DNAJ heat shock protein family member C1 can regulate proliferation and migration in hepatocellular carcinoma

Data collection

Expression data and clinical data were downloaded from The Cancer Genome Atlas database (TCGA, https://cancergenome.nih.gov/)(374 tumor samples and 50 normal samples) (Cancer Genome Atlas Research Network, 2013). The TCGA database is open to the public, and all data have been agreed to be used for analysis and have obtained moral recognition. This study is based on open source data, strictly abides by the release guidelines and access policies of the database, and is not bound by other ethics.

DNAJC1 expression analysis

The Tumor Immune Estimate Resource (TIMER, https://cistrome.shinyapps.io/ TIMER/) database (Li et al., 2017) was used to compare the expression level of DNAJC1 in all TCGA tumors. Subsequently, DNAJC1 mRNA expression analysis was based on TCGA database by R package “Limma”, and the “ggplot2” package was used to draw the box plot for visualization.

Prognostic role of DNAJC1 in HCC

The UALCAN database (https://ualcan.path.uab.edu/) (Chandrashekar et al., 2017) was a website tool for online analysis and extraction based on the clinical data of 31 cancer types in TCGA database. In this study, the correlation between DNAJC1 expression and the prognosis of patients was explored by the UALCAN database.

Gene set enrichment analysis (GSEA)

Gene set enrichment analysis (GSEA) (Subramanian et al., 2005) was used to explore the related signaling pathways. In this study, TCGA samples were divided into high-low expression groups according to the median expression of DNAJC1. GSEA was performed with the package “ClusterProfiler” (Yu et al., 2012) to investigate whether DNAJC1 in the two groups was rich in meaningful tumor hallmarks (h.all.v7.5.symbols.gmt (Hallmarks)). The p-value < 0.05 and FDR < 0.25 were considered significantly enriched.

Immunohistochemistry

Immunohistochemical (IHC) was performed as previously described. We collected 20 pairs of HCC samples and adjacent non-tumor tissues from October 2020 to April 2021 at the Affiliated Hospital of Youjiang Medical University for Nationalities. All procedures were performed according to the Ethics Guidelines for Human Genome/Gene Research and approved by the Ethics Committee of the Affiliated Hospital of Youjiang Medical College for Nationalities (2022090501). Furthermore, we received informed consent from participants in our study. Immunohistochemistry (IHC) was performed on the 20 samples to detect the expression of the DNAJC1 protein.

Cell culture

The human normal liver cell line (LO₂) and three human HCC cell lines (Huh7, MHCC97H and HepG₂) were purchased from the Chinese Academy of Sciences. LO₂, Huh7, MHCC97H and HepG₂ cells were cultured in DMEM (Dulbecco minimum Essential medium) containing 10% FBS (Fetal Bovine Serum). LO₂, Huh7, MHCC97H and HepG2 cells were cultured at 37 °C with 5% CO₂.

RNA extraction and quantitative real-time PCR

Total RNA was extracted from cell lines using RNA isolation kit (Axygen, Suzhou, China). The total RNA was then reverse transcribed to cDNA. qRT-PCR was performed with the SYBR Green kit, and it was performed using a thermocycler (Bio-Rad, Hercules, CA, USA) at 95 °C for 600 s, 95 °C for 10 s,65 °C for 60 s, 97 °C for 1 s and 37 °C for 30 s, for a total of 40 cycles. The GAPDH was used as internal control, 2^−ΔΔct method was used to quantitatively analyze the expression level of DNAJC1.The sequence of primers used in the experiment was as follows: DNAJC1 (forward:5′-TTCTCACAGTGGGTCATTATGC-3′; reverse:5′-ACCGAGTTTTGATACATCCACAC -3′); GAPDH (forward: 5′-GGACCTGACCTGCCGTCTAG-3′; reverse:5′-GTAGCCCAGGATGCCCTTGA-3′).

Western blotting

Total protein was extracted from cells with RIPA lysis buffer solution and protein concentration was determined by the BCA protein determination kit. Proteins were separated by SDS-PAGE and transferred to a polyvinylidene fluoride membrane. The membranes were then sealed with 5% skim milk at room temperature for 1 h, and the membranes combined with the DNAJC1 and GAPDH primary antibody were incubated at 4 °C overnight. The second antibody was incubated for 2 h at room temperature. Protein bands were detected using the ECL chemiluminescence system.

Transfection

Based on DNAJC1 expression in three HCC cell lines, Huh7 and MHCC97H cell lines were selected for transfection. Specific small interfering RNA (siRNA) targeting DNAJC1 and negative control (NC) siRNA were as follows:

siDNAJC1 (forward:5′-GCCAAGCAACUGAAGGAUUTT -3′; reverse:5′-AAUCCUUCAGUUGCUUGGCTT -3′);

Negative Control (forward:5′- UUCUCCGAACGUGUCACGUTT-3′; reverse:5′- ACGUGACACGUUCGGAGAATT-3′).

The vectors and siRNA were transfected into Huh7 and MHCC97H cell lines using Lipofectamine 3000 reagents. The transfection efficiency was evaluated by qRT-PCR and Western blotting, respectively.

Cell counting kit-8 (CCK-8) assay

The transfected cells (5 × 10³/well) were evenly distributed in the 96-well plate with three replicate wells. After transfecting for 24 h, 48 h, 72 h, and 96 h, the 10 µL volume CCK-8 reagent (Dojindo Laboratories, Kumamoto, Japan) was added to each well. The 96-well plate was cultured in an incubator for 1 h, and then its absorbance value at 450 nm was detected in a microplate reader.

Colony formation assay

1. 0 × 10³ transfected cells were distributed into 6-well plates. After 14 days of incubation, 1. 0 × 10³ treated cells were grown into visible colonies. Finally, methanol was used to fix cell colonies and crystal violet was used to stain cell colonies.

Wound healing assay

Cells were seeded, and scratch wounds were made when cell confluence reached 80%–90% after 48 h transfection. The 10 µL pipette tip was used to draw a straight line perpendicular to the bottom of the 6-well plates. The scratches were cleaned 3 times with PBS buffer. The scratches were calculated after 0 and 24 h incubation at room temperature. Finally, the remaining cells were cultured in a medium free of fetal bovine serum. The pictures were taken under the microscope at 0 and 24 h.

Transwell migration and invasion assays

For the transwell migration assay, transfected cells were diluted to 1 × 10⁵ /mL with serum-free RPMI-DMEM medium, and 200 μL cell suspension was added to the upper transwell chamber and 600 μL medium containing 20% fetal bovine serum was added to the lower chamber, respectively. For invasion assays, the matrigel was diluted to 1:9 with serum-free DMEM.  The transwell chambers were then solidified at 37 °C for 2 h. Then, the transwell invasion experiment was carried out on the basis of the transwell migration experiment. Cells were seeded into a matrigel-coated transwell chamber. After 24 h, cells crossed the inserts were stained with crystal violet and counted under phase contrast microscopy.

Hoechst 33342 staining 

Apoptosis of Huh7 and MHCC97H cells was also evaluated using Hoechst 33342 reagents (Beyotime Biotechnology, Shanghai, China) according to the manufacturer’s protocols. The transfected cells were seeded in a 6 well plate and stained with 1 mL Hoechst 33342 dye by incubation in the culture solution for 30 min, and then washed twice with PBS, observed with a fluorescence microscope.

Flow cytometry

Transfected cells were harvested after transfection 48 h and stained with Annexin V-FITC/PI (BD Biosciences) according to the manufacturer’s instructions. Cells were resuspended in 500 µL of experimental buffer, 5 µL of Annexin V-FITC and 5 µL of 100 × PI. Cells were incubated at room temperature for 30min in the dark. Finally, the cells were analyzed with flow cytometry.

Statistical analysis

SPSS 25.0 (SPSS Inc., Chicago, IL, USA) and R 4.04 software (R Core Team, 2021) were used for statistical analysis. The two-tailed Student’s t-test was used for the comparison of samples between two groups, and one-way ANOVA was used for the comparison of samples from more than two groups. All results were presented as mean ± standard deviation (SD). P < 0.05 was considered statistically significant. *P < 0.05, **P < 0.01, ***P < 0.001.

Expression levels of DNAJC1 in HCC

According to TCGA pan-cancer RNA-seq data, DNAJC1 mRNA expression levels in tumor and normal tissues were analyzed and the results were shown in Fig. 1A. The level of DNAJC1 mRNA expression in breast carcinoma (BRCA), cholangiocarcinoma (CHOL), head and neck squamous cell carcinoma (HNSC), clear cell carcinoma of the kidney (KIRC), papillary cell carcinoma of the kidney (KIRP), liver cancer (LIHC), gastric adenocarcinoma (STAD) and other tumor tissues was significantly higher than in normal tissues (P < 0.001). These results suggested that DNAJC1 was up-regulated in most malignant tumors (including HCC) and may be involved in the occurrence and development of cancer.

In particular, based on TCGA database, we observed that the expression of DNAJC1 in HCC tissues was significantly higher than in normal liver tissues (P < 0.001; Fig. 1B). This result suggested that DNAJC1 may play an important role in HCC.

Prognostic role of DNAJC1 in HCC

Based on the UALCAN database, the survival curves revealed that HCC patients with high expression of DNAJC1 had higher mortality (P < 0.05; Fig. 1C). This result indicated that DNAJC1 was a good prognostic biomarker for HCC patients.

Validation of DNAJC1 expression levels

qRT-PCR and Western blotting confirmed that DNAJC1 mRNA and protein expression were up-regulated in liver cancer cell lines compared to normal liver cells (P < 0.05) (Figs. 2A–2C). Furthermore, DNAJC1 expression was higher in HCC tissues than in adjacent non-tumor tissues by IHC (Fig. 2D). These results suggested that DNAJC1 up-regulation may be closely related to the biological characteristics of HCC.

Expression of DNAJC1 mRNA and protein after siRNA transfection

We transfected DNAJC1 siRNA into Huh7 and MHCC97H cells, and the transfection efficiency was evaluated by qRT-PCR and Western blotting, respectively. The results showed that the mRNA and protein levels of the siDNAJC1 group were significantly lower than those of the NC group (P < 0.05) (Figs. 3A–3B).

Knockdown of DNAJC1 reduces the proliferation of HCC cells

The CCK-8 assay showed that the Huh7 and MHCC97H cell inhibition rate of the siDNAJC1 group increased at 24 h, 48 h, 72 h and 96 h compared to the NC group (P < 0.05) (Fig. 4A). Colony formation assay showed that the number of Huh7 and MHCC97H cell clones in the siDNAJC1 group were significantly reduced compared with the NC group (P < 0.05) (Fig. 4B). These results showed that DNAJC1 knockdown could inhibit HCC cell proliferation.

DNAJC1 knockdown promotes HCC cell apoptosis

Hoechst 33342 staining showed that apoptotic cells in the siDNAJC1 group increased significantly and apoptotic cells showed obvious pyknotic deformation and luminous phenomena (Fig. 5A). In addition, flow cytometry showed that the apoptosis rate of NC group was significantly lower than that of siDNAJC1 group (P < 0.05) (Fig. 5B). These results showed that DNAJC1 knockdown could promote HCC cell apoptosis.

DNAJC1 may mediate HCC cell apoptosis via p53 signaling pathway

In the experiments mentioned above, we confirmed that DNAJC1 can affect the biological behavior of HCC cells, but the underlying mechanism remained unclear. The GSEA showed that the high expression of DNAJC1 was related to the p53 signaling pathway (Fig. 6A). To investigate the mechanism of HCC cell apoptosis induced by DNAJC1 knockdown, PARP, Bax, Bcl-2, p21, p53 and p-p53 (Ser20) proteins were evaluated. These proteins were essential for HCC cell apoptosis and were important for the p53 signaling pathway. The results showed that compared to the control group, the expression levels of p21, p53, p-p53 (Ser20) and Bax increased significantly, and the expression levels of Bcl-2 and PARP decreased significantly after DNAJC1 knockdown (P < 0.05) (Figs. 6B–6C). The results indicated that DNAJC1 may mediate HCC cell apoptosis via p53 signaling pathway.

Knockdown of DNAJC1 reduces the migration and invasion of HCC cells

The results of the wound healing experiment showed that the scratch width of the Huh7 and MHCC97H cells was slightly changed in the siDNAJC1 group, while the scratch width of the Huh7 and MHCC97H cells was significantly reduced in the NC group (P < 0.05) (Fig. 7). Transwell migration assay showed that the knockdown of DNAJC1 expression reduced the migration ability of Huh7 and MHCC97H cells (P < 0.05) (Figs. 8A–8B). Furthermore, the transwell invasion assay showed that DNAJC1 expression suppression inhibited the invasion ability of Huh7 and MHCC97H cells (P < 0.05) (Figs. 8C–8D). These results showed that knockdown of DNAJC1 could inhibit the migration and invasion of HCC cells.

DNAJC1 may mediate HCC cell migration through the EMT signaling pathway

To investigate the mechanism of HCC cell migration induced by DNAJC1 knockdown, E-cadherin, N-cadherin, MMP9, Vimentin, and Snai1 proteins were evaluated. These proteins were essential for the migration of HCC cells, and they were important for EMT signaling pathway. The results showed that compared to the control group, the expression level of E-cadherin increased significantly, and the expression levels of N-cadherin, MMP9, Vimentin, and Snai1 decreased significantly after DNAJC1 knockdown (P < 0.05) (Figs. 9A–9B). The results indicated that DNAJC1 may mediate HCC cell migration via EMT signaling pathway.