MicroRNA-154-5p suppresses cervical carcinoma growth and metastasis by silencing Cullin2 in vitro and in vivo

Cell lines and laboratory animals

Human immortalized epidermal cell lines (HaCaT), 293T, and HPV16-positive human cervical cancer cell lines (SiHa) were acquired from the Cell Center of Shanghai Institutes for Biological Sciences (Shanghai, China), which were kept in Dulbecco’s modified Eagle’s medium (DMEM, Hyclone, Logan, UT, USA) supplemented with 10% fetal bovine serum (FBS) (Gibco, Gaithersburg, MD, USA) and 1% penicillin mixture (Solarbio, Beijing, China) in a moisturizing condition involving 5% CO₂ at 37 °C.

A total of 32 female BALB/c nude mice (4–6 weeks) were obtained from Charles River Laboratories (Beijing, China). Groups were divided using random number table method. All nude mice were housed with 12 h light-dark cycles and stored in a specific pathogen-free (SPF) condition. The bedding was changed every 2 days, the room temperature was controlled at 22~26 °C, and the mice were fed with a feed rich in 18~20% protein. The entire experimental process followed the relevant regulations of the Ethics Committee of the Second Hospital of Shanxi Medical University on the welfare ethics of animal experiments (Approval No. DW2022035).

Lentiviral construction and cell grouping

All lentiviral vectors in this study carried green fluorescent protein (GFP) tags, and the construction scheme was completed by Fubio Biotechnology Co., Ltd (Suzhou, China). SiHa cells transfected with an empty lentiviral vector were employed as the control group (Lv-Control). Lentiviral vectors of miR-154-5p precursor (pre-miR-154-5p) and sponge fragments were introduced into 293T cells, packaged with lentivirus, and infected with SiHa cell after purification of the lentivirus solutions. After 48 h, puromycin (Solarbio, Beijing, China) at a concentration of 3.0 µg/mL was added to screen stable overexpressed and silenced cell lines, named Lv-miR-154-5p-OE group and Lv-miR-154-5p-Sp group, respectively. Real-time quantitative polymerase chain reaction (RT-qPCR) was utilized to verify miR-154-5p expression in cells.

FV136 lentiviral vector (Fubio, Suzhou, China) with CUL2 overexpression was prepared and infected into SiHa cells of the Lv-miR-154-5p-OE group. Screening drug hygromycin (Fubio, Suzhou, China) at 300 µg/mL concentration was added to obtain a SiHa stable cell line model of miR-154-5p and CUL2 co-overexpression, named Lv-CUL2-OE group. SiHa cells of the Lv-miR-154-5p-OE group transfected with the empty sequence vector were used as a control group (Lv-CUL2-CO). The expression levels of CUL2 mRNA and CUL2 proteins in the stable cell lines were verified by RT-qPCR and Western blot, respectively.

Real-time quantitative polymerase chain reaction

Overall RNA was extracted from samples using Trizol lysate. A260/A280 ratio of 1.9–2.2 was used to confirm the quality and purity of the RNA, which was reverse transcribed to cDNA. RT-qPCR was employed to measure the mRNA expression using real-time fluorescent quantitative universal reagents (Cat#GMRS-001; GenePharma, Shanghai, China) and real-time fluorescent quantitative PCR instrument (FTC-3000, Funglyn Biotech, Canada). We conducted quantitative PCR reaction program as follows: denaturation at 95 °C for 3 min, followed by 40 cycles of 95 °C for 12 s, 62 °C for 30 s, 72 °C for 30 s. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and U6 were employed as endogenous reference genes, and the specific primer sequences were designed and synthesized by Shanghai GenePharma Company. Every gene expression was computed applying the 2^−∆∆Ct function. The primer sequences of RT-qPCR used in this investigation are as follows:

miR-154-5p forward primer 5′-CGCGTAGGTTATCCGTGTTG-3′ and reverse primer

5′-AGTGCAGGGTCCGAGGTATT-3′;

U6 forward primer 5′-GGAACGATACAGAGAAGATTAGC-3′ and reverse primer

5′-TGGAACGCTTCACGAATTTGCG-3′;

CUL2 forward primer 5′-TATGTGTGGCCTATCCTGAACC-3′ and reverse primer

5′-TGCAAATGCCGAACATGATTTTC-3′;

GAPDH forward primer 5′-GGAGCGAGATCCCTCCAAAAT-3′ and reverse primer

5′-GGCTGTTGTCATACTTCTCATGG-3′.

CCK-8 assay

A 96-well plate was used for the stable SiHa cells growing at a density of 3 × 10³ cells per well, and the plate was removed after pre-culture for 24 h. Then, 10 μL CCK-8 (Absin, Shanghai, China) was put into each well under dark conditions and 1 h in an incubator. We monitored the OD₄₅₀ value at five time-points (0, 24, 48, 72, and 96 h) using a microplate reader.

Cell cycle distribution

The cells were digested with trypsin without EDTA. The collected cells (1.0 × 10⁷) were rinsed twice with PBS, then with pre-cooled 75% ethanol diluted in PBS, and regularized at −20 °C overnight. A 5 min centrifugation was performed on cells at 1,000 g, discarding the supernatant, the cell pellet was rinsed with PBS, and centrifuged again. Next, 500 μL prepared propidium iodide staining solution (Yeasen, Shanghai, China) (supplemented 10 μL propidium iodide stock solution and 10 μL RNaseA to 500 μL staining buffer) was added to cell samples to resuspend the cells. Cell samples were incubated in a 37 °C water bath (Yiheng Instruments Co., Ltd., Shanghai, China) without light for 30 min, and the NovoCyte flow cytometer (ACEA Pharma, Hangzhou, China) was used for detection.

Colony formation assay

Cells were inoculated in a six-well plate at a density of 1,000 cells per well and cultured in an incubator for 2 weeks. The medium was discarded, and every well of the culture plate was gently rinsed twice with PBS and fastened with 4% paraformaldehyde (Meilunbio, Dalian, China) for 20 min and stained with 0.5% crystal violet (Solarbio, Beijing, China) for 20 min. After staining was completed, the colonies were scanned and imaged by 3D HISTECH scanner (Budapest, Hungary).

Would healing assay

The stable SiHa cells were inoculated into a six-well plate during logarithmic growth phase, and when the cell fusion rate reached approximately 80%, a 200 μL pipette tip was employed to streak it vertically. The scratched cells were washed three times gently with sterile PBS, and serum-free medium was supplemented for culture. Images were acquired in the same region of view at 0 and 48 h with an inverted microscope (Leica DMIL LED, Weitzlar, Germany), and the cell migration index was calculated to evaluate cell migration ability. Cell migration index = (0 h scratch void region − 48 h scratch void region)/0 h scratch void region.

Bioinformatic prediction of targets and potential functions of miR-154-5p

Datasets of downstream target genes of miR-154-5p were downloaded from three online tools: TargetScan (https://www.targetscan.org/vert_72/) (Release 7.2, 21 July, 2021 accessed), The Encyclopedia of RNA Interactomes (Encori) (https://starbase.sysu.edu.cn/) (21 July, 2021 accessed) and miRDB (https://mirbase.org/) (21 July, 2021 accessed) (Li et al., 2022). The overlapping genes from the above databases were managed by Venny 2.1.0 (Chen et al., 2022) (https://bioinfogp.cnb.csic.es/tools/venny/) and uploaded to the STRING database (https://www.string-db.org/) (Version 11.5, 1 August, 2021 accessed) to observe protein-protein interactions (Szklarczyk et al., 2021). Meanwhile, the pathway enrichment and functional clustering of the downstream overlapping genes of miR-154-5p were analyzed and visualized based on the Kobas 3.0 website (http://kobas.cbi.pku.edu.cn/) (17 October, 2021 accessed) (Bu et al., 2021). For each group set, the first five terms with the highest enrichment ratio were shown if there were more than five terms. Based on the above analysis, the potential biological effect and target mRNAs of miR-154-5p were further understood.

Access to the open data

Data on the pan-cancer expression profile of CUL2 (Ensembl ID: ENSG00000108094.14) were retrieved from the Gene Expression Profiling Interactive Analysis (GEPIA) database (http://gepia.cancer-pku.cn/) (17 October, 2021 accessed) (Zhang et al., 2023).

Xenograft mouse model

A total of 18 nude mice were chosen, randomly divided into three groups, with six in each group. SiHa stable cell lines in Lv-Control, Lv-miR-154-5p-OE and Lv-miR-154-5p-Sp groups were made into a single cell suspension (PBS: Matrigel = 7:3) and injected subcutaneously into mice at an inoculation density of 1.0 × 10⁷. At 7 days, the modeling was considered successful if the grafted tumor became visible and hard to touch. Tumor growth was observed, the length and width of graft tumor were measured every 4 days, and a curve was drawn. On day 49, we terminated the animal experiment after a clear trend of observable tumor growth sufficient for RNA and protein extraction. The mice were euthanized quickly via cervical dislocation. Tumors were then isolated, weighed, and photographed. Calculation formula for tumor volume: V (volume) = (length × width²)/2 (Zhang et al., 2022a).

Moreover, six additional nude mice were obtained and divided into two groups, with three in each group. Cell suspensions from Lv-CUL2-CO group and Lv-CUL2-OE group were injected into these mice to perform the tumor xenograft assay. Tumor cells were successfully modeled at 7 days, and nude mice in the CUL2-OE and CUL2-CO groups were euthanized at 31 days.

In vivo metastasis assay

We obtained eight additional mice and randomly divided them into four groups: blank group (no cell injection) (n = 1), control group (Lv-Control) (n = 1), miR-154-5p overexpression group (Lv-miR-154-5p-OE) (n = 3), and miR-154-5p sponge group (Lv-miR-154-5p-Sp) (n = 3). They were fixed in a visual tail vein injection fixture (Yiyan, Jinan, China), using an alcohol cotton ball to dilate the vein of the mouse tail by a temperature difference. A 1 mL syringe was used to inject 2 × 10⁶ GFP-labeled SiHa cells (100 μL) into the tail vein. The mice were observed to exclude abnormal states, after which they were kept under SPF conditions. The tumor metastasis ability and extent of SiHa cells in vivo were evaluated. After 60 days, the mice were euthanized, and the liver, spleen, lung, kidney, and uterus were dissected. Then, the GFP signal intensity in each organ was precisely surveyed using the IVIS Spectrum small animal in vivo optical image system (PerkinElmer, Waltham, MA, USA) (Chen et al., 2021).

Western blot

The RIPA lysate (Boster, Wuhan, China) was mixed with protease inhibitors (Boster, Wuhan, China), and animal tumor tissues were lysed and total protein was extracted. The BCA concentration determination kit (Boster, Wuhan, China) detected the protein concentration and determined the loading amount (cell samples: 25 µg, animal samples: 30 µg). SDS-PAGE gel (8%) for electrophoresis was prepared and the protein was transferred to the polyvinylidence fluoride (PVDF), which was sealed with 5% degreased milk powder for 2.5 h. The primary antibodies against CUL2 (Cat# sc-166506, 1:100; Santa Cruz, CA, USA) and β-actin (Cat# 4970, 1:1,000, CST, Boston, MA, USA) were added and cultured overnight in a refrigerator at 4 °C. Then, the secondary antibody was added and the sample was incubated for 1 h. The ECL chemiluminescent solution (Seven, Beijng, China) was used for visualization via Gel imaging system (ChemiDocTM XRS; Bio-Rad, Hercules, CA, USA). Using β-actin as the internal reference control, the correlative expression of CUL2 protein was computed as the quotient of gray value of CUL2 and β-actin via Image Lab 5.2 software.

Hematoxylin and eosin (HE) staining

The dissected tumor samples of the nude mice were fastened with formaldehyde (Meilunbio, Dalian, China), dehydrated, embedded, and sectioned. After HE staining, we used Adobe Photoshop CS6 to monitor the presence of cancerous foci.

Immunohistochemical analysis

After the mice tumor tissues were dehydrated, waxed, embedded, and cut into sections, each section of tissue was deparaffinized in xylene and hydrated using ethanol at different concentration gradients. High pressure was used for antigen retrieval and blocked with goat serum, and CUL2 antibody (Cat#51-1800, 1:200; Invitrogen, Carlsbad, CA, USA) was subjoined and cultured overnight at 4 °C. Secondary antibody was subjoined after PBS rinsing. The secondary antibody was removed, rinsed with PBS, stained with freshly prepared DAB staining solution, and the degree of staining was monitored under a microscope (Leica DM500; Leica, Weitzlar, Germany). After counterstaining with hematoxylin and differentiation with hydrochloric acid and alcohol, tap water was used to reverse the blue color. After dehydration with different concentrations of ethanol and transparent xylene, the slides were mounted with neutral gum. Under electron microscopy, a cytoplasm or nucleus exhibiting brownish yellow coloration was regarded as a positive finding. Five regions of view were randomly chosen for analysis under a 400× field of view. Using Adobe Photoshop (version: CS6) and Image pro (version: plus 6.0), the average value of the integrated optical density (IOD) of the five fields of view was taken as the statistic.

Statistical analysis

SPSS 25.0 and GraphPad Prism (version 9.0) software packages were used for data processing and chart drawing. Data from the research were shown as the mean ± standard deviation (SD). Count data are shown as n. The student t-test was conducted for the comparison of the two groups. One-way analysis of variance was used to compare the three or more groups. P value < 0.05 was considered statistically significant, and all experiments were replicated independently in triplicates.

Construction of SiHa cell lines with stable high and low miR-154-5p expression

To evaluate the effect of miR-154-5p on cervical cancer cells, we first performed RT-qPCR on SiHa and HaCaT cells to detect expression levels of miR-154-5p. The results indicated its expression level was lower in SiHa cells than in HaCaT cells (Fig. 1A). Subsequently, we created SiHa stable cell lines with overexpressed and silenced miR-154-5p, named Lv-miR-154-5p-OE and Lv-miR-154-5p-Sp, respectively. Meanwhile, the SiHa cells transfected with an empty lentiviral vector were employed as the control group (Lv-Control) (Fig. 1B). To ensure that cell models could be used in subsequent experiments, RT-qPCR was used for verification. As shown in Fig. 1C, compared with the Lv-Control group, miR-154-5p expression in the Lv-miR-154-5p-OE group was significantly increased, but there was no difference in the Lv-miR-154-5p-Sp group.

Effect of miR-154-5p on the proliferation and migration of SiHa cells

CCK-8, colony formation, and cell cycle measurements were used to assess the effects of miR-154-5p on the proliferation of SiHa cells. The overexpression group resulted in weaker cell proliferation and lower malignancy rates in comparison to the Lv-Control group, and the proportion of cells in the G1 phase was increased. In contrast, silencing miR-154-5p boosted the proliferation of SiHa cells, enhanced cell colony formation ability, and lowered the scale of cells in the G1 phase (Figs. 2A–2C). Immediately afterward, we assessed the migration capabilities of SiHa cells by scratch assay. As shown in Fig. 2D, miR-154-5p overexpression restrained the migration of SiHa cells, and its knockdown promoted their migration, in comparison to the Lv-Control group.

CUL2 was associated with miR-154-5p and was highly expressed in cervical cancer

To further elucidate the mechanism of miR-154-5p in the tumorigenesis of cervical cancer, we screened the downstream target genes of miR-154-5p and elucidated its function, as shown in Fig. 3.

The three databases (TargetScan, Encori, and miRDB) showed 169, 2,097, and 339 genes, respectively, with targeted binding to miR-154-5p. According to Venny 2.1.0 analysis, a total of 38 genes overlapped in the above database (Fig. 4A). The dataset of genes in the overlapping regions was uploaded to the STRING database to retrieve the interaction relationship between proteins. Homo sapiens, full STRING network, medium confidence (0.400), and FDR stringency (five percent) were selected as the filtering criteria by default. Of 37 other proteins, CUL2 interacted with only three proteins (UBE2H, FBXL16, and SOCS5) (Fig. 4B). Meanwhile, Kobas 3.0 was utilized to perform pathway enrichment and functional clustering analysis of 38 genes. The visual bubble diagram showed that the downstream overlapping genes of miR-154-5p were co-clustered into seven categories (C1–C7): C1 referred to target genes mainly distributed in intracellular components, C2 encompassed protein ubiquitination-related pathways, C3 represented 38 genes with multiple protein kinase activities and protein binding functions, C4 represented congenital disorders, C5 encompassed solid tumor signaling pathways (including renal cell carcinoma and glioma), C6 included glucagon and oxytocin signaling pathways, and C7 included signaling pathways related to metabolism (Fig. 4C). These findings show that miR-154-5p alters certain tumor-related biological processes by modulating the 38 genes.

Next, we searched the pan-cancer expression profile of CUL2 using the online database GEPIA and discovered that CUL2 expression was higher in CESC (cervical cancer, including cervical squamous cell carcinoma and adenocarcinoma) than in normal cervical tissue (Fig. 5A). Subsequently, we detected the level of CUL2 in SiHa cell, HaCaT cell, Lv-Control, Lv-miR-154-5p-OE and Lv-miR-154-5p-Sp SiHa stable cell lines by RT-qPCR. CUL2 mRNA expression was higher in SiHa cells than in HaCaT cells (Fig. 5B). Moreover, in SiHa stable cell lines, compared with the Lv-Control group, the Lv-miR-154-5p-OE group showed reduced CUL2 mRNA expression, while the Lv-miR-154-5p-Sp group showed no statistically significant difference in its expression (Fig. 5C).

MiR-154-5p hindered the growth and metastasis of cervical cancer by targeting CUL2 in vivo

To study whether miR-154-5p was capable of hindering the development and metastasis of SiHa cervical cancer cells in vivo, mice (n = 18) were injected subcutaneously with Lv-Control and Lv-miR-154-5p-OE/Sp cell suspension, and the tumor weight and size were measured. The average tumor volume and weight in the overexpression group were smaller than in the Lv-Control group (Figs. 6A and 6B). RT-qPCR showed that the overexpression group had higher miR-154-5p expression and lower CUL2 mRNA (Figs. 6C and 6D). Western blot and IHC revealed that CUL2 expression in the overexpression group was downregulated compared to the control group, while this phenomenon was opposite in the silenced group (Figs. 6E and 6F).

In the tail vein injection model, we used an in vivo imaging instrument to observe the metastasizing signal intensity in cancer cells in mice. The liver, spleen, lung, kidney, and uterus of the mice showed positive signals compared to the Lv-Control group. Except for the liver, spleen, and kidney, the mean values of the radiant efficiency in the lung and uterus of mice in the Lv-miR-154-5p-OE group were significantly decreased (Figs. 7A and 7B).

Overexpression of CUL2 influenced the effect of miR-154-5p on cervical cancer

To further demonstrate whether the effects of miR-154-5p on cervical cancer were influenced by CUL2, CUL2 was stably overexpressed in the background of the Lv-miR-154-5p-OE group, named Lv-CUL2-OE, and miR-154-5p-overexpressing SiHa cells transfected with an empty vector were considered the control group (Lv-CUL2-CO) (Fig. 8A). They were then verified by RT-qPCR and western blot, the outcomes illustrated that the expression levels of CUL2 mRNA and CUL2 protein were all higher in the Lv-CUL2-OE group than in the Lv-CUL2-CO group (Figs. 8B and 8C). Further, in comparison to the Lv-CUL2-CO group, the Lv-CUL2-OE boosted the proliferation and cloning capability, lowered the proportion of cells in the G1 phase, and promoted the migration capability of SiHa cells (Figs. 8D–8G).

In the in vivo experiment, after successful modeling of nude mice, the mice were killed 31 days after inoculation, and the weight of nude mice transplanted tumors in the CUL2-OE group was greater than that in the CUL2-CO group (Fig. 8H).