Pancreatic cancer (PC) ranks among the most lethal human malignancies, with a 5-year survival rate of only 13% and a median survival of approximately 4 months, primarily due to late diagnosis and limited therapeutic achievements (1). PC is currently the sixth leading cause of death among cancers worldwide (2), however, by 2030, it may overtake breast cancer and become the second most common cause (1, 3).

Approximately 90% of PC cases are classified as pancreatic ductal adenocarcinoma (PDAC), an exocrine tumor originating from noninvasive precursor lesions known as pancreatic intraepithelial neoplasia (PanIN) in which genetic alterations begin (KRAS, CDKN2, TP53) (4, 5).

The poor prognosis associated with PDAC is attributed mainly to the lack of reliable early detection methods, as PC typically remains non-symptomatic at its early stages. Consequently, 80% of patients are diagnosed at advanced stages, with metastasis to regional lymph nodes and the liver often occurring (6, 7). However, surgical resection remains the most effective and curative treatment; its success is limited to cases detected early (10–15%) (8). Modified FOLFIRINOX (mFOLFIRINOX), a combination of 5-fluorouracil (5-FU), leucovorin, oxaliplatin, and irinotecan and nab-paclitaxel in combination with gemcitabine, is regarded as the most effective adjuvant therapy, although it exhibits considerable toxicity and modest survival rate changes. Due to the inter- and intratumor heterogeneity of pancreatic tumors, therapeutic resistance has faced a challenge that extends to chemotherapy and radiotherapy (7). Therefore, identifying gene-targeted therapy strategies has become increasingly important, particularly through the modulation of key molecular drivers. The 2021 FDA approval of Sotorasib, a KRAS^G12C inhibitor for non-small-cell lung cancers (NSCLCs), is a landmark example of targeted therapy (9). Gene therapy, which emerged in the early 1990s, is a novel and potentially curative approach for various inherited or acquired illnesses, including cancer, by directly targeting specific molecules involved in carcinogenesis while minimizing side effects (10, 11). This therapeutic policy involves the delivery of exogenous nucleic acids (such as genes, gene fragments, miRNAs, siRNAs) into cells to modify a target gene, regulate its expression, and alter mRNA levels, or produce an exogenous protein (12). Genetic alterations, such as the dysregulated expression of microRNAs or miRNAs, play a major role in cancer development and progression, and their expression can potentially be restored through gene therapy (12, 13).

miRNAs are small non-coding RNA molecules (~ 22 nucleotides) that post-transcriptionally regulate gene expression by inhibiting translation or mRNA degradation, without altering the gene sequence (14, 15). They play a key role in regulating cellular processes, including proliferation, differentiation, cell survival and apoptosis, DNA repair, and Angiogenesis (12, 13, 15). Extensive research has shown miRNAs are dysregulated in various cancers, including pancreatic cancer, presenting their essential role in tumor development (14, 15). miR-142, located on human chromosome 17q22, give rise to two mature strands: 5p and 3p (16). miR-142 is recognized as a tumor suppressor miRNA, showing decreased expression in pancreatic cancer (14, 17, 18). Beyond cancer, miR-142 is involved in inflammation, virus infections, and immune tolerance (19).

The downregulation of miRNA in cancer results in the overexpression of target genes that promote tumor progression through enhanced cell growth, proliferation, and differentiation (10, 14, 17, 19). Although its biological function has not been recognized yet, previous studies showed that miR-142-5p is downregulated in pancreatic cancer tumor tissues and PanC1, BxPC3, SW1990, and CAPAN-1 cell lines (14, 20). Furthermore, miR-142-5p suppresses pancreatic cancer growth and enhances antitumor immunity by regulating PD-L1 (21).

In this study, we selected potential key target genes involved in carcinogenesis and progression pathways, including MCL1 (an anti-apoptotic protein) (22), CDK6 (a cell cycle promoter) (23), and PIK3CA (a regulator of PI3K/AKT signaling) (24). Based on bioinformatics analyses, miR-142-5p was identified as a key regulator of these genes. To experimentally evaluate the impact of miR-142-5p on the selected genes, we overexpressed pre-miR-142 in two PDAC cell lines (AsPC-1 and PANC-1). Gene expression changes were investigated using RT-qPCR, and cell behavior was analyzed through flow cytometry to assess cell cycle progression and apoptosis. Finally, a dual-luciferase reporter assay was performed for CDK6 and MCL1 to confirm the direct targeting of these oncogenes by miR-142.

This study suggests that miR-142-5p functions as a significant tumor-suppressing miRNA with potential applications in gene therapy to counteract oncogenic activity in pancreatic cancer.

miR-142 (MI0000460) was assessed from the miRbase database http://www.mirbase.org/, and miR-142-5p (MIMAT0000433 ) was selected for further investigation (29). The amplicon was amplified by the polymerase chain reaction (PCR) technique on the genomic DNA template of pre-miR-142 inserted into the cloning site of the pCDH-GFP-Puro lentiviral vector (System Biosciences, USA; Cat. No.CD510B-1). EcoRI and BamHI (Thermo Fisher Scientific, USA,) were selected as restriction enzymes. Finally, the cloning vector was transformed into the Escherichia coli (E. coli) competent cells and the pre-miRNA sequence was aligned by MEGA software to confirm its structure (33).

The cell lines, PANC-1, AsPC-1, and human embryonic kidney cells (HEK293T) were purchased from the Iranian Biological Resource Center (IBRC, Iran). All Cell lines were cultured according to standard protocols. AsPC-1 cells were maintained in RPMI-1640 medium (BIO-IDEA, Iran), while PANC-1 and HEK cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) containing glucose (BIO-IDEA, Iran). Both media were supplemented with 10% fetal bovine serum (FBS) (Gibco,USA), 2 mM L-glutamine (Sigma-Aldrich, USA), 1 mM sodium pyruvate (Sigma-Aldrich, USA), and 1% penicillin-streptomycin (100 U/mL penicillin, 100 µg/mL streptomycin; Sigma-Aldrich, USA). Cell cultures were incubated at 37°C in a humidified atmosphere of 95% humidity and 5% CO₂.

The cloned miR-142 in pCDH-GFP-Puro lentiviral vector (System Biosciences, USA, Cat. No. CD510B-1) was transfected into AsPC-1 and PANC-1 using Lipofectamine® 2000 (Invitrogen, USA, Cat. No.11668019) following the manufacturer’s protocol. For the scramble group, an empty pCDH vector was transfected into both cell lines. A day before transfection, cells were seeded in 6-well sterile culture plates at a density of 2×10⁵ cells/well with 70% to 90% confluency without antibiotics. Cells were incubated at 37°C in a CO2 incubator. After 24 hours of transfection, 50% of the medium was replaced with fresh complete medium with 5% FBS and 1% penicillin-streptomycin. The transfection efficiency was assessed after 48 hours using a fluorescent microscope to visualize GFP expression in cells.

Total RNA was extracted for synthesizing complementary DNAs (cDNA) 48 hours after transfection using TRIzol reagent (RNX-Plus), based on the manufacturer’s protocol (Sinaclon, Cat No EX6101). RNA concentration was determined using a Nano Drop spectrophotometer. For cDNA synthesis, 5 µg of Total RNA was used for target genes and 1µg for miR-142-5p. Reverse transcription was performed using MMLV reverse transcriptase enzyme, Random hexamers were used for target genes, and stem-loop RT primers were used for miR-142-5p and Snord47, based on the manufacturer’s protocol (Parstous, Iran Cat: a101161). RT-qPCR was performed with YTA SYBR GREEN qPCR Master Mix 2x (Yektajhiz, Iran, Cat No: YT2551) following the manufacturer’s protocol. Expression levels were measured based on the 2-ΔΔCt method. Snord47 was used as an endogenous control for miR-142-5p, and β2M (Beta-2-microglobulin) was used for target genes. Each reaction was amplified twice. primers were designed using the Primer-Blast tool and are listed in Table 1.

AsPC-1 and PANC-1 cells were plated in 12-well sterile culture plates and transfected with Lipofectamine® 2000. After harvesting with Trypsin/EDTA and washing with PBS, cells were fixed by adding cold ethanol 70% dropwise on the vortex and kept overnight at -20°C. Fixed cells were centrifuged and washed with PBS. Then, 1ml PI MASTER MIX, containing 1 mg/ml Propidium iodide (PI), 10MG/ML RNase (DNaseFREE) was added. The result was quantified after 30 minutes incubation at 37°C by flow cytometry BD FACS Calibur (BD biosciences, San Jose, CA, USA) and data were assessed by FlowJo v10 software (FlowJo LLC, USA)

The 3' untranslated region (3'-UTR) of CDK6 and MCL1 was previously cloned into the Dual-luciferase reporter psiCHECK 2.0 vectors (Promega, Wisconsin) downstream of the Renilla luciferase coding region. HEK293T Cells were seeded in 48-well plates at a density of 1 × 10⁴ cells per well 24 hours before transfection to reach 70–80% confluency. The day after, cells were co-transfected with psiCHECK/3'-UTR constructs and PCDH/miR-142 using PEI MAX (PolyScience, USA) based on the manufacturer’s protocol. psiCHECK/3’-UTR of the genes was used as a negative control. Luciferase was measured 48 hours post-transfection with the Dual-Luciferase Reporter Assay System (Promega, USA). Renilla luciferase activity normalized to Firefly luciferase activity.

PDAC is a principal cause of death among malignancies, distinguished by its asymptomatic behavior in early stages, and despite extensive studies, effective therapies remain limited. Gene therapy, including miRNAs, may provide a new approach with encouraging outcomes for combating malignancy. miRNAs regulate gene expression post-transcriptionally and before protein synthesis. Altered miRNA expression is recognized as a pivotal molecule in cancer development and progression (34). Notably, miRNAs can significantly modulate oncogenes and overexpressed genes as a tumor suppressor (35). Thus, targeting or modulating miRNAs represents a novel therapeutic strategy in cancer gene therapy. Cellular processes, including apoptosis and cell cycle regulation, along with the PI3K signaling pathway, could have a significant effect on cancer initiation and progression of PDAC (22, 24). Consequently, we aimed to investigate the regulatory effect of miR-142-5p on three important genes in PDAC, MCL1, CDK6, and PIK3CA. MCL1, an antiapoptotic member of the BCL2 family, is frequently overexpressed in PDAC and allows cancer cells to evade apoptosis and survive under stress conditions (hypoxia and chemotherapy), which leads to tumorigenesis and resistance to chemotherapeutic drugs. (22, 36, 37). CDK6 is a key regulatory enzyme that facilitates the transition from G1 to S phase of the cell cycle by phosphorylating the Retinoblastoma (RB) protein, thereby promoting cell proliferation(23). Overexpression of CDK6 has been reported in many malignancies, such as PDAC (38). PIK3CA encodes p110 protein, the catalytic subunit of PI3K, which contributes to the regulation of cell growth, survival, and metabolism. Alterations in PIK3CA hyperactivate the PI3K/AKT/mTOR pathway, promoting tumor growth and therapeutic resistance in PDAC. Therefore, regulating these genes through gene therapy could represent an important approach in pancreatic cancer treatment. Cao et al. demonstrated that CDK6 assists in migration and invasion in pancreatic cancer cell lines AsPC-1 and SW1990, and overexpression of CDK6 suppresses the tumor suppressor miR-3613-5p, thereby enhancing the metastatic potential (39). Lu et al reported that miR-142 is downregulated in pancreatic cancer tissue and PANC-1, SW1990, Hup, and CFPAC-1 cell lines, compared to the control group. They revealed that miR-142 targets HIF-1α and its overexpression inhibits invasion and proliferation in PANC-1 and SW1990 (19). Consistent with our study, Zhu et al and Yoa et al showed in separate studies that miR-142-5p is downregulated in PDAC. Zhu et al analyzed 35 paired pancreatic cancer tissue samples, adjacent normal tissues, as well as four pancreatic cancer cell lines, and validated the tumor suppressor effect of miR-142-5p. They showed that miR-142-5p inhibits cell proliferation in PANC-1 by targeting PIK3CA (14, 20). Likewise, the overexpression of miR-142-5p inhibits cell proliferation and induces apoptosis in PANC-1 cells via targeting RAP1A(20). Neither of the previous studies, however, investigated the effect of miR-142 strands on proliferation through cell cycle assay (14, 19, 20). In contrast, our study not only demonstrated that miR-142 induces apoptosis in PANC-1 cells but also uniquely indicated that miR-142 causes G1-phase cell cycle arrest in both cell lines by targeting MCL1, CDK6, and PIK3CA. Specifically, in AsPC-1 cells, the G0/G1 phase increased from 45.34% ± 0.67 in the control group to 63.55% ± 7.76 in the treated group, and in PANC-1 cells, it increased from 50.68% ± 0.51 to 56.43%, which leads to suppression in proliferation in both cell lines. In our study, GEPIA2 revealed the aberrant expression of MCL1, CDK6, and PIK3CA in pancreatic adenocarcinoma (PAAD), and we confirmed the downregulation of target genes MCL1, CDK6, and PIK3CA by RT-qPCR following miR-142-5p overexpression (26). Additionally, a Dual-luciferase reporter assay suggested that miR-142 reduced the luciferase activity of MCL-1 and CDK6 significantly. Although luciferase validation was not performed for PIK3CA, its regulation was strongly supported by bioinformatic tools and RT-qPCR.

Integrated target prediction tools, Target Scan and microT-CDS, identified potential target genes MCL1, CDK6, and PIK3CA by each containing seed matches for miR-142-5p in their 3′UTRs (27, 29). Consistently, the expression profile in miR-base shows that miR-142-5p is the predominant strand in humans. Therefore, these findings suggest that the effects of apoptosis, cell cycle assay, and luciferase assay on two cell lines are likely mediated by miR-142-5p. Based on the OncomiR database analysis, miR-142-5p expression was significantly reduced in PAAD tissues in comparison with normal pancreatic tissues (30). The mean log2 expression levels were 4.80 in tumor samples and 7.57 in normal samples, indicating an approximately 6.9-fold decrease.

Although our results are promising, apoptosis was not observed in AsPC-1 cells, while it was manifested in PANC-1 cells. This discrepancy could be attributed to the fundamental distinctions between the two cell lines, since they originate from different sources and exhibit various dependencies on anti-apoptotic proteins such as MCL-1. AsPC-1 is derived from the ascitic fluid of a patient with metastatic pancreatic adenocarcinoma, while PANC-1 is established from a primary pancreatic ductal adenocarcinoma (40, 41). Although both exhibit invasive behavior, AsPC-1 has been reported to demonstrate more aggressiveness and metastatic potential compared to PANC-1(42, 43).

According to Takahashi et al, MCL-1 protein is not detectable in the AsPC-1 cell line, and the survival of this cell line mainly depends on Bcl-xl as an anti-apoptotic factor. Therefore, targeting MCL-1 alone could not be sufficient to induce apoptosis in AsPC-1. In contrast, in PANC-1, MCL-1 is detectable by western blot, and although targeting of MCL-1 alone may not always lead to apoptosis in PANC-1 (44). Our study suggests that by targeting oncogenic pathways simultaneously, including cell survival, cell regulation through specific genes, and apoptosis is evident.

In conclusion, our research suggests that miR-142 acts as a multitarget inhibitor in pancreatic cancer by targeting key regulators of cell proliferation and apoptosis as a therapeutic candidate. More investigations are demanded to confirm the safety and therapeutic potential of miR-142-5p. Potential delivery systems should also be investigated in combination with current chemotherapeutic agents.