TM4SF1 promotes the self-renewal of esophageal cancer stem-like cells and is regulated by miR-141

Lei Xue, Xiying Yu, Xingran Jiang, Xin Deng, Linlin Mao, Liping Guo, Jinhu Fan, Qinqxia Fan, Liuxing Wang, Shih-Hsin Lu, Lei Xue, Xiying Yu, Xingran Jiang, Xin Deng, Linlin Mao, Liping Guo, Jinhu Fan, Qinqxia Fan, Liuxing Wang, Shih-Hsin Lu

Abstract

Cancer stem-like cells have been identified in primary human tumors and cancer cell lines. Previously we found TM4SF1 gene was highly expressed in side population (SP) cells from esophageal squamous cell carcinoma (ESCC) cell lines, but the role and underlying mechanism of TM4SF1 in ESCC remain unclear. In this study, we observed TM4SF1 was up-regulated but miR-141 was down-regulated in SP cells isolated from ESCC cell lines. TM4SF1 could stimulate the self-renewal ability and carcinogenicity of esophageal cancer stem-like cells, and promote cell invasion and migration. In miR-141 overexpression cells, the expression of TM4SF1 was significantly reduced. We also found that overexpression of miR-141 could abolish the self-renewal ability and carcinogenicity of esophageal cancer stem-like cells and decrease cell invasion and migration by suppressing TM4SF1. Consequently, TM4SF1 is a direct target gene of miR-141. The regulation of TM4SF1 by miR-141 may play an important role in controlling self-renewals of esophageal cancer stem-like cells. It may also promote the development of new therapeutic strategies and efficient drugs to target ESCC stem-like cells.

Keywords: ESCC; TM4SF1; cancer stem-like cells; esophageal cancer; miR-141.

Conflict of interest statement

CONFLICT OF INTEREST

The authors disclose no potential conflicts of interest.

Figures

Figure 1. The expression of TM4SF1 and…
Figure 1. The expression of TM4SF1 and miR-141 in esophageal cancer stem-like cells
A. Side population (SP) analysis of KYSE150 and KYSE180 cell lines. Incubation with 5pM FTC abolished the SP cell fraction. B, C. In SP and non-SP cells of KYSE150 and KYSE180 cell lines, TM4SF1 expression level was measured by Western blot and real-time PCR, and D. miR-141 expression level was measured by real-time PCR.
Figure 2. The effect of TM4SF1 expression…
Figure 2. The effect of TM4SF1 expression on esophageal cancer stem-like cells, migration and invasion
A. SP analysis of KYSE150 and KYSE180 cells after transfection or RNA interference. B. The expression of TM4SF1 and metastasis related proteins after transfection or RNA interference was measured by Western blot assay. C. IC50 of KYSE150 and KYSE180 cells after transfection or RNA interference to cisplatin by CCK8 assay (n=3, error bars indicate SD, P<0.05). D. Representative images from migration and invasion assay (n=3, error bars indicate SD, P<0.05).
Figure 3. The effect of TM4SF1 expression…
Figure 3. The effect of TM4SF1 expression on carcinogenicity
A. Representative images from colony formation assay of KYSE150 and KYSE180 cells after transfection or RNA interference (n=3, error bars indicate SD, P<0.05). B. The expression of TM4SF1 after infection of KYSE150 cells with lentivirus was measured by Western blot assay. C-D. Tumor weights of lenti-TM4SF1, lenti-shRNA1, lenti-shRNA2 and control KYSE150, lenti-NC and lenti-shNC cells were plotted (n=8, error bars indicate SD, P<0.05). E. And the expression of TM4SF1 of tumor xengrafts was detected by Western blot assay.
Figure 4. TM4SF1 is a direct target…
Figure 4. TM4SF1 is a direct target of miR-141
A. An miR-141 target site resides at +157 to +163 of the TM4SF1 3′UTR and is highly conserved in different species. B. After infection of cells with indicated lentivirus, miR-141 expression level was measured by real-time PCR and C. the expression of TM4SF1 mesured by Western blot after KYSE150 and KYSE180 cells infection with lentivirus. D. Diagram of luciferase reporter plasmids: plasmid with TM4SF1 3′UTR insert (pIS0-TM4SF1-3′UTR) and plasmid with a mutant TM4SF1 3′UTR (pIS0-TM4SF1-3′UTRmut) that carried a substitution of seven nucleotides within the miR-141 binding site. E. Luciferase acitivity assay demonstrates a direct targeting of the 3′UTR of TM4SF1 by miR-141. Cells were transfected with plasmids pIS0-TM4SF1-3′UTR or pIS0-TM4SF1-3′UTRmut. pRL-SV40 was used for normalization of transfection efficiency (n=3, error bars indicate SD, P<0.05).
Figure 5. The effect of miR-141 overexpression…
Figure 5. The effect of miR-141 overexpression on esophageal cancer stem-like cells
A. The expression of miR-141 in human ESCC was measured by real-time PCR (n=36, *P<0.05). B. SP analysis of KYSE150, KYSE180 and their infection cells. C. IC50 of KYSE150, KYSE180 and their infection cells to cisplatin by CCK8 assay (n=3, error bars indicate SD, P<0.05). D. Representative images from migration and invasion assay (n=3, error bars indicate SD, P<0.05). E. The expression of metastasis and invasion related proteins after infection was measured by Western blot assay. F. Representative images from colony formation assay (n=3, error bars indicate SD, P<0.05). G. Tumor weights of KYSE150 cells and its infection cells (lenti-miR-NC and lenti-miR-141 cells) were plotted. And the expression of TM4SF1 of tumor xengrafts was detected by Western blot assay (n=6, error bars indicate SD, P<0.05).
Figure 6. The effect of rescued TM4SF1…
Figure 6. The effect of rescued TM4SF1 expression on esophageal cancer stem-like cells
A. SP analysis of cells after transfection or RNA interference of KYSE150-Lenti-miR-141 and KYSE180-Lenti-miR-141 cells. B. The expression of TM4SF1 after transfection or RNA interference was measured by Western blot assay. C. Representative images from colony formation assay (n=3, error bars indicate SD, P<0.05).

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