Distinct miRNA319a identified from Persicaria chinensis mediates cross-kingdom suppression of cervical cancer targeting ITGA3

Persicaria chinensis, a well-known traditional Chinese medicinal herb that is both edible and medicinal, has been widely acknowledged for its therapeutic effects, such as anti-inflammatory, antioxidant, and antitumor activities. However, the role of miRNAs from this plant in the cross-kingdom regulation of human diseases has not been investigated. In this study, we analyze the miRNA expression profile of P. chinensis using high-throughput sequencing and identify a total of 673 miRNAs, including 422 novel miRNAs that are unique to this plant and 251 conserved miRNAs. Among the conserved miRNAs, pch-miR319a is found to be the most abundant. Moreover, food-oriented pch-miR319a accumulates in the uterus and tumors and exhibits a rich repertoire of target genes within cancer-related pathways, demonstrating significant cross-kingdom regulatory potential. Utilizing the dual-luciferase reporter gene assay, we demonstrate that pch-miR319a from P. chinensis targets the Itga3 gene, which is associated with cervical cancer progression. Overexpression of pch-miR319a significantly decreases the viability, migration, and induces apoptosis of HeLa cervical cancer cells in vitro. Moreover, in a syngeneic mouse tumor model of cervical cancer, treatment with pch-miR319a effectively inhibits tumor growth and downregulates the expression of ITGA3 and the proliferation marker Ki-67. Our study highlights the potential of pch-miR319a from P. chinensis as a novel therapeutic agent for cervical cancer by targeting ITGA3 and provides new insights into the cross-kingdom regulatory mechanisms of plant miRNAs in human diseases. Overall design: 1. Small RNA-seq of Persicaria chinensis: Persicaria chinensis samples were collected from Guangdong Province, China. Total RNA was first extracted from Persicaria chinensis samples by adding Trizol reagent for lysis, followed by chloroform partitioning, isopropanol precipitation, and ethanol washing. The concentration and purity of the RNA were then assessed using a NanoDrop spectrophotometer (Thermo Fisher Scientific, MA, USA), and RNA integrity was evaluated by electrophoresis. Subsequently, miRNA sequencing libraries were constructed using the QIASeq miRNA Library Kit. A pre-adenylated DNA adapter was ligated to the 3' end of miRNAs, and unbound adapters were removed. An RNA adapter was then ligated to the 5' end, followed by reverse transcription using a primer with an integrated unique molecular index to generate cDNA. Impurities were removed by magnetic bead purification, and indexed libraries were created through amplification with sample-specific indices, followed by a final round of magnetic bead purification to obtain the miRNA sequencing libraries. The libraries were typically sequenced on the NextSeq 500 System (Illumina Corp) using a paired-end 150 bp sequencing strategy. After sequencing, bioinformatics analysis was performed on the data. 2. mRNA-seq after pch-miR319a transfection in cervical cancer cells: To further elucidate the regulatory effects of pch-miR319a on its target genes, we conducted a comprehensive RNA-seq analysis to determine the differential expression of target genes in response to pch-miR319a overexpression. Cells were seeded into 24-well plates and transiently transfected of control vector or pch-miR319a plasmid using Lipofectamine 3000 (Invitrogen, CA, USA). After 48 hours of transfection, the transfected cells were selected for RNA-seq analysis.