The cells were cultured in Dulbecco’s modified eagle medium (DMEM) (Gibco, Gaithersburg, MD, United States) with 10% fetal bovine serum (Gibco) at 37°C. The cells were passaged every 2–3 days. miR-92a-3p mimics control and miR-92a-3p inhibitor control were synthesized by Ribobio (Guangzhou, China), and transfected into cells with DharmaFECT 1 (VWR, Radnor, PA, United States) in accordance with the instruction for use provided by the manufacturer. The pCMV6-LATS1 plasmid and empty plasmid were purchased from OriGene Technologies (Rockville, MD, United States), and transfected into cells with Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, United States) according to the manufacturer′s instruction for use.

Quantitative real-time PCR (qRT-PCR) was used to determine mRNA levels. Total RNA of differently treated cells was prepared with TRIZOL (Thermo Fisher Scientific) according to the manufacturer’s instruction for use. The RNA concentration was quantified with NanoDrop 2000c (Thermo Fisher Scientific). The cDNA was obtained by reverse transcription with the PrimerScript RT Reagent Kit (VWR) according to the manufacturer’s instruction for use. SYBR Green PCR Master Mix (Bio-Rad, Hercules, CA, United States) and ABI 7500 real-time PCR system (Thermo Fisher Scientific) were adopted to analyze the RT-PCR products. U6 was used as an internal control. The mRNA levels were measured according to the 2^−△△Ct method (Livak and Schmittgen, 2001). All experiments were repeated for three times.

The viable cell mass was measured with the Cell Counting Kit-8 (CCK8) Assay (Bimake, Houston, TX, United States). The CC stem cells (1 × 10⁵) were transfected with miR-92a-3p mimic/inhibitor and seeded in 96-well plates. The cells were then cultured in an incubator with 5% CO₂ at 37 °C for 1, 2, 3, 4, and 5 days respectively. Totally 10 μl CCK8 solution was added into each well, and the cells were cultured for another 2 h. The absorbance was finally determined at 490 nm with a microplate reader.

The invasion assay was performed with a Transwell chamber (Corning, Corning, NY, United States) and covered with Matrigel (BD Biosciences, San Jose, CA, United States). Serum-free medium and complete medium were added in the upper and lower chambers, respectively. The cells (1 × 10⁵) were seeded in the upper chamber and cultured for 36 h in an incubator with 5% CO₂ at 37°C. Non-invasive cells were removed, and the cells that entered the lower chamber were stained with hematoxylin and counted with a microscope (BX53; Olympus, Tokyo, Japan) at the magnification of ×400.

The CC stem cells were seeded into a 6-well plate and cultured in an incubator with 5% CO₂ at 37°C for 48 h. The cells were then resuspended with phosphate-buffered saline (Thermo Fisher Scientific) and fixed with precooled alcohol (75%). Totally 300 μl of propidium iodide was then added into the tube and the mixed solution was incubated for 15 min in the darkness. The cell cycle was determined with the flow cytometry.

Western Blotting was used to determine the levels of proteins. Briefly, the cells were harvested with lysis buffer containing 1% protease inhibitor and phosphatase inhibitors. Totally 30 μg of total protein was loaded onto a 10% SDS-PAGE gel, and after electrophoresis, the protein was transferred to polyvinylidene fluoride (PVDF) membranes, which was to be blocked with 5% bovine serum albumin and incubated with primary antibodies of cyclin E (4,129; 1:1,000; Cell Signal Technology, Danvers, MA, United States), E-cadherin (3,195; 1:1,000; Cell Signal Technology), vimentin (3,932; 1:1,000; Cell Signal Technology), TAZ (83,669; 1:1,000; Cell Signal Technology), LATS1 (9,153; 1:1,000; Cell Signal Technology), and β-actin (4,970; 1:1,000; Cell Signal Technology) at 4°C for overnight. The PVDF membranes were then washed with TTBS buffer and probed with horseradish peroxidase (HRP)-conjugated secondary antibody (7,074/7,076; 1:2,000; Cell Signal Technology) at 37°C for 2 h. The immunosignal substance was detected with the SuperSignal West Dura Extended Duration Substrate Kit (Thermo Fisher Scientific), and the resulting images were captured with an imaging system (DNR BioImaging System, Jerusalem Israel).

Wild type and mutant sequences of LATS1 3′-UTR with putative miR-92a-3p binding sites were synthesized and sub-cloned into the luciferase vector to generate wild type and mutant luciferase reporters. Then, 100 ng of the luciferase vector was co-transfected with the miR-92a-3p mimic or negative control with Lipofectamine 3000 (Invitrogen, Carlsbad, CA, United States). The luciferase activities were assessed with a Dual-luciferase Reporter Assay Kit (Promega, Madison, WI, United States) according to the manufacturer’s protocol.

TCGA is a landmark cancer genomics program, which molecularly characterizes over 20,000 primary cancer and matched normal samples including 33 different cancer types. The miR-92a-3p mRNA expression profiles for CC patients were obtained from TCGA (https://www.cancer.gov/about-nci/organization/ccg/research/structural-genomics/tcga). Overall survival of CC patients was also downloaded from TCGA data portal. Data acquirement and application were performed in accordance with TCGA publication guidelines and data access policies.

SPSS statistical software for Windows, version 22.0 (SPSS, Chicago, IL, United States) was used for the statistic analysis. The data were compared with the t-test and one-way analysis of variance. The bilateral 95% confidence interval was used for all tests, and a value of p < 0.05 represented statistical significance.

We analyzed miR-92a-3p expression profiles of 307 cases of CC patients from the TCGA database. The results showed that the CC tissues had higher miR-92a-3p expression levels than adjacent normal tissues (Figure 1A, unpaired t-test, p = 0.0002). We also analyzed the correlations of miR-92a-3p levels with patients’ overall survival. Kaplan-Meier survival analysis showed that the overall survival of CC patients with low miR-92a-3p levels was longer than those with high miR-92a-3p levels (Figure 1B, log-rank test, p = 0.0143).

The levels of miR-92a-3p in a normal cervical cell line Ect1/E6E7 and several CC stem cells lines (CaSki, SiHa, Hela, ME-180, MS751, and C-33A) were examined. Figure 1C shows that in five out of six CC cell lines, the levels of miR-92a-3p were higher in 5/6 CC cell lines than that in Ect1/E6E7, which was consistent with its expression pattern in CC tissues. Transfections of the miR-92a-3p mimic and inhibitor were conducted in SiHa and CaSki cell lines, respectively. Figure 1D shows that miR-92a-3p mimic transfection upregulated its endogenous levels, while miR-92a-3p inhibitor transfection downregulated its expression in both SiHa and CaSki cells. The CCK8 assay was then used to determine its role in CC cell growth. The results showed that miR-92a-3p overexpression promoted proliferation, while miR-92a-3p inhibition decreased the growth rate of SiHa and CaSki cells (Figure 2A). The Transwell invasion assay showed that the number of cells passing through the Transwell membrane increased in the miR-92a-3p mimic-transfected SiHa and CaSki cells. miR-92a-3p inhibitor transfection decreased the invading SiHa and CaSki cell numbers (Figure 2B). Changes of the cell cycle were also examined. Figure 2C shows that the miR-92a-3p mimic transfection decreased the percentage in G1 phase but increased it in S phase in the SiHa and CaSki stem cells, however, miR-92a-3p inhibitor was on the contrary, which suggested that miR-92a-3p facilitated cell cycle progression in CC stem cells.

TarBase v7.0 software predicted that miR-92a-3p had potential binding sites in the 3′-UTR of LATS1 (Figure 3A). Their relationship was validated by Western Blotting. Figure 3B shows that the miR-92a-3p mimic downregulated LATS1 protein levels, but the miR-92a-3p inhibitor upregulated them. In addition, a dual-luciferase reporter assay was used to verify whether miR-92a-3p directly targeted LATS1. SiHa cells were co-transfected with the luciferase reporter plasmid containing wild type mutated binding sites of LATS1 3′-UTR and the miR-92a-3p mimic. The results showed that miR-92a-3p downregulated luciferase activities in the cells transfected with the LATS1-wild type reporter plasmid, but there were no significant changes in cells transfected with LATS1-mutant reporter plasmid (Figure 3C).

Then it was determined that whether LATS1 affected proliferation, invasion, and cell cycle of CC stem cells. The CCK8 assay showed that overexpression of LATS1 decreased cell growth in the SiHa and CaSki stem cells, and LATS1 siRNA knockdown increased the growth (Figure 4A). The Transwell invasion assay showed that LATS1 overexpression decreased invading cell numbers, and LATS1 siRNA knockdown increased the number (Figure 4B). Flow cytometry showed that overexpression of LATS1 increased the percentage of cells in G1 phase but decreased it in S phase, and LATS1 knockdown showed the opposite effect (Figure 4C).

In order to identify the mechanism of CC cell invasion and proliferation regulated by LATS1, we screened several proteins which are potentially associated. Expressions of the epithelial-mesenchymal transition (EMT)-related factors, E-cadherin and vimentin were detected in the CaSki and SiHa cells transfected with the LATS1 plasmid and siRNA. Western Blotting showed that overexpression of LATS1 decreased the levels of TAZ, vimentin and cyclin E, but increased the protein levels of E-cadherin and p27 in SiHa and CaSki cells. LATS1 siRNA silencing increased the protein levels of TAZ, vimentin and cyclin E, but it decreased the protein levels of E-cadherin and p27 in SiHa and CaSki cells (Figure 4D).

To further confirm whether miR-92a-3p regulated biological behaviors via targeting LATS1, SiHa cells were transfected with the miR-92a-3p mimics and the LATS1 plasmid. CCK8 assays showed that cell growth increased after transfection with the miR-92a-3p mimics, and restoration of LATS1 reversed the upregulation of growth (Figure 5A). The Transwell assay showed that the number of invading cells increased after transfection of the miR-92a-3p mimics, and LATS1 restoration abolished the positive effect of miR-92a-3p on invasion (Figure 5B). Similarly, LATS1 transfection reversed the effect of miR-92a-3p on G1-S cell cycle progression (Figure 5C). These results indicated that miR-92a-3p regulated the proliferation, invasion, and cell cycle of CC stem cells by targeting LATS1. Next, we examined if restoration of LATS1 reversed the effects of miR-92a-3p on TAZ, cyclinE and the EMT markers. Figure 5D shows that miR-92a-3p overexpression downregulated the protein levels of LATS1 and E-cadherin, but upregulated of TAZ, vimentin and cyclin E, and LATS1 plasmid transfection partly reversed the effects of miR-92a-3p on TAZ, E-cadherin, vimentin and cyclin E.