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. 2021 Jun 8;73(4):585–592. doi: 10.1007/s10616-021-00479-y

miR-185-3p targets Annexin-A8 to inhibit proliferation in cervical cancer cells

Wenfang Zhang 1,✉,#, Dongyan Han 2,#
PMCID: PMC8319227  PMID: 34349348

Abstract

Numerous studies have found that microRNAs (miRNAs) are involved in regulating various tumor-related biological functions. The downregulation of miR185-3p have been identified in various types of cancer but the effect and its underlying molecular mechanism in cervical cancer have not been elucidated. Therefore, it is important to investigate the role of miRNAs associated with cervical cancer and its corresponding molecular mechanism to develop new therapeutic targets. The cell counting kit (CCK-8) assay was performed to measure the cell viability. The quantitative real-time PCR (qRT-PCR) and western blot analyses were carried out to identify mRNA and protein expression levels, respectively. Besides, a luciferase activity assay was conducted to confirm the target miRNA gene predictions. In this study, it is found that miR185-3p expression was potentially downregulated in cervical cancer tissues when compared with normal tissues. The CCK-8 results indicated that miR185-3p overexpression suppressed the cancer cell proliferation and the downregulation of miR185-3p enhanced the cancer cell growth. Further, enhanced miR185-3p expression led to a reduction in Annexin-A8 (Anx-A8) expression but miR185-3p inhibition promoted ANX-A8 levels in cervical cancer cells. The luciferase reporter assay indicated that ANX-A8 was a direct target of miR185-3p in cervical cancer cells.

Keywords: miR-185-3p, Annexin, Inhibition, Cervical cancer, Proliferation

Introduction

Cervical cancer, being a curable disease is considered as second most common malignant type among woman globally (zur Hausen 2002). Worldwide, the cervical cancer incidence is found to be increasing in developing countries and vice versa in the developed nations (Banno et al. 2014). Despite various progress have been made on theranostic techniques, cervical cancer remains a major public concern due to the unsatisfactory clinical outcomes of the disease (Handler et al. 2015). Only 50% response rate have been identified from the currently available therapies with a < 2 years median survival period (Tewari and Monk 2014). Multiple factors are associated with the poor prognosis of patients having cervical cancers and the most difficult part lies in finding out the accurate molecular mechanism (Kogo et al. 2015). Thus, new therapeutic approaches are required to further reveal the exact underlying molecular mechanism for the cause of cervical carcinogenesis. Various treatment trials are tried out to establish an effective significant immunotherapy strategy but none of them is considered as a checkpoint for the most commonly occurring cervical cancer till date.

In recent years, it has been found that miRNAs have become essential for gene expression, which are involved in the maintenance of various pathological and physiological processes. miRNAs are small, single-stranded, non-coding RNA molecules that can maintain balance between the proteome and the transcriptome. These miRNAs are having short nucleotides in the range of 17–23 in length and maintain the gene expression upon limited base-pairing with 3′ untranslated (3′-UTRs) region of their target messenger RNAs (mRNAs), further leads to mRNA cleavage/translation suppression. The sequence pairing of miRNA and its mRNA target is one of the main characteristic features of miRNAs which further end up in casual interactions, for instance, one miRNA pairs with multiple mRNAs, and one transcript can target more than one miRNAs (He 2016). The miRNA disorders have been noticed in various carcinoma including cervical cancer. At the time of cancer progression, many miRNAs are found to be dysregulated and function as a significant biomarker for theranostic molecular targets (Xu et al. 2017).

Number of studies on miRNAs showed that some of the miRNAs such as miR1297, miR21, and miR224 can be overexpressed or some (miR506, miR 143, and miR152) can be downregulated in cervical cancer (Wang et al. 2019). Recent evidence indicated that miRNAs can be potential targets for the treatment of patients having cervical cancer. Currently, the identity and physiological process of target genes are only evident for some miRNAs and the analysis of the whole miRNAome has become possible with the help of microarrays having known human miRNAs. The screening of such miRNAs which are expressed between normal and cancerous tissues helps to identify which miRNA type has participated in cancer pathogenesis. Thus, the consistent analysis of miRNA expression account between normal and cancerous tissue may provide the details of more miRNA candidates that can play a major role in cervical cancer pathogenesis.

Annexins are commonly called cellular proteins that are specifically participated in assembling cytoskeletal proteins and phospholipid membranes. The core structure contains two main domains namely COOH terminal core and NH2 terminal core. These cores act as membrane binding site that helps to bind with phospholipids and maintains Ca2+ binding site. Annexin-A8 (ANX-A8) is the least studied type within the annexin family but seems to describe similar functions in membrane-cytoskeleton dynamics (Pimiento et al. 2015). Annexin dysregulations are related to human disease particularly cancer. Some of the studies reported ANX-A8 overexpression in acute promyelocytic leukemia (Stein et al. 2005) and pancreatic cancer (Karanjawala et al. 2008). However, ANX-A8 role as a therapeutic target for cervical cancer has not yet been elucidated in detail. This study aims to demonstrate that miR185-3p targets ANX-A8 to inhibit proliferation in cervical cancer. Thus, miR185-3p can be used as a novel inhibitor for annexin and hence, is a potential therapeutic target for cervical cancer.

Materials and methods

Cell lines and culture methods

Three human cervical cell lines were utilized to carry out the experimental studies. The cervical cancer cell lines HeLa, SiHa, and C4I and normal human cervical cell line Ect1/E6E7 were from the Type Culture Collection cell bank of the Chinese Academy of Sciences in Shanghai, China. All three cell lines were cultured using supplemented Dulbecco’s Modified Eagle Medium (Corning Life Sciences, Wujiang, China). The supplement contains fetal bovine serum (10%, FBS) Corning Life Sciences, Wujiang, China, and penicillin/streptomycin (1%, mix), and the cells were grown at 37 °C and 5% CO2 in a humidified incubator.

Cell transfection

Synthetic miR185-3p mimic/inhibitor and its negative control were obtained from Shanghai GenePharma Company Limited, Shanghai, China. The oligonucleotide sequences used in the transfection are as follows. miR185-3p-mimic forward, sense, 5′-AGGGGCUGGCUUU CCUCUGGUC-3′ and reverse, 5′-CCAGAGGAAAGCCA GCCCCUUU-3′; miR185-3p-mimic control, forward 5′-UUCUCCGAACGUGUCACGUTT-3′ and reverse, 5′-ACGUGACACG UUCGGAGAATT-3′; miR185-3p-inhibitor, 5′-GACCAGAGG AAAGCCAGCCCCU-3′ and miR185-3p inhibitor control, 5′-CAGUACUUUUGUGUAGUACAA-3′. The cells were cultured in 6-well plates at a density of 5 × 105 cells/well and then transfected withmiR185-3p mimic and control using HiPerFect-Transfection reagent from Qiagen (Hilden, Germany). Cell transfection was done carefully according to the manufacturer’s guidelines. The transfected cells were harvested after 48 h of transfection to conduct further experiments. ThemiR185-3p and protein expression levels were identified.

Extraction of RNA and quantitative qRT-PCR

The total RNA was extracted using RNAios Plus reagent according to the standard protocol given in the RNAiso Plus kit obtained from Takara Bio (Shiga, Japan). The cDNA was synthesized to detectmiR185-3p by using Qiagen miScript reverse transcription kit (Dusseldorf, Germany) and then mRNA expression was analyzed using TaqMan microRNA assay kit (Bio-Tek company, Winooski, VT, USA). The quantitative RT-PCR was carried out using the TaqMan qRT-PCR kit. The U6 small nuclear RNA was used as the internal reference and the mRNA expressions were studied using glyceraldehyde 3-phosphate dehydrogenase (GADPH) as control.

Cell counting kit-8 (CCK-8) assay

The effect of miR185-3p on cervical cancer cell proliferation was studied by performing a CCK-8 assay. In this assay, the transfected cells upon incubation withmiR185-3p mimics or inhibitor were collected after 48 h and treated into 96-well plates at a density of 5 × 103 cells/well at 37 °C and 5% CO2. This is followed by the addition of 10 µl of CCK-8 solution obtained from Dojindo Molecular Technologies (Kumamoto, Japan) into each well and further incubated at dark for an additional 2 h at 37 °C. Finally, the absorbance was recorded at 490 nm after shaking using a microplate reader.

Luciferase reporter assay

TargetScanHuman was used to identify the binding. targets of human miR19b-3p. The predicted results showed that miR185-3p may be involved in the regulation of Annexin-A8 and TargetScanHuman also predicted the putative binding site. The 3'-UTR of ANX-A8 was amplified using PCR and inserted into the vector. The luciferase activity was analyzed based on luciferase reporters that were generated based on firefly luciferase expression vector pGL3 control vector using XbaI site (Promega, USA). A site-directed mutagenesis kit was used for generating the mutated putative binding site of miR185-3p in 3′-UTR of ANX-A8. In this method, the cell lines at a density of 2 × 105 were plated into 24-well plates freshly one day before transfection. Then it is subjected to co-transfection with luciferase reporter, miR185-3p mimics or inhibitor or negative control using HiPerFect-Transfection Reagent. The cells were harvested after 48 h after transfection and assessed luciferase activity using dual-luciferase reporter assay, by following the manufacture’s protocol.

Western blot and antibodies

The cultured cells were collected, washed with PBS, and lysed in radioimmunoprecipitation assay (RIPA) lysis buffer (Shanghai Qianchen Biotechnology Company, Shangai, China) to perform protein extraction. The cell lysates were then centrifuged at 12000×g for 4 min at 4 °C and the concentration. of proteins was measured by using BCA protein. assay kit (BioTeke Corporation, Beijing, China). An equal amount of protein samples (20 µg) was used in SDS-PAGE and then transferred into nitrocellulose membrane. The membrane was blocked with non-fat milk (5%) in TBST. After blocking the membrane was incubated with primary antibodies (anti-ANX-A8 and anti-GAPDH) overnight first at 4 °C and then with horseradish peroxidase-conjugated secondary antibody next for 1 h at room temperature. The immunoreactive proteins were then detected using an enhanced chemiluminescence system (ECL system) (Roche, Basel, Switzerland).

Statistical analysis

The experimental data were expressed as mean ± standard deviation of three independent experiments. The statistical comparisons were made with students' t test (two-tailed) using the software GraphPad Prism version 7.0c (GraphPad Software, San Diego, CA, USA). The values of p < 0.05 were considered statistically significant.

Results

miR185-3p hinders the cervical cancer cell proliferation

The effect of miR185-3p in cervical cancer was examined in vitro by transfection of miR185-3p-mimic or miR185-3p-inhibitor into cervical tumor cell lines HeLa, SiHa, and C4I. The cell viability was determined using the CCK-8 assay. The assay results indicated that transfection with miR185-3p mimics prominently reduced HeLa cell proliferation whereas miR185-3p inhibitor transfection into HeLa cells significantly increased its cell viability. Furthermore, the upregulation of miR185-3p hindered the SiHa cell growth whereas the downregulation of miR185-3p enhanced the cell proliferation. Similar results were noticed in the case of C4I cells and the results are shown in Fig. 1. These results demonstrated that miR185-3p could act as an effective suppressor in cervical cancer.

Fig. 1.

Fig. 1

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Proliferation of cancer cells inhibited by miR185-3p. The HeLa, SiHa, and C4I cells were transfected with miR185-3p-mimic and miR185-3p-inhibitor. The proliferation of HeLa (a), SiHa (b), and C4I (c) cells were measured by CCK-8 assay. **p < 0.01, *p < 0.05

miR185-3p suppresses the expression of ANX-A8 in cervical cancer cells

We used bioinformatics tools to identify the potential targets of miR185-3p. This will help to determine the potential mechanism by which miR185-3p inhibits cervical cancer cell proliferation. This study found the presence of the putative miR185-3p binding site in the ANX-A8 3’-UTR as shown in Fig. 2a. Consistent with the previous experimental reports, a significant increase in the ANX-A8 expression was identified in the cervical cancer tissues was determined by using the real-time PCR and western blot analysis (Fig. 2b–d). Normal human cervical cell line Ect1/E6E7 showed decreased expression of ANX-8 when compared to cervical cancer cell lines (Fig. 2b–d). ANX-A8 was selected for further studies as this gene is expected to have important functions in regulating cervical cancer.

Fig. 2.

Fig. 2

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ANX-A8 is a target gene of miR185-3p. The predicted binding site of miR185-3p in ANX-A8 by TargetScanHuman (a). RT-PCR (b) and Western blot (c and d) showing total RNA and protein expression of ANX-A8 in Ect1/E6E7, HeLa, SiHa, and C4I cell lines. *p < 0.05

To determine whether miR185-3p targets ANX-A8, the cells were transfected with miR185-3p mimic/inhibitor and measured mRNA expression of ANX-A8 using qRT-PCR. From the results, we found that miR185-3p upregulation hinders the ANX-A8 expression at both mRNA and protein levels whereas miR185-3p inhibitor transfection significantly enhanced the ANX-A8 expression in cervical cancer cells (Fig. 3). The results from the luciferase assay revealed that miR185-3p overexpression significantly reduced the luciferase activity whereas the miR185-3p inhibitor treatment potentially enhanced the luciferase activity when treated with cervical cancer cells, demonstrating that ANX-A8 is a direct target of miR138 in cervical cancer cells (Fig. 4). The results from mutant ANX-A8 reporter gene showed that the binding site between miR185-3p and the coding region of ANX-A8 was disrupted. This resulted in miR185-3p mediated inhibition of ANX-A8. The mutation of ANX-A8 binding site shows that miR1185-3p is directly involved in regulating the ANX-A8 protein expression through specific binding to the coding regions of its mRNA.

Fig. 3.

Fig. 3

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ANX-A8 is a target of miRNA185-3p in cervical cancer. MiR185-3p-mimic or miR185-3p-inhibitor was transfected into HeLa cells. The RT-PCR (a and d) and Western blot analysis (b, c, e and f) showing mRNA and protein expression of ANX-A8 in the presence of miR185-3p-mimic or miR185-3p-inhibitor transfected HeLa cells respectively

Fig. 4.

Fig. 4

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Luciferase activity measured in cervical cancer cell line, HeLa with miR185-3p mimic or miR185-3p inhibitor. a A mutation at the complementary site of miR185-3p is highlighted in red color. b Relative luciferase activity after ANX-A8 mutation co-transfected with miR185-3p MIMIC

Discussion

Several studies reported the role of miRNA dysregulations in cervical cancer (Diaz-Gonzalez Sdel et al. 2015; Yang et al. 2018; Shang et al. 2019). These miRNAs also participate in regulating numerous tumor-related biological functions such as cell proliferation, cell cycle analysis, apoptosis, etc., (Banno et al. 2014). To develop novel therapeutic targets, it is important to investigate the function of cervical cancer-related miRNAs and their molecular mechanistic pathway. In this study, the expression levels and the clinical significance of miR185-3p in cervical cancer were determined. Also, the detailed study of miR185-3p roles in cervical cancer development was studied. Further, extensive research on the underlying molecular mechanism on which miR185-3p may alter cervical cancer development was also studied.

Currently, numerous studies on miR-185 were concentrated on miR185-3p, a tumor suppressor related to different features of cancer development such as cell proliferation, cell cycle, apoptosis, and chemoresistance (Li et al. 2014; Ma et al. 2015). The inhibition of colorectal cancer metastasis and breast cancer cell invasion was reported on miR185-3p (Wang et al. 2014; Zhang et al. 2016). Another report indicated that miR185-3p can target 2-T1 neurotrophic tyrosine kinase receptor which is associated with the neurobiological process (Maussion et al. 2012). The binding of miR185-3p with homologous oncogene of avian myelocytomatosis v-myc also inhibits cell proliferation and arrest cell cycle (Liao and Lu 2011). The miR185-3p aimed to target 3′ amino acid coding of c-Myc mRNA and results in cell cycle arrest (Liao and Lu 2011). The in vitro functional analyses suggested that miR185-3p could bind with 3′-UTR-Tr kB-T1 sequences to function on mental disorders (Maussion et al. 2012). miR-138 is known to be downregulated in different cancers (Liu et al. 2009; Yeh et al. 2013).

In this study, we found that miR185-3p was downregulated in cervical cancer tissues when compared to the non-cancerous tissues. The ectopic miR185-3p expression suppressed cell proliferation in Hela, SiHa, and C4I cervical cancer cell lines whereas the miR185-3p overexpression promotes cancer cell growth. Collective results implied that miR185-3p serves as a tumor suppressor miRNA against cervical cancer. To further illustrate the underlying molecular mechanism behind the anti-tumor effect of miR185-3p against cervical cancer, we introduced ANX-A8 as a potential target of miR185-3p using computer-aided bioinformatics studies. ANX-A8 deregulation has been identified in several malignancies including pancreas, melanoma, squamous carcinoma of the cervix uterine. It is found that ANX-A8 expression was potentially increased in cervical cancer tissues which is consistent with previous results. The miR185-3p ectopic expression led to downregulation in ANX-A8 expression both in mRNAs and protein levels. Further, a remarkable reduction was observed in firefly luciferase activity. This is followed by transfection with miR138-3p-mimic. When the cervical cancer cells co-transfected with miR185-3p-inhibitor the luciferase activity is found to be increased and this demonstrated that ANX-A8 is a direct target of miR185-3p in cervical cancer cells.

Conclusions

This study demonstrated that miR185-3p inhibits cervical cancer cell proliferation by targeting ANX-A8. The current study provides basic results for the application of miR185-3p in the ANX-A8 pathway for the treatment of cervical cancer patients. The combined evidence suggests that miR185-3p can represent a potential therapeutic target for cervical cancer. In our future work, we will focus on the influence of miR185-3p on the cell cycle analysis of cervical cancer in the future.

Acknowledgements

The authors thank Huangshi Central Hospital and The Third People’s Hospital of Kaifeng City for providing the core facilities to conduct the experiments.

Declarations

Conflict of interest

The author declares that they have no Conflict of interest.

Ethical approval

The protocols used and conducted studies were approved by the Ethical Committee of Huangshi Central Hospital and The Third People’s Hospital of Kaifeng City.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Wenfang Zhang and Dongyan Han are contributed equally to this work.

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