Exosomal miR-93 promotes proliferation and invasion in hepatocellular carcinoma by directly inhibiting TIMP2/TP53INP1/CDKN1A

https://doi.org/10.1016/j.bbrc.2018.05.208Get rights and content

Highlights

  • Exosomal miR-93 is up-regulated both in HCC cell lines' media and HCC patients' serum.

  • Exosomal miR-93 promotes HCC proliferation and invasion by regulating TIMP2/TP53INP1/CDKN1A.

  • Exosomal miR-93 is correlated with clinical information including stage, tumor size and predict patients' survival rate.

Abstract

Hepatocellular carcinoma (HCC) is a malignant cancer worldwide; lacking biomarkers for early prognostication contributes to its high lethality. Herein, we report a novel biomarker, exosome delivered miR-93, is up-regulated in HCC cell line media and serum samples of HCC patients. We measured the proliferation and invasion ability of HCC cell lines following exosomal miR-93 treatment. After prediction with online algorithms, we further confirmed that TP53INP1, TIMP2 and CDKN1A are direct targets of miR-93 by dual-luciferase reporter assay. In addition, the diagnostic value of exosomal miR-93 was evaluated by qPCR and ROC analysis. The significant correlation between serum exosomal miR-93 and clinical information including stage, tumor size were observed. Furthermore, the survival differences of HCC patients with high or low miR-93 were statistically significant using Kaplan-Meier analysis. In summary, our work identified exosomal miR-93 as a novel biomarker for both diagnosis and prognosis in HCC.

Introduction

Hepatocellular carcinoma (HCC) is among the top five common cancer worldwide, with more than 400,000 deaths in China [1]. Accumulated studies have demonstrated that infection of hepatitis B/C virus, alcohol consumption and fatty liver disease were contributed to high incidence of HCC in China. HCC is an aggressive tumor often with metastasis and it is also a highly heterogeneous disease [2]. The late diagnosis and treatment contribute to a low survival rate of HCC in China. Therefore, early diagnostic and prognostic are urgently needed.

In the last decades, efforts have been made to identify novel serum biomarkers for HCC. For example, AFP is a widely used diagnostic biomarker for early identification of HCC. In addition, several non-coding RNAs have been identified as new biomarker for HCC diagnosis using liquid biopsy, including micorRNAs (miRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) [3,4].

MiRNAs are approximately 22-nucleotide long, non-coding RNAs that modulate essential cellular processes in tumorigenesis at the post-transcriptional level [5]. Exosomes are extracellular vesicles approximately 20–100 nm in diameter which packed with non-coding RNAs and proteins. A lot of studies indicated cancer associated exosomes delivering miRNAs participate in the regulation of cancer proliferation, migration and invasion [[6], [7], [8]]. Those naturally existed exosomes are involved in cellular communications and tumor microenvironment. There is a significant difference of exosomal miRNAs in many types of tumors, which offer opportunities for developing serum derived exosomal miRNAs as new biomarkers [[9], [10], [11]]. Besides, natural exosomes also facilitate the therapeutic targeting of oncogenic KRAS in pancreatic cancer, suggesting the role of exosome in cancer treatment [12]. Very recently, we reported that upregulated expression of miR-106a promotes HCC cell invasion by targeting CDKN1A and TP53INP1 [13]. A set of serum exosomal microRNAs can be serviced as candidate diagnostic biomarkers for Kawasaki disease [14]. Decreased levels of serum exosomal miR-638 predict poor prognosis in hepatocellular carcinoma [4]. Further, miR-93 promotes cancer cell proliferation by regulating PTEN in several types of cancer [15,16]. Considering the important role of exosomal miRNA in HCC tumorigenesis, we'd like to know whether exosome-derived miR-93 can be used as a new diagnostic and prognostic biomarker.

In the present study, we purified exosomes with a median size close to 40–100 nm from cell culture media as well as patient serum, and described that serum exosomal miR-93 is upregulated in both HCC cell line media and serum samples. Functional studies demonstrated that exosome derived miR-93 involved in tumor proliferation and invasion by targeting CDKN1A, TP53INP1 and TIMP2. At last, serum exosomal miR-93 correlated with clinical features of HCC, including tumor stage, size and survival rate. Collectively, exosomal miR-93 promotes tumorigenesis and can be used as a new diagnostic and prognostic biomarker.

Section snippets

Materials

PET track-etched membranes (12 well) and matrigel were purchased from BD bioscience (USA). Antibodies against CD63, CD9, CDKN1A, TIMP2 and TP53INP1 were got from Proteintech (Wuhan, China). MiR-93 mimics and inhibitor were obtained from GenePharma (Suzhou, China). RPMI-1640, fetal bovine serum (FBS) was from Sigma-Aldrich (St.Louis, USA). Total exosome isolation kits for plasma and cell culture media were got from Thermo Fisher Scientific (Waltham, USA).

Cell culture and transfection

WRL68, HepG2, SMMC7721, SKHEP1 and HUH7

Exosome isolation and validation

We first assessed the exosomes isolated from the culture media by transmission electron microscopy (TEM) and Photon Cross Correlation Spectroscopy (PCCS, SYMPATEC NANOPHOX, German). The results revealed that spherical vesicles are approximately 40–100 nm in diameter in all samples (Fig. 1A), little micro-vesicle with the diameter larger than 100 nm (Fig. 1B), which is consistent with previously described [13], suggesting that we had sufficiently purified exosome from the culture media. We also

Discussion

HCC is the fifth common malignant tumors and the second leading cause of cancer related death worldwide. Late diagnosis, metastasis and recurrence account for the high mortalities. Although exosomal miRNAs were proposed as a potential resource of biomarkers in several studies, little has been investigated and implemented in clinical practice for the management of HCC. In this study, we isolated exosomes with high and stable purity, ensuring a reliable miRNA source (Fig. 1). This unbiased

Acknowledgments

We'd like to thank Prof. Jiarui Wu and Dr. Peizhuo Zhang for advice. This study was supported by a grant from the National Youthful Science Foundation of China (No.81302145), the project of Jiangsu Provincial Medical Youth Talent (QNRC2016706) and Suzhou Youthful Science and technology project (KJXW2017001).

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