis further confirmed a significant increase in PPARG protein levels following miR-27b-3p inhibition (P < 0.001, Fig. 4E). Collectively, these results demonstrate that miR-27b-3p directly targets the 3’UTR of PPARG and negatively regulates its expression at both the mRNA and protein levels in pancreatic cancer.

PPARG knockdown rescues the suppressive effects of miR-27b-3p Inhibition on PC cell malignant phenotypes

To validate whether the antitumor effect of miR-27b-3p inhibition is dependent on PPARG upregulation, a PPARG knockdown rescue experiment was performed in PANC-1 cells.

First, PPARG expression levels across experimental groups were assessed. Compared with the miR-27b-3p inhibitor + si-NC group, PPARG expression in the miR-27b-3p inhibitor + si-PPARG group was significantly reduced (P < 0.001, Fig. 5A).

Fig. 5.

PPARG knockdown rescues the suppressive effects of miR-27b-3p inhibition on PC cell malignant phenotypes. A qRT-PCR indicated that PPARG mRNA expression was significantly reduced in the miR-27b-3p inhibitor + si-PPARG group compared to the inhibitor + si-NC group, ***P < 0.001; B The proliferation capacity of PANC-1 cells (suppressed by miR-27b-3p inhibitor) was restored in the inhibitor + si-PPARG group at 72 h, ***P < 0.001 vs.; C PPARG knockdown reversed the migratory suppression induced by miR-27b-3p inhibition, **P < 0.01; D The invasive capacity of PANC-1 cells (suppressed by miR-27b-3p inhibitor) was restored in the inhibitor + si-PPARG group, ***P < 0.001

Subsequently, cell viability was evaluated using the CCK-8 assay. At 72 h, the OD value in the miR-27b-3p inhibitor + si-PPARG group was significantly higher than that in the control group (P < 0.001, Fig. 5B), indicating restored proliferative capacity.

Transwell migration assays revealed that the migration rate in the miR-27b-3p inhibitor + si-PPARG group was significantly elevated relative to the control group (P < 0.01, Fig. 5C), suggesting partial reversal of migratory suppression. Similarly, Transwell invasion assays demonstrated a significantly higher invasion rate in the si-PPARG group compared to the control (P < 0.001, Fig. 5D), consistent with the restoration of invasive potential.

Discussion

PC has long posed a severe threat to human health [19]. Due to its asymptomatic early stage and the difficulty in achieving timely diagnosis, most patients are not diagnosed until the disease progresses to an advanced stage, this delay results in the loss of the optimal period for surgical treatment [20, 21]. Although surgical resection remains the only therapeutic modality with potential curative effects for PC, the disease is characterized by an extremely high postoperative recurrence rate. In turn, this high recurrence rate contributes to a significantly lower 5-year survival rate of PC compared with other malignant tumors [22]. Therefore, identifying reliable biomarkers capable of early prediction and detection of metastasis is crucial for improving patient survival and guiding personalized therapeutic strategies [23]. Numerous studies have demonstrated that miR-27b-3p acts as a diagnostic biomarker in various diseases, including breast cancer, acute ischemic stroke, and Parkinson’s disease [24–26]. The findings of this study indicate that miR-27b-3p is significantly overexpressed in PC tissues, and its expression level is associated with lymph node metastasis, distant metastasis, and elevated serum CA19-9 levels. Furthermore, high expression of miR-27b-3p is significantly correlated with poor prognosis in PC patients. Thus, miR-27b-3p may drive PC progression and has potential as a candidate prognostic biomarker for this malignancy.

miRNAs are key regulators in the development and progression of malignant tumors [27, 28]. A growing body of evidence has shown that miR-27b-3p exerts diverse, context-specific functions in different types of cancer. For instance, it can regulate the invasive and metastatic abilities of endometrial cancer cells by modulating MAECHE7 expression [29]. In lung cancer tissues, miR-27b-3p is frequently underexpressed and acts as a tumor suppressor t [30]. In contrast, in colorectal cancer, miR-27b-3p augments cancer cell migration and invasion via HOXA10 targeting, thus manifesting oncogenic characteristics [11]. Our data demonstrate that treating PC cells with miR-27b-3p inhibitors leads to a significant reduction in their proliferative, migratory, and invasive capacities. These findings further validate that miR-27b-3p may be an oncogenic miRNA in PC.

Under normal physiological conditions, PPARG is widely involved in the regulation of various biological processes, including cell proliferation, differentiation, metabolism, and inflammatory responses [31]. Accumulating evidence has demonstrated that PPARG exerts a significant effect on the progression of multiple types of cancer, and its functional roles vary by tumor type [32, 33]. For example, in breast cancer, PPARG can suppress tumor progression by modulating the immune microenvironment [34]. Whereas in colorectal cancer, it can both promote cancer cell proliferation and enhance sensitivity to the chemotherapeutic agent 5-FU [35]. The current study offers additional confirmation that PPARG is a direct target gene of miR-27b-3p. Specifically, miR-27b-3p downregulates PPARG expression at the post-transcriptional level, and a significant inverse correlation exists between the expression levels of these two molecules. These findings clearly validate the direct regulatory interaction between miR-27b-3p and PPARG, thereby providing new molecular insights into the mechanisms underlying PC initiation and progression.

This research merely constitutes an initial investigation into the regulatory mechanism of the miR-27b-3p/PPARG axis in PC and is associated with certain limitations. Future studies will perform transcriptome sequencing to investigate downstream pathways regulated by miR-27b-3p following PPARG modulation, and conduct in vivo animal experiments to validate the oncogenic role of this axis, thereby enhancing the depth and translational relevance of this research.

In brief, this study has clearly established that miR-27b-3p is markedly upregulated in PC and fulfills oncogenic functions. It further confirms that PPARG is a direct downstream target of miR-27b-3p. Mediated by their reciprocal regulatory interactions, it contributes to the initiation and progression of PC and is closely tied to patient prognostic outcomes. These results provide novel perspectives on the molecular pathogenesis of PC and indicate that targeting the miR-27b-3p/PPARG axis could serve as a potential therapeutic approach.

Author contributions

All authors designed this study. LJ Z, CP L, YX T, CX W and LX W conducted the experiment and analyzed the data. LJ Z wrote the manuscript. T Yand J C revised the manuscript. All authors reviewed and approved for publication.

Funding

No funding was received to assist with the preparation of this work.

Data availability

Corresponding authors may provide data and materials.

Declarations

Ethics approval and consent to participate

The study protocol was approved by The Ethics Committee of Shanghai Baoshan Luodian Hospital. All procedures performed in studies involving human participants were in accordance with the 1964 Helsinki Declaration and later versions. For investigations involving human subjects, informed consent has been obtained from the participants involved.

Consent for publication

All patients provided written informed consent.

Competing interests

The authors declare no competing interests.