ONCOLOGY REPORTS 45: 95-106, 2021 Erb‑B2 Receptor Tyrosine Kinase 2 is negatively regulated by the p53‑responsive microRNA‑3184‑5p in cervical cancer cells HONGLI LIU1, YUZHI LI1, JING ZHANG1, NAN WU2, FEI LIU2, LIHUA WANG1, YUAN ZHANG1, JING LIU1, XUAN ZHANG3, SUYANG GUO1 and HONGTAO WANG4 Departments of 1Gynecological Oncology and 2Respiration and Anhui Clinical and Preclinical Key Laboratory of Respiratory Disease, First Affiliated Hospital of Bengbu Medical College; Departments of3 Gynecological Oncology and 4Immunology and Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu, Anhui 233030, P.R. China Received November 30, 2019; Accepted October 2, 2020 DOI: 10.3892/or.2020.7862 Abstract. The oncogenic role of Erb-B2 Receptor Tyrosine Introduction Kinase 2 (ERBB2) has been identified in several types of cancer, but less is known on its function and mechanism of Among women, cervical cancer is ranked 4th in global action in cervical cancer cells. The present study employed cancer-associated deaths (1), with over half a million deaths a multipronged approach to investigate the role of ERBB2 in in 2012 (2). Cervical cancer can be broadly categorized into cervical cancer. ERBB2 and microRNA (miR)-3184-5p expres- squamous cell carcinoma, which constitutes the majority of sion was assessed in patient-derived cervical cancer biopsy cases (70-80%) or adenocarcinoma, which comprises 10-15% tissues, revealing that higher levels of ERBB2 and lower levels of cases (3). Cervical cancer is frequently caused by the of miR-3184-5p were associated with clinicopathological indi- oncovirus human papillomavirus (HPV), mainly by types cators of cervical cancer progression. Furthermore, ERBB2 HPV-16 and -18 in ~70% of patients with cervical cancer (3). stimulated proliferation, migration and sphere-formation of The molecular drivers of the formation and development of cervical cancer cells in vitro. This effect was mediated by cervical cancer are often due to recurrent mutations within enhanced phosphatidylinositol-4,5-bisphosphate 3-kinase the PI3K/AKT signaling pathway (4-7) or from deactivating, catalytic subunit α activity. Additionally, it was revealed that loss-of-function mutations in the tumor suppressor TP53 miR-3184-5p directly suppressed ERBB2 in cervical cancer gene (p53 protein), which produces a particularly aggres- cells. The p53 activator Mithramycin A stimulated p53 and sive phenotype (8). Additionally, altered expression levels miR-3184-5p expression, thereby lowering the levels of ERBB2 of microRNAs (miRNAs/miRs), which are short, regula- and attenuating proliferation, migration and sphere-formation tory, non-coding RNAs, have been implicated in metastatic of cervical cancer cells. In conclusion, the findings of the cervical cancer (9,10). Among miRNAs associated with present study suggested ERBB2 as an oncogenic protein that cervical cancer (11-13), miR-3184 has been identified as a may promote invasiveness in cervical cancer cells. Treatment p53-responsive miRNA (14). However, its mechanistic role in of cervical cancer cells with the p53 activator Mithramycin cervical oncogenesis remains unknown. A restored the levels of the endogenous ERBB2 inhibitor Erb-B2 Receptor Tyrosine Kinase 2 (ERBB2), also known miR-3184-5p and may represent a novel treatment strategy for as human epidermal growth factor receptor 2, is an established cervical cancer. oncogenic protein associated with tumorigenesis, cancer progression and acquisition of therapy resistance in breast, esophagogastric and colorectal cancer (15-17). However, the involvement of ERBB2 in cervical cancer development is less well known (18). Mithramycin A is an antibacterial and anticancer agent that was first purified from Streptomyces bacteria (19-21). It binds to G/C-rich stretches of DNA within minor grooves (19), which is one of its possible mechanisms for its antitumor properties (22). Mithramycin A possesses a Correspondence to: Mr. Hongtao Wang, Department of Immunology and Anhui Key Laboratory of Infection and favorable safety profile and inhibits tumor proliferationin vivo Immunity, Bengbu Medical College, 2600 Donghai Road, Bengbu, in cervical cancer murine xenograft models (23). Another Anhui 233030, P.R. China possible mechanism for the antitumor effects of Mithramycin E-mail: [email protected] A is via p53 activation (24); however, in cervical cancer, the influence of Mithramycin A on p53 and ERBB2 has not been Key words: cervical cancer, Erb-B2 Receptor Tyrosine Kinase 2, examined. microRNA-3184-5p, p53 Phosphatidylinositol-4,5-bisphosphate 3-kinase catalytic subunit α (PIK3CA) is the catalytic subunit of PI3K (25). In humans, PIK3CA acts as a proto-oncogene that supports 96 LIU et al: ONCOGENIC ERBB2 REGULATED BY p53/miR-3184-5p AXIS proliferation and metastasis of tumor cells (25), and PIK3CA plasmid vector (cat. no. PCMV6XL5) were obtained from copy number gains are associated with higher-grade tumors OriGene Technologies, Inc. For plasmid transfections, cells and poor patient prognosis in several malignancies, including were seeded in 6-well plates (1.5x105 cells/well) and tran- gastric, thyroid and prostate cancer (25). In patients with siently transfected with 1 µg plasmid using Lipofectamine® cervical cancer, PIK3CA mutations are significantly associ- 3000 transfection reagent (Invitrogen; Thermo Fisher ated with shorter survival times (PIK3CA mutant median Scientific, Inc.) for 20 min at room temperature and then survival of 67.1 months vs. non-mutant median survival of incubated in fresh medium at 37˚C for an additional 48 h 90.3 months) (26). The aforementioned evidence suggests prior to subsequent experimentation. siRNAs targeting human that targeting PIK3CA activity may be a rational approach ERBB2 (ERBB2-siRNA; cat. no. sc-29405), human PIK3CA in combating cervical cancer. Therefore, the present study (PIK3CA-siRNA; cat. no. sc-39127) and scrambled control aimed to examine the association between the effects of the (scr-siRNA; cat. no. sc-37007) were obtained from Santa p53-responsive miR-3184-5p, ERBB2 and PIK3CA in cervical Cruz Biotechnology, Inc. For siRNA transfections, cells were cancer. seeded in 6-well plates (2x105 cells/well) and transiently transfected with 80 pmol siRNA using Lipofectamine 3000 Materials and methods transfection reagent for 7 h at 37˚C and then incubated in fresh medium at 37˚C for an additional 48 h prior to subse- Patient samples. The present study was approved by the quent experimentation. miR-3184-5p (miRBase accession no. Ethics Review Committee of the First Affiliated Hospital MIMAT0015064) mirVana® miRNA mimic (cat. no. 4464066) of Bengbu Medical College (approval no. 201858; Bengbu, and mirVana® miRNA inhibitor (cat. no. 4464084), as well as China). Patients who donated cervical cancer tissues gave the corresponding controls (cat. nos. 4464058 and 4464078, their informed consent in writing prior to their involvement respectively), were obtained from AmbionÒ (Thermo Fisher in the study. All specimens were harvested following biopsy Scientific, Inc.). For miRNA mimic/inhibitor transfec- or surgery (cone biopsy, radical trachelectomy or radical tions, HeLa and SiHa cells were seeded in 6-well plates hysterectomy) at the aforementioned hospital between (1x105 cells/well) and transiently transfected with 3 nM mimic March 2000 and March 2002 from female patients (median or 10 nM inhibitor using Lipofectamine 3000 transfection age, 53 years; age range, 35-65 years) that had never received reagent for 48 h at 37˚C prior to subsequent experimentation. chemo- or radiotherapy, yielding 65 pairs of primary cervical cancer samples and matched healthy cervical tissue samples RT‑qPCR. TRIzol® (Invitrogen; Thermo Fisher Scientific, Inc.) (>2 cm from the tumor edge). Tumor specimens were staged was used to extract total RNA from cells or tissue samples, by a licensed pathologist according to the International of which a 100-ng aliquot from every sample was used for Federation of Gynecologists and Obstetricians (FIGO) staging cDNA preparation using the PrimeScript™ RT Reagent Kit system (27). The specimens were immediately kept in liquid according to the manufacturer's protocol (Takara Bio, Inc.). nitrogen and frozen at ‑80˚C until further use. A SuperScript™ III Platinum™ SYBR™ Green One-Step qRT-PCR kit or NCode™ SYBR™ Green miRNA qRT-PCR Cell lines, culture conditions and general materials. Human kit (both from Invitrogen; Thermo Fisher Scientific, Inc.) was cervical cancer HeLa and SiHa cell lines were obtained from used to quantify mRNA or miR-3184-5p expression, respec- the American Type Culture Collection. The non-cancerous, tively, on an ABI PRISM™ 7000 Sequence Detection System HPV-immortalized H8 cell line of cervical epithelial squa- (Applied Biosystems; Thermo Fisher Scientific, Inc.). The mous cells was obtained from The Cell Bank of Type Culture mRNA qPCR thermocycling conditions were as follows: 95˚C Collection of the Chinese Academy of Sciences. Cells were for 3 min, followed by 45 cycles of 95˚C for 15 sec and 60˚C grown in DMEM/F12 with 10% FBS (both from Invitrogen; for 45 sec. The miRNA qPCR thermocycling conditions were Thermo Fisher Scientific, Inc.) at 37˚C in a 5% CO2 incubator. as follows: 50˚C for 2 min and 95˚C for 2 min, followed by Reverse transcription-quantitative PCR (RT-qPCR) was 40 cycles of 95˚C for 15 sec and 60˚C for 50 sec. The following regularly employed to check for contaminating mycoplasma primers were obtained from OriGene Technologies, Inc., and