CMTM7 is down-regulated in HCC tissues
We previously measured CMTM7 expression in 75 paired HCC and adjacent non-tumor tissues by immunohistochemistry and found that the positive expression of CMTM7 was significantly lower in HCC tissues than that of CMTM7 in adjacent non-tumor tissues [8]. We also conducted bioinformatics analysis to check CMTM7 expression in HCC tissues from GEO and TCGA databases. As shown in Figure 1A, we found a lower expression of CMTM7 in HCC tissues than normal liver tissues in GSE3500 (P<0.001), but CMTM7 expression had no difference between normal liver tissues and HCC tissues in TCGA database.
To further confirm the expression of CMTM7 in HCC tissues, we chose another 20 paired HCC and adjacent non-tumor tissues to perform Western blot. As Figure 1B shown, down-regulated CMTM7 was determined in 80% (16/20) of HCC tissues. We also detected significantly decreased mRNA expression of CMTM7 in 75.7% (53/70) of HCC tissues by qRT-PCR (Figure 1C). In consistent with the previous studies, we detect a down-regulated expression of CMTM7 in HCC tissues.
CMTM7 expression is correlated with the clinical characteristics of HCC patients
We first explored the relationship between CMTM7 expression and the clinical characteristics of HCC patients from online databases. It was found that the expression of CMTM7 was highly expressed in cirrhosis liver tissues than normal liver tissues in GSE6764 (P<0.001, Figure 2A). However, we found no difference between the copy number of CMTM7 in HCC tissues and normal liver tissues in GSE32649 (Figure 2B). The relationship between CMTM7 expression and the prognosis of HCC patients was also analyzed from the TCGA database by Kaplan-Meier method. As Figure 2C and 2D shown, the overall survival time of HCC patients in CMTM7 low expression group was significant shorter than that in CMTM7 high expression group both in five-year and ten-year of survival analysis (P<0.05).
We also collected clinical information from the 70 HCC patients for qRT-PCR detection to analyze the correlation between CMTM7 expression and the clinical characteristics of HCC patients. The 70 HCC patients were divided into CMTM7 high and low expression groups according to the qRT-PCR results (LgT/N>0 means high expression and LgT/N<0 means low expression). The result showed that CMTM7 expression was significantly correlated with HCC metastasis (P<0.05), while had no relationship with other clinicopathological features of HCC patients (Table 1). These results suggest that CMTM7 expression had a relationship with HCC metastasis and prognosis.
Over-expression of CMTM7 inhibits HCC cell proliferation
In order to explore the biological functions of CMTM7 in HCC cells, we detected CMTM7 expression in seven cell lines, SMMC-7721, HCC-97H, HCC-LM3, SK-Hep-1, Bel-7402, Huh-7 and HepG2 by Western blot. Compared with the hepatocytes L02, the expression of CMTM7 was decreased significantly in SK-Hep-1, Bel-7402, Huh-7, and HepG2 cells (Figure 3A). There were only 20% of CMTM7 expression in SK-Hep-1 and 10% in Bel-7402 cells as compared with L02 cells. SK-Hep-1 and Bel-7402 cell lines were then selected for subsequent cell function test.
To next study the role of CMTM7 in cell proliferation, CMTM7 was over-expressed in SK-Hep-1 and Bel-7402 cell lines by exogenous introduction with a lentiviral wild-type CMTM7 plasmid (named as SK-Hep-1-CMTM7 and Bel-7402-CMTM7 cells). As shown in Figure 3B, CMTM7 was increased by 1.5 fold in Bel-7402 cells and 1 fold in SK-Hep-1 cells, compared with the negative control group (named as SK-Hep-1-Vector and Bel-7402-Vector cells). After CMTM7 was over-expressed in SK-Hep-1 and Bel-7402 cell lines, we detected the proliferation ability of these cells by conducting CCK8 and colony formation assay. We found that the proliferation ability of SK-Hep-1-CMTM7 and Bel-7402-CMTM7 cells was decreased significantly than SK-Hep-1-Vector and Bel-7402-Vector cells, respectively (Figure 3C and 3D, P<0.05). In addition, the colony formation test showed the result as same as the CCK8 assay (Figure 3E, P<0.01). These results suggest that over-expressed CMTM7 inhibits the proliferation of HCC cells.
The function of CMTM7 in HCC cell metastasis
To find out whether CMTM7 plays a role in HCC cell metastasis, we performed a wound healing assay to measure the metastatic ability of SK-Hep-1-CMTM7 and Bel-7402-CMTM7 cells (Figure 4A and 4B). As compared with SK-Hep-1-Vector and Bel-7402-Vector cells, the cell migration distance was wider in SK-Hep-1-CMTM7 and Bel-7402-CMTM7 cells after 48 h of wound (P<0.01).
Cell invasion and migration were the major features of most malignant tumors. Transwell assay was performed to detect the invasion and migration ability of HCC cells after exogenous expression of CMTM7. As shown in Figure 4C and 4D, CMTM7 over-expression caused an obvious reduced invasion and migration of Bel-7402 cells and SK-Hep-1 cells (P<0.01). These results show the exogenous expression of CMTM7 disturbs the metastasis of HCC cells, further support that CMTM7 expression is correlated with tumor metastasis of HCC patients.
Over-expression of CMTM7 inhibits HCC metastasis via up-regulating CMTM3
Epithelial mesenchymal transition (EMT) is a critical process in HCC metastasis [15]. To determine the role of CMTM7 in HCC metastasis, the expression of EMT factors were measured in CMTM7 over-expressed SK-Hep-1 cells. As compared to SK-Hep-1-Vector cells, the epithelial markers E-cadherin and β-catenin in SK-Hep-1-CMTM7 cells were induced by 100%±32% and 281%±72%, while the mesenchymal markers N-cadherin and Vimentin in SK-Hep-1-CMTM7 cells were redued by 47%±11% and 40%±12%, respectively (Figure 5A). These results show an inhibitory role of over-expressed CMTM7 in the EMT process.
To further explore the mechanism of CMTM7 in HCC metastasis, we next searched CMTM7 co-expressed genes in TCGA liver cancer data. As shown in Figure 5B, of the topmost 10 positively and 10 negatively CMTM7-correlated genes, a CMTM family member, CMTM3 attracted our interest. We detected the expression of CMTM3 in CMTM7 over-expressed SK-Hep-1 cells and found that CMTM3 was increased by 35%±15% than SK-Hep-1-Vector cells (Figure 5A). Moreover, in the same 30 paired HCC and adjacent non-tumor tissues as CMTM7, the mRNA expression of CMTM3 was down-regulated in 73.3% (22/30) of HCC tissues by qRT-PCR (Figure 5C). In addition, CMTM3 also had a relationship with metastasis of HCC patients (P<0.05, Table 2). In consistent with the results of HCC cells, we found a significantly positive correlation between CMTM7 and CMTM3 in HCC tissues (r=0.489, P<0.01, Table 3). These results show that down-regulated expression of CMTM7 might promote HCC metastasis via its family member CMTM3.