Original article
Identification of autophagy-associated miRNA signature for the cervical squamous cell cancer and high-grade cervical intraepithelial lesions

https://doi.org/10.1016/j.repbio.2021.100536Get rights and content

Highlights

  • miR-30e, miR-30a and miR-30c, miR-143, miR-372, miR-375, miR-520e were downregulated in the SCC.

  • miR-96, miR-17, miR-130a and miR-520b were upregulated in the SCC.

  • miR-30a, miR-520e, miR-548c and miR-372 associated with the overall survival of SCC.

Abstract

Cervical cancer markedly threatens women's health worldwide and currently ranks fourth leading cause of cancer mortality in women according to recent global cancer statistics. Recent advances have proven that not only tumor suppressor and oncogenes but also non-coding RNAs including micro RNAs (miRNAs) have significant impact in the development and progression of cervical cancers. Previous studies have identified many cancer-specific miRNAs for the early detection of cervical cancers. However, the diagnostic and prognostic use of autophagy-associated miRNAs for the cervical squamous cell cancer (SCC) cases and high-grade squamous intraepithelial lesion (HSIL) have not been uncovered. In the present study, we revealed that miRNAs are differentially expressed in both cervical SCC and HSIL. A total of 35 HSIL, 35 cervical SCC and 30 healthy controls were enrolled for the present study. Total RNA including miRNAs were isolated from the FFPE tissue samples and miRNA expression levels were quantified by quantitative PCR. Predicted miRNA targets of autophagy related genes were determined using miRNA-target prediction algorithms. MiR-143, miR-372, miR-375 and miR-30c were markedly downregulated in HSIL and cervical SCC. MiR-130a was significantly upregulated in the cervical SCC group compared to HSIL and control groups. MiR-30a, miR-520e, miR-548c and miR-372 were significantly associated with the overall survival of cervical SCC patients and these miRNAs were determined to be significant diagnostic markers as revealed by ROC analysis. Together, these results indicate that autophagy-associated miRNAs are potentially valuable for the differential diagnosis and targeted therapy to cervical cancer.

Introduction

Cervical cancer is the fourth ranking malignancy for both incidence and mortality among females worldwide [1]. Furthermore, it is the second leading cause of cancer death in women aged 20–39 years and responsible for the significant number of premature deaths in United States [2]. The incidence and mortality of cervical cancer vary dramatically across countries and among populations depending on the socio-economic status and life style [1]. The emergence of cervical cancer is a multifactorial and multi-step process that involves the transformation of the normal cervical epithelium into cervical intraepithelial neoplasia and subsequently to invasive cervical cancer [3]. The incidence and mortality rates have substantially reduced with the implementation of the cervical cancer screening programs by cytopathological smear test [4]. Thus, cytopathological smear test is accepted as a gold standard technique for the cervical cancer screening [5]. However, specificity of cytopathology is relatively low compared to its high sensitivity.

Cervical cancer originates from non-invasive premalignant lesions called pre-existing squamous intraepithelial lesions or cervical intraepithelial lesions. These lesions are classified histologically according to the atypia of epithelial cells that spread through the lower parabasal layer of the squamous epithelium and spread to cover the entire epithelial layer [6]. Cervical intraepithelial lesions and low-grade cervical intraepithelial lesion corresponds to mild dysplasia, while cervical intraepithelial lesions II correspond to moderate dysplasia and cervical intraepithelial lesions III corresponds to severe dysplasia and carcinoma in situ. High-grade cervical intraepithelial lesions reflects the combination of cervical intraepithelial lesion II and cervical intraepithelial lesion III [7]. Histologically, cervical cancer is divided into subtypes of squamous cell carcinoma (cervical squamous cell cancer; about 80 % of cases) and adenocarcinoma (about 5–20 % of cases) [6,7]. While cervical squamous cell cancer (SCC) develop from well-defined precursor lesions called cervical intraepithelial neoplasia (cervical intraepithelial lesions; grade1−3), the precursor stages of cervical adenocarcinoma have not been fully characterized [8]. Some cases of cervical intraepithelial lesions progress to a more severe form of dysplasia or invasive cancer, therefore, it is extremely important to identify novel biomarkers with a high predictive value for disease progression [3]. Although radiotherapy, chemotherapy and surgery are standard treatment modalities in cervical cancer, disease remission and clinical outcomes vary widely among patients and can be difficult to predict. Therefore, understanding the cellular origin, causes, genetics and molecular biology of the cervical cancer is very important for identifying novel prognostic biomarkers and developing advanced treatment strategies. Since squamous cell cancer, which constitutes the majority of cervical cancer cases, develops as a result of progression of precursor lesions, early detection of the precursor lesion is of great importance for both determining the prognosis of patient and providing treatment options based on the severity of the disease, taking into account the age and fertility desires of the patients. Thus, identification of novel biomarkers with strong prognostic potential to differentiate precursor lesion from invasive cancer is very important.

Furthermore, discovery of microRNAs (miRNAs) has revolutionized our understanding of cancer genetics [9,10]. These tiny molecules are approximately 22 nucleotides in length and do not code for proteins, rather they code for functional regulatory RNAs [11,12]. MiRNAs fine tune the regulation of almost every cellular process such as proliferation, apoptosis, stress response as well as autophagy by interfering the translation of their target mRNAs [13,14,15,16]. Due to their crucial physiological and clinical significance in human cancers, a great effort has been made to understand biological importance of miRNAs by using several high-throughput screening assays [17]. Many studies have demonstrated that changes in miRNA expression have great impact in cancer development and progression of human cancers including cervical cancer [11,18,19]. Moreover, autophagy which stands for self (auto)-eating (phagy), is a mechanism by which cells break down structures within the cell to obtain nutrients in case of starvation [20]. Thus, intracellular molecules are recycled, and homeostasis is preserved. In recent studies, autophagy has been shown to play key roles in the regulation of metabolism, morphogenesis, differentiation, aging, cell death and the immune system dynamics [[20], [21], [22]]. Abnormalities in autophagy mechanism have been implicated in the development and progression of several diseases, especially cancer [23,24]. The most important stimuli in autophagy regulation are starvation, hypoxia and cellular stress conditions [23,24]. Various oncogenes and tumor suppressor genes as well as miRNAs have also been found to play a role in the regulation of autophagy [25,26].

In the present study, we aimed to identify differentially expressed miRNAs that are involved in the regulation of autophagy in patients with cervical SCC and high-grade squamous intraepithelial lesion (HSIL), and to determine the impact of these miRNAs in the clinical course of cervical cancer development. Consequently, we address, for the first time, the diagnostic use of autophagy-associated miRNAs for the differentiation of cervical SCC cases and HSIL.

Section snippets

Study population and sample collection

A total of 35 HSIL (median age, 44 years; range, 30–74) and 35 cervical SCC (median age, 52 years; range, 34–74) patients, and 30 healthy controls (median age, 47 years; range, 29–68) were enrolled for the study. All patients were pathologically confirmed for the presence of indicated disease. Formalin-Fixed Paraffin-Embedded (FFPE) tissue samples of patients were obtained from the routine pathology archive of Gaziantep University Hospital. The study was ethically approved by the institutional

Demographic and clinical characteristics of patients

The clinical (stage, metastasis, lymph node, age of onset) and demographic (age, gravida, smoking and alcohol habits) characteristics of patients were summarized in Table 1, Table 2. Age distributions in HSIL and cervical SCC groups were normal compared to the control group (p > 0.05). Also, distribution of the age of onset between groups were normal (p > 0.05). The distribution of the number of gravidae in the control group and the HSIL group was normal (p = 0.8395), while it was higher in the

Discussion

Squamous cell cancer of cervix constitutes the major type of cervical cancer cases and develops as a result of the progression of precursor lesions. Therefore, early identification of precursor lesion and determination of the molecular changes in these lesions is very important. Herein we demonstrated that miRNAs involved in the regulation of autophagy were differentially expressed in cervical squamous cell cancer and high-grade cervical intraepithelial lesions tissues compared to healthy

Conclusions

Previous studies have shown that miRNAs involved in autophagy are dysregulated in cervical cancer. To the best of our knowledge, this study, for the first time, addresses the diagnostic use of autophagy-associated miRNAs for the differentiation of cervical SCC cases and HSIL among the Turkish population. We believe that identified miRNAs will provide a better concept for the treatment plan in patients with cervical cancer. Moreover, in addition to their diagnostic and the prognostic potentials,

Funding

The present study was supported by the grant TF.ALT.19.43 from Gaziantep University.

Availability of data and material

The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions.

Authors’ contributions

Neslihan Bayramoglu Tepe; Conceptualization, Supervision, Funding acquisition, Writing - review & editing Esra Bozgeyik; Investigation, Methodology, Data curation, Software, Writing - original draft, Writing - review & editing Zehra Karadag; Methodology, Writing - review & editing Ozcan Balat; Methodology, Writing - review & editing Huseyin Caglayan Ozcan; Methodology, Writing - review & editing Mete Gurol Ugur; Methodology, Writing - review & editing.

Declaration of Competing Interest

All authors declare that they have no conflict of interest.

References (45)

  • Y. Cheng et al.

    Has-miR-30a regulates autophagic activity in cervical cancer upon hydroxycamptothecin exposure

    Biomed Pharmacother

    (2015)
  • A.-M. Gao et al.

    Apigenin sensitizes hepatocellular carcinoma cells to doxorubic through regulating miR-520b/ATG7 axis

    Chem Biol Interact

    (2018)
  • F. Bray et al.

    Global cancer statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36 cancers in 185 countries

    CA Cancer J Clin

    (2018)
  • R.L. Siegel et al.

    Cancer statistics, 2020

    CA Cancer J Clin

    (2020)
  • K. Zeng et al.

    Dysregulated microRNAs involved in the progression of cervical neoplasm

    Arch Gynecol Obstet

    (2015)
  • S.D. Isidean et al.

    Embracing a new era in cervical cancer screening

    Lancet (London, England)

    (2013)
  • I.I.I.J. Thomison et al.

    Human papillomavirus: molecular and cytologic/histologic aspects related to cervical intraepithelial neoplasia and carcinoma

    Hum Pathol

    (2008)
  • L.P. Howell et al.

    Significance of subclassifying high‐grade squamous intraepithelial lesions into moderate dysplasia/CIN II versus severe dysplasia/CIN III/CIS in the Bethesda system terminology

    Diagn Cytopathol

    (2004)
  • S. Wilting et al.

    Altered microRNA expression associated with chromosomal changes contributes to cervical carcinogenesis

    Oncogene

    (2013)
  • V. Ambros

    The functions of animal microRNAs

    Nature

    (2004)
  • G.A. Calin et al.

    MicroRNA signatures in human cancers

    Nat Rev Cancer

    (2006)
  • D. Gozuacik et al.

    Autophagy-regulating microRNAs and cancer

    Front Oncol

    (2017)
  • View full text