Apolipoprotein M could inhibit growth and metastasis of SMMC7721 cells via vitamin D receptor signaling

Abstract

Objective: Hepatocellular carcinoma (HCC) is one of the most common malignant tumors with high mortality-to-incidence ratios. Apolipoprotein M (ApoM), a member of the apolipoprotein family, is mainly synthesized in the liver, whereas its role in HCC has not been elucidated. Here, we examined the effect of ApoM on the biological behavior of HCC cells and the possible mechanisms. Methods: We used CRISPR/Cas9 technology to knock out ApoM in SMMC7721 cells. Differentially expressed genes before and after ApoM knockout (KO) were analyzed by GeneChip microarrays and confirmed by qRT-PCR. Cell assays of proliferation, apoptosis, migration and invasion were performed in SMMC7721 cells, and the expression of epithelial-mesenchymal transition (EMT) markers was performed by western blot. And we performed functional recovery experiments by overexpressing vitamin D receptor (VDR) in SMMC7721. Results: The ApoM-KO SMMC7721 cell line was successfully constructed using the CRISPR/Cas9 technology. Our results showed that silencing ApoM suppressed apoptosis and promoted proliferation, migration, invasion and EMT of SMMC7721 cells. The microarray data revealed that a total of 1,868 differentially expressed genes were identified, including VDR. The qRT-PCR and western blot verification results demonstrated that knocking out ApoM could significantly reduce the expression of VDR. The functional recovery experiments indicated that VDR overexpression could offset the inhibition of cell apoptosis and the promotion of cell proliferation, migration, invasion and EMT caused by knocking out ApoM in SMMC7721 cells. Conclusion: ApoM could function as a tumor suppressor to inhibit the growth and metastasis of SMMC7721 cells via VDR signaling in HCC.

Keywords: CRISPR/Cas9; GeneChip microarrays; apolipoprotein M; hepatocellular carcinoma cells; vitamin D receptor.

Figure 1

CRISPR/Cas9-mediated ApoM knocking out in SMMC7721 cells. (A) The optical microscopy images of SMMC7721 cells transfected by the GFP-labeled plasmid vector in bright and fluorescence field. Scare bar =100 μm. (B) After limited-dilution culture for 10 days, the optimal plating density for monoclonal formation of SMMC-7721 cells in 96-well plates was recorded with Celigo. (C) Sequencing peak map of sgRNA, mixed clones and monoclone. Red boxes marked the PAM sequence, red arrowhead denoted the Cas9 cutting site, green box marked the inserted base. (D) The amino acid sequence translated from ApoM gene before (NC.seq) and after (ApoM-KO.seq) CRISPR/Cas9 treatment. Yellow shades represent the same amino acid sequence and green shades represent the different one. (E) ApoM protein expression was analyzed using western blot in ApoM-KO and NC group. GAPDH was a loading control. Abbreviations: ApoM, Apolipoprotein M; GFP, green fluorescent protein; sgRNA, single-guide RNA; NC, negative control; KO, knock-out; GAPDH, glyceraldehyde 3‑phosphate dehydrogenase.

Figure 2

Changes of biological behavior after silencing ApoM. (A) Apoptosis rate in ApoM-KO group was lower than the NC group in SMMC7721 cells. Q1 represents necrotic cells, Q2 represents late apoptosis cells, Q3 represents viable cells and Q4 represents early apoptosis cells. Apoptosis is designated Q2+Q4. (B) Caspase3/7 activity of ApoM-KO group was lower than the NC group in SMMC7721 cells. (C) OD 450-fold values of ApoM-KO group were higher than NC group in SMMC7721 cells at day 2, 3, 4 and 5. (D) Cell migration of ApoM-KO and NC group was measured using a wound healing assay. Wound images were captured using a digital camera and an inverted microscope at 0, 24, 48 and 72 hrs after wounding. (E) Cell invasion of ApoM-KO and NC group was measured with transwell assays. Cells that invaded were stained with Giemsa and imaged and counted under a microscope. Scare bar =150 μm. (F) E-cadherin, fibronectin, Twist and Snail protein levels were analyzed using western blot in ApoM-KO and NC groups. GAPDH was a loading control. Data are means ± SEM. Student’s t test was used to analyze significant differences, * P<0.05, ** P<0.01, *** P<0.001 versus NC. Abbreviations: ApoM, Apolipoprotein M; KO, knock-out; NC, negative control; GAPDH, glyceraldehyde 3‑phosphate dehydrogenase.

Figure 3

Differentially expressed genes between ApoM-KO and NC group. (A) Volcano plot, which demonstrated the distribution of the differentially expressed genes between ApoM-KO and NC group. The X-axis represents the logarithm conversion of the fold difference to base 2 and the Y-axis represents the logarithm conversion of the corrected significant levels to base 10. The red color represents all the probes with fold difference >2 and FDR <0.05. (B) Scatter plot, which exhibited the distribution of the signals between ApoM-KO and NC group in the Cartesian coordinate plane. The X-axis represents the NC group and the Y-axis represents ApoM-KO group. The ordinate value and the abscissa of each spot represent the expression values of one probe in ApoM-KO and NC group. The red spots above the green lines represent the relatively upregulated probes in ApoM-KO group. The green spots underneath the green lines represent the relatively upregulated probes in the NC group. (C) VDR mRNA was quantified by qRT-PCR in ApoM-KO and NC group, normalized to GAPDH. Fold change was calculated using 2^−ΔCt. (D) Quantification of VDR protein expression with western blot presented as a ratio relative to expression of the NC group. GAPDH was a loading control. Data are means ± SEM. Student’s t test was used to analyze significant differences, ** P<0.01 versus NC. Abbreviation: ApoM, Apolipoprotein M; KO, knock-out; NC, negative control; FDR, false discovery rate; VDR, vitamin D receptor; GAPDH, glyceraldehyde 3‑phosphate dehydrogenase.

Figure 4

Effect of VDR on the biological function of ApoM-KO SMMC7721 cells. (A) VDR mRNA was quantified by qRT-PCR in VDR-OE and NC group, normalized to GAPDH. (B) OD 450-fold values of VDR-OE group were lower than NC group in ApoM-KO SMMC7721 cells, especially at day 4 and 5. (C) Caspase3/7 activity of VDR-OE group was higher than NC group in ApoM-KO SMMC7721 cells. (D, E) Cell migration and invasion of VDR-OE and NC group were measured with transwell assays. Cells that invaded were stained with Giemsa and imaged and counted under a microscope. Scare bar =150 μm. (F) E-cadherin, fibronectin, Twist and Snail protein levels were analyzed using western blot. GAPDH was a loading control. Data are means ± SEM. Student’s t-test was used to analyze significant differences, * P<0.05, ** P<0.01, *** P<0.001 versus NC. Abbreviations: VDR, vitamin D receptor; ApoM, Apolipoprotein M; KO, knock-out; OE, overexpression; NC, negative control; GAPDH, glyceraldehyde 3‑phosphate dehydrogenase.