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MiR-181c-5p Mitigates Tumorigenesis in Cervical Squamous Cell Carcinoma via Targeting Glycogen Synthase Kinase 3β Interaction Protein (GSKIP)

Authors Li N, Cheng C, Wang T

Received 8 January 2020

Accepted for publication 6 May 2020

Published 21 May 2020 Volume 2020:13 Pages 4495—4505

DOI https://doi.org/10.2147/OTT.S245254

Checked for plagiarism Yes

Review by Single anonymous peer review

Peer reviewer comments 2

Editor who approved publication: Dr Leo Jen-Liang Su

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Niuniu Li,1,* Chun Cheng,2,* Tieyan Wang3

1Department of Gynecology and Obstetrics of Shiyan, Taihe Hospital of Hubei Province, Shiyan, Hubei 442000, People’s Republic of China; 2Department of Pediatrics, Huangshi Central Hospital, Affiliated Hospital of Hubei Polytechnic University, Edong Healthcare Group, Huangshi, Hubei 435000, People’s Republic of China; 3Clinical Pathology Department of Shiyan, Taihe Hospital of Hubei Province, Shiyan, Hubei 442000, People’s Republic of China

*These authors contributed equally to this work

Correspondence: Tieyan Wang Email [email protected]

Background: Cervical cancer (CC) is a highly prevalent cancer and one of the main causes of death among women worldwide. The miR-181 family has turned out to be associated with tumorigenesis in a variety of tumors by regulating the expression of tumor-related genes. However, the mechanisms and biological function of miR-181c-5p in cervical squamous cell carcinoma (SCC) have not been well elucidated.
Materials and Methods: SiHa cell lines with specific gene overexpression vectors were constructed. Targetscan was used to predict the binding site of miR-181c-5p and GSKIP. MTT assay was used to detect the clone formation rate. Flow cytometry was used to detect the apoptosis rate and to separate the cell markers. The Transwell test was used to detect cell invasion. Immunohistochemistry was used to detect protein expression in tumor tissues. Western Blotting was used to detect the expression levels of related proteins.
Results: GSKIP was predicted to be the target gene of miR-181c-5p in cervical SCC. MiR-181c-5p overexpression suppressed SiHa cells proliferation and promoted apoptosis; the protein expressions of Ki67 and PCNA were decreased, but the expressions of Caspase-3 and Bax/Bcl-2 were increased. The overexpression of miR-181c-5p inhibited the stem-like properties of SiHa cells; the expressions of SOX2, OCT4 and CD44 were decreased. Furthermore, miR-181c-5p upregulation limited the invasion of SiHa cells; the expression of E-cadherin was higher, but the expressions of N-cadherin and Vimentin were lower. MiR-181c-5p overexpression inhibited tumorigenesis in cervical SCC tissues; the expressions of Ki67, Caspase-3, CD44 and Vimentin in vivo were consistent with those in vitro.
Conclusion: Taken together, miR-181c-5p was able to mitigate the cancer cell characteristic and invasive properties of cervical SCC through targeting GSKIP gene.

Keywords: apoptosis, cancer stem cell, epithelial-mesenchymal transition, kinase 3β interaction protein, miR-181c-5p

Introduction

Cervical cancer (CC) is the third most common gynecological malignancy worldwide and the second leading cause of cancer-related mortality in women, with an estimated 530,000 female dying each year.1 About 87 percent of cervical cancer occurs in developing countries, where cervical cancer is the leading form of gynecological cancer.2,3 Cervical squamous cell carcinoma (SCC) is one of the most common types of CC, accounting for about 80–90% of CC, and the most important risk factor for cervical SCC is persistent human papillomavirus (HPV) infection.4 Epidemiological studies have reported that more than 99% of patients with cervical SCC are positive for high-risk HPV (HPV16, HPV18 and HPV31).5,6 High-risk HPV contains oncoproteins, E6 and E7, which promote cervical SCC by silencing tumor-suppressing p53 and Rb proteins, as well as some cancer-related genes.7 The molecular mechanism of the occurrence, development and metastasis of cervical SCC has not been fully explained. Therefore, it is necessary to further understand the molecular targets and pathways for progression and metastasis of cervical SCC.

Previous studies have reported that miRNAs affected multiple biological pathways of cervical cancer, by analyzing 246 dysregulated miRNAs and 40 confirmed CC target genes.8 Evidences have indicated that miR-181 family contains four highly conserved mature miRNAs: miR-181a, miR −181b, miR −181c and miR −181d, which come from six precursors on three different chromosomes.9 MiR-181a-1 and miR-181b-1 are located on chromosome 1, miR-181a-2 and miR-181b-2 on chromosome 9, and miR-181c and miR-181d on chromosome 19.10 Studies have found that the aberrant expression of miR-181s in tumor tissues suggest a crucial role in cancer development and progression.11

Glycogen synthase kinase 3 beta interacting protein (GSKIP) is a scaffolding protein in the cytoplasm, which binds to a protein kinase (PKA) and glycogen synthesis kinase 3β (GSK3β).12 As one of the A-kinase anchoring proteins (AKAPs), GSKIP is a good substrate of GSK3β. The interaction between GSKIP and GSK3β can block the phosphorylation of β-catenin protein at ser-33/ser-37/thr-41, and can negatively regulate the Wnt signaling pathway of GSK3β.13 Wnt signaling modulates different biological processes and its deregulation is linked to diseases such as type 2 diabetes, inflammatory, and cancer.14

It has been found that miRNA-758 inhibited cell proliferation and metastasis of CC by targeting the HMGB3 Wnt/β-catenin signaling pathway.15 MiR-150-5p significantly inhibited Wnt/β-catenin signaling by simultaneously targeting GSKIP and β-catenin in NSCLC cells.16 However, the role of miR-181c-5p in cervical SCC has been rarely reported. In this study, the expression level of miR181c-5p in tumor cell lines and tissues was profiled. Furthermore, the role of miR181c-5p in tumorigenesis in cervical SCC and the underlying mechanism was investigated.

Materials and Methods

Cell Culture

Ect/E6E7 cell line was purchased from Mingzhou biotechnology co., LTD. SiHa, HEC-1-A, ME-180, Hela cell lines were purchased from Procell life sciences LTD. All cell lines were maintained in Dulbecco’s Modified Eagle Medium (DMEM) (Thermo Fisher Scientific, Inc., Waltham, MA, USA) containing 10% fetal bovine serum (FBS) (Gibco, Rockville, MD, USA) at 37°C in a humidified incubator containing 5% CO2.

Cell Transfection

MiR-181c-5p mimics and scramble (antisense inhibitors) were purchased from RiboBio (Guangzhou, China). GSKIP overexpression plasmid and control vectors were also purchased from RiboBio (Guangzhou, China). SiHa cell lines were assigned to the control group.

Transfection of miRNA mimics: the miR-181c-5p mimics and scramble were transfected into SiHa cell lines by Lipofectamine® 2000 (Invitrogen, Carlsbad, CA, USA) in strict line with the manufacturer’s protocol. After 48 hours of transfections, the transfection efficiency was detected by qRT-PCR. Recombinant plasmid transfection: the mixture of miRNA mimics and the recombination vector (mimic+ pc-GSKIP), the recombination vector (pc-GSKIP) and the empty vector (pcDNA-NC) were fully mixed with transfection reagent, respectively, and incubated with cells for 8h after 25 minutes in a CO2 incubator (Forma, Thermo, USA). After 48 hours of transfections, the SiHa cells were collected for further analysis.

Reverse Transcription Quantitative Real-Time PCR (qRT-PCR)

Total RNA was extracted from cells and tissues with TRNzol Universal Reagent (TIANGEN BIOTECH, Beijing, China) following the manufacturer’s protocol. The qRT-PCR reaction was performed with Quant One Step qRT-PCR Kit (Probe, TIANGEN BIOTECH, Beijing, China) by using the Bio-Rad CFX96 PCR System (Bio-Rad, CA, USA). The thermocycling conditions were as follows: A holding step at 95°C for 30 sec, and 40 cycles at 95°C for 5 sec and 60°C for 30 sec. All primers were designed as follows: GSKIP, forward 5ʹ -CATGGTGGTACCTGCTTGTG-3ʹ, reverse 5ʹ -GAAAGGGTCTTGCTCTGTGG-3ʹ; miR-181c-5p, forward 5ʹ -UUGUAAGUUGGACAGCCACUCA-3ʹ, reverse 5ʹ -TGGTGTCGTGGAGTCG-3ʹ, GAPDH, forward 5ʹ-TAGCTGAACCGTAACTGGC-3ʹ, reverse 5ʹ-CCTAGGAACTAACGGCGTT-3ʹ. CT value was normalized to GAPDH and calculated with the 2−ΔΔCt.

Luciferase Reporter Assay

Targets between GSKIP and miR-181c-5p were speculated by Targetscan7.0 (http://www.targetscan.org). 3ʹUTR fragments of GSKIP (wt and mut) were constructed into a luciferase reporter vector (pmirGLO; Promega, USA). Thereafter, cells were transfected with Luc-GSKIP-wt or Luc- GSKIP -mut solely or combination with miR-181c-5p mimic. Luciferase activity was determined by the Dual-Luciferase Reporter Assay kit (Promega, USA).

Cell Proliferation Assay

Methyl thiazolyl tetrazolium (MTT; Solarbio, China) assays were carried out to detect proliferation in SiHa cells.Cells were seeded in 96-well plates at a density of (5–7) × 103 cells per well. Cells from the different treatment groups were cultured for another 24 h, 48 h or 72 h, and 10 μL of MTT (5 mg/mL) was added to each well and incubate for 4 h at 37 °C. Then, the reaction was stopped with 100 mL of dimethyl sulfoxide (DMSO), and the absorbance was measured at 490 nm on a microplate reader (Bio-Tek Instruments, Winooski, VT, USA).

Flow Cytometry

The ALDEFLUOR (ALDH) kit (StemCell technologies Inc) was used to identify and sort ALDH+ cells with high ALDH enzymatic activity. PI/AV-fluorescein isothiocyanate (BD Biosciences) was used to detect cell apoptosis rate. All assays were determined by the flow cytometry FACSCalibur instrument (BD Biosciences) according to the manufacturer’s recommendations.

Western Blots Assay

Proteins isolated from cells and tumors were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, MA, USA). After being blocked by 5% skim milk, proteins were incubated with the primary antibodies overnight at 4 °C. The primary antibodies were as follows: Ki67 (sc-23900, 1:1000 Santa Cruz Biotechnology, USA), PCNA (#2586, 1:1000, CST, USA), cleaved caspase-3 (#9660, 1:1000, CST, USA), Bcl-2 (#13-8800, 1 µg/mL, invitrogen, USA), Bax (#2772,1:1000, CST, USA), SOX2 (#2748, 1:1000, CST, USA), OCT4 (#2750, 1:1000, CST, USA), CD44 (#3578, 1:1000, CST, USA), E-cadherin (#14472, 1:1000, CST, USA), N-cadherin (#14215, 1:1000, CST, USA), Vimentin (#39325, 1:1000, CST, USA) and GAPDH (#97166, 1:1000, CST, USA). Then, the samples were washed with PBS and incubated with anti-rabbit IgG (#7074, 1:2000, CST, USA) and anti-biotin HRP-linked Antibody (#7074, 1:1000, CST, USA) for 1h. The bands were visualized with the ECL chemiluminescent detection kit (Perkin Elmer Cetus, Foster City, CA, USA).

Transwell Assay

Transwell assay was conducted to monitor invasive capacity. The cells were trypsinized and seeded in a serum-free medium supplemental with 10% FBS at 3×104 cells/well. After incubation for 24 h, the remaining cells on the upper surface were dislodged by a cotton swab. The migrated cells were fixed with 95% ethanol at room temperature and then stained with 1% crystal violet. The staining in five random fields of each well was examined under an optical microscope.

Animal Models

All animal experiments were performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals and were approved by the Ethical Committee of Taihe Hospital. A total of 20 BALB/c nude mice (male, 7-week old) were obtained from Taihe Hospital and housed in a controlled environment at 25 ± 3°C, humidity 60%, in a 12-h light/dark cycle with free access to food and water. SiHa cells (purchased from Procell BioTech Inc., China) transfected with miR-181c-5p mimic were subcutaneously injected into the right thigh to form xenograft tumors. The mice were divided into 2 groups with 9 in each group: control group, miR-181c-5p mimic group. Tumor weight was measured after 30 days post injection. Mice were euthanized by intraperitoneal injection of pentobarbital sodium (200 mg/kg body weight). Tumors were harvested for subsequent experiments.

Immunohistochemistry

After dewaxing, rehydration and repair, the paraffin section was sealed with 5% normal goat serum for 1h and co-cultured with primary antibodies Ki67 (#9449S, 1:400, CST, USA), cleaved caspase-3 (#9661, 1:200, CST, USA), CD44 (#3570, 1:50, CST, USA), Vimentin (#5741, 1:200, CST, USA) overnight at 4 °C. Sections were then washed with TBST and incubated with SignalStain® Boost IHC Detection Reagent (#8125, CST, USA) for 30 min at room temperature. After that, sections were stained with a SignalStain DAB Substrate Kit (#8059, CST, USA) and observed under an optical microscope.

Statistical Analysis

The statistical analysis was conducted with SPSS 21.0 (SPSS, Inc, Chicago, IL, USA). Data were expressed as mean ± S.D. and analyzed by t-test. Multiple sets of data were analyzed by One-way ANOVA followed by Bonferroni post-hoc test. P < 0.05 was considered statistically significant.

Results

The Expression of miR-181c-5p Is Down-Regulated in Different Cervical Cancer Cell Lines

As shown in Figure 1, we detected the expression level of miR-181c-5p in Ect/E6E7 (cervical immobilized cell line), Hela (cervical cancer cell line), SiHa (cervical squamous cell carcinoma cell line), HEC-1-A (endometrial adenocarcinoma cell line), and ME-180 (cervical epidermal cancer cell line). The results showed that miR-181c-5p was down-regulated in all tumor cell lines, especially in SiHa cell line, compared with that in the normal Ect/E6E7 cell line. And because the SiHa cell line was derived from human cervical squamous cell carcinoma, it was selected for subsequent studies.

Figure 1 miR-181c-5p was low expressed in multiple CC cell lines. RT-qPCR assay was used to detect the expression level of mir-181c-5p in Ect/E6E7, SiHa, Hela, HEC-1-A and ME-180 cell lines. The experiments were repeated three times and the data are represented as means ± SD; *P<0.05, **P<0.01 vs.Ect/E6E7 group.

GSKIP Is the Target Gene of miR-181c-5p

Bioinformatics software predicted that the putative target gene of miR181c-5p might be GSKIP, and that the binding site of miR-181c-5p and GSKIP was on the position 664–671 of GSKIP 3ʹUTR (Figure 2A). In order to explore how miR181c-5p regulated GSKIP, SiHa cells were transfected with miR-181c-5p mimics (mimic group). The relative expression levels of miR-181c-5p and GSKIP mRNA were detected by qRT-PCR. The results suggested that the relative level of miR-181c-5p in the mimic group was significantly increased; but the relative level of GSKIP mRNA in the mimic group was obviously decreased compared with that in the control group (Figure 2B). Next, we built the overexpression vector for GSKIP (pc- GSKIP). SiHa cells were co-transfected with mimic and pc- GSKIP, and investigated the relationship between miR-181c-5p and GSKIP again. As shown in Figure 2C, compared with the control group, the GSKIP mRNA level of mimic group decreased significantly, while that of pc-GSKIP group was increased significantly; Compared with the pc- GSKIP group, the level of GKSIP mRNA in mimic+ pc-GSKIP group decreased significantly. Thus, miR-181c-5p significantly offset the overexpression of GSKIP. To confirm the targeting relationship between mir181 and GSKIP, we performed luciferase reporter gene detection. The result demonstrated that, in the GSKIP wild type, the luciferase activity was significantly reduced after the addition of miR-181c-5p mimic; but in the GSKIP mutation type, there was no obvious change in the luciferase activity even if miR-181c-5p mimic was added. In summary, these data indicated that miR-181c-5p directly targeted the 3ʹUTR of the GSKIP gene in cervical squamous carcinoma cells (Figure 2D).

Figure 2 GSKIP was a direct target gene of miR-181c-5p. (A) TargetScan was used to predict the binding site between GSKIP 3ʹ UTR and miR-181c-5p. (B) RT-qPCR was used to detect the levels of miR-181c-5p and GSKIP mRNA in SiHa cells after transfection with a mimic. (C) RT-qPCR was used to detect the levels of GSKIP mRNA in SiHa cells after transfection with a mimic and a pc-GSKIP. (D) The luciferase reporter gene assay was used to verify the targeting relationship between miR-181c-5p and GSKIP. The experiments were repeated three times and the data are represented as means ± SD; *P<0.05 vs control (B, C) or GSKIP wt (D), #P<0.05 vs pc-GSKIP.

miR-181c-5p Overexpression Inhibited Proliferation and Promoted Apoptosis in SiHa Cells by Targeting the GSKIP Gene

As can be seen from the MTT assay, the clone formation rate of the mimic group was significantly decreased, that of the pc-GSKIP group was increased, both of which compared with that of the control group; but the clone formation rate of the mimic + pc-GSKIP group was significant decreased compared with that of the pc-GSKIP group (Figure 3A). Flow cytometry was used to detect the apoptotic cells. The results showed that, compared with that of the control group, cell apoptosis rate of the mimic group was significantly increased,that of the pc-GSKIP group was significantly decreased; however, cell apoptosis rate of mimic+ pc-GSKIP group were significantly elevated compared with that of the pc-GSKIP group (Figure 3B). In Western blotting assay, the expressions of Ki67 and PCNA related to proliferation and Caspase-3, Bcl-2 and Bax related to apoptosis were detected. The results showed that the expressions of Ki67 and PCNAof the mimic group were decreased and those of Caspase-3 and Bax/bcl-2 were increased, both of which compared with those of the control group; The expressions of Ki67 and PCNA of the pc-GSKIP group were greatly increased and those of Caspase-3 and Bax/bcl-2 were greatly decreased, both of which compared with those of the control group. In the mimic+ pc-GSKIP group, Ki67and PCNA were obviously decreased and Caspase-3 and Bax/Bcl-2 were significantly increased, both of which compared with those of the pc-GSKIP group (Figure 3C). All the above experiments indicated that miR-181c-5p overexpression limited the proliferation of SiHa cells growth by targeting GSKIP.

Figure 3 miR-181c-5p inhibited cell proliferation and promoted cell apoptosis in SiHa cells. Cells were divided into four groups: control group, untransfected SiHa cells; mimic group, SiHa cells transfected with miR-181c-5p; pc-GSKIP group, cells transfected with overexpression vector of GSKIP; mimic+pc-GSKIP group, cells co-transfected with mimic and pc-GSKIP. (A) MTT was used to detect the rate of cell cloning rate. (B) Flow cytometry was used to detect the apoptosis rate. (C) Western Blot was used to detect levels of proteins associated with proliferation and apoptosis. The experiments were repeated three times and the data are represented as means ± SD; *P<0.05 vs control, #P<0.05 vs pc-GSKIP.

miR-181c-5p Overexpression Limited the Stem-Like Properties of SiHa Cells by Targeting the GSKIP Gene

In the present study, ALDH positive cells were separated by FACS. Compared with the control group, ALDH positive cells of the mimic group were significantly decreased, and those of the pc- GSKIP group were significantly increased; However, ALDH positive cells of the mimic+pc-GSKIP group were obviously decreased compared with those of the pc-GSKIP group (Figure 4A). Subsequently, molecules associated with stem cell properties such as SOX2, OCT4 and CD4 were detected by West Blotting. The results showed that, compared with the control group, the expressions of SOX2, OCT4 and CD44 of the mimic group were significantly down-regulated compared with those of the mimic group and those of the pc-GSKIP group were significantly up-regulated; the expressions of SOX2, OCT4 and CD44 of the mimic+ pc-GSKIP group were obviously down-regulated compared with those of the pc-GSKIP (Figure 4B). The above two experiments showed that miR-181c-5p overexpression which targeted GSKIP could inhibit stem cell-like characteristics in SiHa cells.

Figure 4 miR-181c-5p blocked stem-like properties in SiHa cells. (A) Flow cytometry was used to detect ALDH positive cells in SiHa cells. (B) Western Blot was performed to detect protein levels of stemness-related SOX2, OCT4 and CD44 in SiHa cells. The experiments were repeated three times and the data are represented as means ± SD; *P<0.05 vs control, #P<0.05 vs pc-GSKIP.

miR-181c-5p Overexpression Repressed the Invasion of SiHa Cells by Targeting the GSKIP Gene

As can be seen from the transwell assay, compared with the control group, the invasion cells of the mimic group were significantly reduced and those of the pc-GSKIP group were significantly increased; However, the invasion cells of the mimic+ pc- GSKIP group were significantly decreased compared with those of the pc-GSKIP group (Figure 5A). Expressions of proteins related to epithelial mesenchymal transition (EMT), such as E-cadherin, N-cadherin and Vimentin, were detected. The data suggested that, compared with the control group, E-cadherin expression of the mimic group was significantly increased and N-cadherin and Vimentin expressions were significantly decreased; E-cadherin expression of the pc-GSKIP group was significantly reduced and N-cadherin and Vimentin were significantly increased. In mimic+pc-GSKIP group, E-cadherin expression was largely increased, but N-cadherin and Vimentin expressions were significantly decreased, both of which compared with those of the pc-GSKIP group (Figure 5B). These data indicated that miR-181c-5p overexpression restrained the invasion of SiHa cells by targeting GSKIP.

Figure 5 miR-181c-5p suppressed the invasion in SiHa cells. (A) Transwell assay was used to detect the invasion capacities of SiHa cells. (B) Western Blot was performed to detect protein levels of invasion-related E-cadherin, N-cadherin and Vimentin in SiHa cells. The experiments were repeated three times and the data are represented as means ± SD; *P<0.05 vs control, #P<0.05 vs pc-GSKIP.

miR-181c-5p Mitigates the Tumor Growth in vivo

In order to verify the results of in vitro experiments, we also conducted a mice xenograft experiment. The result demonstrated that the tumor weight in the mimic group was significantly reduced compared with that in the control group (Figure 6A and B). The expressions of miR-181c-5p and GSKIP mRNA in tumor tissues were detected by qRT-PCR. The result showed that the relative level of miR-181c-5p in mimic group was significantly increased and the relative level of GSKIP mRNA was significantly decreased, both of which compared with those in the control group (Figure 6C). At the protein level, expressions of Ki67, Caspase-3, CD44 and Vimentin in tumor tissues were consistent with the results of assays in vitro (Figure 6D and E). In a word, in vivo experiments demonstrated that miR-181c-5p overexpression inhibited the growth of squamous cell carcinoma in vivo.

Figure 6 miR-181c-5p inhibited cervical SCC tumor growth in vivo. (A) Tumor tissue from subcutaneous xenotransplantation of mice. (B) The tumor weight. (C) qRT-PCR was used to detect expression levels of miR-181c-5p and GSKIP from the mice. (D) IHCs were carried out to detect the impacts of the processes on proliferation, apoptosis, stem cell-like property and EMT from the mice. (E) The proportion of Ki67, caspase-3, CD44 or Vimentin positive cells in vivo. The experiments were repeated three times and the data are represented as means ± SD; *P<0.05 vs control.

Discussion

In recent years, various studies have clarified that microRNA regulated gene expression by affecting important regulatory genes, which thus resulted in the occurrence of CC.8 It has been indicated that the aberrant expression of the miR-181s in tumor tissues played a potentially important role in tumor development and progression.17

However, what roles miR-181c-5p plays in cervical SCC has been rarely reported. In this study, we first investigated the expression of miR-181c-5p in the normal cervical cell line (Ect/E6E7) and other four cervical cancer cell lines (SiHa, Hela, HEC-1-A and ME-180). The results showed that the expression of miR-181c-5p was down-regulated in all cervical tumor cell lines and lowest in SiHa cell lines from cervical squamous cell carcinoma. Therefore, we focused on the effect of miR-181c-5p on SiHa cells. The prediction of target gene of miR-181c-5p indicated that GSKIP mRNA was potentially regulated by miR-181-5p. Then, it was found that mir-181c-5p was negatively correlated with the expression of GSKIP in SiHa cells through experiments, and further luciferase detection confirmed that GSKIP was the target gene of miR-181c-5p.

The upregulation of miR-181 family inhibited the proliferation of different types of tumor cells and promoted apoptosis.11 Reports showed that transfection of the precursor miR-181c molecule blocked growth of two gastric cancer cell lines.18 Studies showed that miR-181c exhibited tumor-suppression via the regulation of NCAPG levels in hepatocellular carcinoma tissue samples.19 It has been elucidated that miR-181c inhibited the biological progression of osteosarcoma via targeting SMAD7 and regulating TGF-β signaling pathway.20 Consistent with these reports, this study found that the colony forming rate of SiHa cells with increased expression of miR-181c-5p was obviously reduced. These results indicated that miR-181c-5p suppressed the viability of cervical SCC.

Studies have shown that microRNA plays an important role in the stem cell-like characteristics of a variety of tumors. Liu et al reported that miR-612 inhibited the stem cell-like property of HCC by regulating the Sp1/Nanog pathway.21 Zhou’s study found that miR-590-5p inhibited breast cancer cell stemness and metastasis by targeting SOX2.22 Other study has shown that miR-106b-5p promoted stem cell-like characteristics of HCC by targeting the PTEN via PI3K/Akt pathway.23 In this paper, the effect of mir-181c-5p on the stem cell-like characteristics of SiHa cells was investigated. Cancer stem cells have the ability to form tumors after transplantation, as opposed to massive tumor cells or non-tumorigenic cancer cells.24 Overactivation of Wnt-mediated signals is widely recognized as one of the most common events in CSCs of various tumor types.25 Here, we identified GSKIP gene as the target of miR-181c-5p in cervical SCC. GSKIP is an activator of Wnt/β-catenin signals and leads to upregulation of β-catenin by negatively modulating the β-catenin inhibitor GSK3β.16,26 We found that miR-181c-5p attenuated the stemness of SiHa cells by inhibiting the expression of GSKIP. In addition, at the protein level, miR-181c-5p down-regulated the levels of SOX2, OCT4 and CD44 with stem cell characterization. This suggested that miR-181c-5p reduced the stemness in SiHa cells by targeting the GSKIP gene.

Malignant tumors are usually characterized by tumor migration and invasion.27 An epithelial-mesenchymal transition (EMT) is a biologic process which allows polarization of epithelial cells. EMT normally works with the basement membrane through its basal surface to undergo multiple biochemical changes that lead it to consider mesenchymal cell phenotypes, including enhanced migration, invasion, enhanced anti-apoptosis, and significantly increased production of extracellular matrix (ECM) components.28 Completion of an EMT indicates the potential degeneration of basement membranes and the formation of mesenchymal cells, which can migrate from their origin in the upper cortex.29 Studies have shown that EMT plays a crucial role in tumor migration and invasion. For example, He et al confirmed that upregulation of miR-181c inhibited EMT, with E-cadherin increased, and N-cadherin and Vimentin decreased in glioblastoma cells.27 Han et al demonstrated that miR-137 could significantly suppress the migration and invasion of MCF-7 cells, which might be correlated with its suppressive effects on the EMT procedure.30 In this study, Transwell assay was used to investigate whether miR-181c-5p regulated migration and invasion of cervical SCC. The results showed that the number of invasive cells was clearly very low. At the protein level, the expressions of E-cadherin, N-cadherin and Vimentin associated with the EMT process were also detected. We found that the expression of E-cadherin was high while both of N-cadherin and Vimentin were low in SiHa cells transfected with miR-181c-5p mimics. It is well known that E-cadherin expression is up-regulated and N-cadherin and Vimentin expressions are down-regulated during the EMT process.29 Obviously, the data showed that EMT-like changes could be inhibited by miR-181c-5p overexpression. In addition, in vivo experiments in mice further verified that miR-181c-5p overexpression can significantly inhibit cell proliferation and metastasis. In ICH assay, it was also further confirmed that miR-181c-5p inhibited the expressions of Ki67, CD44 and Vimentin but improved the expression of Caspase-3 in cervical SCC tissues.

In conclusion, the miR-181c5p overexpression was considered to be negatively correlated with the expression of GSKIP in cervical SCC tissues. MiR-181c-5p inhibited the migration and invasion of cervical SCCs as well as EMT via Wnt/β-catenin signaling. These results offer basic data on the role of miR-181-5p in the progress of cervical SCC. Meanwhile, the role of miR-181c-5p as a gene target is valuable for further study.

Abbreviations

GSKIP, glycogen synthase kinase 3β interaction protein; CC, cervical cancer; SCC, squamous cell carcinoma; CSC, cancer stem cell; HPV, human papillomavirus; PKA, protein kinase; GSKIP, GSK3β glycogen synthesis kinase 3β; AKAP, A-kinase anchoring protein; EMT, epithelial-mesenchymal transition.

Acknowledgments

We would like to give our sincere gratitude to the reviewers for their constructive comments.

Disclosure

The authors declare that they have no competing interests.

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