miR-4319 Reduces Tumorigenicity of Cervical Cancer Cells by Targeting TUFT1

Background: In the present study, a new tumor suppressor function of miR-4319 was disclosed in CC. Methods: Up-regulation of miR-4319 suppressed cell viability, proliferation, migration, invasion, and induced cell apoptosis in CC cells were measured by cell transfection, CCK-8, colony formation, EdU, ow cytometer, wound healing, transwell migration and invasion and western blot assays. Moreover, Tuftelin 1 (TUFT1) was veried as a direct target of miR-4319 by binding its 3’-UTR, conrmed by dual-luciferase reporter assay. Result: The expression of miR-4319 was obviously decreased in clinical CC tissues and CC cell lines. TUFT1 was remarkably increased in clinical CC tissues and CC cell lines, and was negatively associated with miR-4319 expression. Furthermore, over-expression of TUFT1 partially restored the effects of miR-4319 mimic on cell viability, proliferation, migration, invasion, and cell apoptosis in CC cells. Conclusion: miR-4319 played an anti-cancer role in the occurrence and development of CC, which might be achieved by targeting TUFT1.


Introduction
Cervical cancer (CC) is one of the most common malignant tumors in women, which occurs in the cervix and vagina (1).According to worldwide statistics, there are about 500, 000 new cases of CC every year, accounting for 5% of all new cancer cases, of which more than 80% are in developing countries (2,3).Despite the increasing awareness of cancer prevention and treatment in recent years, and the popularity of related vaccines, the incidence rate of CC is still rising, and the age of audience becomes younger and younger (4).A large number of studies have shown that persistent high-risk human papilloma virus (HPV) infection is a relatively clear risk factor for CC.In women's life, cervical HPV infection is quite common, genital tract infection rate of HPV is more than 75%, but most of them can subside by themselves (5,6).It is worth emphasizing that the early symptoms of CC are not obvious, some patients are in advanced stage at the time of diagnosis, and lose the best opportunity for treatment, which seriously threatens the health and quality of women's life (7).Therefore, it is necessary to explore the potential mechanisms of CC for early diagnosis and effective treatment.
MicroRNAs (miRNAs) are a kind of non-coding single stranded RNA molecule with about 18-25 nucleotides in length (8).MiRNAs cause degradation or translation inhibition of target gene mRNA through speci cally pairing with 3'-UTR region of target gene mRNA, further regulating the expression of target genes at post transcriptional level (9).According to statistics, miRNAs regulate at least 30% of human genes and play an important role in the normal development of the body (10).However, the disorder of miRNA expression is also common in many malignant tumors, including CC.For example, miR-21 and miR-155 are signi cantly over-expressed in CC tissues, and may be helpful in the prediction of both HPV positive and HPV negative cases of CC (11).MiR-501 is up-regulated, whereas CYLD protein is down-regulated in CC tissues, and down-regulation of miR-501 inhibits proliferation, migration and invasion, and promotes apoptosis of CC cells via targeting CYLD mediated with NF-κB p65 signaling pathway (12).MiR-1284 is down-expressed in CC tissues and CC cell lines.Up-regulation of miR-1284 suppresses proliferation and invasion, promotes apoptosis of CC cells, and enhances sensitivity of CC cells to cisplatin achieved by targeting HMGB1 (13).These ndings suggest that miRNAs, acted as oncogenes or tumor suppressor genes, exhibit essential functions in the occurrence and development of CC.
MiR-4319, a newly identi ed cancer-related miRNA, is aberrantly expressed and becomes a predictor of patient survival in cancers (14).For instance, miR-4319 is low-expressed in colorectal cancer tissues, and over-expression of miR-4319 markedly reduces proliferation, and induces cell cycle distribution of colorectal cancer cells via targeting PLZF (15).Up-regulation of miR-4319inhibits triple-negative breast cancer (TNBC) initiation and metastasis in vivo, and suppresses the self-renewal in TNBC cancer stem cells (CSCs) through targeting E2F2 (16).Moreover, miR-4319 expression is lower in esophageal squamous cell carcinoma (ESCC) cells, and over-expression of miR-4319 represses cell growth and induces cell cycle arrest via regulating NLRC5 (17).However, the possible effects and underlying mechanisms of miR-4319 on CC remained unknown.Therefore, our intention was designed to elucidate the biological impacts and associated mechanisms of miR-4319 on CC cells.

Tissue Samples
The clinical CC tissues and adjacent tissues were obtained from 30 CC patients who underwent surgery at the Second A liated Hospital of Soochow University.No radiotherapy or chemotherapy was performed before the surgery.All fresh tissues surgically resected were immediately stored at -80 o C. The signed written informed consents were obtained from all the participants before the study.
HeLa and C33A cells were cultured into 6-well plates and transfected with Lipofectamine 2000 Reagent (Invitrogen, USA) according to the manufacturer's protocols.

Colony formation assay
Transfected HeLa and C33A cells (1 × 10 3 /well) were placed in a 6-well plate, and the medium was replaced with fresh culture medium every 2-3 days for 10 days.The colonies were xed in 10% formaldehyde for 30 min and stained with 0.5% crystal violet for 5 min.The Image-Pro Plus 6.0 (Silver Springs, MD, USA) was used for data analysis.

Ethynyl deoxyuridine (EdU) incorporation assay
Transfected HeLa and C33A cells (1 × 10 4 /well) were cultured in 96-well plates and transfected for 48 h.Then HeLa and C33A cells were xed with 4% paraformaldehyde, Triton X-100 was used to permeabilize the nuclear membrane, and HeLa and C33A cells were blocked with goat serum for 1 h.Furthermore, HeLa and C33A cells were stained according to the manufacturer's manual.

Cell apoptosis by ow cytometry
The apoptosis rate of transfected HeLa and C33A cells was detected by ow cytometry.Transfected HeLa and C33A cells were collected after 48-h transfection.Washed with PBS, FITC-labeled Annexin V and PI (5 µL each) were added to 500 µL of the cell suspension, mixed and incubated at room temperature for 5-15 min in the dark.Then, the rate of apoptosis was detected by ow cytometry.The experiment was repeated for 3 times.

Wound healing assay
Transfected HeLa and C33A cells at a density with 1 × 10 5 /mLwere seeded in a 6-well plate.Then the cultured cells grew to con uence 80%, the wound was made on the surface of the cell with 200 µL tip gun heads.After 0 and 48 h respectively, HeLa and C33A cells were observed with an inverted microscope with an inverted microscopy (Tokyo, Japan), and the distance between the wounds was recorded.

Transwell migration and invasion assays
Transwell migration and invasion assays (Corning Inc.; USA) were performed to determine the migration and invasion capacities of HeLa and C33Acells.For transwell invasion assay, Matrigel (Becton-Dickinson Pharmingen, San Diego, CA, USA), dissolved at 4°C overnight, was diluted with serum-free medium and added to the upper chambers to form a gel for 30 min at 37 o C with 5% CO 2 .Then for transwell migration and invasion assays, transfected HeLa and C33A cells (1 × 10 6 cells/well) were seeded into upper chamber with serum-free DMEM medium, and DMEM medium containing with 20% FBS was added in the lower chamber.After 48-h incubation, the migration and invasion cells were treated with 70% ethanol, stained with 0.5% crystal violet, and counted by an inverted microscope (Tokyo, Japan).
2.9 RNA Extraction and qRT-PCR TRIzol reagent (Invitrogen, Carlsbad, CA, USA) was utilized to extract the total RNA from clinical CC tissues and cell lines.Through the reverse Transcription Kit (TaKaRa Biotechnology Co., Ltd., Dalian, China), the total RNA was reversely transcribed to complementary deoxyribose nucleic acids (cDNAs).

Western blot analysis
The protein of CC cells was lysed in RIPA lysis buffer.After 12, 000 g centrifugation for 15 min at 4°C, the total protein concentrations were determined by BCA protein assay kit (Beyotime, Haimen, China).

Statistical analysis
Statistical analysis was conducted by the Statistical Product and Service Solutions (SPSS) 17.0 (SPSS Inc., Chicago, IL, USA).The Student's t-test was performed to compare inter group differences.Data were presented as mean ± standard deviation (SD).P < 0.05 was considered of statistical signi cance.

miR-4319 expression is down-regulated in clinical tissues and cancer cell lines
To explore the possible effects and underlying mechanisms of miR-4319on CC, rstly, qRT-PCR assay was performed to evaluate the expression of miR-4319 in clinical CC tissues and cell lines.The data of Fig. 1A showed that miR-4319 expression was signi cantly down-regulated in CC tissues compared with that in normal tissues.Besides, as expected, the expression of miR-4319 was also decreased in CC cells (especially in HeLa and C33A cells) relative to that in H8 cells (Fig. 1B).These data suggested that miR-4319 was down-regulated in CC tissues and cell lines, and functioned as a tumor suppressor gene in the occurrence and development of CC.To further investigate the functional role of miR-4319 in CC cells, miR-4319 mimic and NC mimic were transfected into HeLa and C33A cells, and transfection e ciency was assessed.As shown in Fig. 1C and 1D, the expression of miR-4319 was signi cantly increased in HeLa and C33A cells transfected with miR-4319 mimic compared with NC mimic group.

Up-regulation of miR-4319 inhibits viability, proliferation, and induces apoptosis of CC cells
Firstly, CCK-8 assay was adopted to evaluate the effects of miR-4319 on the viability of HeLa and C33A cells, and the data of Fig. 2A displayed that miR-4319 mimic signi cantly inhibited the viability of HeLa and C33A cells in a time-dependent manner compared with NC mimic group.Besides, colony formation and EdU assays were carried out to assess the effects of miR-4319 on the proliferation of HeLa and C33A cells, and the data of Fig. 2B and 2C showed that up-regulation of miR-4319 obviously suppressed the proliferation of HeLa and C33A cells compared with NC mimic group.Moreover, the effects of miR-4319 on the apoptosis of HeLa and C33A cells was determined by ow cytometry, and the data of Fig. 2D indicated that compared with NC mimic group, over-expression of miR-4319 remarkably promoted the apoptosis of HeLa and C33 cells.Furthermore, western blot was performed to evaluate the effects of miR-4319 on the expression levels of apoptosis-related proteins including Bax, Bcl-2, Cleaved-caspase-3 and Cleaved-caspase-9 in HeLa and C33A cells.The data of Fig. 2E showed that relative to NC mimic group, miR-4319 mimic notably reduced the protein expression of Bcl-2, but promoted the protein levels of Bcl-2, Cleaved-caspase-3 and Cleaved-caspase-9 both in HeLa and C33A cells.These data suggested that upregulation of miR-4319 inhibited viability, proliferation, and induced apoptosis of CC cells.

Up-regulation of miR-4319 inhibits migration and invasion of CC cells
Firstly, wound healing assay was performed to further explore the functional effects of miR-4319 on the migration of CC cells.As shown in Fig. 3A, compared with the NC mimic group, miR-4319 mimic obviously inhibited the migration of HeLa and C33A cells.Then transwell migration and invasion assays were also carried out, and the data of Fig. 3B suggested that compared with NC mimic group, overexpression of miR-4319 signi cantly inhibited the migration and invasion of HeLa and C33A cells.Furthermore, western blot was performed to evaluate the effects of miR-4319 on the expression levels of metastasis-related proteins, including Cox-2, MMP-2 and MMP-9.The data of Fig. 3C revealed that relative to NC mimic group, miR-4319 mimic notably decreased the protein expressions of Cox-2, MMP-2 and MMP-9 in HeLa and C33A cells.These data suggested that up-regulation of miR-4319 inhibited migration and invasion of CC cells.

TUFT1 is direct target gene of miR-4319, and negatively associated with miR-4319
In order to study on the possible target genes of miR-4319 involved in the occurrence of CC, rstly, TargetScan was carried out to predict that TUFT1 was potential candidate of miR-4319 (Fig. 4A).TUFT1, as an important regulator, has been reported to control the occurrence and development of various tumors (18, 19).In addition, dual-luciferase reporter gene analysis was performed to verify whether miR-4319 directly regulated TUFT1, and the results of Fig. 4B indicated that co-expression with miR-4319 mimic signi cantly inhibited the activity of a re y luciferase reporter containing wild-type TUFT1 3'-UTR, while this did not be detected on a reporter with a mutated TUFT1 3'-UTR.The data suggested that TUFT1 was a direct target of miR-4319.To further con rm the targeting relationship between miR-4319 and TUFT1, qRT-PCR and western blot assays were used to determine the mRNA and protein expressions of TUFT1 in HeLa and C33A cells transfected with miR-4319 mimic.The data of Fig. 4C and 4D showed that compared with NC mimic group, the mRNA and protein expression levels of TUFT1 were obviously downregulated in HeLa and C33A cells transfected with miR-4319 mimic.Moreover, qRT-PCR assay was carried out to assess the TUFT1 in clinical CC tissues and CC cell lines, and the data of Fig. 4E and 4F illustrated that TUFT1 was obviously up-regulated in clinical CC cancer tissues and CC cell lines.These data suggested that TUFT1 was direct target gene of miR-4319, and was negatively associated with miR-4319 in CC.

Up-regulation of miR-4319 inhibits viability, proliferation, migration and invasion, and induced apoptosis of CC cells via targeting TUFT1
Pc-TUFT1 was transfected into HeLa and C33A cells, and qRT-PCR was performed so as to explore whether miR-4319 functioned as a tumor suppressor gene through targeting TUFT1.The data of Fig. 5A showed that after transfection with pc-TUFT1, the expression of TUFT1 was obviously increased in HeLa and C33A cells.Then, CCK-8 was performed to evaluate the effects of TUFT1 on the viability of HeLa and C33A cells.As shown in Fig. 5B, up-regulation of TUFT1 obviously restored the inhibitory effects of miR-4319 mimic on the viability of HeLa and C33A cells.Besides, EdU assay showed that over-expression of TUFT1 obviously retarded the inhibitory effects of miR-4319 mimic on the proliferation of HeLa and C33A cells (Fig. 5C).In addition, the results of Fig. 5D indicated that up-regulation of TUFT1 obviously inhibited promoting effects of miR-4319 mimic on the apoptosis of HeLa and C33A cells.Furthermore, transwell migration and invasion results displayed that the up-regulation of TUFT1 obviously antagonized the inhibitory effects of miR-4319 mimic on the migration and invasion of HeLa and C33A cells.These data suggested that up-regulation of miR-4319 inhibited viability, proliferation, migration and invasion, and induced apoptosis of CC cells via targeting TUFT1.

Discussion
The etiology of CC involves complex molecular mechanisms, including activation of oncogenes and inhibition of tumor suppressor genes (20).With the continuous research on the molecular mechanisms of CC, more and more genes and proteins involved in the occurrence of CC have been reported, which promotes the diversity of treatment methods for CC, including gene targeted therapy, hormone therapy and so on (21)(22)(23).In view of this, to explore the molecular mechanisms of the occurrence, metastasis and recurrence of CC, to nd markers for clinical diagnosis and prognosis of CC, and to identify effective molecular targets have become the research hotspot of medical personnel in various countries.In view of the extensive existence of miRNAs and the diversity of gene regulation, much more attention has been paid to the role of miRNAs in the development and tumorigenesis in recent years (24-26).Among them, miR-4319 has been con rmed as a regulatory factor of gene mRNA transcription, regulating the post transcriptional expression of speci c target genes, and then participating in the pathological process of many cancers (27)(28)(29).However, the expression of miR-4319 in human CC and its biological mechanisms remained unclear.
It has been con rmed that miR-4319 is involved in the pathological process of many diseases, and is recognized as a tumor suppressor gene, which can be used as an independent factor of clinical prognosis of many cancers.For example, miR-4319 acts as a tumor suppressor in non-small cell lung cancer (NSCLC) progression via restraining cell proliferation and migration as well as boosting apoptosis through inhibiting LIN28-mediated RFX5 stability (30).MiR-4319 is notably down-regulated in thyroid cancer tissues and cells.Up-regulation of miR-4319 attenuates proliferation, migration, and epithelial-tomesenchymal transition (EMT) in thyroid cancer via targeting SMURF1 (31).Moreover, miR-4319 level is decreased in prostate cancer (PC) specimens, and re-expression of miR-4319 in PC cells inhibits Her-2dependent cell growth (32).Similarly, our study displayed that miR-4319 was low-expressed in CC tissues and cell lines.Up-regulation of miR-4319 repressed viability, proliferation, migration, invasion, and induced apoptosis of CC cells, suggesting that miR-4319 was an inhibitory factor of CC, which might participate in the occurrence and development of CC.
MiRNAs produce a marked effect through inducing mRNA degradation or translation inhibition by base pairing with 3'-UTR of target genes (33,34).At present, post transcriptional regulation mechanisms of miRNAs on target genes play an important role in tumor development including CC (35)(36)(37).In this study, TUGT1 was highly expressed in CC tissues and cell lines.TUGT1 was predicted as a candidate target of miR-4319 by TargetScan, which was further con rmed by dual-luciferase reporter assay.TUFT1 gene is identi ed, cloned and sequenced from ameloblast cDNA library by Deutsch et al (38,39).TUFT1 is located at 1dq21.3, which is involved in the growth and maturation of extracellular enamel (39).At present, studies have focused on its relationship with enamel mineralization and tooth development.However, TUFT1 exists in many tissues, such as kidney, adrenal gland, liver and testis (40).Furthermore, recent studies have shown that TUFT1 is abnormally expressed in many kinds of tumors, and is closely related to the development of multiple tumors.TUFT1 is over-expressed in pancreatic cancer (PC), and depletion or over-expression of TUFT1 correspondingly inhibits or promotes PC cell migration and metastasis in vitro and in vivo, through regulation of EMT (18).TUFT1 is elevated in hepatocellular carcinoma (HCC), and promotes HCC cell growth, metastasis and EMT in vitro and in vivo via activation of Ca 2+ /PI3K/AKT pathway (19).TUFT1 expression is increased in breast cancer samples.Inhibition of TUFT1 expression in T-47D and MDA-MB-231 breast cancer cells signi cantly affects cell proliferation, induced apoptosis, and leads to G1-phase cell cycle arrest (39).These ndings were closely in line with our studies.Through rescue experiment, over-expression of TUFT1 partially restored the effects of miR-4319 mimic on viability, proliferation, migration and invasion of CC cells.
In conclusion, the expression ofmiR-4319 was decreased, while the expression of TUFT1 was increased in clinical CC tissues and cell lines.Up-regulation of miR-4319 inhibited the growth and metastasis of CC cells via targeting TUFT1, which provided research basis for gene targeted therapy of CC in the future.

Declarations Ethic approval
The experimental protocol was established, according to the ethical guidelines of the Helsinki Declaration and was approved by the Animal Ethics Committee of A liated Huai'an Hospital of Xuzhou Medical University

Consent for publication
This has no consent for publication

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