Exosomal miR-21 Regulates TETs/PTENp1/PTEN Signaling Pathway to Promote Hepatocellular Carcinoma Growth

Abstract

Background: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in the world, and ranks fifth in incidence and third in mortality. As an important means of communication, exosome plays an important role in the occurrence and development of HCC,and regulats tumor invasion and metastasis. Methods: Fluorescence microscopy was used to analyze the internalization of the exosomes. Quantitative RT-PCR analysis was applied to determine the expression level of miR-21, PTEN, PTENp1, TET1, TET2 and TET3. Western blotting analysis was performed to detect the relative proteins expression. Moreover, the cell proliferation was examined by BrdU assay, the cell apoptosis was detected by flow cytometric analysis , and the invasion of cells was evaluated by Transwell matrigel invasion assay, the nude mice model was used to determine the effect of exosomal miR-21 on tumor formation. Findings: Detection of microRNAs (miRNAs) in exosomes of HCC showed that miR-21 expression in exosomes was positively correlated with the expression level of miR-21 in cells, and negatively correlated with the expression of its target genes Phosphatase and tensin homolog (PTEN), PTEN pseudogene 1 (PTENp1) and Tet methylcytosine dioxygenases (TETs). HCC cell-derived exosomes could increase miR-21 expression in HCC cells, and down-regulate the expression of PTEN, PTENp1 and TETs. MiR-21 inhibitors or PTENp1 over-expression vector could weaken the effect of the above-mentioned exosomes, and at the same time weaken their role in promoting cell proliferation, migration and inhibiting apoptosis. Further studies have shown that miR-21 not only directly regulates the expression of PTEN, PTENp1 and TETs, but also increases the methylation level of PTENp1 promoter by regulating the expression of TETs, thereby inhibiting the expression of PTENp1 and further down-regulating the expression of PTEN. Iterpretations: Exosomal miR-21 can regulate the expression of tumor suppressor genes PTEN and PTENp1 in various ways and affect the growth of HCC cells. These findings may provide new targets for the prevention and treatment of HCC, and provide new evidence for the mechanism of exosomal miRNAs. Funding Statement: This work was supported by grants from Natural Science Foundation of Guangdong Province (2015A030313466)，and Science and Technology Project of Guangdong Province (2014A020212326). Declaration of Interests: The authors declared that they have no conflicts of interest to this work. Ethics Approval Statement: Animal care and use followed the ethical guidelines of the Chinese Council on Animal Care, and were reviewed and approved by the Institutional Animal Care and Use Committee.