MOLECULAR MEDICINE REPORTS 21: 209-219, 2020 miR‑218 functions as a tumor suppressor gene in cervical cancer ZHEN LIU1, LIN MAO2, LINLIN WANG1, HONG ZHANG2 and XIAOXIA HU1 1Department of Gynecology, The People's Hospital of Guangxi Zhuang Autonomous Region, Nanning, Guangxi 530021; 2Department of Gynecology, Renmin Hospital of Wuhan University, Wuhan, Hubei 430060, P.R. China Received October 23, 2018; Accepted April 12, 2019 DOI: 10.3892/mmr.2019.10809 Abstract. Previous microRNA (miR) microarray analysis in regulating cell proliferation, adhesion and migration, and revealed that miR‑218 is downregulated in cervical cancer the cell cycle. In conclusion, the findings of the present study tissues. The present study aimed to further evaluate the suggested that miR‑218 may possess antitumor activities in expression of miR‑218 in cervical cancer specimens, deter- cervical cancer. mine the association between its expression with disease progression, and investigate the roles of miR‑218 in cervical Introduction cancer cells. Tissue specimens were obtained from 80 patients with cervical squamous cell carcinoma, 30 patients with Cervical cancer remains a significant global health problem, high-grade cervical intraepithelial neoplasia [(CIN) II/III] and particularly in developing countries, where cervical cancer is 15 patients with low-grade CIN (CINI); in addition, 60 plasma a leading cause of cancer-associated death in women (1). The samples were obtained from patients with cervical cancer, and risk factors for cervical cancer development primarily include 15 normal cervical tissue specimens and 30 plasma samples human papillomavirus (HPV) infection (accounting for ~90% were obtained from healthy women. These samples were used of cervical cancer cases), tobacco smoking, long-term use of for analysis of miR‑218 expression via reverse transcription‑­ oral contraceptives, multiple pregnancies, and genetic and quantitative PCR. In addition, tumor cells were transfected immunological factors (2-4). The treatment of cervical cancer with miR‑218 mimics, human papillomavirus (HPV)16 E6/E7 depends on the stage; early-stage cancer can be effectively small interfering RNA, or their respective negative controls treated with surgery, whereas late-stage cancer can only be to determine the viability, colony formation, migration and managed by surgery plus radiotherapy and cisplatin-based invasion of cells using MTT, colony formation, wound healing chemotherapy, with variable effectiveness (5-7). The prognosis and Transwell assays, respectively. Target genes of miR‑218 of patients with cervical cancer depends on the tumor stage were bioinformatically predicted and analyzed using Gene and treatment effectiveness. For example, the 5-year overall Ontology (GO) terms. The results revealed that miR‑218 was survival of patients with early-stage cervical cancer can reach downregulated in the tumor tissues and plasma of patients with 92%, whereas the rate for late-stage disease, such as stage IV, cervical cancer, with expression associated with the advanced is <15% (8). Thus, prevention, early diagnosis and novel treat- clinicopathological characteristics of patients, including HPV ment strategies are required to successfully control cervical positivity, tumor size, blood vessel invasion and lymph node cancer. Therefore, improved understanding of the underlying metastasis. Furthermore, miR‑218 overexpression reduced molecular mechanisms and associated gene alterations in the tumor cell viability and xenograft growth, and suppressed pathogenesis of cervical cancer may lead to the development tumor cell migration and invasion. HPV was detected in of novel approaches for the prevention, early detection and 75% of the 80 patients with cervical cancer, and HPV posi- treatment of patients with cervical cancer. tivity was inversely associated with miR‑218 expression. In MicroRNAs (miRNAs/miRs) are a class of noncoding RNA addition, bioinformatics analysis predicted that roundabout in the human genome, which are 22-25 nucleotides in length. guidance receptor 1 (ROBO1) was a target gene of miR‑218; miRNAs function to regulate gene expression, primarily via miR‑218 overexpression significantly reduced ROBO1 levels. binding to the 3'-untranslated region of target mRNAs; upon Furthermore, GO analysis revealed that ROBO1 was involved binding, miRNAs can degrade the target mRNA transcripts and/or inhibit their translation into protein. Furthermore, miRNAs can act as oncogenes or tumor suppressors in human tumorigenesis (9,10). At present, studies have reported that Correspondence to: Dr Xiaoxia Hu, Department of Gynecology, cancer cells express specific miRNA profiles, and that HPV is The People's Hospital of Guangxi Zhuang Autonomous Region, able to modulate miRNA expression in infected cells (11,12). 6 Taoyuan Road, Nanning, Guangxi 530021, P.R. China In cervical cancer, a number of studies have detected altered E‑mail: [email protected] expression of miRNAs, and their functions in suppressing or promoting cervical epithelial cell carcinogenesis (13-19). In Key words: cervical cancer, microRNA, miR‑218, biomarker our previous miRNA microarray study, it was observed that miR‑218 expression is decreased or absent in cervical cancer tissues compared with normal epithelia (20). 210 LIU et al: miR-218 IS A TUMOR SUPPRESSOR IN CERVICAL CANCER Human genome database data has localized the miR‑218 reagent (Invitrogen; Thermo Fisher Scientific, Inc.) according gene to two different human chromosomes (miR‑218-1 at to the manufacturer's protocols. The RNA samples were stored chromosome 4p15.31, and miR‑218-2 at chromosome 5q35.1), at ‑80˚C until required. which are embedded in the intron region of Slit homolog 2 For RT-qPCR analysis, RNA samples were reverse tran- (SLIT2) and SLIT3, respectively (21). Furthermore, previous scribed into cDNA using a Quantscript RT kit (Tiangen Biotech studies have demonstrated that miR‑218 is able to suppress the Co., Ltd.) according to the manufacturer's protocols. Each RT progression of various types of human cancer by targeting the reaction was conducted in a mixture containing 150 ng RNA expression of roundabout guidance receptor 1 (ROBO1) (21-24). and a pool of RT primers from the kit in accordance with the Therefore, in the present study, miR‑218 expression was manufacturer's instructions. The RT primer sequences were as detected in normal, precancerous and cancerous tissues, follows: miR‑218, 5'‑CTC​AAC​TGG TGT CGT GGA​GTC​GGC​ and in plasma obtained from patients with cervical cancer. AAT TCA​GTT GAG​AGC​TAT GC‑3'; U6, 5'‑AAC​GCT TCA​ Subsequently, the association between miR‑218 expression CGA​ATT TGC​GT-3'; and Caenorhabditis elegans miR‑39 and various clinicopathological features was determined. The stem-loop (cel-miR‑39), 5'‑CTC​AAC​TGG TGT CGT GGA​GTC​ role of miR‑218 in regulating cervical cancer cell proliferation, GGC​AAT TCA​GTT GAG CTA​TGC‑3'. Subsequently, qPCR migration and invasion, and the underlying molecular events, was performed using a SuperReal PreMix Plus kit (SYBR® were also investigated. Green; Tiangen Biotech Co. Ltd.) and an ABI7500 thermocy- cler (Applied Biosystems; Thermo Fisher Scientific, Inc.). The Materials and methods qPCR primers were miR‑218, 5'‑CGA​GTG​CAT TTG TGC​TTG ATC​TA‑3' and 5'-TGG TGT CGT GGA​GTC​G-3';U6, 5'‑CTC​ Patients and samples. The present study collected 140 cervical GCT TCG GCA​GCA​CA‑3' and 5‑AAC​GCT TCA​CGA​ATT tissues samples from patients who received medical care in the TGC​GT-3'; cel-miR‑39, 5'‑ACA​CTC​CAG CTG GGT GGC​AAG Department of Gynecology, The People's Hospital of Guangxi ATG CTG GC‑3' and 5'-TGG TGT CGT GGA​GTC​G-3'; ROBO1, Zhuang autonomous region between January 2011 and January 5'-TCC​ACA​CAG​CAA​TAG CGA​AG-3', and 5'‑CCT GTA​ACA​ 2015. These specimens included four groups of patients: i) Tissue TGG GCT GGA​GT-3'; and GAPDH, 5'‑CAT GAG​AAG TAT specimens obtained from 80 patients with cervical squamous GAC​AAC​AGC​CT-3' and 5'‑AGT​CCT TCC​ACG ATA​CCA​AAG cell carcinoma (SCC); ii) 30 patients with high-grade squa- T-3'. All primers were designed and synthesized by Invitrogen mous intraepithelial lesions (HSIL) or cervical intraepithelial (Shanghai, China). The qPCR conditions were: Following an neoplasia (CIN)II/III, and 15 patients with low-grade squamous initial denaturation step at 95˚C for 15 min, amplifications were intraepithelial lesions (LSIL) or CINI; iii) Plasma specimens carried out with 40 cycles of a melting temperature of 95˚C obtained from 60 patients with pathologically diagnosed for 10 sec and an annealing temperature of 60˚C for 32 sec. cervical cancer (Stage IB1‑IIA); and iv) control cervical tissue The relative mRNA expression levels were calculated using samples collected from 15 patients who underwent hysterec- the 2-ΔΔCq method (27) following normalization to GAPDH or tomy due to benign gynecological diseases, and plasma samples U6 mRNA. Cel-miR‑39 was used as an exogenous control for obtained from 30 healthy females who visited the hospital for a plasma miR‑218, according to a previous study (28). routine health check-up, and had no cervical lesions or history of malignancy. All diagnoses of these patients were pathologically Detection of HPV‑DNA and HPV infection status. Genomic confirmed. None of the patients with cervical cancer and CIN DNA was extracted from tissue samples using a TIANamp had received any prior treatment intervention, including radio- FFPE DNA kit (Tiangen Biotech Co., Ltd.), in order to genotype therapy, chemotherapy,