Gu and Zhang Cancer Cell Int (2020) 20:517 https://doi.org/10.1186/s12935-020-01601-4 Cancer Cell International

PRIMARY RESEARCH Open Access High‑throughput sequencing identifcation of diferentially expressed microRNAs in metastatic ovarian cancer with experimental validations Yang Gu and Shulan Zhang*

Abstract Background: Ovarian cancer (OC) is a common gynecological cancer and characterized by high metastatic poten- tial. MicroRNAs (miRNAs, miRs) have the promise to be harnessed as prognostic and therapeutic biomarkers for OC. Herein, we sought to identify diferentially expressed miRNAs and mRNAs in metastatic OC, and to validate them with functional experiments. Methods: Diferentially expressed miRNAs and mRNAs were screened from six pairs of primary OC tissues and metastatic tissues using a miRStar™ Human Cancer Focus miRNA and Target mRNA PCR Array. Then, gene expression profling results were verifed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and western blot assays. The binding afnity between miR-7-5p and TGFβ2 was validated by dual-luciferase reporter assay. Expres- sion of miR-7-5p and TGFβ2 was manipulated to assess their roles in malignant phenotypes of highly metastatic HO-8910PM cells. Results: MiRNA profling and sequencing identifed 12 miRNAs and 10 mRNAs that were diferentially expressed in metastatic tissues. Gene ontology and Pathway analyses determined that 3 diferentially expressed mRNAs (ITGB3, TGFβ2 and TNC) were related to OC metastasis. The results of RT-qPCR confrmed that the decrease of miR-7-5p was most signifcant in OC metastasis, while TGFβ2 was up-regulated in OC metastasis. Moreover, miR-7-5p targeted and negatively regulated TGFβ2. MiR-7-5p overexpression accelerated HO-8910PM cell viability and invasion, and TGFβ2 overexpression reversed the results. Meanwhile, simultaneous miR-7-5p and TGFβ2 overexpression rescued the cell activities. Conclusions: This study characterizes diferentially expressed miRNAs and mRNAs in metastatic OC, where miR-7-5p and its downstream target were most closely associated with metastatic OC. Overexpression of miR-7-5p targets and inhibits TGFβ2 expression, thereby inhibiting the growth and metastasis of OC. Keywords: High-throughput sequencing, MicroRNA sequencing, MicroRNA-7-5p, TGFβ2, Ovarian cancer, Metastasis, In vitro validation

Background Ovarian cancer (OC) is the deadliest gynecological can- *Correspondence: [email protected] cer, due to the absence of symptoms at the early stage Department of Obstetrics and Gynecology, Shengjing Hospital [1, 2]. Te non-specifc symptoms of OC contribute to of China Medical University, No. 36, Sanhao Street, Heping District, Shenyang 110004, Liaoning, P. R. China a delayed diagnosis at advanced stages of cancer, which

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causes poor 5-year overall survival rate that is about high-pressure treatment, and rapidly placed in liquid 30–40% [3]. Moreover, the widespread and distant metas- nitrogen for quick freezing, and then transferred to a tases are observed in 59% OC patients, which are clearly − 80 ˚C cryogenic freezer for preservation. All patients correlated with unfavorable prognosis of OC patients [4]. were diagnosed with ovarian serous cystadenocarcinoma Terefore, it is pivotal to unveil invasive and metastatic by postoperative parafn pathology, and their clinical mechanisms of OC. Of note, accurate prognostic strate- staging was made according to the International Federa- gies with the assistance of biomarker targets would aid in tion of Gynecology and Obstetrics (FIGO) staging system the diagnosis and prognosis of OC patients [5]. However, for OC (2009). None of the patients had undergone pre- the understanding of mechanisms behind biomarkers operative radiotherapy, chemotherapy or other special and signaling pathways controlling invasive and meta- treatments. Patients who provided tissue specimens for static potential of OC should been thoroughly probed [6]. expression profling experiments were aged 48–64 years MicroRNAs (miRNAs), small RNA molecules [21–23 with the mean age of 57.5 years, and those providing nucleotides (nt)], function as potent modulators of gene samples for RT-qPCR validation were aged 38–80 years expression through mRNA translation blockade or RNA with the mean age of 57 years. Data and tissues were har- interference [7]. MiRNAs have become a hotspot of vested upon receiving the informed consent of patients tumor researches over the recent years. Interestingly, and approval by the ethics committee of Shengjing Hos- the diferential expression of miRNAs has been reported pital of China Medical University (Approval number: in OC. For example, in a previous study conducted by 2016PS040K). Chong et al. [8] diferential miRNA expression profles showed that miR-551b, miR-19b, miR-196b and miR- RNA extraction from tissues 3198 were upregulated and miR-8084, miR-3201, miR- A Trizol Kit (Invitrogen Inc., Carlsbad, CA, USA) was 3613 and miR-7515 were downregulated in OC. Besides, applied for isolation of total RNA from metastatic and another study revealed that there were 17 diferentially primary tissues of OC. RNA purity was estimated by expressed miRNAs in metastatic OC, among which miR- adopting a ­NanoDrop® ND-1000 spectrophotom- 21, miR-150, and miR-146a were upregulated in meta- eter (Termo Fisher Scientifc, Waltham, MA, USA), static OC tumors [9]. and assessed by the ratio of optical density (OD) Moreover, the dysregulated miRNAs may act as novel 260 nm/280 nm. A ratio between 1.8 and 2.0 signifed the oncogenes or anti-oncogenes in OC by regulating target sample to be highly pure. genes [10]. For instance, miR-7 was found to block the EGFR/ERK pathway to decrease OC cell invasion [11]. MiRNA profling and sequencing Stated thus, a hypothesis can be made that miRNAs may Six pairs of metastatic and primary tissues of OC were participate in OC metastasis via modulating their target obtained from Shengjing Hospital of China Medical Uni- genes. Notably, emerging evidence has demonstrated the versity. Total RNA was extracted from tissues using a Tri- importance of the miRNA-mRNA interplay in the patho- zol Kit. cDNA was synthesized, purifed, and hybridized genesis and metastasis of OC [12, 13]. Terefore, difer- based on the instructions of an Arraystar miRNA First- entially expressed miRNAs and mRNAs were screened Strand cDNA Synthesis Kit (Cat# AS-MR-004), which from primary and metastatic tissues of OC using high- was fully compatible with an miRStar™ Human Cancer throughput sequencing technique. We found 12 miRNAs Focus miRNA and Target mRNA PCR Array (Array- and 10 mRNAs that were diferentially expressed in and star, Rockville, MD, USA). After standardization of the related to metastatic potential of OC. Te expression original data from fuorescence quantitative PCR, fold profling results were validated by functional experimen- changes in miRNAs and mRNAs were calculated based tal validations. on a comparison between metastatic and primary tissues of OC. A fold change > 1.5 and p < 0.05 were considered as Methods a noteworthy up-regulation, while fold change < − 1.5 and Study participants p < 0.05 were considered as a striking down-regulation. All 31 pairs of metastatic tissues (lymph node metasta- sis tissue) and primary tissues of OC were harvested Gene ontology (GO) functional enrichment and pathway from patients receiving surgery at Shengjing Hospital of analyses China Medical University from 2014 to 2016. Of these, GO analysis and Kyoto Encyclopedia of Genes and 6 pairs were used for expression profling experiments, Genomes (KEGG) enrichment analysis were employed to and 25 pairs were employed for RT-qPCR validation. Tis- analyze diferentially expressed mRNAs in OC based on sue samples resected during surgery were immediately a David database (https​://david​.ncifc​rf.gov/). GO analy- placed into 1.5-mL centrifuge tubes with RNase-removal sis provided annotations and function classifcations for Gu and Zhang Cancer Cell Int (2020) 20:517 Page 3 of 15

diferentially expressed mRNAs by analyzing the clas- Table 1 Primer sequences for RT-qPCR sifcations of Biological Processes, Cellular Components Genes Primer sequences and Molecular Function. Pathway analysis was based on KEGG results combined with Fisher’s exact test and a t miR-141-3p Forward: 5′-GGG​GTA​ACA​CTG​TCT​GGT​AA-3′ test. A p < 0.05 was regarded statistically distinct. Reverse: 5′-TGC​GTG​TCG​TGG​AGTC-3′ miR-187-5p Forward: 5′-GGG​GAG​GCT​ACA​ACA​CAG​GA-3′ The miRNA‑mRNA network analysis Reverse: 5′-TGC​GTG​TCG​TGG​AGTC-3′ Using a miRStar™ Human Cancer Focus miRNA and miR-7-5p Forward: 5′-GGG​GGT​GGA​AGA​CTA​GTG​ATTT-3′ Target mRNA PCR Array expression profling, we carried Reverse: 5′-GTG​CGT​GTC​GTG​GAG​TCG​-3′ out the screening of 6 pairs of metastatic and primary tis- miR-584-5p Forward: 5′-TGG​GTT​ATG​GTT​TGC​CTG​G-3′ sues of OC in 184 cancer-related miRNAs and 178 target Reverse: 5′-GTG​CGT​GTC​GTG​GAG​TCG​-3′ mRNAs. Te miRNA-mRNA networks with a potential miR-200a-3p Forward: 5′-TAA​CAC​TGT​CTG​GTA​ACG​ATGT-3′ targeting relationship were screened out from miRNA Reverse: 5′-CAT​CTT​ACC​GGA​CAG​TGC​TGGA-3′ microarray analysis and then plotted with the assistance miR-200b-3p Forward: 5′-GCT​GCT​GAA​TTC​CAT​CTA​ATT​TCC​AAAAG-3′ of online tools targetscan (https​://www.targe​tscan​.org/ Reverse: 5′-TAT​TAT​GGA​TCC​GCC​CCC​AGG​GCA​ATGGG-3′ vert_71/) and miranda (https​://34.236.212.39/micro​rna/ miR-9-3p Forward: 5′-GAG​GCC​CGT​TTC​TCT​CTT​TG-3′ home.do). Reverse: 5′-AGC​TTT​ATG​ACG​GCT​CTG​TG-3′ TGFβ2 Forward: 5′-CTG​CTA​ATG​TTA​TTG​CCC​TCC​TAC​-3′ RT‑qPCR Reverse: 5′-CCT​GTA​ACA​ACG​CAT​CTC​ATATT-3′ Te metastatic and primary tissues of OC frozen at TNC Forward: 5′ GTG​ACA​GAA​GTG​ACG​GAA​GAGAC3′ − 80 °C were collected. TRIzol reagent (Invitrogen) was Reverse: 5′ GAT​GGC​AAA​TAC​ACG​GAT​AAAGT3′ applied for total RNA isolation from tissues and cells. HOXB5 Forward: 5′-GGC​GCA​TGA​AGT​GGAA-GAA​GGA​CA-3′ A mixture (14.5 µL) of 2.0 µg RNA, 1 µL Oligo, 1.6 µL Reverse: 5′-GAG​AGG​GAG​CCA​CAG​GAA​GACC-3′ deoxy-ribonucleoside triphosphate Mix and ­H2O was U6 Forward: 5′-GCT​TCG​GCA​GCA​CAT​ATA​CTA​AAA​T-3′ incubated at 65 °C in water for 5 min and then put on ice Reverse: 5′-CGC​TTC​ACG​AAT​TTG​CGT​GTCAT-3′ for 2 min. After centrifugation, the mixture was mixed β-actin Forward: 5′-GTG​GCC​GAG​GAC​TTT​GAT​TG-3′ with reverse transcription reaction liquid, and placed at Forward: 5′-CCT​GTA​ACA​ACG​CAT​CTC​ATATT-3′ 37 °C for 1 min. Te mRNA was reversely transcribed miR microRNA, TGFβ2 transforming growth factor beta 2, TNC Tenascin-C, HOXB5 into cDNA, and the samples were then temporarily homeobox B5, RT-qPCR reverse transcription quantitative polymerase chain placed on ice. All cDNA template samples were prepared reaction in a PCR amplifcation system. Te fuorescence value of SYBR Green was detected. PCR primer sequences (Table 1) were designed and synthesized by Sangon primary antibody overnight at 4 °C. Afterwards, the Biotech (Shanghai, China). Te relative expression of membrane was then re-probed with goat anti-rabbit sec- mRNAs or miRNAs, normalized to β-actin or U6, was ondary antibody (1:5000; Shanghai Kangchen Biological ΔΔCt calculated using ­2− method. Engineering Co., Ltd., Shanghai, China) complexed to horseradish peroxidase at RT for 1 h. After rinsing with Western blots TBST, the membrane was soaked in electrogenerated Tissues were immersed in radioimmunoprecipitation chemiluminescence (P0018, Beyotime) for 1 min, devel- assay lysis bufer (P0013B; Beyotime, Shanghai, China) oped, photographed and recorded. β-actin (1:10,000; Cell containing phenylmethyl sulfonylfuoride to extract total Signaling Technology, Danvers, MA, USA) served as an protein. Te samples were allowed to stand on ice for endogenous control for TGFβ2. A Gel-Pro Analyzer 4.0 30 min, followed by 20-min centrifugation at 12,000×g. program (Media Cybernetics, Silver Spring, MD, USA) Te supernatant was collected and transferred to an EP was adopted for gray value analysis. Te gray value ratio tube to determine the total protein concentration accord- of each target protein band to the endogenous control ing to the instructions of a bicinchoninic acid kit (KC- band was calculated as the relative expression of each 430, Shanghai Kangchen Biological Engineering Co., protein. Experiments were independently conducted Ltd., Shanghai, China). Te samples were separated at three times. 100 V for 1.5 h, and electroblotted onto a polyvinylidene fuoride membrane using a sandwich method at 80 V for Dual‑luciferase reporter assay 1.5 h. After 1-h blocking in 5% bovine serum albumin- Binding sites for miR-7-5p and TGFβ2-3′untranslated Tris-bufered saline (TBST), the membrane was incu- region (UTR) were predicted using Genebank and Tar- bated with diluted (1:1000) rabbit anti-human TGFβ2 getscan combined with expression profling analysis Gu and Zhang Cancer Cell Int (2020) 20:517 Page 4 of 15

′ ′ 4 results. TGFβ2-3 -UTR wild type (WT), TGFβ2-3 -UTR with trypsin to prepare into 3 × 10 cells/mL cell suspen- mutant type (MUT) luciferase plasmid and miR-7-5p sion. Cell suspension (2 mL) was seeded into 6-well plates mimic/negative control (NC) were constructed by for further culture. Te infection medium (Eni.S + poly- Shanghai GeneChem Co., Ltd (Shanghai, China). After brene) was renewed, and the cell density was 20%/well plasmid transfection, commonly used tool HEK293T at the time of infection. Te optimal amount (infection cells in the logarithmic growth phase were prepared into MOI = 10) was added for infection. Twelve hours after a suspension that was then cultured in a 24-well plate so infection, the conventional medium was renewed to con- as to achieve 85% confuence at 24 h. Four groups were tinue culture cells. used in the experiment (NC mimic + TGFβ2-WT, miR- Cell counting kit (CCK‑8) assay 7-5p mimic + TGFβ2-WT, NC mimic + TGFβ2-MUT, and miR-7-5p mimic + TGFβ2-MUT). After 48-h trans- Te 80% confuent cells were dispersed into single cell fection, the cells were lysed to obtain the supernatant suspension with the reaction of 0.25% trypsin. After that was centrifuged for 3–5 min. Te luciferase activ- counting, the cells were separately seeded in a 96-well ′ 4 ity of TGFβ2 3 UTR in response to miR-7-5p mimic was plate with 100 μL (2 × 10 cells/mL) per well, and set with detected by utilizing a Dual-Luciferase® Reporter Assay 6 duplicated wells. Cells were cultured in the incubator, System (E2910; Promega, Fitchburg, WI, USA). Te and 10 μL CCK-8 solutions (Sigma-Aldrich, St. Louis, luminescence intensity was measured by ­GloMax®20/20 MO, USA) were supplemented to each well after culture luminometer (Promega, Madison, WI, USA). for 24 h, 48 h, 72 h, and 96 h. After further 2-h culture, OD value at 450 nm was measured using an enzyme- Cell treatment and grouping linked immunometric meter. Cell viability curve was Te cells used for the cell phenotype detection were the drawn with time as X-axis and OD value as Y-axis. human highly metastatic OC cell line HO-8910PM, which has also been previously reported to establish a highly Transwell assay metastatic animal model [14, 15]. Besides, the human After transfection, cells were suspended in 0.25% trypsin normal ovarian cell line HUM-CELL-0088 was selected (C0205, Beyotime), followed by centrifugation at 400 rpm as a control. Both cell lines were purchased from Shang- for 5 min with the supernatant discarded. Subsequently, hai Institute of Biochemistry and Cell Biology (Shanghai, cells were resuspended in RPMI-1640 medium. Cell den- 5 China). HEK293T cells (used for dual-luciferase reporter sity was adjusted to 10­ cells/mL. Before the experiment, assay) were cultured in Dulbecco minimum essen- Transwell chambers (3413, Millipore, Massachusetts, tial medium (DMEM; 12800017; Gibco, Carlsbad, CA, USA) were coated with Matrigel. Te 100 μL cell suspen- USA) containing 10% fetal bovine serum (FBS; FBS500- sion was supplemented into the 24-well apical chamber 5 S; Ausgenex, Brisbane, Australia) in an incubator (BB15; ­(10 cells/well). RPMI-1640 medium (600 μL) contain- Termo Fisher Scientifc, Waltham, MA, USA) with satu- ing 30% FBS was added into the basolateral chamber. rated humidity and 5% CO­ 2 at 37 °C. HO-8910-PM cells Each group was set 3 duplicated wells. After 48-h culture, and HUM-CELL-0088 cells were cultured in RPMI-1640 cells not invading through Matrigel were discarded using medium (Gibco-BRL, Life Technologies, Gaithersburg, cotton swabs, and cells invading through Matrigel were MD, USA) containing 10% FBS in an incubator with stained with 0.1% Giemsa (32884, Sigma-Aldrich) for saturated humidity and 5% ­CO2 at 37 °C. Te medium 10 min. Te number of cells invading through Matrigel was renewed every 24 h, and subculture was performed was counted in fve random felds under the microscope. every 72 h. After removal of medium, cells were treated Te number of cells invading through Matrigel was con- with 0.25% trypsin for 3 min, and then the reaction was sidered as an indicator of the invasion ability. ended by adding RPMI-1640 medium containing 10% FBS. Afterwards, cells were pipetted into single cell sus- Scratch test pension. Ten HO-8910-PM cells were transduced with Even horizontal linear scratches were made in the back lentiviral vectors containing miR-7-5p mimic, NC mimic, of 6-well plate by a marker pen with 3 scratches in each miR-7-5p inhibitor, NC inhibitor, TGFβ2 overexpression well. Six pairs of cells at logarithmic growth phase were 5 plasmid (oe-TGFβ2) or oe-NC. Lentiviruses were pur- seeded in the culture plate (5 × 10 cells/well). Following chased from Shanghai Genechem Co., Ltd. (Shanghai, 24-h culture, when cells settled at the bottom of the well, China). Te negative control and miR-7-5p mimic and 200 μL pipette tips was adopted to make even vertical lin- inhibitor were obtained from GenePharma (Shanghai, ear scratches in the back of the plate with 3 scratches in China). Te sequence of the negative control, miR-7-5p each well. Afterwards, cell incubation was conducted in mimic and inhibitor were provided in Additional fle 1: RPMI-1640 medium with 5% CO­ 2 at 37 °C. Te migra- Table S1. Cells in logarithmic growth phase were treated tion distance was observed and photographed under Gu and Zhang Cancer Cell Int (2020) 20:517 Page 5 of 15

inverted microscope at 0 h and 24 h after scratch. Tree Table 2 Diferentially expressed miRNAs in metastatic replicates were made in each group. Te number of cells tissue compared with primary OC tissue passing through scratches was considered as an indicator Genes Metastatic Primary P value Fold change ΔCt ΔCt of the migration ability. (2­ − ) (2­ − ) miR-10a-5p 9.10E 03 3.20E 03 0.014557 2.85 Flow cytometry − − miR-15a-5p 2.90E 01 1.30E 01 0.008198 2.24 − − Overnight cell fxative was conducted in 70% chilled miR-16-5p 7.30E 01 4.00E 01 0.042838 1.84 − − ethanol at 4 °C. After centrifugation at 800 g and 4 °C miR-195-5p 1.00E 00 6.10E 01 0.047072 1.66 × + − with the removal of supernatant, cells were washed with miR-584-5p 5.40E 02 8.50E 02 0.032832 1.58 − − − phosphate bufer saline (PBS) containing 1% FBS. Cells miR-27a-3p 7.60E 01 4.90E 01 0.003931 1.54 − − were resuspended with 400 μL binding bufer, followed miR-141-3p 3.10E 02 1.70E 01 0.013728 5.64 − − − by 30-min incubation with 50 μL RNAase at 37 °C. After miR-187-5p 9.50E 02 1.80E 01 0.031732 1.90 − − − that, cells were added with 50 μL propidium iodide (PI) miR-200a-3p 5.60E 03 3.40E 02 0.023611 5.99 − − − (50 mg/L, Sigma-Aldrich) for 30-min incubation in dark. miR-200b-3p 1.20E 02 5.20E 02 0.022363 4.53 − − − Flow cytometry was used to detect cell cycle. miR-7-5p 2.20E 01 3.60E 01 0.020499 1.65 − − − miR-9-3p 1.90E 03 3.40E 03 0.040069 1.76 Statistical analysis − − − miR microRNA, OC ovarian cancer All data were summarized as mean ± standard deviation and analyzed using SPSS 21.0 statistical software (IBM Corp., Armonk, New York, USA). Data between two GO functional enrichment and Pathway analyses groups were compared using unpaired t-test. Statistical for the identifed diferentially expressed mRNAs analysis in relation to time-based measurements within To determine which mRNAs were involved in the regu- each group was realized using repeated measures analysis latory efect of miRNAs in metastatic OC, GO analysis of variance (ANOVA), followed by a Bonferroni’s post- (Fig. 2) was performed to investigate the function of 10 hoc test. Pearson correlation coefcient was adopted for diferentially expressed mRNAs. For Biological Processes, correlation analyses. Signifcance was defned as p < 0.05. 10 diferentially expressed mRNAs mostly participated Results in anatomical structure morphogenesis, embryo devel- opment, cell diferentiation and cellular developmental Diferentially expressed miRNAs and mRNAs processes (p < 0.001, Enrichment scores > 8; Table 4). For with metastatic OC cellular components, the diferentially expressed mRNAs Diferentially expressed miRNAs and mRNAs in meta- ™ mostly participated in rufe, adherens junction, anchor- static OC were screened using miRStar Human Cancer ing junction and cell junction (p < 0.001, Enrichment Focus miRNA and Target mRNA PCR Array analysis. As scores > 3.3; Table 5). For molecular functions, the dif- shown in Table 2, 12 diferentially expressed miRNAs ferentially expressed mRNAs mostly participated in RNA were screened out from metastatic and primary tissues of polymerase II distal enhancer sequence-specifc DNA OC (Fig. 1a): (in sequence of absolute fold change) miR- binding transcription factor activity involved in posi- 200a-3p, miR-141-3p, miR-200b-3p, miR-10a-5p, miR- tive regulation of transcription (p < 0.001, Enrichment 15a-5p, miR-187-5p, miR-16-5p, miR-9-3p, miR-195-5p, scores > 3; Table 6). Furthermore, the Pathway analysis miR-7-5p, miR-584-5p and miR-27a-3p (p < 0.05). Te based on KEGG method (Fig. 3) indicated that, among expression of miR-10a-5p, miR-15a-5p, miR-16-5p, miR- the 10 diferentially expressed mRNAs, ITGB3, TGFβ2 195-5p and miR-27a-3p in metastatic tissues from OC and TNC were linked to the pathway of MicroRNAs in was increased, as compared with primary OC tissues Cancer. (p < 0.05, fold change > 1.5), while that of miR-141-3p, miR-7-5p, miR-187-5p, miR-200a-3p, miR-200b-3p, miR-584-5p and miR-9-3p was lower than that in pri- The down‑regulation of miR‑7‑5p is the most signifcant mary OC tissues (p < 0.05, fold change < − 1.5). As shown in metastatic tissue of OC in Table 3, a total of 10 diferentially expressed target Next, we moved to assess expression of the screened dif- mRNAs were identifed. Relative to primary OC tissues, ferentially expressed miRNAs as mentioned above, so as the gene expression of BCL2L1, TGFβ2, TNC, HOXB5, to determine the research focus for this present study. As HOXB7, HOXB8, ITGB3 and MTSS1 in metastatic tis- illustrated in Fig. 4, when compared with primary OC tis- sues from OC was substantially up-regulated (p < 0.05, sues, there were 5 miRNAs miR-7-5p, miR-141-3p, miR- fold change > 2; Fig. 1b), while that of PAK1 and FOXA2 187-5p, miR-200a-3p and miR-200b-3p were appreciably was down-regulated (p < 0.05, fold change < − 2). Gu and Zhang Cancer Cell Int (2020) 20:517 Page 6 of 15

a Color Key b Color Key and Histogram and Histogram 6 Count Count 06 03 -2 02 -1 1 Row Z-Score Row Z-Score Primary Metastatic Primary Metastatic

hsa-miR-195-5p BCL2L1

hsa-miR-27a-3p HOXB8

hsa-miR-16-5p TNC hsa-miR-15a-5p

PAK1 hsa-miR-7-5p

hsa-miR-187-5p TGFβ2

hsa-miR-141-3p HOXB5

hsa-miR-200b-3p FOXA2

hsa-miR-200a-3p MTSS1 hsa-miR-10a-5p

ITGB3 hsa-miR-9-3p

HOXB7 hsa-miR-584-5p 1 2 3 4 5 6 A1 A2 A3 A4 A5 A6 B1 B2 B3 B4 B5 B6 A A A A A A B1 B2 B3 B4 B5 B6 Fig. 1 Diferentially expressed miRNAs and mRNAs with metastatic OC. a Heat map of OC-related diferentially expressed miRNAs in metastatic tissues and primary tissues of OC. b Heat map of OC-related diferentially expressed mRNAs in metastatic tissues and primary tissues of OC. The color red represents up-regulated expression and the color green represents down-regulated expression

Table 3 Diferentially expressed mRNAs in metastatic The up‑regulation of TGFβ2 is the most signifcant tissue compared with primary OC tissue in metastatic tissue of OC

ΔCt ΔCt Genes Metastatic (2­ − ) Primary (2­ − ) P value Fold change Further, we proceeded to probe the downstream tar- get genes of aforementioned miR-7-5p. A diferentially BCL2L1 4.70E 02 2.30E 02 0.047085 2.07 − − expressed miRNA-mRNA network (Fig. 5a) was plot- TGFβ2 2.00E 03 3.50E 04 0.017231 5.68 − − ted, where we found that the target genes of miR-7-5p TNC 5.20E 02 2.50E 03 0.006661 20.7 − − were TGFβ2 and HOXB5. Terefore, mRNA expres- HOXB5 6.10E 04 1.00E 04 0.035099 6.11 − − sion of TGFβ2 and HOXB5 was measured by RT- HOXB7 1.20E 02 2.50E 03 0.033375 4.9 − − qPCR (Fig. 5b, c). Relative to the primary OC tissues, HOXB8 4.10E 02 5.70E 03 0.046631 7.19 − − expression of TGFβ2 mRNA was enhanced in meta- ITGB3 5.60E 03 1.40E 03 0.021938 3.9 − − static tissues (p < 0.05), while no signifcant diference MTSS1 5.10E 03 1.00E 03 0.034451 5.08 − − was witnessed in the slightly up-regulated expression FOXA2 1.70E 05 1.10E 04 0.034065 6.48 − − − of HOXB5 mRNA (p > 0.05). Tis may be implicated PAK1 2.60E 03 5.40E 03 0.006704 2.04 − − − in factors, such as sample size and ethnic diferences. BCL2L1 B-cell CLL/lymphoma 2-like 1, TGFβ2 transforming growth factor beta In addition, western blot assay (Fig. 5d) further con- 2, TNC tenascin-C, HOX homeobox, ITGB3 integrin beta-3, MTSS1 metastasis suppressor 1, FOXA2 forkhead box A2, PAK1 p21-activated kinase, OC ovarian frmed that the protein level of TGFβ2 was elevated in cancer metastatic tissues versus that in primary OC tissues (p = 0.0014). Accordingly, we selected miR-7-5p and its downstream target TGFβ2 for further investigations. decreased in metastatic tissues, where miR-7-5p expres- sion presented with the most signifcant decrease. Tus, miR-7-5p was selected as a research focus for following experiments. Gu and Zhang Cancer Cell Int (2020) 20:517 Page 7 of 15

10

Biological Process Cellular Component 9 Molecular Function

8

7 )

6

5

4 Enrichment Score (-log10 (Pvalue) 3

2

1

0 ] ] ] ] ] ] ] ] ] ] ] ] ] 5] 9] 0] 5] 2] 29 09 29 53 4 792 0 29 7 6 90 8731 8 886 979 09 43 35 05 04 00 :00 :00 :00 :004 :004 O:0060 O O O:0009 O:0030175] GO:0 GO:0030154GO:0 [GO:000592 G t [G . [GO t[GOt [G t[ G [GO:00 n .. n n n nt[ n m[ ne[GO:0032587 sis[GOess[ e sis [G o u a tion[GO:0070161 me e di c me pm ne villus n p pme en roc e hesi po Ruffle [GO:0001726] lo lopme g ing edge[GO:0031252] binding [GO:0042802ranscr…[GO:0003700] og d n binding [GO:0043565] ve velo ve h ferentiation[ ho Micro Filo e rp ntal p if evelo membr lpha granule[GO:0031091]Cell junction[GO:0030054] g in birth or o rp cal ad a th factor re…[GO:0005172] de n ffle ll lea w e de m ll d Fo di b Ru Ce molecule binding[GO:0050839 lium stem d Ce re mo Adherens junction[GO:0005912] binding t en ryonieTu develop elopme u Anchoring ju BH domain binding [GO:0051400]A nt Sy ev Embryoct d Platelet N Tissue d u Epitbe Identical protei pme o ata enb lar llu evel ord Ce d Ch omical str Protein domain specificCell binding[GO:0019904] adhession Sequence-specific DNA scular endothelial gro polymerase polymerase ǁ distal ǁenhancer distal enhancer seq…[GO:0003705 seq…[GO:0001205 Anat A A mbryo Va Nucleic acid binding transcription fa…[GO:0001071] E Sequence-specificRN D RN Fig. 2 GO functional enrichment analysis for 10 diferentially expressed mRNAs. The DAVID database (https​://david​.ncifc​rf.gov/; version: 6.8) was used to perform GO analyses. The top 10 items of these enrichment analyses are conducted by using the “ggplot2” package in R software. P < 0.05 was considered statistically signifcant; DAVID database for annotation, visualization and integrated discovery, GO gene ontology. The top 10 items of GO categories including biological process (BP), cellular component (CC), and molecular function (MF) were then sorted and displayed in the form of maps

Table 4 Nodes in biological processes found by gene ontology GO.ID Term Count P value Enrichment score

GO:0009653 Anatomical structure morphogenesis 12 7.67E 11 10.11505 − GO:0009790 Embryo development 9 1.88E 09 8.72576 − GO:0030154 Cell diferentiation 12 4.76E 09 8.322078 − GO:0048869 Cellular developmental process 12 9.76E 09 8.010594 − GO:0048729 Tissue morphogenesis 7 2.67E 08 7.573257 − GO:0048731 System development 12 3.86E 08 7.412918 − GO:0035295 Tube development 7 5.16E 08 7.287302 − GO:0043009 Chordate embryonic development 7 6.66E 08 7.176411 − GO:0009792 Embryo development ending in birth or egg 7 7.21E 08 7.142 hatching − GO:0060429 Epithelium development 8 1.02E 07 6.989655 − Gu and Zhang Cancer Cell Int (2020) 20:517 Page 8 of 15

Table 5 Nodes in cellular components found by gene was diminished in response to miR-7-5p mimic and ontology elevated in response to miR-7-5p inhibitor in highly GO.ID Term Count P value Enrichment score metastatic HO-8910PM cells (Fig. 6c, d). As shown in Fig. 6c, we have also tested the transfection efciency of GO:0001726 Rufe 3 0.000125 3.901568 miR-7-5p inhibitor and miR-7-5p mimic. Besides, miR- GO:0005912 Adherens junc- 4 0.000167 3.777312 tion 7-5p expression was inversely correlated with TGFβ2 GO:0070161 anchoring junc- 4 0.000195 3.709575 expression in metastatic tissues from OC (Fig. 6e). tion Additionally, we also examined the expression of GO:0030054 Cell junction 5 0.000474 3.324369 miR-7-5p and TGFβ2 in normal ovarian cell line HUM- GO:0031091 Platelet alpha 2 0.000817 3.087852 CELL-0088 and highly metastatic OC cell line HO- granule 8910PM. Te RT-qPCR results (Fig. 6f) showed that, GO:0031252 Cell leading edge 3 0.001105 2.956574 compared with normal ovarian cells, miR-7-5p was GO:0030175 Filopodium 2 0.00113 2.946761 poorly expressed in highly metastatic OC cells, while GO:0032587 Rufe membrane 2 0.00113 2.946761 TGFβ2 was highly expressed in highly metastatic OC GO:0005902 Microvillus 2 0.001389 2.857181 cells. In conclusion, miR-7-5p directly bound to TGFβ2 GO:0005925 Focal adhesion 3 0.001841 2.73493 3′UTR and negatively regulated its expression.

Overexpression of miR‑7‑5p inhibits OC cell viability, miR‑7‑5p targets and inhibits TGFβ2 migration and invasion by targeting TGFβ2 We used the commonly used tool cell HEK293T cells After determining the negative correlation between miR- to conduct a dual luciferase reporter gene assay to 7-5p and TGFβ2, we shifted to validate their roles func- verify the targeting relationship between miR-7-5p tionally in malignant phenotypes of highly metastatic OC and TGFβ2. It was found that the luciferase activity cell line HO-8910PM by using CCK-8 assay (Figs. 7a, 8b), of TGFβ2-WT was reduced in response to miR-7-5p Scratch test (Fig. 7b), Transwell invasion assay (Figs. 7c, mimic transfection, while there was no signifcant dif- 8c), fow cytometric detection (Fig. 7d), Annexin V-APC ference in luciferase activity of TGFβ2-MUT (Fig. 6a, staining (Fig. 7e) and western blots (Fig. 7f). In the pres- b), suggesting that miR-7-5p could target TGFβ2. ence of miR-7-5p mimic, HO-8910PM cells presented Furthermore, we transfected miR-7-5p mimic or with attenuated viability (Fig. 7a), migration (Fig. 7b) miR-7-5p inhibitor into highly metastatic OC cell after 24 h (no signifcant diference observed at 8 h), and line HO-8910PM to validate its regulatory efect on invasion (Fig. 7c). Tese results indicated that up-regu- TGFβ2. As shown in Fig. 6c, we have also tested the lated miR-7-5p appreciably restricted the proliferative, transfection efciency of miR-7-5p inhibitor and miR- migratory and invasive capacities of OC cells in vitro. 7-5p mimic. Te RT-qPCR detection presented that Moreover, the quantitative fow cytometric analy- miR-7-5p expression was upregulated in the pres- sis (Fig. 7d) showed that the percentages of cells in ence of miR-7-5p mimic and downregulated in the G1 phase of the NC mimic group and the miR-7-5p presence of miR-7-5p inhibitor. In addition, Western mimic group were (63.62 ± 0.85)% and (68.32 ± 0.62)%, blot analysis manifested that protein levels of TGFβ2 respectively; the percentages of cells in S phase of

Table 6 Nodes in molecular functions found by gene ontology GO.ID Term Count P value Enrichment score

GO:0001205 RNA polymerase II distal enhancer sequence-specifc DNA binding transcription factor activity 2 0.000122 3.911881 involved in positive regulation of transcription GO:0003705 RNA polymerase II distal enhancer sequence-specifc DNA binding transcription factor activity 2 0.001717 2.765282 GO:0043565 Sequence-specifc DNA binding 4 0.001875 2.727084 GO:0003700 Sequence-specifc DNA binding transcription factor activity 4 0.006255 2.203762 GO:0001071 Nucleic acid binding transcription factor activity 4 0.006277 2.202281 GO:0042802 Identical protein binding 4 0.006603 2.180266 GO:0050839 Cell adhesion molecule binding 2 0.006752 2.170542 GO:0005172 Vascular endothelial growth factor receptor binding 1 0.007614 2.118391 GO:0051400 BH domain binding 1 0.007614 2.118391 GO:0019904 Protein domain specifc binding 3 0.008101 2.091458 Gu and Zhang Cancer Cell Int (2020) 20:517 Page 9 of 15

Sig pathway of DE gene

Focal adhesion [3 genes]

Proteoglycans in cancer [3 genes]

Malaria [2 genes]

Renal cell carcinoma [2 genes]

Pancreatic cancer [2 genes]

HTLV-l infection [3 genes]

Chronic myeloid leukemia [2 genes]

MicroRNAs in cancer [3 genes]

ECM-receptor interaction [2 genes]

Hypertrophic cardiomyopathy (HCM) [2 genes]

00.5 11.5 22.5 EnrichmentScore (-log10(Pvalue)) Fig. 3 Pathway analysis based on KEGG method for 10 diferentially expressed mRNAs. The DAVID database (https​://david​.ncifc​rf.gov/; version: 6.8) was used to perform KEGG analyses. The top 10 items of these enrichment analyses are conducted by using the “ggplot2” package in R software. P < 0.05 was considered statistically signifcant. KEGG: Kyoto Encyclopedia of Genes and Genomes; DAVID: database for annotation, visualization and integrated discovery

* * * 2.0 8 2.0 levels levels 1.5 6 levels 1.5 on on on si si si 1.0 4 1.0 pres pres pres miR-7-5p miR-141-3p ex miR-187-5p ex ex of of 0.5 2 of 0.5 relative relative relative 0.0 0 0.0 primary metastasis primary metastasis primary metastasis

ns * * 1.2 0.7 2.2

1.1 2.0 p levels levels levels 0.6 1.0 on on on 1.8 si essi essi 0.9 0.5 pr pr pres

miR-9-3p 1.6 ex ex ex miR-200b-3 miR-200a-3p

of 0.8 ve of of 0.4 ti 1.4 la 0.7 lative re relative re 0.6 0.3 1.2 primary metastasis primary metastasis primarymetastasis Fig. 4 The expression of the 6 identifed down-regulated miRNAs (miR-7-5p, miR-187-5p, miR-141-3p, miR-200a-3p, miR-200b-3p, and miR-9-3p) in metastatic tissues and primary tissues of OC as detected by RT-qPCR. Relative expression of miRNA was measurement data and analyzed by unpaired t test. N 25. *p < 0.05 compared with primary OC tissues. ns no signifcance = Gu and Zhang Cancer Cell Int (2020) 20:517 Page 10 of 15

P = 0.3427 a miR-187-5p b c

miR-7-5p P = 0.0065 0.005 0.10

HOXB5 TNC 0.004 0.09 TGFβ2 5 0.003 0.08 HO XB of of TGFβ 2 0.002 0.07

miR-141-3p 0.001 0.06 Relative expression levels miR-200a-3p Relative expression levels MTSS1 0.000 0.05 Primary Metastasis Primary Metastasis

P = 0.0014 d TGFβ2 50 KDa 2.0

n 1.5 β-actin 43 KDa

1.0 s

Primary

TGFβ2/β-acti 0.5 Metastasi

0.0 Primary Metastasis Fig. 5 The up-regulation of TGFβ2 is the most signifcant in metastatic tissue of OC. a Diferentially expressed miRNA-mRNA network. b, c The mRNA expression of TGFβ2 (b) and HOXB5 (c) in metastatic tissues and primary tissues of OC as measured by RT-qPCR. d Western blots and quantitation of protein level of TGFβ2 in metastatic tissues and primary tissues of OC. Relative expression of miRNA and protein was measurement data and analyzed by unpaired t test. N 25. *p < 0.05 compared with primary OC tissues = the NC mimic group and the miR-7-5p mimic group Additionally, we conducted rescue experiments (Fig. 8) were (15.82 ± 0.79)% and (14.12 ± 0.52)%, respec- on HO-8910PM cells overexpressing miR-7-5p and tively; and the percentages of cells in G2/M phase of TGFβ2 simultaneously. Firstly, western blot analysis the NC mimic group and the miR-7-5p mimic group (Fig. 8a) suggested that expression of TGFβ2 protein was were (20.56 ± 0.11)% and (17.57 ± 0.11)%, respectively. elevated in response to oe-TGFβ2 alone and reduced to Accordingly, the miR-7-5p mimic treatment prolonged miR-7-5p mimic alone, while the combined treatment G1 phase (increased cell percentage) and shortened S of oe-TGFβ2 and miR-7-5p mimic rescued its expres- and G2/M phases (reduced cell percentage). Tese data sion. Furthermore, miR-7-5p mimic alone diminished suggested that up-regulated miR-7-5p blocked OC cell viability and invasion of HO-8910PM cells, while TGFβ2 cycle progression. overexpression alone facilitated viability and invasion of In terms of cell apoptosis detected by Annexin V-APC HO-8910PM cells. However, simultaneous miR-7-5p and staining method (Fig. 7e), the apoptosis rate of HO- TGFβ2 overexpression rescued cell viability and invasion 8910PM cells in the NC mimic group and miR-7-5p in response to miR-7-5p mimic or TGFβ2 overexpression mimic group was lower than 5%, and there was no sig- alone (Fig. 8b, c). nifcant diference in the apoptosis rate between the two groups. Western blot assay further verifed the Discussion efect of miR-7-5p mimic on cyclin D1 protein expres- MiRNAs confer pivotal roles in multiple cellular func- sion and caspase-3 activity in HO-8910PM cells. Te tions, such as proliferation, apoptosis and diferentiation. results showed that overexpression of miR-7-5p up-reg- However, aberrant expression of miRNAs appears in car- ulated cyclin D1 protein expression, but had no efect cinogenesis and metastasis during cancer progression on caspase-3 (Fig. 7f). Moreover, as shown in Fig. 7g, [16]. Mounting evidence highlights a crucial role of miR- HO-8910PM cells transfected with miR-7-5p mimic pre- NAs in OC progression. For instance, alterations in miR- sented with elevated expression of E-cadherin and low- 101, miR-206, miR-200a and miR-203 are involved with ered expression of Vimentin. Tis result indicated that OC cell proliferation and invasion [17–19]. Terefore, overexpression of miR-7-5p suppressed epithelial–mes- this current study presented diferentially expressed miR- enchymal transition (EMT) of highly metastatic OC cell NAs and mRNAs related to metastatic OC and revealed line HO-8910PM. a tumor-inhibiting role of miR-7-5p in the restriction of Gu and Zhang Cancer Cell Int (2020) 20:517 Page 11 of 15

a b NC mimic 1.5 miR-7-5p mimic 2D Structure Local AU < 0.0001 2180 2202 Predicted By M T 8mer 1.0 5’ -g ttgattATTTTTTTGTCTTCCa- 3’ UTR

GTCTTCCA hsa-miR-7-5p 3’ -uguuguuUUAGUGAUCAGAAGGu- 5’ miRNA 0.5 16 15 14 13 76 5 432 8mer

3’pairing Seed Relative luciferase activity 0.0 c d

4 1.5 *** 2.5 * 7-5p 50 KDa R- * 2.0 mi 3

of 1.0 43 kDa 1.5 2 ic r * m 1.0 pression mi hibitor 0.5 mimic ex 1 in NC -5p 0.5 NC inhibito7 tive iR-7-5p m 0 miR- 0.0 0.0 Rela c c NC mimicmiR-7-5p mimic NC inhibitor miR-7-5p inhibitor

mimi 1.5 2.5 * NC mimi e f * NC inhibitor 0.006 r = -0.5246 2.0 miR-7-5p miR-7-5p miR-7-5p inhibitor P = 0.0071 of 1.0 0.004 1.5

1.0 0.002 0.5 expression 0.5 0.000 0.0 0.0 0.51.0 1.52.0 2.5 Relative HUM-CELL-0088HO-8910PM HUM-CELL-0088HO-8910PM -0.002 miR-7-5p Fig. 6 MiR-7-5p targets and inhibits TGFβ2 expression. a Predicted binding sites between miR-7-5p on TGFβ2 3′-UTR. b Dual luciferase reporter assay verifying the binding of miR-7-5p to TGFβ2 in commonly used tool HEK293T cells. c RT-qPCR detection (left) of relative expression of miR-7-5p and Western blots (right) of protein level of TGFβ2 in highly metastatic HO-8910PM cells in the presence of miR-7-5p mimic or inhibitor. d The mRNA expression of TGFβ2 in highly metastatic HO-8910PM cells in the presence of miR-7-5p mimic or inhibitor as measured by RT-qPCR. e The negative correlation between miR-7-5p and TGFβ2. f The expression of miR-7-5p and TGFβ2 in normal ovarian cell line HUM-CELL-0088 and highly metastatic OC cell line HO-8910PM as measured by RT-qPCR. Relative expression of miRNA and protein was measurement data and analyzed by unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with NC mimic group or HUM-CELL-0088 cells. The cell experiment was repeated three times

OC metastasis, which was associated with the regulation expressed in adult and juvenile granulosa cell tumors, of TGFβ2. and that these could be used as markers for the diagnosis Diferent types of cancer samples show varying gene and recurrence of ovarian granulosa cell tumors. expression patterns, and even the same miRNA often Te high-throughput sequencing screening of this regulate various target genes in diferent types of cancers. study found 12 diferentially expressed miRNAs (miR- In studies on OC, the application of a miRNA expression 195-5p, miR-27a-3p, miR-16-5p, miR-15a-5p, miR- profling technique is helpful in identifying diferentially 10a-5p, miR-584-5p, miR-7-5p, miR-187-5p, miR-141-3p, expressed miRNAs afecting malignant phenotypes and miR-200b-3p, miR-200a-3p and miR-9-3p) and 10 dif- metastasis in OC in a more rapid and comprehensive ferentially expressed mRNAs (BCL2L1, TGFβ2, TNC, manner. In the present study, we analyzed the miRNA HOXB5, HOXB7, HOXB8, ITGB3, MTSS1, PAK1 and expression profles of primary OC tissues and metastatic FOXA2) in OC. GO analysis and Pathway analysis fnally tissues using a miRStar™ Human Cancer Focus miRNA determined that 3 diferentially expressed mRNAs and Target mRNA PCR Array expression profling. With (ITGB3, TGFβ2 and TNC) were related to OC metasta- the application of a miRCURY LNA™ microRNA Array sis. Te results of RT-qPCR confrmed that the decrease expression profling technique, Liu et al. [20] identifed of miR-7-5p was most signifcant in OC metastases, 31 diferentially expressed miRNAs from another two while TGFβ2 was signifcantly up-regulated in OC metas- pairs of cisplatin-resistant OC cell lines, of which the tases. Terefore, miR-7-5p and its downstream target expression of 21 miRNAs was up-regulated and that of gene TGFβ2 were selected as research focuses. A wide 10 miRNAs was down-regulated. Moreover, Cheng et al. array of miRNAs have been reported to present difer- [21] found by using an Afymetrix miRNA 3.0 Array ential expression in OC [22]. For instance, miR-187 has expression profling that 37 miRNAs were diferentially been found to exert a dual role in OC by regulating the Gu and Zhang Cancer Cell Int (2020) 20:517 Page 12 of 15

abNC-mimicmiR-7-5p mimic NC-mimic 0.5 NC-mimic 0.8 0 h miR-7-5p mimic miR-7-5p mimic

) 0.4 250μm 250μm ) 0.6

0.3 * 0.4 8 h 0.2

250μm 250μm 0.2 0.1 Migration rate (% OD Value (450 nm

0.0 0.0 24 h 24 h 48 h72 h 96 h 8 h 24 h

250μm 250μm

c NC-mimicmiR-7-5p mimic d

300 ) 80

s ** NC-mimic 60 miR-7-5p mimic 200

*** 40

100 20 * ** Number of invasive cell Percentage of cell cycle (% 0 0 NC-mimicmiR-7-5p mimic GS1 G2/M e f 6 2.0 Cyclin D1 33 kDa * NC mimic 1.5

%) 4 Caspase-3 32 kDa miR-7-5p mimic expression e(

at 1.0 sr β-actin 43 kDa

2 protein 0.5 ic ic im tive la Apoptosi m

NC mim 5p Re 0 -7- 0.0 R NC-mimic miR-7-5p mimic mi Cyclin D1 Caspase-3 3 n g E-cadherin 135 kDa * NC mimic

Vimentin 54 kDa xpressio 2 miR-7-5p mimic ne

β-actin 43 kDa protei 1 c c

i i tive m * mi mim la

5p Re NC 0 R-7- mi E-cadherin Vimentin Fig. 7 Overexpression of miR-7-5p inhibits OC cell viability, migration and invasion. a The viability of HO-8910PM cells after miR-7-5p overexpression as determined by CCK-8. b The migration of HO-8910PM cells after miR-7-5p overexpression as measured by Scratch test. c The invasion of HO-8910PM cells after miR-7-5p overexpression as determined by Transwell assay. d The cell cycle distribution of HO-8910PM cells after miR-7-5p overexpression as determined by fow cytometry. e The cell apoptosis rate of HO-8910PM cells after miR-7-5p overexpression as determined by Annexin V-APC staining. f Western blots and quantitation of protein level of cyclin D1 and caspase-3 in HO-8910PM cells after miR-7-5p overexpression. g Western blots and quantitation of protein level of epithelial–mesenchymal transition-related factors (E-cadherin and Vimentin) in HO-8910PM cells after miR-7-5p overexpression. Measurement data were presented as mean standard deviation, analyzed ± by unpaired t test. Statistical analysis in relation to time-based measurements within each group was realized using repeated measures ANOVA, followed by a Bonferroni’s post-hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 compared with NC mimic group. The cell experiment was repeated three times disabled homolog-2 gene [23]. Gao et al. [24] also found in silico prediction results. Of interest, underexpressed that miR-141 acted as a potential diagnostic and prog- miR-7-5p has been documented in metastatic breast nostic biomarker for OC, which was consistent with our cancer [25] and invasive pancreatic cancer [26]. Another Gu and Zhang Cancer Cell Int (2020) 20:517 Page 13 of 15

a b 2.0 TGFβ2 50 KDa oe-NC ) β-actin 43 KDa 1.5 oe-TGFB2 *# c c 2 NC-mimic

1.0 * oe-NC # miR-7-5p mimic NC mimi oe-TGFβ2 miR-7-5p mimic + oe-TGFβ2 miR-7-5p mimi 0.5 OD value (450 nm

0.0 miR-7-5p mimic + oe-TGFβ 24 h48 h72 h 96 h

c oe-NC oe-TGFβ2 NC mimic #

400 *

s

l

l e

c 300

e

v * #

i

s a

v 200

n

i

f

o

r

e 100

b

m u 100μm 100μm 100μm N 0

miR-7-5p mimic miR-7-5p mimic + oe-TGFβ2 oe-NC oe-TGFβ2NC mimic

miR-7-5p mimic

miR-7-5p mimic + oe-TGFβ2

100μm 100μm Fig. 8 Overexpression of miR-7-5p inhibits OC cell viability and invasion by targeting TGFβ2. a miR-7-5p overexpression and TGFβ2 overexpression alone or in combination. b The viability of HO-8910PM cells in response to miR-7-5p overexpression and TGFβ2 overexpression alone or in combination as determined by CCK-8. c The invasion of HO-8910PM cells in response to miR-7-5p overexpression and TGFβ2 overexpression alone or in combination as determined by Transwell assay. *p < 0.05 compared with NC mimic group or oe-NC group. #p < 0.05 compared with miR-7-5p mimic group or oe-TGFβ2 group. Data between two groups were analyzed using unpaired t test. The cell experiment was repeated three times

study also claimed that miR-7-5p defciency was associ- miR-7-5p was verifed using a luciferase activity assay of ated with recurrence in glioblastoma patients, and that the present study. Moreover, there was a negative corre- its overexpression decreased glioblastoma cell stemness lation between miR-7-5p expression and TGFβ2 expres- [27]. Furthermore, it was also elucidated that ectopic sion in OC tissues, and miR-7-5p targeted and negatively expression of miR-7 functioned as an anti-oncogene in regulated TGFβ2. MiR-7-5p has been found to target OC by repressing cell invasion and proliferation [11]. and regulate target genes as SATB1 and PARP1 in some Recently, a growing number of studies elucidated that malignancies [31, 32]. For instance, Zhu et al. [33] dem- miRNAs are implicated in the invasive and metastatic onstrated that miR-7-5p suppresses cell migratory and potential of tumors. One miRNA can regulate nearly invasive potential by targeting SOX18 in pancreatic 100 diferent mRNAs, and a single mRNA can bind to ductal adenocarcinoma. several diferent miRNAs, thus forming a miRNA-target In addition, through gain- and loss-of-function gene network regulatory relationship [28, 29]. Of note, experiments in highly metastatic OC cells, our results miRNAs can post-transcriptionally mediate a number of revealed that miR-7-5p overexpression reduced OC genes through the binding of specifc sequences in tar- cell viability and invasion, and blocked cell cycle entry get mRNA molecules [30]. TGFβ2 as a putative target of by targeting TGFβ2. In addition, we found that OC Gu and Zhang Cancer Cell Int (2020) 20:517 Page 14 of 15

metastatic tissues presented higher levels of TGFβ2 Acknowledgements We acknowledge and appreciate our colleagues for their valuable eforts and relative to primary OC tissues. TGFβ2 is an isoform of comments on this paper. TGFβ in mammals [34] and is abnormally expressed in various cancers, such as human melanoma and hepato- Authors’ contributions SLZ designed the study. YG conducted the experiments and wrote the paper. cellular carcinoma [35, 36]. Furthermore, TGFβ2 and SLZ collected the data, and prepared all tables. Both authors reviewed the the TGFβ pathway could interact with miRNAs, such as manuscript. Both authors read and approved the fnal manuscript. miR-187 and miR-141 identifed in this study, to afect Funding growth of tumors. Ectopic miR-187 expression could This study was supported by the Free Researcher Project of Shengjing Hospital negate the contribution of TGFβ to aggressiveness in (Grant No. 201302). metastatic colorectal cancer [37]. Besides, the interplay Availability of data and materials of miR-141 and oncogenic TGFβ2 was found to orches- The datasets generated and/or analyzed during the current study are available trate malignancy in gastric cancer [38]. Additionally, from the corresponding author on reasonable request. many researchers have also explored the role of TGFβ Ethics approval and consent to participate in OC. Cao et al. [39] reported that TGFβ-induced Data and tissues were harvested upon receiving the informed consent of transglutaminase accelerated EMT and a cancer stem patients and approval by the ethics committee of Shengjing Hospital of China cell phenotype that consequently enhanced ovarian Medical University (Approval number: 2016PS040K). tumor metastasis. TGFβ induced EMT and a more Consent for publication invasive phenotype in epithelial OC cells in collabora- Not applicable. tion with the EGF pathway, indicating TGFβ may be a Competing interests promising target candidate for the treatment of meta- The authors declare that they have no competing interests. static OC in future [40]. Received: 5 February 2020 Accepted: 12 October 2020

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