Desmocollin-2 inhibits cell proliferation and promotes apoptosis in hepatocellular carcinoma via the ERK/c-MYC signaling pathway

DSC2 expression levels in HCC cell lines

To understand the expression of DSC2 in HCC cell lines, we detected the expression of DSC2 in normal hepatocyte cells (L02) and HCC cell lines (SMMC-7721, Huh7, HCC-LM3, and MHCC-97H). The results showed that the expression of DSC2 protein in HCC cell lines SMMC-7721 (P < 0.01), Huh7 (P < 0.001), HCC-LM3 (P < 0.001), and MHCC-97H (P < 0.001) were all significantly down-regulated compared with L02 cells (Figure 1). In addition, in the HCC cell lines above, DSC2 was highly expressed in 7721 cells, while the expression level was the lowest in LM3 cells (highly metastatic cell line). So we chose 7721 and LM3 for subsequent experiments.

Figure 1. DSC2 expression in HCC cell lines. DSC2 expression levels in L02 and HCC cell strains were detected by Western blot analysis (repeated three times). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Selecting overExp-DSC2 and shRNA-DSC2-1 plasmids for transfections

Since the expression of DSC2 in LM3 cells was lower than that in Huh7, 97H, and 7721 cells, we overexpressed DSC2 in LM3 cells and interfered with the expression of DSC2 in 7721 cells. We found that after transfection with an overexpression vector (overExp-DSC2), the expression of DSC2 in LM3 cells was significantly up-regulated (P < 0.05). Then, we confirmed that the expression of DSC2 in 7721 cells had successfully interfered with the specific shRNA (shRNA-DSC2-1 and shRNA-DSC2-2) (P < 0.001), while the expression level of DSC2 in 7721 cells transfected with the shRNA-DSC2-3 vector was not significantly altered (P > 0.05) (Supplementary Figure 1). Because of the lowest expression of DSC2 in cells after shRNA-DSC2-1 interference, the shRNA-DSC2-1 plasmid was selected for subsequent experiments.

DSC2 inhibited the ability of cell proliferation

To explore the effect of DSC2 on cell proliferation, we detected the proliferation ability of transfected cells by using colony formation, CCK-8, and EdU assays. Results indicated that the formation of tumor cell colonies was significantly inhibited in overExp-DSC2-transfected LM3 cells (P < 0.01), while promoted in shRNA-DSC2-transfected 7721 cells (P < 0.05) (Figure 2A). Similarly, CCK8 assay showed that there were significantly lower rates of cell proliferation in LM3 cells with overExp-DSC2, but higher proliferation rates in 7721 cells with shRNA-DSC2 (Figure 2B, P < 0.001). We further verified the effect of DSC2 on proliferation by using the EdU experiment. It was also found that the proliferation capacity of the overExp-DSC2-transfected LM3 cells was noticeably decreased, whereas it was increased for 7721 cells transfected with shRNA-DSC2 (Figure 2C, P < 0.01).

Figure 2. DSC2 inhibits the cell proliferation ability in vitro and affects cell cycle-related proteins. (A) The clone formation ability of overExp-DSC2-transfected LM3 and shRNA-DSC2-1-transfected 7721 cells was shown by clone formation assay (repeated three times). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (B, C) The cell viability of overExp-DSC2-transfected LM3 and shRNA-DSC2-1-transfected 7721 cells were detected by CCK-8 and EdU assay (repeated three times for both). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001. (D) Western blot analysis of the protein levels of Cyclin D1, Cyclin B1, CDK1, and CDK2 from LM3 and 7721 cells transfected with overExp-DSC2 and shRNA-DSC2-1 (repeated three times). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

To understand the effect of DSC2 on the cell cycle, we detected the expression levels of cell cycle-related proteins in transfected cells by Western blotting and the results demonstrated that the expression levels of Cyclin D1, Cyclin B, CDK1, and CDK2 were significantly down-regulated in overExp-DSC2-transfected LM3 cells (P < 0.001), while up-regulated in shRNA-DSC2-transfected 7721 cells (P < 0.05) (Figure 2D). These results indicated that DSC2 overexpression suppressed HCC cell proliferation, while DSC2 knockdown promoted HCC cell proliferation.

DSC2 promoted cell apoptosis and regulated apoptosis-related proteins expression

Transfected cells were stained with Acridine Orange/Ethidium Bromide (AO/EB) to detect cell apoptosis, and the results indicated that DSC2 overexpression significantly increased apoptosis in overExp-DSC2-transfected LM3 cells (P < 0.001), while the knockdown of DSC2 decreased apoptosis in shRNA-DSC2-transfected 7721 cells (P < 0.01) (Supplementary Figure 2A).

To further clarify the role of DSC2 on cellular apoptosis, we assessed the expression of key apoptosis-related proteins in the transfected cells. The results showed that the expression levels of Bax, c-Caspase-3, and Caspase-8 were noticeably increased in overExp-DSC2-transfected LM3 cells (P < 0.01), whereas the expression levels of Bcl-2 and Survivin were decreased (P < 0.01). Furthermore, the expression levels of Bcl-2 (P < 0.001) and Survivin (P < 0.05) were elevated in shRNA-DSC2-transfected 7721 cells, while the expression levels of Bax and c-Caspase-3 were reduced significantly (P < 0.001) (Supplementary Figure 2B). In conclusion, DSC2 overexpression could promote apoptosis of HCC cells.

DSC2 inhibited HCC cells migration and invasion

To investigate whether the DSC2 affected the migration and invasion of HCC cells, cell wound healing and Transwell assays were performed. Wound healing assays demonstrated that DSC2 overexpression in LM3 cells inhibited the cell motility (P < 0.05 for 24 h and P < 0.01 for 48 h), while DSC2 knockdown exhibited the opposite effect in 7721 cells (P < 0.05 for 24 h and P < 0.01 for 48 h) (Supplementary Figure 3A). Moreover, Transwell assay showed that the migration and invasion abilities were decreased dramatically in overExp-DSC2-transfected LM3 cells (P < 0.01), while increased in shRNA-DSC2-transfected 7721 cells (P < 0.05) (Supplementary Figure 3B). These findings suggested that DSC2 overexpression could inhibit the cell migration and invasion abilities of HCC cells and vice versa.

DSC2 expression levels affected the ERK/c-MYC signaling pathway

To explore the molecular mechanism of DSC2 inhibiting proliferation and promoting apoptosis in HCC cells, we detected the ERK1/2 signaling pathway. The results showed that the overexpression of DSC2 inhibited the expression of p-ERK1/2 (p-ERK1-T202/Y204+p-ERK2-T185/Y187) and c-MYC in LM3 cells, while DSC2 knockdown increased the expression levels of p-ERK1/2 and c-MYC in 7721 cells (Figure 3). These data indicate that DSC2 may affect the proliferation, apoptosis, migration, and invasion of HCC cells by regulating the ERK/c-MYC signaling pathway.

Figure 3. DSC2 inhibits the ERK1/2 signaling pathway. The expression levels of ERK1/2, p-ERK1/2, and c-MYC in overExp-DSC2-transfected LM3 and shRNA-DSC2-1-transfected 7721 cells were determined by Western blot analysis (repeated three times). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

DSC2 inhibited the growth of tumors in vivo

To identify in-depth the effect of DSC2 on tumor growth in vivo, the transfected cells (vector, overExp-DSC2, shControl, and shRNA-DSC2 respectively) were subcutaneously injected into the right back of 5-week-old nude mice. Tumor volume was measured and recorded every 4 days. Mice were sacrificed 28 days later for sampling. Subsequently, tumor volume and weight were determined. As shown in Figure 4A, the volume and weight of the tumors from the mice injected with DSC2-overexpressing LM3 cells were significantly lower than those from the mice injected with the vector-transfected cells (P < 0.001 for both), indicating that DSC2 overexpression inhibited the tumor growth dramatically. Additionally, as shown in Figure 4B, we found that the volume and weight of tumors in mice injected with the DSC2-knockdown cells were markedly higher than those in the mice injected with shControl-transfected cells (P < 0.001 for both), indicating the down-regulation of DSC2 promoted the tumor growth.

Figure 4. DSC2 inhibits HCC tumor growth in vivo. (A) LM3 cells transfected with vector and overExp-DSC2 were injected into the right back of 5-week-old male nude mice with 6 rats in each group. After 28 days, the tumor was collected, and the volume and weight of the tumor were measured. (B) 7721 cells transfected with shControl and shRNA-DSC2-1 were injected into the right back of 5-week-old male nude mice with 6 rats in each group. After 28 days, the tumor was collected, and the volume and weight of the tumor were measured.

Cell culture and transfection

HCC cell lines SMMC-7721, Huh7, HCC-LM3 (highly metastatic cell line), MHCC-97H (highly metastatic cell line), and human immortalized hepatocyte line L02 obtained from Chinese Academy of Sciences Cell Bank (Chinese Academy of Sciences, Shanghai, China) were identified by STR and detected by mycoplasma. The cells were cultured in high glucose DMEM (Invitrogen, CA, USA), which was supplemented with 100 U/ml penicillin, 100 U/ml streptomycin, and 10% fetal bovine serum (FBS). The cells were cultured in 37°C, 5% CO₂ incubator, and had been subcultured after digestion with 0.05% EDTA-trypsin. The cells in the logarithmic growth phase were inoculated in a 6-well plate with 2 × 10⁴ cells/well. 16–18 hours later, the cells were transfected with vector, overExp-DSC2, shControl, shRNA-DSC2-1, shRNA-DSC2-2, and shRNA-DSC2-3 (Shanghai Jikai Gene Co., Ltd., Shanghai, China). Puromycin (2 ug/ml) was used to screen the cell lines. The expression of DSC2 was detected by RT-qPCR and Western blot to determine the transfection effect.

Colony formation assay

The transfected cells were seeded into a 6-well plate with 500 cells/well for routine culture. The cells were cultured for 2–3 weeks and terminated when visible cell clones emerged in the 6-well plate. Washed twice with PBS, the clones were fixed with 4% paraformaldehyde for 15 min and then stained with Wright-Giemsa compound staining solution for 30 min. After washing and drying, the clones were observed and photographed.

Cell counting kit-8 (CCK-8)

The transfected cells were collected and inoculated into 96-well plates (8000 cells/well) for routine culture. After the cells adhered to the wall, 10 ul CCK-8 (Yuheng Biotechnology Co., Ltd; Jiangsu, China) was added at 0 h, 24 h, 48 h, and 72 h, respectively, and incubated at 37°C for 2 hours in dark. The optical density (OD) at 450 nm was measured by Thermo Multiskan FC (Thermo Fisher, MA, USA).

Edu assay

To determine the rate of cell proliferation, we visualized the incorporation of the thymidine analog 5-Ethynyl-2′-deoxyuridine (EdU) into newly synthesized DNA using the Click-it EdU kit (Yuheng Biotechnology Co., Ltd; Jiangsu, China). Cells were seeded on 96-well plates (1 × 10⁴ cells/well) and processed according to the instructions of the EDU reagent the next day. After Hoechst re-staining of nuclear for 5 min, it was observed and photographed under the fluorescence microscope. Counting was performed automatically using ImageJ (https://www.nih.gov).

Subcutaneous tumorigenesis

This experiment has been approved by the Ethics Committee of the second affiliated Hospital of Nanchang University. Transfected cells (4 × 10⁶ cells/0.1 mL) were subcutaneously inoculated into the right back of 5-week-old nude mice, six mice in each group. Tumor volume was measured every 4 days. After tumor-bearing mice were sacrificed 28 days later, tumor tissue was harvested and weighed, and the tumor volume was measured.

Wound healing assay

The transfected cells were seeded in a 6-well plate for conventional culture. After the cells were fused completely, a 10 μl pipette tip was used to scratch in a straight line vertically at the bottom of the wells. The monolayer cell fragments and residual medium were washed off by PBS, and the scratches were observed and photographed under the microscope at 0 h, 24 h, and 48 h, respectively. ImageJ (https://www.nih.gov) was used to quantify the degree of wound closure at the above-specified time points.

Transwell assay

The bottom of the chamber was evenly smeared with a matrix to form a film. The transfected cells were collected and re-suspended in a serum-free DMEM medium to prepare a single-cell suspension. The upper chambers (no matrix glue/matrix glue) were added with 200 ul cell suspension containing 1 × 10⁵/2 × 10⁵ cells respectively, and 750 μl DMEM medium containing 10% fetal bovine serum was added into the lower chamber. The upper chamber was taken out and the culture medium was discarded after continuous culture for 24 hours. The chamber was fixed in 4% paraformaldehyde for 10 min, stained with 10 g/l crystal violet for 30 min, and gently wiped the cells on the upper chamber’s inner surface with a cotton swab. The cells were observed and photographed by the microscope.

Acridine orange/ethidium bromide (AO/EB) assay

The transfected cells were collected and inoculated into a 96-well plate (1 × 10⁴ cells/well) for routine culture. The next day, the cells were incubated with 200 ul staining buffer and 2 ul AO/EB staining solution at room temperature for 30 min. The cells were observed and photographed by the fluorescence microscope after washing.

Western blot analysis

As the transfected cells reached 90% fusion, the mixture of RIPA and protease inhibitor (400 ul) was added to the cells for 30 minutes (always performed on ice). The liquid was placed into an Eppendorf tube and centrifuged at 12,000 g for 15 min at 4°C. Protein concentration was estimated using the BCA protein assay kit (Beyotime Biotech, China). Equal amounts of protein (30 μg/lane) were resolved by 8% SDS-PAGE and transferred onto PVDF membranes. The membrane was then washed with Tris-buffered saline containing 0.05% Tween-20 (TBST) and blocked in TBST containing 5% non-fat dry milk or 5% BSA, and further incubated with the respective specific antibodies to DSC2, Bax, Bcl-2, Caspase-3, c-Caspase-3, Caspase-8, Survivin, Cyclin D1, Cyclin B1, CDK1, CDK2, ERK1/2, p-ERK1/2 (p-ERK1-T202/Y204+p-ERK2-T185/Y187) and c-MYC and β-actin. The membranes were then incubated with appropriate horseradish peroxidase (HRP)-conjugated secondary antibodies and developed using ECL western blotting detection reagents (Yuheng Biotechnology Co., Ltd; Jiangsu, China). All experiments were repeated three times.

Statistical analyses

The statistical analysis was performed by using SPSS 25.0 (SPSS, Inc., Chicago, IL, USA). All data were expressed as the mean ± standard deviation. Comparisons between data sets were performed using the χ² test and a two-tailed independent sample t-test when appropriate. P value < 0.05 was considered significant difference.

Data availability statement

The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding authors.