The novel miR-1269b-regulated protein SVEP1 induces hepatocellular carcinoma proliferation and metastasis likely through the PI3K/Akt pathway

Abstract

Decreased intercellular adhesion is a key step in the metastasis and recurrence of many cancers, including hepatocellular carcinoma (HCC). SVEP1 is an important cell adhesion molecule that plays a key role in regulating intercellular adhesion and embryonic lymphatic development. However, the expression patterns and roles of SVEP1 in HCC are still largely unknown. We identified SVEP1 expression by analyzing 220 HCC samples from our cancer center. TCGA and GEO online-databases were used for data calibration and validation. SVEP1 was differentially expressed in two groups of HCCs with different risks of recurrence and was deemed as an independent risk factor for the prognosis of HCC. The expression of SVEP1 is negatively related to the proliferation and metastasis of HCC. Downregulation of SVEP1 expression promoted in vitro HCC cell migration, chemotaxis, invasion and proliferation, as well as in vivo tumor growth, local invasion and metastasis in a mouse model. Bioinformatic analysis and RT-PCR results showed that miR-1269b expression is negatively correlated with the SVEP1 expression and the prognosis of HCC patients. Further experiments showed that miR-1269b directly targets and downregulates the expression of SVEP1, which further induces the phosphorylation of Akt at thr308. These regulatory effects ultimately mediate the proliferation and metastasis of HCC cells. SVEP1 could serve as a promising prognostic marker of HCC. MiR-1269b downregulates SVEP1 expression and promotes HCC proliferation and metastasis likely through the PI3k/Akt signaling pathway.

Fig. 1. SVEP1 expression pattern and its correlation with prognosis in human HCC.

a. Full transcriptome sequencing of two groups of HCC patients with different risk of recurrence; revealed the signaling pathways in which DEGs are enriched. b Heatmap of the top 20 DEGs in these two groups. c IHC and HE staining of SVEP1 in HCC and para-tumor tissues (the images shown are representative, the IHC scores of para-tumor, SVEP1 high expression tumor and SVEP1 low expression tumor are 6, 3, and 1, respectively). d. WB showing the differences in SVEP1 protein levels between four pairs of HCC tissues and para-tumor tissues. e, f. Kaplan–Meier survival curve analysis of the correlation between SVEP1 IHC scores and HCC patient OS and DFS (n = 207).

Fig. 2. The relationship between SVEP1 expression and clinicopathological features of HCC.

a GSEA validating the correlation between SVEP1 expression and the downregulated expression of genes in HCC tissues. b, c. GSE45436 and TCGA validation of the expression of SVEP1 in HCC and para-tumor tissues. d, e Kaplan–Meier survival curve of TCGA data to validate the correlation between the expression of SVEP1 and HCC patient OS and DFSs. f, g. Correlation analysis between the SVEP1 IHC score and clinicopathological parameters (tumor size and satellite nodules). h, i. Kaplan–Meier survival curve showing the correlation between the SVEP1 IHC score and the OS of a subgroup of HCC patients tumor size ≥3 cm and satellite nodule positive patients. j, k Kaplan–Meier survival curve showing the correlation between the SVEP1 IHC score and DFS of a subgroup of HCC patients (tumor size ≥3 cm and satellite nodules positive patients).

Fig. 3. Knock down of SVEP1 promotes HCC cell migration, invasion, and proliferation in vitro.

a WB showing the expression of SVEP1 at the protein level in 7 HCC cell lines (Hep3B, PLC, MHCC97L, MHCC97H, and MHCCLM3 are hepatitis B positive, Huh7 and HLE are hepatitis B negative). b The construction of the SVEP1 control group and SVEP1 knockdown Hep3B HCC cell line. c GSEA of the relationship between the expression of SVEP1 and tumor metastasis. d High-throughput microscopy comparing the migration ability of the SCR and SVEP1/KD groups. e Comparison of the chemotaxis potential the SVEP1/KD and SCR groups. f The invasive ability of the SVEP1/KD and SCR groups in the invasion assay. g Wound-healing assay comparing the translocation distance between the SVEP1/KD and SCR groups. h GSEA of the correlation between the expression level of SVEP1 and tumor proliferation. i CCK-8 assay showing the proliferation ability of the SVEP1/KD and SCR groups.

Fig. 4. SVEP1 is a direct target of miR-1269b.

a Differentially expressed miRNAs in the high-throughput RNA sequencing. b The expression of miR-1269b in HCC tissues and para-tumor tissues in the TCGA database. c, d The correlation between the expression of miR-1269b and HCC patient OS/DFS in the TCGA miRNA database. e The correlation between the mRNA level of miR-1269b and SVEP1 in HCC patients by RT-PCR. f The sequence of the 3′UTR of SVEP1. g Luciferase reporter assays of SVEP1 and miR-1269b. h, i Establishment of different miR-1269b expression levels in Hep3B and MHCCLM3. j, k The verification of the expression level of the target gene (SVEP1) by WB in Hep3B and MHCCLM3. l, m Different migration abilities associated with different expression levels of miR-1269b in Hep3B and MHCCLM3 cells.

Fig. 5. Knockdown of SVEP1 promotes proliferation and metastasis through the PI3K/Akt signaling pathway in HCC.

a Signal pathway enrichment analysis of SCR and SVEP1/KD groups by RNA-seq in Hep3B cells. b Heatmap of 15 DEGs in the microarray. c The differential expression of the 15 DEGs in the SCR and SVEP1/KD groups by RT-PCR. d, e The effect of the downregulation of SVEP1 expression on PI3K/Akt signaling pathway activation was verified by WB in Hep3B and MHCCLM3 cells. f, g The effect of SVEP1 expression downregulation on proliferation in Hep3B and MHCCLM3 cells assessed by CCK-8 assay. h, i The effect of SVEP1 expression downregulation on chemotaxis in Hep3B and MHCCLM3 cells.

Fig. 6. Decreased SVEP1 expression promotes HCC cell proliferation and metastasis in vivo.

a Images of tumors from NOD-SCID nude mice in the SCR and SVEP1/KD groups. b, c The tumor volume and weight of the two groups. d HE and representative IHC staining of SVEP1, Akt^Thr308 and PKCζ in tumor sections derived from SCR and SVEP1/KD HCC cell-derived models (the images shown are representative). e Statistical analyses of the bone invasion and multiple in situ metastases of the two groups. f, g Representative images and statistical analysis of the lung metastatic nodules in the two groups.

Fig. 7. The proposed mechanistic scheme of SVEP1 induces proliferation and metastasis in HCC.

SVEP1 is one of the most important cell adhesion molecules as well as extracellular matrix proteins that participates in cell-cell adhesion. There are several subclones with different characteristics in HCC. Some of them are highly expressing miR-1269b which is the key cause of SVEP1 degradation. Notably, decreased expression of SVEP1 in HCC further leads to the activation of PI3K/Akt signaling pathway, which may be the potential mechanism for the recurrence and metastasis of HCC.