miRNA-101 Regulates Filamentogenesis During Migration and Invasion of Cervical Cancer Cells


 Background: miRNAs play critical roles in cervical cancer (CC) progression. miRNA-101 is a tumour suppressor in several cancers. The aim of this study was to investigate the in vitro and in vivo effect of miRNA-101 on the biological behaviour of CC cells by analysing the formation of filamentous pseudopodia.Methods: Quantitative reverse-transcription-polymerase chain reaction was performed to analyse miRNA-101 expression in CC cells before and after its transfection. Cell counting kit-8, transwell migration, and wound healing assays were performed to investigate the effect of miRNA-101 on the malignant behaviour of CC cells in vitro. The impact of miRNA-101 on filopodia initiation in CC cells was investigated via scanning electron microscopy and TRITC-labelled phalloidin staining evaluation. In vivo analysis was performed using a nude mouse model of CC, established via subcutaneous tumour transplantation, to validate the role of miRNA-101 on CC malignant behaviour. One-way ANOVA with Fisher’s least significant difference post hoc test and t-test were used to evaluate statistical significance of differences between groups.Results: miRNA-101 was significantly downregulated in CC cell lines. Overexpression of miRNA-101 inhibited the malignant behaviours of CC cells in vitro, while it inhibited the formation of filopodia in CC cells. Similar results were observed using immunofluorescence and confocal microscopy.Conclusions: These results indicate that miRNA-101 inhibits CC migration and invasion. The present findings, combined with future research, will help provide a theoretical basis and novel insight for the clinical management of CC.


Background
Cervical cancer (CC) is one of the most common malignancies in women worldwide. In 2012, 527,000 new cases and 265,700 CC-related deaths were reported [1,2]. In recent years, owing to the use of various diagnostic screening methods and surgical procedures, the incidence of CC showed a decreasing trend [3]; however, it remains the primary cause of mortality among women. The incidence of CC is particularly high among Uyghur women in the Xinjiang Uygur Autonomous Region of China, where most patients have advanced CC with a high mortality rate [4]. Metastasis, especially to the pelvic lymph node, is a major pathognomonic hallmark of CC that is associated with poor prognosis and high mortality. Therefore, the early detection of metastasis and relapse, as well as improvement of prognosis, are critical for CC outcome.
Some recent studies have reported that the rearrangement of cytoskeleton proteins forms the cellular basis of tumour adaptation to new environments and their migration [5]. Changes in cytoskeletal morphology prominently in uence tumour motility, permeability, and angiogenesis, potentially causing tumour cells to invade the surrounding tissues and gradually develop distant metastasis. Filopodia, located at the anterior region of the cell, guides the posterior region of the cell to move forward and serves as a 'sensory organ' for communication with the extracellular microenvironment. miRNAs are non-coding single-stranded RNAs of 18-24 nucleotides [6] that contribute to the regulation of tumorigenesis and tumour behaviour in humans [7]. We previously reported 12 differentially expressed miRNAs (all down-regulated) in CC tissues derived from Uyghur women, wherein miRNA-101 was downregulated more than 3-fold and was associated with lymph node metastasis in CC. miRNA-101 was signi cantly down-regulated in HeLa and SiHa CC cell lines, and its up-regulation inhibited CC cell invasion [8,9].
The aim of this study was to: (1) construct a lentiviral vector harbouring miRNA-101 to infect CC cell lines, (2) observe lamentogenesis in CC cells via scanning electron microscopy (SEM) and TRITC-labelled phalloidin staining, (3) perform scratch healing, transwell, and matrigel assays to examine CC cell invasion and migration, and (4) establish a mouse model of CC lung metastasis using BALB/c nude mice and investigate the effect of miRNA-101 on subcutaneously transplanted CC cell lines in the nude mice.

Materials And Methods
Cell lines and cell culture protocols The human CC cells lines HeLa and SiHa were purchased from the Cell Culture Collection of the Shanghai Branch of the Chinese Academy of Sciences. HeLa and SiHa cells were cultured at 37 °C and 5% CO 2 in Dulbecco's Modi ed Eagle Medium (Gibco, Waltham, MA, USA) supplemented with 10% foetal bovine serum (Gibco) and 2% penicillin-streptomycin (Beyotime Biotechnology, Haimen, China). On approaching 80%-90% con uence, cells were trypsinised.

Construction, packaging, and transduction of lentiviral vectors
Lentivirus GV-miRNA-101 (Genechem, Shanghai, China) was used to infect SiHa and HeLa cells at a multiplicity of infection (MOI) of 10 and lentivirus GV-EGFP (Genechem) was used as the control vector.
After 72 h of transduction, cells were harvested and used for subsequent tests.
RNA isolation and quantitative reverse-transcription-polymerase chain reaction (qRT-PCR) Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) and 1 µg of total RNA was retrieved using the miScript RT Kit (Qiagen, Hilden, Germany). The Bio-Rad CFX96 system (Hercules, CA, USA) with miScript SYBR Green (Qiagen) was used for qRT-PCR analysis and to determine the expression levels of the mRNA-101 and genes of interest. The qRT-PCR protocol was as follows: 95 °C for 15 min, followed by 40 cycles at 94 °C for 15 s, 55 °C for 30 s, and 70 °C for 30 s. After 40 ampli cation cycles, the melting curve was examined, the Ct value of each sample was automatically calculated, and relative mRNA expression levels were determined using the 2 -ΔΔCT algorithm. The samples were analysed in triplicate and U6 was used as an internal control for miRNA ampli cation. miRNA-101 and U6 were reverse-transcribed (RT) with Bulge-loop miRNA qRT-PCR primers (one pair of RT primers and one pair of qPCR primers in each group) designed by Ribo Bio (Guangzhou, China).

Cell counting kit (CCK)-8 assay
The lentivirus-transduced CC cells were seeded in 96-well plates at 5 × 10 3 cells/well and cultured overnight at 37 °C and 5% CO 2 . Thereafter, 10 μL of CCK-8 solution was added into each well and incubated for 24, 48, and 72 h and the absorbance was measured at 450 nm, using a microplate reader (Bio-Rad). The control group comprised human CC cells transduced with a sham lentivirus. Cell viability was determined using the formula: cell viability = (absorbance of the test well -absorbance of the blank well)/(absorbance of the control well -absorbance of the blank well) × 100%.

Scanning electron microscopy (SEM) analysis
The same number of cells (HeLa and SiHa) was seeded on a glass coverslip (0.8 × 0.8 cm 2 ), placed in a 48 well plate, and cultured in Dulbecco's Modi ed Eagle Medium at 37 °C and 5% CO 2 . When the cell fusion rate approached 50%, the coverslips were removed, xed with 3.7% formaldehyde for 10 min, and washed thrice with phosphate-buffered saline (PBS), followed by sequential dehydration, air-drying, and placement on a short column. Thereafter, the slides were placed in the vacuum chamber of an SEM gold coating apparatus (plating instrument) and gold-coated at 2.5 kV, 20-25 mA for 2 min. The cells were then observed using a SEM (JEOL JSM-5800, Japan) at an accelerating voltage of 20 kV.

Wound healing assay
The wound healing assay was performed to evaluate the migration potential of CC cells. Brie y, human CC cell lines were inoculated onto 6-well plates (1 × 10 6 cells/well). Upon 70%-80% cell fusion, arti cial wounds with the same width were obtained by scratching the the cell layer with sterile plastic micropipette tips. Thereafter, cell debris were eliminated by washing with PBS. Cell migration was observed and photographed at 24, 48, and 72 h after wounding and compared with the 0 h image.

Combinatorial transwell and matrigel assay
Cell invasion was assessed using matrigel invasion chambers (BD Bioscience, San Diego, CA, USA).
Brie y, 3 × 10 4 cells were seeded in serum-free medium in the upper chamber. The medium, supplemented with 10% foetal bovine serum, was added to the lower chamber. After 24 h of culturing, the cells at the bottom of the membrane were xed and stained with 0.1% crystal violet. Five visual elds of invasive cells were randomly selected, counted, and photographed under a microscope.

Xenograft mouse model of CC
All procedures involving mice were approved by the Experimental Animal Ethics Committee of Xinjiang Medical University and met all regulatory standards. Forty-ve female (4-6 weeks old) BALB/c nude mice were divided into three groups (n = 15/group). A 0.2 mL cell suspension (5 × 10 6 cells) was subcutaneously injected into the right forelimb capsule. The blank group comprised mice injected with HeLa cells, the control group comprised mice injected with HeLa cells transfected with empty-load lentivirus, and the experimental group comprised mice injected with HeLa cells transfected with miRNA-101 lentivirus. The physiological features (including mental state, activity, diet, body weight, appearance, and tactile responses to touching the inoculated area) of nude mice were evaluated every 2-3 days.

Histopathological analysis
Tumour, lung, liver, heart, kidney, and brain tissues were xed with normal neutral formalin, embedded in para n, sectioned, stained with haematoxylin and eosin, and the histological characteristics of the transplanted tumour and critical organs were observed using a light microscope.

Statistical analyses
The experimental data were analysed using SPSS19.0 software (IBM Inc, Armonk, NY, USA) and all graphs were plotted using GraphPad Prism for Windows version 5.0 (GraphPad Software, San Diego, CA, USA). Continuous variables were expressed as mean ± standard deviation values and the data were assessed for normality and conformance with a normal distribution. The one-way ANOVA was performed to compare the homogeneity of the assessed differences among groups and independent samples t-test was used for between-group comparisons. Each experiment was performed in triplicate. A P-value < 0.05 was considered statistically signi cant. Tumour volume among groups was simultaneously analysed using a one-way ANOVA and Fisher's least signi cant difference test (variance uniformity) for post hoc pairwise comparison.

Results
Page 6/20 E ciency of miRNA-101 lentiviral transduction in CC cells The miRNA-101 lentiviral vector was used to infect HeLa and SiHa cells. After 48-72 h of infection, uorescence expression was observed with a laser-scanning confocal microscope to determine the transduction e ciency, as the GFP-encoding gene was incorporated into the lentiviral vector to produce green uorescence after CC cell infection. MOI values of the miRNA-NC group (infected with the control uorescent lentiviral vector) and miRNA-101 group (infected with the lentiviral vector harbouring miRNA-101) were screened via laser-scanning confocal microscopy. At an MOI of 10, the uorescence abundance of HeLa and SiHa cells in both groups was > 80%. After sub-culturing, HeLa and SiHa cells in the two groups displayed non-signi cant uorescence attenuation. At an MOI of 10, the lentivirus stably transfected CC cells and subsequent experiments were conducted.

miRNA-101 overexpression inhibits the growth and proliferation of CC cells
To further investigate the effect of miRNA-101 on human CC cells, we performed functional assays using CC cells overexpressing miRNA-101. qRT-PCR analysis revealed that after transduction, miRNA-101 was signi cantly up-regulated (P < 0.05) in CC cells compared to miRNA-NC transfected cells (Fig. 1a, b), whereas it was not signi cantly expressed (P > 0.05) in the untreated group and miRNA-NC group. The CCK-8 assay revealed that miRNA-101 overexpression signi cantly decreased the viability and proliferation (P < 0.05) of Hela cells (Fig. 1c). Similarly, miRNA-101 forced expression in SiHa cells inhibited cell proliferation compared to the control group (Fig. 1d).

miRNA-101 overexpression inhibits CC cell migration and invasion
We performed the wound healing and combinatorial transwell and matrigel assays to examine the effect of miRNA-101 on CC cell migration and invasion. The wound healing rate of miRNA-101-overexpressing cells was signi cantly lower (P < 0.05) than that of the normal control group, and no difference was observed between the miRNA-101-untreated group and the normal control group (Fig. 2a, c). Similarly, miRNA-101 up-regulation markedly inhibited the invasiveness of CC cells in the combinatorial transwell and matrigel assays (Fig. 2b, d). Table 1 shows the number of miRNA-NC, miRNA-101-transfected, and untransfected HeLa and SiHa cells subjected to the migration assay.

miRNA-101 regulates lopodia initiation in CC cells
We stained and labelled cytoskeletal element F-actin with TRITC-labelled phalloidin and observed the uorescent staining via confocal microscopy. The staining revealed that HeLa and SiHa cells had more lopodia than untransfected cells, whereas miRNA-NC-transfected HeLa and SiHa cells displayed no obvious change in the number of lopodia (Fig. 4a, b). Table 2 shows the number of lopodia in untransfected, miRNA-NS-transfected, and miRNA-101-transfected HeLa and SiHa cells. Similar results were obtained in ultrastructural analysis of the cytoskeleton via SEM (Fig. 4a, b).
miRNA-101 inhibits subcutaneous xenograft formation from CC cells in nude mice Subcutaneous xenografts visibly developed in all three groups of nude mice and the tumorigenesis rate was 100%. The mice administered miRNA-NC-transfected and untransfected CC cells displayed a generally poor condition, with a weak mental state, limited activity, longer reaction times, and decreased water intake at later stages of the cancer, whereas those administered miRNA-101-transfected CC cells displayed a good physical and mental state, as well as adequate physical activity, and food and water intake. No signi cant difference was observed in body weight among the three groups of nude mice (P > 0.05) ( Table 3).
The transplanted tumours were papillary and tough, and most had a pseudo-envelope. The transplanted tumour cells displayed low adhesion to the surrounding tissues and had a rich blood supply. In mice administered miRNA-101-transfected CC cells, some grey-white necrotic areas were observed in the centre of the transplanted tumour (Fig. 5).
Microscopically, tumour cells from mice administered miRNA-NC-transfected CC cells or untransfected CC cells showed an ovoid morphology and were markedly heteromorphic. The nucleus was spherical and stained purple-brown with a discernible nucleolus and the cytoplasm was unevenly stained, revealing pathological mitosis to a large extent. The tumour cells displayed marked atypia, purple-brown nuclei, discernible nucleoli, uneven cytoplasmic staining, and numerous pathological mitotic features. In mice administered miRNA-101-transfected cells, different degrees of haemorrhage and necrosis were observed at the centre of the transplanted tumour and necrotic tissue was red and granular (Fig. 8).
A comparison of the growth inhibition rates of subcutaneous xenografts in the three groups revealed that tumour growth was signi cantly inhibited in mice administered miRNA-101-transfected CC cells compared to mice administered miRNA-NC-transfected CC cells (P < 0.05) or untransfected CC cells (P < 0.05; Table 5).

Discussion
Numerous miRNAs are aberrantly expressed in CC and play a key role in CC pathogenesis and progression [10]. We previously reported the association between miRNA-101 down-regulation and lymph node metastasis in CC in Uyghur women in China, as well as its signi cant down-regulation in HeLa and SiHa cells [4]. miRNA-101 contains 21 nucleotides and is highly conserved among various species. It is down-regulated in several solid tumours including prostate, breast, lung, bladder, ovarian, and cervical cancers [11,12]. However, studies focusing on the regulatory mechanism of miRNA-101 in different tissues have revealed inconsistent ndings.
To show that miRNA-101 markedly contributes to the pathogenesis and occurrence of CC, we constructed a lentiviral vector harbouring miRNA-101 and transfected HeLa and SiHa CC cell lines. Consequently, the proliferation potential was signi cantly reduced in these miRNA-101 transfected CC cells compared to the control group, suggesting that miRNA-101 potentially inhibits the proliferation of CC cells. A previous study had showed similar results, with miRNA-101 overexpression signi cantly decreasing proliferation and invasion of ovarian cancer cells in vitro by down-regulation of SOCS-2 [13].
We performed the scratch healing test and a combinatorial transwell and matrigel assay to examine CC cell migration and invasion before and after miRNA-101 transfection. Consequently, the wound healing rate and invasiveness of miRNA-101 overexpressing cells signi cantly decreased, suggesting that miRNA-101 inhibits CC cell migration and invasion.
Cancer cells can use different approaches to support their migration, such as aky pseudopodia, lamentous pseudopodia, and vesicles [14]. The formation of these processes depends on the actin cytoskeleton, which can be rapidly reconstituted and participates in the formation, movement, intracellular transport, and division of cells [15,16]. Pseudopodium formation is the initial step of tumour cell invasion and migration. Filamentous pseudopods are usually less than 200 nm in diameter and are located at the apical end of the cell, leading the basal end of the cell to move forward [17].
Herein, SEM and TRITC-labelled phalloidin staining were performed to observe the formation of lamentous pseudopodia in CC cells transfected with the lentiviral vector harbouring miRNA-101. SEM revealed that lamentous pseudopods were amorphous lamentous processes on the cell surface, indicating that the number of lamentous pseudopods in HeLa and SiHa cells decreased signi cantly upon miRNA-101 transfection. Furthermore, slight differences were observed between the two cell lines.
The number of amorphous processes on the surface of HeLa cells did not signi cantly decrease; however, the length of the processes did decreased. Therefore, this was not classi ed as lamentous pseudopodia. The number of amorphous processes on the surface of SiHa cells signi cantly decreased.
Taken together, miRNA-101 overexpression inhibits the formation of lamentous pseudopods in CC cells. Four types of cells were observed upon laser-scanning confocal microscopy and TRITC-labelled phalloidin staining -polygonal/slender, non-polygonal/non-slender, unclear/early diffusion, and small spherical. Only polygonal/slender cells could be considered lamentous pseudopodia. The number of polygonal/slender cells in HeLa and SiHa CC cells signi cantly decreased upon transfection with the lentiviral vector harbouring microRNA-101, indicating a reduction in the number of lamentous pseudopods in HeLa and SiHa CC cells. The F-actin in lamentous pseudopodia was labelled with TRITClabelled phalloidin, with the uorescence intensity of different groups of cells could re ect the formation of lamentous pseudopodia at the same excitation intensity. The observed results followed the same trend as the above mentioned data on polygonal slender cells. These results indicated that miRNA-101 overexpression potentially inhibits the formation of lamentous pseudopods in CC cells, thus inhibiting migration and invasion in CC cells.
Bioinformatics analysis and pathway analyses revealed that RAC1, STMN1, and CAPN2 are associated with the regulation of the actin cytoskeleton. The Rac/WAVE/Arp2/3 pathway is essential for generating and maintaining lamentous pseudopods [18][19][20][21]. The Arp2/3 complex-mediated network stabilizes the protrusions and makes them robust [22]. However, it remains unclear whether miRNA-101 targets Rac1 in the complex pathway and affects the formation of lamentous pseudopods.

Conclusion
Altogether, these results demonstrate that miRNA-101 inhibits CC migration and invasion. Further detailed studies on the effect of miRNA-101 on lamentous pseudopodia formation, and its target genes and associated signalling pathways would elucidate the mechanism underlying invasion and metastasis in CC and provide a theoretical basis and novel insight for the clinical management of CC. Availability of data and materials All data generated or analyzed in this study are included in this published article. Further details are available from the corresponding author upon request.

Competing interests
The authors have no competing interests to declare.

Funding
This study was supported by the National Natural Science Foundation of China (grant number 81660428).
Authors' contributions CL and PZ conceived and designed the study. PZ, ZQH, YL, and AE performed the experiments. CL, PZ, and WW wrote the article. CL, PZ, and WW reviewed and edited the manuscript. All authors read and approved the manuscript.      Figure 1 Up-regulation of miRNA-101 signi cantly inhibited the growth and proliferation of cervical cancer cells.
The expression levels of miRNA-101 in HeLa (A) and SiHa(B) cells of the normal control group, the miRNA-NC group, and the miRNA-101 group. The proliferation of cervical cancer HeLa(C) and SiHa(D) cells in the normal control group, the miRNA-NC group and the miRNA-101 group was decreased compared with the normal control group, but no signi cant difference was found in the miRNA-NC group. * * P < 0.01.     The growth curve of subcutaneous xenografts Figure 7 Volume comparison of subcutaneous xenografts in three groups of nude mice Figure 8 HE staining of subcutaneous xenografts From left to right in turn is the non-transfected group,miRNA-NC transfected, miRNA-101 transfected group. (upper row was x100, lower row was x400)