As an oncogenic virus, HPV16 can lead to the dysfunction of cervical epithelial cells and contribute to the progression of cervical cancer. Components from the cervical-vaginal fluid (CVF) could be used as the basis for cervical cancer screening. Exosomes are widely present in various body fluids and participate in intercellular communication via its cargos of proteins, mRNAs, and miRNAs. This study was conducted to explore the changes of miRNAs in exosomes isolated form the cervical-vaginal fluid during HPV16 infection and to predict the potential effects of exosomal miRNAs on the development of cervical cancer. CVF was collected from volunteers with or without HPV16 infection. The exosomes in CVF were identified by electron microscopy. Microarray analysis was subjected to find the differentially expressed miRNAs in CVF exosomes. To confirm the results, 16 miRNAs were randomly selected to go through real-time quantitative polymerase chain reaction. In addition, GO and pathway analyses were conducted to reveal potential functions of differentially expressed miRNAs. A total of 2548 conserved miRNAs were identified in the cervical-vaginal fluid-derived exosomes. In response to HPV16 infection, 45 miRNAs are significantly upregulated and 55 miRNAs are significantly downregulated (
Infections with certain HPV types have a high risk for cervical cancer [
In this study, the expression profiles of miRNAs in CVF-derived exosomes from women with or without HPV16 infection were detected by the microarray technology. Some of the differentially expressed miRNAs were randomly selected and validated by quantitative reverse transcriptase PCR (qRT-PCR). Moreover, bioinformatics analysis was explored to describe the potential functions of the related miRNAs. The study on miRNAs in CVF-derived exosomes with or without HPV16 infection will help us to better understand the pathological implications of HPV16 in cervical cancer progression.
CVF samples were collected from 6 HPV-positive and 6 HPV-negative women aged 20–35 years in Women’s Hospital of Nanjing Medical University. All women had no cervical cancerous disease and abstained from sexual activity at least 3 days prior to sample collection. The samples of CVF were collected with a softcup collection device as described [
CVF samples were centrifuged at 300×
To observe exosome morphology, an exosome suspension was mixed with an equal volume of 2% paraformaldehyde. The mixture was subsequently applied to a formvar-coated copper grid. The sample was then stained with 1% aqueous uranyl acetate for 2 min. After being wicked off with filter paper, sample was finally observed under transmission electron microscope (FEI. Hillsboro, USA).
The concentration and size distribution profile of exosomes were analyzed applying a NanoSight NS300 system (Malvern Instruments Ltd., Malvern, United Kingdom) and evaluated with NTA 3.1 Dev Build 3.1.54 software. The exosome preparations were resuspended with 800
Western blotting was performed to detect the presence of exosomal surface markers. We lysed CVF exosomes by RIPA lysis buffer on ice. Then, the concentration of the protein was measured using Pierce BCA Protein Assay Kit (Thermo Scientific, Massachusetts, America). Each sample was run on SDS–PAGE and transferred and blotted with exosome marker antibodies CD9 (SBI, California, America) and CD63 (SBI, California, America). The protein blots were detected by a detection system.
Total RNA was extracted and purified applying Qiagen serum/Plasma Kit (Qiagen#217184), following the instructions of the manufacturer. The RIN number to inspect RNA integration was checked applying an Agilent Bioanalyzer 2100 (Agilent Technologies, California, America). All RNA samples used for miRNA microarrays exhibited a RIN of 6.0. miRNA molecular in total RNA was labeled by miRNA Complete Labeling and Hyb Kit (Agilent Technologies, California, America) following the manufacturer’s instructions and labeling section. Each slide was hybridized with 100 ng Cy3-labeled RNA using miRNA Complete Labeling and Hyb Kit (Agilent Technologies, California, America). Slides were scanned by Agilent Microarray Scanner (Agilent technologies, California, America) and Feature Extraction software 10.7 (Agilent technologies, California, America) with default settings. Raw data were normalized by Quantile algorithm, included in the R package AgimicroRNA (López-Romero, P. BMC Genomics 2011).
Total RNA was obtained from exosome samples using the TRIzol reagent (Tiangen, Beijing, China) as described in the manufacturer’s instructions. The quality and quantity of the extracted RNA were confirmed by the One Drop OD-1000 + Spectrophotometer (One Drop Technologies, Nanjing, China). TaqMan miRNA reverse transcriptase kit (Applied Biosystems; Thermo Fisher Scientific, Inc.) was used to synthesize cDNA with 200 ng total RNA as a template. RNA integrity was assessed by standard denaturing agarose gel electrophoresis. The qRT-PCR analysis was performed using TaqMan Universal Master Mix II no UNG (Thermo Fisher Scientific, Inc.) and commercial primers on the Applied Biosystems (Carlsbad, California, America) 7500 Real-Time PCR System. The experimental data was analyzed using the 2−
Differentially expressed miRNAs were identified by a standard Student
Statistical differences were analyzed using Student’s
TEM, NTA, and western blot analysis were used to examined and confirm the exosomes isolated from CVF. Under TEM, round vesicle structures with sizes varying between 30 and 150 nm were observed (Figure
Characterization of CVF-derived exosomes. (a) Representative microscope images of CVF-derived exosome morphology were analyzed by electron microscopy. Scale bar, 200 nm. (b) Exosomal average size and intensity were measured through NanoSight analysis. (c). Expression of exosome markers CD9, CD63, and HSP70 was detected via western blot.
The microarray technology was used to compare miRNA expression profiles of CVF-derived exosomes between HPV16-positive and HPV16-negative women. Based on the results of the microarray analysis, 2548 miRNAs from various chromosomes were found. Mostly, deregulated miRNAs were located in chromosome 1, 19, and X (Figure
Differentially expressed miRNAs in exosomes of HPV16-infected CVF and control samples. (a) Upregulated or downregulated miRNAs were marked in red or blue, respectively. The number of all conserved miRNAs located in the human chromosomes was shown in the histogram. The circular diagram on the right represents total conserved miRNAs in the human chromosome, with 1293 upregulated and 1255 downregulated. Heatmap analysis showing the fold change of the top 15 (b) highest and C. lowest miRNAs in the HPV16-infected group. Color scale is from -1.25 (blue, lower than mean) to 2.41 (red, higher than the mean). Each column represents one sample, and each row indicates a transcript.
To confirm the differential expression of miRNAs, 16 different miRNAs were randomly selected and qRT-PCR was conducted. The expression patterns of these dysregulated miRNAs in HPV-16 infected CVF exosomes shows consistency with the results of microarray analysis (Figures
Validation of the differentially expressed miRNA using qRT-PCR. (a). Eight significantly upregulated miRNAs were randomly selected and validated in exosomes of HPV16-infected CVF and control samples by qRT-PCR. (b). Eight significantly downregulated miRNAs were also validated.
GO and KEGG pathway analyses were applied to reveal potential functions of differentially expressed miRNA target genes (listed in Supplementary Table
Potential function of differentially expressed miRNA. GO and KEGG analyses of differential genes can identify functional or metabolic pathways of differentially expressed miRNAs. Elucidate differences between samples at the level of gene function and metabolic pathways. According to the function annotation of genes, the number of differential genes belonging to different functions is displayed in the form of a bar graph. GO knowledgebase was applied to analyze biological process, cellular component. and molecular function of miRNA predicted genes. KEGG database was applied to investigate the potential functions in the given pathways. (a). The biological process categories. (b). The cellular component categories. (c). The molecular function categories. (d). KEGG signaling pathways. Line length indicates the strength of data support.
Heatmap of KEGG pathways. KEGG pathways of differentially expressed miRNA were involved in multiple pathways, especially proteoglycans in cancer, TNF signaling pathway, and TGF-
The persistent infection of HPV is causally linked with the development of cervical cancer [
According to the results of microarray assays, there were 45 upregulated miRNAs and 55 downregulated miRNAs in HPV16-infected CVF-derived exosomes compared to the uninfected samples. Among these, hsa-miR-5590-3p was the most significantly downregulated miRNA. Previous studies have demonstrated that hsa-miR-5590-3p acted as a negative regulator of the TGF
Notably, bioinformatics analysis of differentially expressed miRNAs target genes revealed that these miRNAs were also actively involved in oncogenic pathways through targeting specific genes. The results of GO analysis demonstrated that the differentially expressed miRNAs target genes such as RPL14, NUP205, and POLR2B were associated with viral transcription, which is a typical feature of HPV infection. Moreover, the results of the KEGG pathways including the Notch signaling pathway, ErbB signaling pathway, and Wnt signaling pathway were mainly associated with the progression of cervical cancer. the Notch signaling pathway was demonstrated to be highly correlated with carcinogenesis, including the cervical cancer development [
In conclusion, the expression of miRNAs in CVF-derived exosomes was significantly different after HPV16 infection. Bioinformatics analysis of the dysregulated miRNAs showed that these differentially expressed miRNAs may be tightly involved in the progression of oncogenesis. Our study provides a foundation for understanding the mechanism of the oncogenic process of HPV16 infection. We will expand the sample size and explore the detailed mechanism of these miRNA changes. Further studies will be proceeded to evaluate whether the exosomal miRNA in CVF can potentially serve as useful biomarkers for cervical cancer diagnosis.
The data that support the findings of this study are available from the corresponding author upon reasonable request.
The authors declare that they have no competing interests.
Ying Wu, Xinyan Wang, Li Meng, and Wenqu Li collected and analyzed the data. Chunyan Li performed the experiments and collected important background information. Ping Li and Siliang Xu designed the study and wrote the manuscript. All authors approved the final submission. Ying Wu, Xinyan Wang, and Li Meng contributed equally.
This work was supported by grants from the Natural Science Foundation of Jiangsu Province (No. BK20181089), Key Project for the Science and Technology Development Fund of Nanjing Medical University (No. 2017NJMUZD077), and Six Talent Peaks Project in Jiangsu Province (No. WSW-119).
Supplementary Table 1: all of the differentially expressed miRNAs. Supplementary Table 2: list of qPCR primers used in this study. Supplementary Table 3: target genes of differentially expressed miRNAs.