The expression of miR-324-5p in pancreatic cancer and its effects and mechanisms on proliferation and migration of pancreatic cancer cells / 中华肝胆外科杂志

ABSTRACT

Objective:To investigate the expression and clinical relevance of micro RNA (miR)-324-5p in pancreatic cancer tissues, and to explore the effects and potential mechanisms of miR-324-5p on the proliferation and migration of pancreatic cancer cells.Methods:Real-time quantitative PCR was used to detect the expression of miR-324-5p in 34 pairs of pancreatic cancer and adjacent normal tissues resected at Peking University First Hospital from October 2018 to September 2019. The correlations between miR-324-5p expression and clinicopathological characteristics and prognosis of pancreatic cancer were analyzed using data from the Cancer Genome Atlas (TCGA) database. Real-time PCR was used to detect the expression of miR-324-5p in pancreatic cancer cell lines, and PANC-1 cell was used for functional study by overexpressing miR-324-5p via mimic transfection. CCK8 assay was used to evaluate cell proliferation. Both transwell and scratch wound healing assay were used to assess the cancer cell migration ability. Related proteins were detected by Western blot. The potential downstream target genes of miR-324-5p were selected using data from miRNA target genes predicted webs, in combination with functional analysis and their expressional correlation with miR-324-5p.Results:Data from TCGA database showed that the expression of miR-324-5p in tumor tissues was significantly lower than that in normal pancreatic tissues. And low level of miR-324-5p in pancreatic cancer was correlated with poor prognosis. Analysis of 34 pairs pancreatic cancer and adjacent normal tissues showed that miR-324-5p expression in tumor tissues (11.7±2.0) was significantly lower than that in adjacent normal tissues (70.9±14.4), and the pancreatic cancer patients who had the nerve invasion cancer showed low level of miR-324-5p (82.1%, 23/28) was significantly higher than that patients with high level of miR-324-5p (33.3%, 2/6). The expression of miR-324-5p in human pancreatic cancer cell line was also significantly lower than that in normal pancreatic ductal cells. CCK-8 assay showed that the proliferation ability of PANC-1 cell was significantly decreased when miR-324-5p was overexpressed. Transwell and wound healing assays showed that the capabilities of vertical migration and the horizontal movement were significantly inhibited in PANC-1 cell with miR-324-5p overexpressed [(30.11±5.2) and (174.6±27.0) μm, respectively] than those in control groups [(63.6±4.2) and (458.3±22.3) μm, respectively]. Moreover, Western blots showed a significant overexpression of miR-324-5p inhibited epithelial-mesenchymal transition (EMT). According to the data from miRNA target genes prediction and the functional analysis we found KLF3, MGAT3, PBX1 and ZNRF2 were considered as the potential downstream target genes of miR-324-5p.Conclusions:Our results indicated that miR-324-5p is lowly expressed and acts as the tumor suppressor gene in pancreatic cancer, and low level of miR-324-5p is correlated to a higher rate of nerve invasion and poor prognosis. In human pancreatic cancer cell, miR-324-5p may regulate EMT by directly inhibiting target genes such as KLF3, MGAT3, PBX1, ZNRF2, which in turn suppresses cancer cell proliferation and migration.