TSPAN9 inhibits malignant progression of hepatocellular carcinoma

Abstract

Background: Tetraspanins (TSPANs) are a critical family for cell migration, which have been implicated in a variety of activities including cancer. Previous study showed that tetraspanin 9 (TSPAN9) plays an important role in gastric cancer. In this study, we aim to explore the biological functions of TSPAN9 in hepatocellular carcinoma (HCC).

Methods: The Cancer Genome Atlas (TCGA), Genotype-Tissue Expression (GTEx) and Gene Expression Profiling Interactive Analysis (GEPIA) databases were used to analyze TSPAN9 expression, prognostic mutations, somatic copy number alterations (sCNAs), and tumor immune characteristics in 33 tumors. Immunohistochemistry images of TSPAN9 in HCC tissues were obtained from the Human Protein Atlas (HPA) database. Survival curves were generated using the Kaplan-Meier Plotter database. The proliferation, migration, and invasion abilities of HCC cells were assessed using Cell Counting Kit-8 (CCK-8), wound healing, and Transwell assays.

Results: TSPAN9 was deregulated in various tumor types, and its expression was lower in HCC tissues than that of normal liver tissues (P<0.05). Moreover, TSPAN9 expression had a correlation with the prognosis of tumor patients, and HCC patients with low TSPAN9 expression showed a poor prognosis (Log-rank P<0.05). In addition, we detected that TSPAN9 was significantly decreased in HCC cells (P<0.01). As compared to the control cells, overexpression of TSPAN9 in HCC cells significantly reduced cell proliferation, slowed the wound healing rate, and inhibited the invasive and migration ability (all P<0.05). TCGA database analysis revealed a relationship between the expression of TSPAN9 and epithelium-mesenchymal transformation (EMT) factors.

Conclusions: Downregulated expression of TSPAN9 indicates a poor prognosis of HCC patients. TSPAN9 can inhibit the proliferation and metastasis of HCC cells.

Keywords: Hepatocellular carcinoma (HCC); metastasis; prognosis; proliferation; tetraspanin 9 (TSPAN9).

Figure 1

The expression level and prognostic value of TSPAN9 in 33 tumors. (A) The expression of TSPAN9 in pan-cancer was analyzed according to TCGA and GTEx database. (B) Relationship between TSPAN9 expression and tumor prognosis. (C) Prognostic value of TSPAN9 in pan-cancer based on TCGA database. ns, not significant; *, P<0.05; **, P<0.01; ***, P<0.001. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; CHOL, cholangiocarcinoma; CI, confidence interval; CESC, cervical squamous cell carcinoma; COAD, colon adenocarcinoma; DFI, disease-free interval; DLBC, lymphoid neoplasm diffuse large B cell lymphoma; DSS, disease-specific survival; ESCA, esophageal carcinoma; GBM, glioblastoma; GTEx, Genotype-Tissue Expression; HNSC, head and neck squamous cell carcinoma; HR, hazard ratio; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; NS, not significant; OV, ovarian serous cystadenocarcinoma; OS, overall survival; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PFI, progression-free interval; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SKCM, skin cutaneous melanoma; STAD, stomach adenocarcinoma; TCGA, The Cancer Genome Atlas; TGCT, testicular germ cell tumor; THCA, thyroid carcinoma; THYM, thymoma; TSPAN9, tetraspanin 9; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma.

Figure 2

Association analysis of TSPAN9 expression with tumor mutations, somatic copy number changes, and immunity in pan-cancer. (A) Percentage of TSPAN9 mutation samples. (B) Somatic copy number changes in the tumor immune assessment resource database. (C) Correlation analysis between immune checkpoint genes and TSPAN9 expression. ACC, adrenocortical carcinoma; BLCA, bladder urothelial carcinoma; BRCA, breast invasive carcinoma; BTLA, B and T lymphocyte associated; CESC, cervical squamous cell carcinoma; CHOL, cholangiocarcinoma; COAD, colon adenocarcinoma; CTLA-4, cytotoxic T lymphocyte-associated protein 4; DLBC, lymphoid neoplasm diffuse large B cell lymphoma; ESCA, esophageal carcinoma; GBM, glioblastoma; HAVCR2, hepatitis A virus cellular receptor 2; HNSC, head and neck squamous cell carcinoma; KICH, kidney chromophobe; KIRC, kidney renal clear cell carcinoma; KIRP, kidney renal papillary cell carcinoma; LAG3, lymphocyte activating 3; LAML, acute myeloid leukemia; LGG, brain lower grade glioma; LILRB2, leukocyte immunoglobulin like receptor B2; LILRB4, leukocyte immunoglobulin like receptor B4; LIHC, liver hepatocellular carcinoma; LUAD, lung adenocarcinoma; LUSC, lung squamous cell carcinoma; MESO, mesothelioma; OV, ovarian serous cystadenocarcinoma; PAAD, pancreatic adenocarcinoma; PCPG, pheochromocytoma and paraganglioma; PDCD1, programmed cell death 1; PRAD, prostate adenocarcinoma; READ, rectum adenocarcinoma; SARC, sarcoma; SIGLEC7, sialic acid binding Ig like lectin 7; SIRPA, signal regulatory protein alpha; SKCM, skin cutaneous melanoma; sCNA, somatic copy number alteration; STAD, stomach adenocarcinoma; TGCT, testicular germ cell tumor; THCA, thyroid carcinoma; THYM, thymoma; TIGIT, T cell immunoreceptor with Ig and ITIM domains; TSPAN9, tetraspanin 9; UCEC, uterine corpus endometrial carcinoma; UCS, uterine carcinosarcoma; UVM, uveal melanoma; VSIR, V-set immunoregulatory receptor.

Figure 3

Expression and prognostic value of TSPAN9 in hepatocellular carcinoma. (A) Analysis of TSPAN9 gene expression difference between HCC tissue and normal liver tissue. (B) The relationship between TSPAN9 expression and overall survival of HCC patients. (C) Expression of TSPAN9 protein in normal liver tissue (https://www.proteinatlas.org/ENSG00000011105-TSPAN9/tissue/liver#img) and HCC tissue (https://www.proteinatlas.org/ENSG00000011105-TSPAN9/cancer/liver+cancer#img) by immunohistochemical method in the HPA database (photographs downloaded when the scale bar at 200 µm). *, P<0.05. CI, confidence interval; HCC, hepatocellular carcinoma; HPA, Human Protein Atlas; HR, hazard ratio; LIHC, liver hepatocellular carcinoma; N, normal; NOS, not otherwise specified; T, tumor; TSPAN9, tetraspanin 9.

Figure 4

Correlation analysis of TSPAN9 expression with immune subtypes and immune cells. (A) Expression analysis of TSPAN9 in different immune subtypes of hepatocellular carcinoma. (B) The correlation between six common immune cells and TSPAN9 expression in hepatocellular carcinoma. (C) GEPIA database was used to analyze the correlation between TSPAN9 expression and common immune checkpoints. CPM, counts per million; GEPIA, Gene Expression Profiling Interactive Analysis; IFN, interferon; LIHC, liver hepatocellular carcinoma; PD-L1, programmed cell death ligand 1; TGF, transforming growth factor; TPM, transcripts per million; TSPAN9, tetraspanin 9.

Figure 5

Effects of upregulation of TSPAN9 on proliferation, invasion and migration of HCC cells. (A) qRT-PCR was used to detect the expression of TSPAN9 in human HCC cells. (B) The overexpression efficiency of TSPAN9 in HCC cells detected by immunofluorescence microscopy. (C) The effects of upregulation of TSPAN9 on the proliferation of HCC cells were detected by CCK-8. (D) Wound healing test showed that cell migration ability decreased after upregulation of TSPAN9. (E,F) Transwell assay showed that cell invasion and migration ability decreased after upregulation of TSPAN9 by crystal violet staining. *, P<0.05. CCK-8, Cell Counting Kit-8; HCC, hepatocellular carcinoma; NC, negative control; OD, optical density; OE, overexpressed; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TSPAN9, tetraspanin 9.

Figure 6

Correlation between TSPAN9 expression and EMT factors. (A) qRT-PCR was used to detect the expression of EMT factors after TSPAN9 overexpression in HCC cells. *, P<0.05 as compared with NC group. (B) Relationship between TSPAN9 expression and EMT factors in HCC tissues through GEPIA database. CDH1, E-cadherin; CDH2, N-cadherin; CTNNB1, catenin beta 1; EMT, epithelium-mesenchymal transformation; GEPIA, Gene Expression Profiling Interactive Analysis; HCC, hepatocellular carcinoma; LIHC, liver hepatocellular carcinoma; NC, negative control; OE, overexpressed; qRT-PCR, quantitative reverse transcription polymerase chain reaction; TPM, transcripts per million; TSPAN9, tetraspanin 9; ZEB1, zinc finger E-box binding homeobox 1.