miR-34a-5p inhibits the malignant progression of KSHV-infected SH-SY5Y cells by targeting c-fos

Cell culture

SH-SY5Y and 293T cell lines were purchased from Shanghai Cell Bank, Chinese Academy of Sciences (Beijing, China). SH-SY5Y and 293T cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM; Gibco, Life Technologies, Carlsbad, CA, USA) containing 10% heat-inactivated fetal bovine serum (FBS; Hyclone, GE Healthcare, Little Chalfont, UK), with 1% 100× penicillin-streptomycin solution (Biosharp, Beijing, China). SH-SY5Y infection by recombinant KSHV (rKSHV.219) was performed previously (Kong et al., 2021). The KSHV-infected SH-SY5Y cells were named SK-RG. SK-RG cells were grown in DMEM supplemented with 10% heat-inactivated FBS, 1% 100× penicillin-streptomycin solution, and 6 μg/ml puromycin (Sigma-Aldrich, St. Louis, MO, USA), in an incubator at 37 °C and 5% CO₂.

Screening the differentially expressed miRNAs

RNA was extracted from KSHV-infected (SK-RG) or uninfected SH-SY5Y cells. Transcriptome sequencing was performed in BGI Biotech, Shenzhen, China. Each group had three parallel samples. After filtering the original sequencing data, high-quality data were compared. The data were integrated to identify and analyze the miRNAs found to be differentially expressed, and the meaningful upregulated and downregulated miRNAs were shown in Table 1. In a data set of miRNAs downregulated in KSHV-infected neurons, we predicted some miRNAs potentially associated with mRNA profilings using TargetScan/miRanda/RNAhybrid websites for bioinformatics analyses. Altogether, the fold change value was taken to select the differential miRNAs with high statistical significance. miR-34a-5p was selected for further studies. Moreover, we performed further experiments to determine the exact interactions because miR-34a-5p might regulate c-fos expression.

RNA extraction and real-time PCR

SH-SY5Y and SK-RG cells were seeded in 60-mm culture dishes and grown to 70–90% confluence for further study. Total RNA was extracted using TRIzol reagent (Invitrogen, Camarillo, CA, USA). RNA concentration and purity were detected. A RevertAid First Strand cDNA Synthesis kit (Thermo Fisher, Carlsbad, CA, USA) was utilized for cDNA synthesis. Real-time PCR was used to detect cDNA recovered (SYBR Green PCR Kit; Qiagen, Hilden, Germany). The following conditions were used to detect mRNA expression: 95 °C, 300 s; 95 °C, 10 s; 55 °C, the 30 s; and 72 °C, 35 s; for a total of 35 cycles. We used the comparative 2^−ΔΔCt way to analyze the relative mRNA expression. Table 2 contained the primer sequences.

Cell transfection

Lipofectamine 2000 was used to transfect SK-RG cells at the logarithmic growth stage. (Invitrogen, Camarillo, CA, USA). miRNA negative control (miR-NC) and miR-34a-5p mimics (miR-34a-5p) were generated by GenePharma (Shanghai, China). pcDNA3.1 vector (pcDNA3.1) and the c-fos eukaryotic expression vector (c-fos) were generated by Gene Create (Wuhan, China), which were diluted and transfected into SK-RG cells.

Cell viability assay

Cell proliferation was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5- diphenyltetrazolium bromide (MTT) assay (Solarbio, Beijing, China). In 96-well plates, 1 × 10³ transfected SK-RG cells were seeded with a 100 µL suspension in each well. Cell proliferation was measured at different time points (24, 48, 72, 96, and 120 h). After incubating for 4 h with MTT, 150 µL dimethyl sulfoxide (DMSO, Solarbio, Beijing, China) was added and shaken for 15 min. A microplate reader was used to measure the absorbance at 490 nm.

Plate clone assay

Cell proliferation was assessed by plate clone assay. Transfected SK-RG cells were digested and seeded into six-well plates (310 cells/well) in 1.5 ml of complete medium. The cells were incubated for 13 days, with the solution being changed every 3–4 days throughout the process. After discarding the culture media, it was washed twice with phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde for 40 min, then stained with 0.1% crystal violet and washed twice with PBS after 30 min. The culture plates were dried at room temperature and observed under an inverted microscope to count the number of cell clones formed in each group.

Wound healing assay

Cells were spread into cell culture dishes and transfected. After culturing for 24 h, the density of cells was about 90%. We used a sterile pipette tip (1 ml) to scratch the monolayer. The cells were washed twice with PBS and incubated in 2% serum DMEM for further culture. Plates were photographed using a microscope 0, 12, 36, and 48 h after scratching at an identical location.

Transwell migration assay

Cell migration was assessed by transwell assay. After 24 h of transfection, the cells were digested. Then, 1 ml of PBS and 1 ml of DMEM were added sequentially for washing. The cells were resuspended in DMEM and counted until a 2 × 10⁵ cells/ml density was reached. Moreover, 200 µL of cell suspension was added to the upper chamber, and 600 μl of the medium with 10% FBS was added to the basolateral chamber. The chambers were incubated for 36 h at 37 °C. The washed cells were fixed with paraformaldehyde for 45 min and stained with 0.1% crystal violet for 35 min. Penetrating cells in three randomly selected fields of each sample were captured for counting under a light microscope. The experiment was repeated three times.

Dual-luciferase reporter assay

The binding sites of miR-34a-5p and c-fos 3′-UTR were predicted using StarBase 2.0 (http://starbase.sysu.edu.cn/). Luciferase reporter constructs encoding the wild-type 3′-UTR of c-fos (pmirGLO-c-fos-wt) or mutant 3′-UTR of c-fos (pmirGLO-c-fos-mut) were synthesized by GenePharma (Shanghai, China). Then, pmirGLO-c-fos-wt, pmirGLO-c-fos-mut, and 293T cells were co-transfected with miR-34a-5p mimics or miR-NC. After 24 h of incubation, the cells were collected and lysed. Using Renilla luciferase activity as an internal reference, dual-luciferase reporter gene analysis was used to calculate the relative activity of luciferase.

Western blot analysis

After transfection for 48 h, SK-RG cells were collected and lysed for total protein extraction. The proteins were electrophoresed in sodium dodecyl sulfate-polyacrylamide gels and subsequently transferred to polyvinylidene difluoride membranes. The membrane was blocked with 5% skimmed milk for 2 h, and primary antibodies, including β-actin (1:1,000; ZSGB-Bio, Beijing, China), c-fos (1:1,000; Bioss, Beijing, China), CDK4, CDK6 (1:1,000; Bioworld Technology, St Louis Park, MN, USA), cyclin D1 (1:500; Bioworlde Technology, St Louis Park, MN, USA), matrix metalloproteinase (MMP)2 (1:1,000; Bioss, Beijing, China), and MMP9 (1:1,000; Boster, Wuhan, China), were added overnight at 4 °C. The membranes were washed with 1 × Tris-buffered saline with Tween solution. Following that, secondary goat anti-rabbit or goat anti-mouse antibodies conjugated with horseradish peroxidase (1:10,000; ZSGB-Bio, Beijing, China) were added for 2 h at room temperature. After washing the membranes three times with 1 × PBS, the bands were detected by electrochemiluminescence and analyzed using ImageJ software (1.48v; NIH, Bethesda, MD, USA).

Flow cytometry analysis

Cell cycle distribution was detected using flow cytometry (FCM) assay. After transfection for 24 h, SK-RG cells were digested with trypsin, centrifuged at 1,000 r/min for 5 min, and then the cells were collected and fixed in 4 ml cooled 70% anhydrous ethanol. After being stored at −20 °C for 2 days, the fixed cells were centrifuged for 5 min and then hydrated with 3 ml PBS at room temperature for 15 min. After adding 1 ml of DNA staining solution, cells were incubated at 37 °C in the dark for 30 min, and the cell cycle was examined by FCM (BD Bio-sciences, San Jose, CA, USA).

Taq-man real-time PCR

Taq-man real-time PCR was used to determine virus DNA copy number. The experiment was performed after transfection in SK-RG cells for 48 h, and each group DNA was extracted according to the instructions of the TIANGEN Cell Genome Kit. DNA concentration was measured and detected using a Taq-man real-time PCR kit (Takara Biomedical Technology, Beijing, China). The sensitivity was determined by testing decreasing DNA quantities of the ORF26 plasmid (10-fold dilutions from 10³ to 10⁷ ng/µl). The temperature was set to 95 °C 20 s; 95 °C 3 s, 60 °C 30 s, a total of 40 cycles. Primer sequences are shown in Table 2.

Nude mice xenograft tumor assay

The xenograft tumor assay and ethics were approved by the institutional guidelines of the Animal Care and Use Committee at Laboratory Animal Research Center, Shihezi University (Xinjiang, China). BALB/c female nude mice were aged 4–6 weeks (mature body organs and good vital health index) were purchased and fed in the Experimental Animal Research Center of Xinjiang Medical University. The individual mouse was considered the experimental unit within the studies. The nude mice were housed in a dedicated SPF facility and raised in a laminar air chamber with the food and drinking water sterilized. We estimated the degree of freedom (E) of an OVA and initially selected 14 mice for the experiment. The xenograft mouse tumor model was established by injecting 3 × 10⁶ SK-RG cells into one side of the spine of nude mice. Before the intervention, six mice did not develop tumors and were euthanized. Then, tumor-bearing mice were randomized into two groups by computer generation of random numbers. The two groups were isolated and cultured in two culture rooms in the same environment to reduce confounding factors. In addition, the experimenters were blinded to the grouping. The mice in the experimental group (n = 4) were injected with miR-34a-5p agomir, while the control group (n = 4) were injected with miR-NC agomir. Injections were given every other day for 2 weeks. All mice were monitored closely. The tumor sizes and body weights of nude mice were measured before injection. Tumor growth was monitored continuously, and all mice were sacrificed by cervical dislocation after injection for two weeks, which was properly performed by trained professionals to avoid pain to subjects. After sacrificing the mice, tumors were collected and photographed.

Immunohistochemical analysis

Tissues isolated from different groups of nude mice were fixed with 10% formalin and paraffin-embedded to produce tissue sections. After dewaxing the slices, they were boiled for 8 min in sodium citrate buffer (pH = 6.0). The sections were cooled to 30 °C and incubated in 3% hydrogen peroxide at room temperature for 10 min while being protected from light. The sections were washed with PBS (3 × 5 min). Primary antibodies c-fos (1:100; Bioss, Beijing, China), CDK6, and cyclin D1 (1:50; Bioworld Technology, St Louis Park, MN, USA) were applied overnight at 4 °C. The sections were washed with PBS. The secondary antibody (ZSGB-Bio, Beijing, China) was added and incubated for 30 min at 37 °C. After washing again, the slides were colored with 3,3′-diaminobenzidine (DAB) working solution for 60 s at room temperature, and finally, the slides were re-stained with hematoxylin and sealed.

Statistical analyses

Experimental results were repeated at least three times in every group. The differences between the two groups were compared by the Student t-test. GraphPad Prism 5.0 was used to conduct all statistical analyses (GraphPad Software, San Diego, CA, USA). A statistically significant difference was marked by a P-value < 0.05.

The miR-34a-5p level was reduced in SK-RG cells

We infected the SH-SY5Y cells as described previously, and the infected cells were named SK-RG (Fig. 1A). The differentially expressed miRNAs were screened in KSHV-infected and uninfected SH-SY5Y cells by RNA sequencing, of which 1,462 miRNAs were upregulated, and 152 were downregulated in SK-RG cells (Fig. 1B). We screened out some miRNA names with significant up-regulation and down-regulation, as shown in Table 1. The relationship between miRNA and mRNA expression profiles was analyzed using the TargetScan/miRanda/RNAhybrid software. miR-34a-5p expression was downregulated in SK-RG cells due to the intersection (Fig. 1C). Furthermore, we performed real-time PCR to confirm that miR-34a-5p was down-regulated in SK-RG cells (Fig. 1D).

miR-34a-5p overexpression suppressed the proliferation and cell cycle of SK-RG cells

miR-34a-5p mimics were transfected into SK-RG cells to evaluate the effects of miR-34a-5p overexpression on cell proliferation and the cell cycle. MTT assay results showed that miR-34a-5p overexpression inhibited the proliferation of SK-RG cells compared with that in the miR-NC group (P < 0.05, Fig. 2A). To investigate whether miR-34a-5p affected the SK-RG cell cycle, we detected the cell cycle by FCM. The percentage of cells in the G0/G1 phase increased significantly after overexpression of miR-34a-5p compared with miR-NC. The results indicated that miR-34a-5p arrested the cell cycle of SK-RG cells in G0/G1 phase (P < 0.05, Figs. 2B and 2C). The expression of related cyclins was evaluated by Western blot analysis. The levels of CDK4, CDK6, and cyclin D1 were decreased when miR-34a-5p was overexpressed (P < 0.05, Figs. 2D–2F). Using the plate clone assay, we also found that miR-34a-5p overexpression reduced the number of colonies in SK-RG cells (P < 0.05, Fig. 2G).

The miR-34a-5p inhibitor was transfected into SH-SY5Y cells to investigate the effect of miR-34a-5p knock-down on the proliferation of SH-SY5Y cells. The real-time PCR results indicated that the miR-34a-5p inhibitor successfully decreased its level (P < 0.05, Fig. S1A). MTT assay results showed increased cell proliferation when miR-34a-5p was knock-downed in SH-SY5Y cells (P < 0.05, Fig. S1B). These results suggested that miR-34a-5p can influence cell proliferation ability.

miR-34a-5p overexpression inhibited the migration of SK-RG cells

We next explored whether miR-34a-5p mimics impacted the cell migration of SK-RG cells. The cell wound healing assay results showed the relative gap width was wider in miR-34a-5p mimics group than that in the miR-NC group (P < 0.05, Figs. 3A and 3B). In the transwell assay, the transmembrane cell number was fewer in miR-34a-5p mimics group than that in the miR-NC group (P < 0.05, Figs. 3C and 3D). Western blot results also revealed miR-34a-5p overexpression reduced the expression of MMP2 and MMP9 (P < 0.05, Figs. 3E and 3F). These data indicated that miR-34a-5p overexpression inhibited cell migration.

The miR-34a-5p inhibitor was transfected into SH-SY5Y cells to investigate the effect of miR-34a-5p knock-down on the migration of SH-SY5Y cells. Compared with the inhibitor-NC group, the miR-34a-5p inhibitor group accelerated the migration abilities of SH-SY5Y cells in wound-healing and transwell assays (P < 0.05, Figs. S1C–S1E). These results showed that miR-34a-5p could influence cell migration ability.

miR-34a-5p regulated the expression of c-fos through the 3′-UTR

We employed the StarBase 2.0 database to predict the potential binding sequence of miR-34a-5p and c-fos 3′-UTR (Fig. 4A). In the dual-luciferase reporter gene assay, the co-transfection of miR-34a-5p mimics and the c-fos 3′-UTR-wt decreased luciferase activity. However, the co-transfection of the c-fos 3′-UTR-mut and miR-34a-5p mimics did not change any activity (P < 0.05, Fig. 4B). We compared the expression levels of c-fos in KSHV-infected and uninfected SH-SY5Y cells by Western blot assay. The results showed that c-fos was upregulated in SK-RG cells (P < 0.05, Fig. 4C). Furthermore, the real-time PCR displayed that the c-fos mRNA level was negatively correlated with the level of miR-34a-5p in SK-RG cells (P < 0.05, r = −0.9990, Fig. 4D). Besides, Western blot analysis showed that c-fos protein expression was down-regulated after miR-34a-5p overexpression (P < 0.05, Fig. 4E). These data indicated that miR-34a-5p had a targeting effect on the expression of c-fos.

c-fos rescued the suppressive effect of miR-34a-5p on the proliferation of KSHV-infected SH-SY5Y cells

c-fos was upregulated in miR-34a-5p-overexpressing SK-RG cells (P < 0.05, Fig. 5A). Then the effect on the cell cycle was detected by FCM. After overexpression of c-fos, the percentage of cells in the G0/G1 phase was reduced compared with the control group, which indicated that c-fos reversed the cycle arresting effect of miR-34a-5p on SK-RG cells (P < 0.05, Figs. 5B and 5C). Subsequently, we explored the c-fos rescue effect on cell proliferation. MTT assays showed that c-fos overexpression rescued and reduced the inhibitory effect of miR-34a-5p on cell proliferation (P < 0.05, Fig. 5D). The protein levels of CDK4, CDK6, and cyclinD1 were increased when c-fos was overexpressed (P < 0.05, Figs. 5E–5G). Using the plate clone assay, we found that overexpression c-fos increased the colony number of SK-RG cells (P < 0.05, Figs. 5H and 5I). These results suggested c-fos rescued the effect of miR-34a-5p on the proliferation of SK-RG cells.

c-fos rescued the suppressive effect of miR-34a-5p on the migration of KSHV-infected SH-SY5Y cells

We upregulated c-fos in miR-34a-5p-overexpressing SK-RG cells, and the cell migration ability was detected. The cell wound healing assay showed that the gap was narrower in the c-fos rescue group than in the control group (P < 0.05, Figs. 6A and 6B). Similarly, the transwell assay showed that the overexpression of c-fos rescued this effect, although miR-34a-5p suppressed the migration of SK-RG cells. (P < 0.05, Figs. 6C and 6D). Western blot analysis showed that the protein levels of MMP2 and MMP9 increased when c-fos was overexpressed (P < 0.05, Figs. 6E and 6F). In summary, c-fos rescued the effect of miR-34a-5p on the migration of SK-RG cells.

miR-34a-5p decreased the KSHV gene expression and copy number, while c-fos rescued the suppressive effects

We transfected miR-NC and miR-34a-5p to investigate the effect of miR-34a-5p on KSHV gene expression and copy number. The results indicated that miR-34a-5p overexpression suppressed the mRNA expression of KSHV genes LANA, RTA, K8.1, and v-GPCR. Furthermore, we upregulated c-fos in miR-34a-5p-overexpressing SK-RG cells and found that the overexpression of c-fos reversed the effects of miR-34a-5p on these KSHV genes’ expressions (P < 0.05, Fig. 7A). We determined the KSHV DNA copy number after overexpression of miR-34a-5p by Taq-man real-time PCR. The results showed miR-34a-5p overexpression decreased the KSHV copy number (P < 0.05, Fig. 7B). However, after we upregulated the c-fos, the c-fos rescued the restraint effect of miR-34a-5p on KSHV copy number (P < 0.05, Fig. 7C).

miR-34a-5p inhibited tumor proliferation in vivo

Xenograft tumor mouse models were established by injecting SK-RG cells into one side of the spine of nude mice to investigate the efficacy of miR-34a-5p in vivo (Fig. 8A). The mice were treated with miR-34a-5p agomir and miR-NC agomir. miR-34a-5p agomir treatment led to a suppression of the growth of tumors, compared with that in the miR-NC group (Figs. 8B and 6C). This consistent trend was also demonstrated by the visual inspection of the isolated tumor size and quantitative measurement of the tumor weight and volume. The tumor volume decreased in mice treated with miR-34a-5p compared with that in the miR-NC group (P < 0.05, Fig. 8D). Moreover, we used immunohistochemistry (IHC) to evaluate the expression levels of c-fos, CDK6, and cyclin D1 in the tumor tissues. The results showed that the levels of c-fos, cyclin D1, and CDK6 were lower in the tissue sections treated with miR-34a-5p than those in the miR-NC group (P < 0.05, Figs. 8E–8H). Tumor weight decreased in mice treated with miR-34a-5p compared with that in the miR-NC group (P < 0.05, Fig. 8I). The data suggested that miR-34a-5p inhibited proliferation in vivo.