Guo et al. Cancer Cell Int (2019) 19:235 https://doi.org/10.1186/s12935-019-0953-4 Cancer Cell International PRIMARY RESEARCH Open Access Efect of hyperoside on cervical cancer cells and transcriptome analysis of diferentially expressed genes Weikang Guo1†, Hui Yu2†, Lu Zhang1, Xiuwei Chen1, Yunduo Liu1, Yaoxian Wang1* and Yunyan Zhang1* Abstract Background: Hyperoside (Hy) is a plant-derived quercetin 3-D-galactoside that exhibits inhibitory activities on vari- ous tumor types. The objective of the current study was to explore Hy efects on cervical cancer cell proliferation, and to perform a transcriptome analysis of diferentially expressed genes. Methods: Cervical cancer HeLa and C-33A cells were cultured and the efect of Hy treatment was determined using the Cell Counting Kit-8 (CCK-8) assay. After calculating the IC50 of Hy in HeLa and C-33A cells, the more sensitive to Hy treatment cell type was selected for RNA-Seq. Diferentially expressed genes (DEGs) were identifed by comparing gene expression between the Hy and control groups. Candidate genes were determined through DEG analysis, pro- tein interaction network (PPI) construction, PPI module analysis, transcription factor (TF) prediction, TF-target network construction, and survival analysis. Finally, the key candidate genes were verifed by RT-qPCR and western blot. Results: Hy inhibited HeLa and C33A cell proliferation in a dose- and time-dependent manner, as determined by the CCK-8 assay. Treatment of C-33A cells with 2 mM Hy was selected for the subsequent experiments. Compared with the control group, 754 upregulated and 509 downregulated genes were identifed after RNA-Seq. After functional enrichment, 74 gene ontology biological processes and 43 Kyoto Encyclopedia of Genes and Genomes pathways were obtained. According to the protein interaction network (PPI), PPI module analysis, TF-target network construc- tion, and survival analysis, the key genes MYC, CNKN1A, PAX2, TFRC, ACOX2, UNC5B, APBA1, PRKACA , PEAR1, COL12A1, CACNA1G, PEAR1, and CCNA2 were detected. RT-qPCR was performed on the key genes, and Western blot was used to verify C-MYC and TFRC. C-MYC and TFRC expressions were lower and higher than the corresponding values in the control group, respectively, in accordance with the results from the RNA-Seq analysis. Conclusion: Hy inhibited HeLa and C-33A cell proliferation through C-MYC gene expression reduction in C-33A cells and TFRC regulation. The results of the current study provide a theoretical basis for Hy treatment of cervical cancer. Keywords: Hyperoside, Cervical cancer, RNA-Seq, Diferentially expressed genes, Gene ontology, Kyoto Encyclopedia of Genes and Genomes, Protein–protein interactions network, Survival analysis *Correspondence: [email protected]; [email protected] †Weikang Guo and Hui Yu contributed equally to this article 1 Department of Gynecology, Harbin Medical University Cancer Hospital, No. 150 Haping Road, Nangang District, Harbin 150081, Heilongjiang Province, China Full list of author information is available at the end of the article © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/ publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Guo et al. Cancer Cell Int (2019) 19:235 Page 2 of 14 Background treated with 0.25, 0.5, 1, 2, 4, or 8 mM Hy (Solarbio, Bei- Cervical cancer is a malignant epithelial tumor that jing, China) for 24, 48, or 72 h. occurs in the cervix. Most cervical cancers can be screened early by cervical cytology and virology. More- Cell viability and IC50 determination over, human papillomavirus (Hpv) vaccination has Cell viability was determined using the Cell Counting emerged as an efective method for cervical cancer Kit-8 (CCK-8) assay at 24, 48, and 72 h. At each time prevention [1]. However, due to inadequate screening point, 100 µL CCK-8 (Beyotime Bio, Shanghai, China) programs in many parts of the world, cervical cancer was added to each well of a 96-well plate, which was then remains one of the most common cancer types in females placed in a 37 °C, 5% CO2 incubator. HeLa and C-33A [2, 3]. Surgery is the main method for early treatment. cells were incubated for 0.5 and 2 h, respectively, in Radiotherapy and chemotherapy are further therapy the dark. Te absorbance of each well was measured at options. Women with cervical, especially advanced or 450 nm using an EPOCH microplate reader (Gene Com- recurrent, cancer are commonly treated using chemo- pany Limited). Te half-inhibitory concentration (IC50) therapy [4]. Recently, several reports have implicated was calculated with GraphPad (version 5.0), and the cell traditional Chinese medicines in the treatment of cervi- line exhibiting higher sensitivity to Hy treatment was cal cancer. For example, ferulic acid inhibits the prolifera- selected for follow-up experiments. tion, invasion, and autophagy of cervical cancer cells, and induces cell cycle arrest [5]. Moreover, casticin induces RNA‑Seq and data preprocessing G0/G1 cell cycle arrest and apoptosis in gallbladder can- Cells were divided into two groups for this experiment: cer [6]. Hyperoside (Hy) is a favonoid found mainly in a Hy-treated and a blank control group; the experiment Chinese herbal medicines. It exhibits anti-infammatory, was repeated three times with independent biological anti-oxidative, and vascular protective efects. Several samples. Total RNA was extracted using TRIzol (TaKaRa recent studies demonstrated an anticancer efect of Hy Bio, Dalian, China), and the extracted RNA was sent to in a variety of tumor types. Tus, Hy increased apopto- Shanghai New Bioinformatics Co., Ltd. to construct a sis and autophagy in pancreatic cancer cells [7]. Another cDNA library using an Illumina HiSeqTM 2000 platform study described Hy-mediated inhibition of human osteo- for double-end PE150 sequencing with 6G data per sam- sarcoma cell proliferation and promotion of osteogenic ple. Unreliable bases and reads were fltered out to obtain diferentiation [8]. Yet another study implicated Hy in clean data for the six samples. Te TopHat software (ver- the caspase-3, p53, and nuclear factor-kappa B (NF-κB) sion 2.1.0) was used to locate clean reads on the human signaling pathways, which induce apoptosis and inhibit reference genome (GENCODE download, GRCh38) [12]. lung cancer cell proliferation [9, 10]. In gynecological Te read count information on each gene alignment was oncology, Hy induces endometrial cancer cell apoptosis obtained using the htseq-count tool (version 0.9.1) based through the mitochondrial pathway [11]. However, Hy on the human gene annotation information provided by efect on cervical cancer development and the molecular GENCODE (Release 25). mechanism implicated are unclear. In the current study, the efect of Hy on two cervical Inter‑sample expression level and principal component cancer cell lines was determined using cytological meth- analysis ods, to detect changes in the cell proliferation index. Dif- Te cor function of the R software (version: 3.4.1) was ferentially expressed genes (DEGs) were identifed by used to calculate the similarity between the two sam- RNA sequencing (RNA-seq), comparing untreated and ples in each experiment. Te prcomp function of the R Hy-treated cells. Further analyses of the DEGs were con- software was utilized to reduce the dimensionality of the ducted to explore the specifc mechanism of Hy action on data. Te ggfortify package (version: 0.4.6) created PCA cervical cancer cells. plots for principal component analysis. Methods DEG screening Cell culture First, the raw count was normalized using the TMM HeLa and C-33A cells (both acquired from the Chinese algorithm in the edgeR package [13, 14] (version: 3.4). Academy of Sciences Shanghai Cell Bank) were cultured Second, the mean–variance relationship was modeled in Dulbecco’s modifed Eagle’s medium (Gibco,Waltham, with the exact weighting method (voom) provided by the MA, USA) supplemented with 10% fetal bovine serum. limma package [15] (version: 3.36.2). Ten, using linear Tey were inoculated in 96-well plates, cultured at 37 °C regression and empirical Bayesian methods provided by for 24 h, and then divided into seven groups. One group the limma package, diferential expression analysis was was untreated, whereas the remaining groups were performed on the Hy and control groups. Te diferential Guo et al. Cancer Cell Int (2019) 19:235 Page 3 of 14 expression threshold for DEGs was set to P < 0.05, in the TF-target regulatory network were utilized as |logFC| > 0.585. candidate genes, and candidate gene expression val- ues were screened from the TCGA. Te median values Kyoto Encyclopedia of Genes and Genomes (KEGG) were divided into two groups (high expression and low and Gene Ontology (GO) expression). A log-rank statistical test was performed, GO [16] functional annotation and KEGG [17] enrich- and the threshold P value was set to < 0.05. Te rela- ment analysis of the DEGs were performed using the tionship between candidate genes and patient progno- DAVID (version 6.8, https ://david -d.ncifc rf.gov/) [18]. sis was analyzed, and a Kaplan–Meier survival curve P < 0.05 and enrichment count of at least 3 were consid- was plotted. ered thresholds for signifcant enrichment results. Protein–protein interaction network (PPI) and PPI module RT‑qPCR analysis analysis Key genes for RT-qPCR verifcation were selected based Te STRING (version 10.0, http://www.strin g-db.org/) on the PPI networks, topological properties, TF analy- database [19] was used to predict whether gene-encoded ses, logFC, and degree ranking data.