Inhibition of Microrna-766-5p Attenuates the Development of Cervical Cancer Through Regulating SCAI.

PURPOSE: MicroRNAs (miRNAs) are considered to play anti-tumor roles in cancers. This study is designed to illustrate the role and potential mechanism of miR-766-5p in cervical cancer (CC) progression.
METHODS: MiR-766-5p expression in tissues and serum of CC patients was detected by quantitative reverse-transcription PCR (qRT-PCR). Receiver operating characteristic (ROC) curve was performed to evaluate the diagnostic value of serum miR-766-5p in CC. The 5-ethynyl-2'-deoxyuridine (EdU) assay, flow cytometry, wound healing as well as transwell assay were utilized to detect the proliferation, apoptosis, migration and invasion of CC cells, respectively. The interaction between miR-766-5p and SCAI was confirmed by dual-luciferase reporter gene assay. Xenografted tumor model was established to measure the growth of tumor xenograft in vivo.
RESULTS: MiR-766-5p was significantly increased in tissues and serum of CC patients. ROC curve suggested that serum miR-766-5p could serve as a biomarker for the diagnosis of CC. Inhibition of miR-766-5p suppressed the proliferation, migration and invasion, and promoted the apoptosis of CC cells. SCAI was proved to be a target of miR-766-5p. Silencing of SCAI eliminated the inhibiting effects of miR-766-5p inhibitor on the proliferation, migration and invasion of CC cells in vitro. Additionally, down-regulation of SCAI also reversed the inhibitory effect of miR-766-5p inhibitor on the growth of tumor xenograft in vivo.
CONCLUSIONS: Inhibition of miR-766-5p restrains the cell proliferation, migration and invasion, and promotes the apoptosis in CC through negatively regulating SCAI.

Introduction

Cervical cancer (CC), a malignant tumor of the cervix, usually occurs in the female reproductive tract.[1] According to the previous study, CC is estimated to cause approximately 509,590 new cases and 311,365 deaths annually.[2] Currently, traditional treatments for CC are surgery and chemotherapy, whereas these methods are usually effective for patients with CC in early stage. Shockingly, a large number of CC patients with advanced stage still suffer from metastasis and recurrence.[3] Hence, it is urgent to explore the regulatory mechanism in CC and characterize a new strategy for treating CC.

MicroRNAs (miRNAs) are some short single-stranded RNA molecules approximately 22 nucleotides in length, and defined as a class of non-coding RNAs that play key roles in the regulation of gene expression.[4,5] MiRNAs have been reported to act as important mediators in multiple cellular processes, such as the proliferation, differentiation, apoptosis and cell cycle.[6] Until now, a variety of researches have been indicated that dysregulation of miRNAs are involved in regulating many human cancers, including CC. For instance, miR-92a stimulates the proliferation and invasion of CC cells through negatively regulating FBXW7.[7] Up-regulation of miR-31 facilitates the proliferation, migration and invasion of CC cells.[8] More importantly, miR-766-5p has been also reported to be participated in modulating multiple cancers, such as breast cancer,[9] colorectal cancer (CRC),[10] lung cancer[11] and hepatocellular carcinoma (HCC).[12] Jia et al.[10] have indicated that inhibition of miR-766-5p suppresses the proliferation and induces apoptosis of SW480 cells. Yang et al.[12] have revealed that miR-766 exerts an carcinogenic effect in progression of HCC by modulating NR3C2. However, the exact role and possible mechanism of miR-766-5p in CC are rarely studied.

MiRNAs have been discovered to modulate the expression of genes through binding to the target sites that located in the 3′-untranslated regions (3′-UTRs) of mRNAs.[13] Thus, researches on tumor-related miRNAs and their targets are of great significance to clarify their biological functions in the development of cancers.[14] Suppressor of cancer cell invasion (SCAI) is a new highly conserved protein, which mediates metastasis via a 3-dimensional matrix.[15] SCAI has been reported to function as a target gene for several miRNAs in human cancers. For example, miR-1228 functions as an onco-miRNA, and promotes the progression of breast cancer through targeting SCAI.[16] MiR-1270 contributes to the proliferation, migration and in vivo transplantation in papillary thyroid cancer (PTC) via inversely regulating SCAI.[17] Nevertheless, the regulatory mechanism between SCAI and miR-766-5p in CC is still unclear.

In our study, miR-766-5p expression in tissues and serum of CC patients, and the regulatory mechanism between miR-766-5p and SCAI on the occurrence and development of CC were investigated. Our data revealed that miR-766-5p may serve as a potential therapeutic-target for CC.

Materials and Methods

Patients and Specimens

This study was permitted by the ethics committee of Weifang Yidu Central Hospital (Approval ID: JN191). All patients in this study had signed the informed consent. From September 2017 to September 2018, a total of 54 paired CC tumor tissues and adjacent normal tissues were collected from CC patients by resection in our hospital. All patients were pathologically diagnosed as CC. The blood samples were collected from 67 CC patients (including the 54 patients prior) and 67 age-matched healthy volunteers in our hospital. These blood samples were centrifuged at 3000 rpm for 15 min at 4°C, then the supernatants were immediately collected and stored at −80°C.

Cell Culture

Human normal cervical epithelial cells (HCerEpiC) and CC cell lines (CaSki, HT-3, C33A and SiHa) were all obtained from Shanghai Cell Bank of the Chinese Academy of Science (Shanghai, China). The cells were cultured in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) containing with 10% fetal bovine serum (FBS; Thermo Fisher Scientific, Waltham, MA, USA) and 1% penicillin-streptomycin (Hyclone, South Logan, UT, USA) at 37°C.

Cell Transfection

CaSki and SiHa cells (1 × 105 cells/well) were cultured in 24-well plates. MiR-766-5p mimics and mimics negative control (mimics NC) (GeneCopoeia, Rockville, MD, USA), as well as miR-766-5p inhibitor and inhibitor negative control (inhibitor NC) were transfected into CaSki and SiHa cells using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA) to detect the transfection efficiency. To explore the interaction between miR-766-5p and SCAI, CaSki and SiHa cells were co-transfected with inhibitor NC or miR-766-5p inhibitor and siRNA negative control (si-NC) or SCAI siRNA (si-SCAI) for 48 h. After transfection, cells were harvested for the follow-up experiments.

Quantitative Reverse-Transcription PCR (qRT-PCR)

Total RNA was extracted from tissues, blood supernatants or cells using TRIzol Reagent (Invitrogen). Moloney Murine Leukaemia Virus (M-MLV) Reverse Transcriptase (Invitrogen) was utilized to reverse transcribe the extracted RNA into cDNA. qRT-PCR was carried out using SYBR ® Premix exTaq™ (Takara, Dalian, Liaoning, China) on StepOnePlus™ Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) under the following reaction conditions: 95°C for 3 min, followed by 40 cycles at 95°C for 15 s, 60°C for 30 s and 72°C for 20 s. U6 or β-actin was used as the internal control. The paired data (Figure 1A, C and 2A) were calculated using the 2−ΔCt method and normalized to the mean value of 2−ΔCt in normal tissues. The grouping data (Figure 1B, 3A, 4C, 2B-C and 5C) were calculated using the 2−ΔΔCt method, and the mean Ct of one repeat in the control group was used as ΔCt2. Primer sequences were shown in Table 1.

Figure 1.

MiR-766-5p is up-regulated in tissues and serum of CC patients. A, Relative expression of miR-766-5p in CC tissues (n = 54) and adjacent normal tissues (n = 54) was detected by qRT-PCR. B, Relative expression of miR-766-5p in HCerEpiC and CC cell lines (CaSki, HT-3, C-33A and SiHa). C, Comparison of miR-766-5p expression in the serum of healthy controls (n = 67) and CC patients (n = 67). D, ROC curve analysis was performed to distinguish between healthy people and CC patients. P < 0.0001 vs. normal tissues (A); ** P < 0.01 vs. HCerEpiC (B); P < 0.0001 vs. healthy (C).

Figure 2.

Inhibition of miR-766-5p alleviates the development of CC through regulating SCAI in vitro. A and B, SCAI expression in CC tissues and cell lines was detected by qRT-PCR. C, Transfection efficiency was assessed by qRT-PCR. D, EdU assay was used to evaluate the proliferation of the transfected SiHa cells. E, The apoptosis of SiHa cells was assessed by flow cytometry. F, Wound healing analysis was conducted to detect the migration of the transfected SiHa cells. G, Transwell analysis was utilized to measure the invasion of the transfected SiHa cells. inhibitor NC + si-NC, SiHa cells co-transfected with inhibitor negative control and siRNA negative control; inhibitor + si-NC, SiHa cells co-transfected with miR-766-5p inhibitor and siRNA negative control; inhibitor + si-SCAI, SiHa cells co-transfected with miR-766-5p inhibitor and SCAI siRNA. P < 0.0001 vs. normal tissues (A); ** P < 0.01 vs. HCerEpiC (B); ** P < 0.01 vs inhibitor NC + si-NC; # P < 0.05 vs. inhibitor + si-NC (C-G).

Figure 3.

Inhibition of miR-766-5p suppresses the proliferation and promotes the apoptosis of CC cells in vitro. A, MiR-766-5p expression in miR-766-5p transfected CaSki SiHa cells. B, EdU assay was used to assess the proliferation of CaSki and SiHa cells (scale bar = 100 μm). C, The apoptosis of CaSki and SiHa cells was detected by flow cytometry. Mock, CaSki or SiHa cells without transfection; mimics-NC, CaSki or SiHa cells transfected with mimics negative control; miR-766-5p mimics, CaSki or SiHa cells transfected with miR-766-5p mimics; inhibitor-NC, CaSki or SiHa cells transfected with inhibitor negative control; miR-766-5p inhibitor, CaSki or SiHa cells transfected with miR-766-5p inhibitor. ** P < 0.01 vs. Mock (A); ** P < 0.01 vs. inhibitor-NC (B, C).

Figure 4.

SCAI is a direct target of miR-766-5p. A, TargetScan software was utilized to predict the binding site of miR-766-5p and SCAI. B, Dual-luciferase reporter gene assay was performed to verify the target correlation between miR-766-5p and SCAI. C, SCAI expression in miR-766-5p inhibitor-transfected CaSki cells and SiHa cells. D, Pearson’s correlation analysis was used to detect the relationship between the expression of miR-766-5p and SCAI. Mock, CaSki or SiHa cells without transfection; inhibitor NC, CaSki or SiHa cells transfected with miR-766-5p inhibitor negative control; miR-766-5p inhibitor, CaSki or SiHa cells transfected with miR-766-5p inhibitor. ** P < 0.01 vs. mimics NC (B); ** P < 0.01 vs. Mock (C-D); P < 0.0001, r = −0.6554 (E).

Figure 5.

Inhibition of miR-766-5p suppresses the growth of tumor xenograft through regulating SCAI in vivo. A, Growth curves of xenograft tumors at 5 weeks post-injection. B, Tumor weight was determined at 5 weeks post-injection. C, qRT-PCR was used to examine the relative mRNA expression of SCAI in tumor. inhibitor NC + si-NC, mice injected with SiHa cells co-transfected with inhibitor negative control and siRNA negative control; inhibitor + si-NC, mice injected with SiHa cells co-transfected with miR-766-5p inhibitor and siRNA negative control; inhibitor + si-SCAI, mice injected with SiHa cells co-transfected with miR-766-5p inhibitor and SCAI siRNA. * P < 0.05, ** P < 0.01 vs. inhibitor NC + si-NC; # P < 0.05, ## P < 0.01 vs. inhibitor + si-NC.

Table 1.

Primer Sequences.

Name of primer	Sequences
miR-766-5p F	5′-TCGAGTACTTGAGATGGAGTTTT-3′
miR-766-5p R	5′-GGCCGCGTTGCAGTGAGCCGAG-3′
SCAI F	5′-CGGGAAACACGAAATTATCC-3′
SCAI R	5′-GCTTCTGGAGATGAGGATTCTC-3′
U6 F	5′-CATTGCACTTGTCTCGGTCT-3′
U6 R	5′-GGTCCGAGGTATTCGCACT-3′
GAPDH F	5′-AGCCTCCCGCTTCGCTCTCTGC-3′
GAPDH R	5′-ACCAGGCGCCCAATACGACCAAA-3′

5-Ethynyl-2′-deoxyuridine (EdU) Proliferation Assay

The proliferation of CaSki and SiHa cells was assessed using an EdU assay kit (Ribobio, Guangzhou, China) based on the manufacturer’s instructions. In brief, cells were cultured with 50 μM EdU for 2 h at 37°C. Then, the cells were fixed with 4% formaldehyde for 15 min and permeabilized using 0.5% Triton X-100 for 20 min at room temperature. Next, cells were incubated with 1 × Apollo reaction cocktail. After incubation for 30 min, DNA was stained with DAPI (4′,6-diamidino2-phenylindole) for another 30 min. The EdU-positive cells were observed under a fluorescence microscope (Carl Zeiss, Oberkochen, Germany).

Flow Cytometry Assay

CaSki and SiHa cells (1×105 cells) were suspended in 500 µl binding buffer. Then the cells were stained with 5 µl Annexin V-EGFP and 5 ml Propidium Iodide (PI) at room temperature for 10 min in the dark. The apoptosis rate of cells was calculated by a flow cytometer (BD Biosciences).

Wound Healing Assay

CaSki and SiHa cells (1×105 cells/μl) were cultured in 12-well plates and grown until 100% confluence in RPMI-1640 medium. Then the wounds on cell monolayer layers were created using a pipette tip. After that, the cells were incubated for 24 h, and the photomicrographs of the scratch wounds were captured. The relative migration rate was analyzed using Image J software.

Transwell Invasion Assay

Cell invasion was measured by transwell membranes (BD, Franklin Lakes, NJ, USA). First, cells (2 × 105/μl) were seeded into the upper chamber coated with Matrigel. RPMI-1640 medium (600 μl) containing 100 ng/mL SDF-1 was added to the lower chamber as a chemoattractant. After incubation for 48 h, the invasive cells were stained with 0.1% crystal violet for 30 min. Four random visual fields of each insert were counted under an inverted microscope (Olympus Ckx53).

Western Blot Assay

Total protein was extracted from CaSki and SiHa cells using RIPA lysis buffer. The proteins were separated by 10% SDS-PAGE and then transferred to PVDF membranes. After that, membranes were incubated with primary antibodies against SCAI (1:1,000; Abcam) and α-tubulin (1:1,000; Abcam) for overnight at 4˚C. Then, tris-buffered saline Tween-20 (TBST) was used to wash the membranes for 3 times. Subsequently, at room temperature, the HRP-conjugated anti-mice IgG secondary antibody (1:5,000; Santa Cruz, Waltham, MA, USA) was added to incubate for 1 h. α-tubulin was used as the internal reference. The membranes were developed using an ECL reagent (Thermo Fisher Scientific) under Gel-Pro analyzer (version 4.0, USA).

Luciferase Reporter Gene Assay

The binding site between miR-766-5p and SCAI was predicted using TargetScan software (http://www.targetscan.org/). According to the predication, SCAI Mutant (5′-CUUUCUUUAGCACUCAGGAGGA-3′) and SCAI Wild type (5′-CUUUCUUUAGCACUCUCCUCCAA-3′) were cloned and combined with pmirGLO vector (Promega, Madison, WI, USA), named SCAI-WT and SCAI-MUT, respectively. Then, CaSki and SiHa cells were all co-transfected with SCAI-WT or SCAI-MUT and miR-766-5p mimics or mimics NC using Lipofectamine 2000. After 48 h of transfection, a dual-luciferase reporter gene assay system (Promega) was utilized to assess the luciferase activity.

Xenografted Tumor Model in Mice

Animal testing procedures were performed on the basis of the “Guidelines for the Care and Use of Laboratory Animals” published by the National Institutes of Health. This study was permitted by the ethics committee of Weifang Yidu Central Hospital (Approval ID: JN191). Male nude mice (BALB/c, 4 weeks old, weighing 20 ± 2 g) were obtained from Shanghai experimental animal center, Chinese academy of sciences (Shanghai, China). Mice were randomly divided into 3 groups (n = 4): inhibitor-NC + si-NC, inhibitor + si-NC, inhibitor + si-SCAI. SiHa cells at logarithmic growth phase were extracted (1 × 107 cells/nude mice), and 0.2 ml of extracted cells were injected into the intradermal left axilla. The longest diameter (L) and the shortest diameter (W) of the transplanted tumor were measured with Vernier caliper every 1 week after injection. Tumor volumes were calculated using the following formula: V = L×W2/2. At the end of 5 weeks, the mice were anesthetized with chloral hydrate and killed by decapitation. The tumor xenograft was separated from mice and weighted.

Statistical Analysis

The data were analyzed by the SPSS 22.0 statistical software (SPSS Inc., Chicago, IL). Data were presented as mean ± standard deviation (SD). Student’s t-test was used to compare the significant difference between 2 groups, and the One-way ANOVA test was utilized to analyze more than 2 groups. Pearson’s correlation analysis was used to determine the correlation between the expression levels of miR-766-5p and SCAI in CC tissues. The diagnostic analysis was performed through receiver operating characteristic (ROC) curve analysis with healthy controls as true negative cases and patients with CC as true positive cases. Differences were considered statistically at P < 0.05. All experiments were performed in triplicate.

Results

Up-Regulation of miR-766-5p Is Observed in Tissues and Serum of CC Patients

qRT-PCR demonstrated that miR-766-5p expression in CC tissues was remarkably enhanced than that in normal tissues (P < 0.0001, Figure 1A). Consistently, in comparison to the HCerEpiC, miR-766-5p expression in CC cell lines (CaSki, HT-3, C33A and SiHa) was remarkably elevated (P < 0.01, Figure 1B). As illustrated in Table 2, miR-766-5p expression was strongly correlated with tumor size, Federation of Gynecology and Obstetrics (FIGO) stage and lymph node metastasis. The expression of miR-766-5p in the serum of CC patients was also detected by qRT-PCR. We found that miR-766-5p expression was significantly increased by contrast to that in the serum of healthy volunteers (P < 0.0001, Figure 1C). Additionally, ROC curve further revealed that serum miR-766-5p could accurately discriminate CC patients from healthy controls with an area under the ROC curve (AUC) of 0.851 (95% CI: 0.787-0.915) (Figure 1D). The results implied that miR-766-5p might serve as an onco-miRNA in CC.

Table 2.

Correlation Between miR-766-5p Expression and Clinicopathological Features of Patients With Cervical Cancer (CC).

Clinicopathological features	No. of cases (n = 54)	miR-766-5p expression		p-value
		Low	High
Age				0.1724
<45	25	15	10
≥45	29	12	17
Tumor size (cm)
<4	33	21	12	0.0120*
≥4	21	6	15
Histology
Squamous	36	21	15	0.0833
Adenocarcinoma	18	6	12
FIGO stage
Ib-IIa	35	23	12	0.0017*
IIb-IIIa	19	4	15
Differentiation
Well/Moderate	34	18	16	0.5730
Poor	20	9	11
Lymph node metastasis
Negative	45	26	19	0.0106*
Positive	9	1	8

Note: * P < 0.05.

Inhibition of miR-766-5p Suppresses the Proliferation and Promotes the Apoptosis of CC Cells in Vitro

To investigate the role of miR-766-5p on the biological functions of CC cells, the transfection efficiency of miR-766-5p was firstly detected. qRT-PCR results showed that miR-766-5p expression was significantly up-regulated by transfection of miR-766-5p mimics, whereas was down-regulated by transfection of miR-766-5p inhibitor (P < 0.01, Figure 3A). EdU assay indicated that the EdU positive cells (green) were remarkably declined in the miR-766-5p inhibitor group compared to the inhibitor-NC group (P < 0.01, Figure 3B). As presented in Figure 3C, flow cytometry analysis demonstrated that the apoptosis rate of CC cells was significantly elevated in the miR-766-5p inhibitor group in contrast to that in inhibitor-NC group (P < 0.01). Above all, inhibition of miR-766-5p suppressed the proliferation, and induced apoptosis of CC cells.

Inhibition of miR-766-5p Restrains the Migration and Invasion of CC Cells in Vitro

The effects of miR-766-5p on the migration and invasion of CaSki and SiHa cells were assessed by wound healing and transwell assay, respectively. The results showed that the relative migration and invasion rate of CC cells were all markedly decreased by transfection of miR-766-5p inhibitor (P < 0.01, Figure 6A and B). Taken together, inhibition of miR-766-5p restrained the migration and invasion of CC cells.

Figure 6.

Inhibition of miR-766-5p restrains the migration and invasion of CC cells in vitro. A, Wound healing assay was utilized to measure the migration of CaSki and SiHa cells. B, Transwell analysis was conducted to assess the invasion of CaSki and SiHa cells. Inhibitor NC, SiHa cells transfected with miR-766-5p inhibitor negative control; miR-766-5p inhibitor, SiHa cells transfected with miR-766-5p inhibitor. ** P < 0.01 vs. inhibitor-NC.

SCAI Is a Target Gene of miR-766-5p

To explore whether miR-766-5p regulates SCAI in the development of CC, we predicted the potential binding site between miR-766-5p and SCAI using Targetscan software (Figure 4A). Dual-luciferase reporter gene assay revealed that overexpression of miR-766-5p reduced the luciferase activity of SCAI-WT, whereas had no effect on that of SCAI-MUT (P < 0.01, Figure 4B). qRT-PCR results indicated that SCAI expression in CC cells was markedly up-regulated by transfection of miR-766-5p inhibitor (P < 0.01, Figure 4C), whereas transfection of inhibitor NC did not influence the expression of SCAI. Similarly, Western blot assay demonstrated that the protein level of SCAI in both CaSki and SiHa cells was elevated by transfection of miR-766-5p inhibitor (P < 0.01, Figure 4D). In addition, Pearson’s correlation analysis illustrated a negative correlation between the expression of miR-766-5p and SCAI (P < 0.0001, r = −0.6554; Figure 4E). The above data suggested that SCAI was a target gene of miR-766-5p and was negatively modulated by miR-766-5p.

Inhibition of miR-766-5p Alleviates the Development of CC Through Regulating SCAI In Vitro

SCAI expression was significantly decreased in CC tissues than that in normal tissues (P < 0.0001, Figure 2A). Consistently, in comparison to HCerEpiC, the expression of SCAI was down-regulated in CC cell lines (CaSki, HT-3, C33A and SiHa) (P < 0.01, Figure 2B). To investigate whether SCAI was involved in the action mechanism of miR-766-5p in CC progression, miR-766-5p inhibitor and SCAI siRNA were transfected into SiHa cells. We found that si-SCAI reversed the promoting effect of miR-766-5p on SCAI expression (P < 0.05, Figure 2C). As presented in Figure 2D-G, the EdU positive cells, relative migration and invasion rate were all decreased, while the apoptosis rate was significantly enhanced in the inhibitor + si-NC group in comparison to the inhibitor-NC + si-NC group (P < 0.01). However, si-SCAI reversed the inhibiting effects of miR-766-5p inhibitor on the proliferation, migration and invasion, and the promoting effect on the apoptosis of CC cells (P < 0.05). These data indicated that inhibition of miR-766-5p alleviates the development of CC through regulating SCAI in vitro.

Inhibition of miR-766-5p Suppresses the Growth of Tumor Xenograft Through Regulating SCAI In Vivo

In order to further validate the effect of miR-766-5p on CC tumorigenesis, in vivo experiments were conducted in mice. As illustrated in Figure 5A-B, 5 week later, tumor volume and tumor weight were both decreased in the inhibitor + si-NC group by contrast to those in inhibitor NC + si-NC group (P < 0.01). In addition, in comparison to the inhibitor NC + si-NC group, the expression of SCAI was also remarkably declined in inhibitor + si-NC group, (P < 0.01, Figure 5C). However, si-SCAI reversed the suppressive effect of miR-766-5p inhibitor on the xenograft tumor growth as well as the expression of SCAI (P < 0.01). These results implied that down-regulation of miR-766-5p suppressed the growth of transplanted tumors in mice through regulating SCAI.

Discussion

The biological role of miR-766-5p in tumors is controversial. Previous researches have revealed that miR-766 is up-regulated in colorectal cancer (CRC),[18] hepatocellular carcinoma (HCC),[12] whereas is down-regulated in lung cancer.[11] Jia et al.[10] have illustrated that miR-766-5p expression is increased in CRC tissues, and serves as an onco-miRNA role in the progression of CRC. However, another study conducted by Bai et al.[11] have demonstrated that the expression of miR-766-5p is decreased in lung cancer tissues, and exerts an anti-tumor role in lung cancer. In this study, a high expression of miR-766-5p was observed in CC tissues as well as in cell lines. Our findings revealed that miR-766-5p might serve as an morbigenous factor in CC. Additionally, statistical data suggested that miR-766-5p expression was markedly relevant to tumor size, FIGO stage and lymph node metastasis. Our data illustrated that the expression of miR-766-5p was closely related to the development of CC.

Numerous studies have suggested that serum miRNA levels can be used in the diagnosis of CC. Jiang et al.[19] have revealed that under-expressed serum miR-101 is closely correlated with metastasis and prognosis of CC, and act as a prognostic biomarker for CC. MiR-486-5p is highly expressed in the serum of CC patients, and serum miR-486-5p can be utilized as a diagnostic biomarker for CC (with an AUC = 0.90).[20] Here, consistent with the high expression level in CC tissues, miR-766-5p expression was also significantly increased in serum of CC patients compared with the healthy controls. Moreover, ROC curve indicated that serum miR-766-5p could accurately discriminate CC patients from healthy controls (with AUC = 0.851). We conjectured that serum miR-766-5p could serve as a diagnostic biomarker for CC.

Growing evidence indicates that miRNAs exert their functions in CC through affecting the cellular processes, such as the proliferation, migration, invasion and apoptosis. Overexpression of miR-141-3p facilitates the cell proliferation, invasion and EMT in CC via inhibition of FOXA2.[21] Down-regulation of miR-155 suppresses the proliferation, migration and invasion of CC cells in vitro.[22] Up-regulation of miR-543 promotes the proliferation, and inhibits the apoptosis of HeLa and SiHa cells.[23] In this study, in vitro experiments demonstrated that down-regulation of miR-766-5p inhibited the proliferation, migration and invasion, and induced the apoptosis of CC cells. Meanwhile, the results of in vivo trails further proved the suppressive effect of miR-766-5p inhibitor in CC progression.

In current research, to further elucidate the potential mechanism of miR-766-5p in CC development, SCAI was confirmed as a target gene of miR-766-5p. SCAI acts as an anti-tumor gene and is down-regulated in many types of cancers.[24-26] Consistent with these studies, we found that SCAI expression was decreased in CC tissues and cell lines. More importantly, SCAI has been reported to function as the target gene of other miRNAs in various human cancers. Lin et al.[16] have indicated that SCAI is directly targeted miR-1228, and overexpression of SCAI reverses the promoting effect of miR-1228 on the migration and invasion of breast cancer cells. Zheng et al.[27] have illustrated that miR-625-3p enhances the ability of migration and invasion in CRC cells via negatively regulating SCAI. Here, we discovered that down-regulation of SCAI reversed the inhibiting effects of miR-766-5p inhibitor on the proliferation, migration and invasion, and the promoting effect on the apoptosis of CC cells. In addition, in vivo test further indicated that down-regulation of SCAI eliminated the suppressive effects of miR-766-5p inhibitor on the growth of transplanted tumors in mice. All these data revealed that inhibition of miR-766-5p might play an anti-tumor role in CC progression via regulating SCAI.

In conclusion, we found that inhibition of miR-766-5p could attenuate the occurrence and development of EC through regulating SCAI in vitro and in vivo. However, this study did not investigate the upstream regulators of miR-766-5p and the relationships among them. This may be a limitation of the current study. Further studies to elucidate these issues will be performed in the future. Even so, we also hope these findings will provide a potential target for treating CC.