Integrated analysis of miRNA–mRNA regulatory crosstalk: Insights from the miR-296-3p and DDHD2 network in breast cancer.

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Background: Breast cancer remains a leading cause of cancer-related morbidity and mortality among women worldwide, with exceptionally high incidence and death rates reported in Pakistan. Molecular biomarkers have gained considerable attention for their potential in improving early detection, prognostic evaluation, and therapeutic targeting. Among these, microRNAs and lipid-regulating genes play significant roles in tumor biology. The miR-296-3p has been described as both a tumor suppressor and an oncogenic regulator depending on cancer type, while DDHD2, a phospholipase A1 family gene involved in intracellular lipid metabolism, has recently been implicated as a proto-oncogene contributing to breast cancer aggressiveness. However, their expression patterns and clinical relevance in the Pakistani population remain largely unexplored. This study analyzed the expression of miR-296-3p and DDHD2 in fifty breast cancer and their adjacent normal tissue samples collected from hospitals in Rawalpindi, Islamabad, and Khyber Pakhtunkhwa. Methods: Ethical approval and informed consent were obtained prior to tissue collection. Total RNA was extracted, converted to cDNA by using commercially available kits, and quantified using quantitative real-time PCR. Relative expression was determined using the 2 ^{^} −ΔΔCt method, and statistical analyses including t-tests and Pearson correlations—were performed using SPSS software. Results: The results demonstrated that miR-296-3p expression was significantly overexpressed in breast cancer tissues compared with normal tissues (mean fold change: 62.34 vs. 1.65; p = 0.02). Similarly, DDHD2 expression was markedly upregulated in tumor tissues (20.39 vs. 1.16; p = 0.03).Correlation analyses revealed weak associations between these biomarkers and clinical variables such as estrogen receptor, progesterone receptor, HER2 status, tumor grade, age, and BMI. The correlation between miR-296-3p and DDHD2 expression was minimal, suggesting that their dysregulation occurs independently rather than through a shared pathway. Conclusions: Overall, the findings highlight that both miR-296-3p and DDHD2 are significantly elevated in breast cancer tissues in this regional cohort, indicating their potential involvement in tumor progression. Although clinical correlations were weak, the observed dysregulation underscores their possible utility as emerging molecular markers. Further functional investigations are required to clarify their biological roles and evaluate their suitability for diagnostic or therapeutic applications.