TLX3 repressed SNAI1-induced epithelial-mesenchymal transition by directly constraining STAT3 phosphorylation and functionally sensitized 5-FU chemotherapy in hepatocellular carcinoma

Abstract

TLX3 has an established role as a sequence-specific transcription factor with vital functions in the nervous system. Although several studies have shown that TLX3 is aberrantly up-regulated in leukemia, its expression and function in hepatocellular carcinoma (HCC) remain unknown. We found that TLX3 expression was decreased in 68/100 (68%) HCC cases and negatively correlated with the expression of p-STAT3, SNAI1, and Vimentin, while it was positively associated with E-cadherin expression. ITRAQ proteomic profiling revealed significantly less TLX3 expression in primary HCC tumors than in portal vein tumor thrombi. Comparison of Kaplan-Meier curves showed that down-regulation of TLX3 in HCC was associated with poor post-surgical survival. TLX3 over-expression inhibited HCC cell viability, proliferation, migration, invasion and enhanced 5-FU treatment, whereas silencing TLX3 produced the opposite results. Further experiments showed that TLX3 attenuated the EMT phenotype. In vivo experiments showed that knockdown of TLX3 promoted the growth of HCC xenografts and attenuated the anti-tumor effects of 5-FU treatment. Gene expression microarray analysis revealed that TLX3 inhibited IL-6/STAT3 signaling. In additional mechanistic studies TLX3 reversed the EMT phenotype of HCC cells by binding to STAT3, inhibiting STAT3 phosphorylation, and down-regulating SNAI1 expression. Taken together, loss of expression of TLX3 induces EMT by enhancing IL-6/STAT3/SNAI1 signaling, and accelerates HCC progression while also attenuated the effect of 5-FU on HCCs.

Keywords: EMT; HCC; SNAI1; STAT3; TLX3.

Figure 1

Aberrant decreased expression of TLX3 was found in HCC tissues and predicted poor post-surgical prognosis of HCCs. (A) IHC staining showed that there was more TLX3 expression in adjacent liver tissues (a) compared to HCC tissues (b), which was also confirmed by Mann-Whitney U test. Western immunoblotting also displayed that TLX3 expression was decreased in tumor tissues from 4 HCC patients compared to matched adjacent liver tissues; (B) Decreased expression of TLX3 in tumor tissues predicted more rapid tumor recurrence and shorter survival time of HCC patients after surgical resection; (C) IHC staining assay showed that there was more expression of p-STAT3, SNAI1 and N-cadherin, and less E-cadherin expression in HCC tissues in contrast to adjacent liver tissues; (D) ITRAQ quantitative proteomic profiling revealed that there was less TLX3 protein (accession No.: Q96AD3) expression in primary HCC lesion than PVTT.

Figure 2

TLX3 overexpression repressed IL-6/STAT3 pathway and attenuated cell migration, invasion, viability, proliferation and colony formation abilities of MHCC97h cells. (A) After analyzing data of gene expression microarray by PathArray^TM system, enhanced expression of TLX3 was found to inhibit IL-6/STAT3 pathway in MHCC97h cells significantly; (B) Scratch wound healing assay showed that migration capacity of MHCC97h cells was restrained by TLX3 overexpression apparently; (C) Transwell chamber coated with Matrigel assay confirmed that overexpression of TLX3 inhibited invasion capacity of MHCC97h cells clearly; (D) Cell apoptosis of MHCC97h cells was strengthened by TLX3 overexpression notably, which was found by flow cytometry assay; (E) MTT assay showed that cell viability of MHCC97h cells was decreased remarkably by TLX3 over-expression; (F)ELISA assay revealed that there was more BrdU incorporation in MHCC97h Vector cells than MHCC97h TLX3 cells; (G) Soft agar colony formation assay demonstrated that colony formation of MHCC97h cells was repressed by enhanced expression of TLX3 magnificently.

Figure 3

TLX3 over-expression reversed EMT of MHCC97h cells and inhibited growth of HCC xenografts. (A) As assessed by Western immunoblotting, it was found that enforced expression of TLX3 increased E-cadherin expression and decreased expression of SNAI1, N-cadherin, and Vimentin in MHCC97h cells, while TLX3 over-expression weaken STAT3 phosphorylation distinctly; (B) Double-label immunofluorescent staining assay showed that there was more E-cadherin (green) expression and less Vimentin (red) expression in MHCC97h TLX3 cells than MHCC97h Vector cells; (C) TLX3 over-expression repressed the growth of HCC xenografts apparently; (D) HCC xenografts was examined by Western immunoblotting and it was found that xenografts driven from MHCC97h TLX3 cells had more E-cadherin expression and less expression of Vimentin, SNAI1 and p-STAT3, which suggested that TLX3 over-expression repressed phosphorylation of STAT3 and EMT in vivo.

Figure 4

Silencing TLX3 increased STAT3 phosphorylation and induced EMT in HCC cells, while abating cell apoptosis and up-regulating cell viability, proliferation, migration and invasion. (A) Western immunoblotting assay displayed that knockdown of TLX3 increased expression of N-cadherin, Vimentin, SNAI1 and p-STAT3 whereas decreased E-cadherin expression significantly in both Huh7 and Hep3B cells; (B) Annexin V/PI flow cytometry assay showed that down-regulation of TLX3 attenuated cell apoptosis in both Huh7 and Hep3B cells; (C) Cell viability of both Huh7 and Hep3B cells was found by MTT assay to be accentuated by knockdown of TLX3; (D) BrdU ELISA assay showed that TLX3 repression resulted in up-regulation of cell proliferation in both Huh7 and Hep3B cells; (E) As assessed by Scratching wound healing assay, it was found that cell migration of both Huh7 and Hep3B cells was increased by knockdown of TLX3; (F) Invasion capacity of Huh7 cells was found reinforced by silencing TLX3 by Transwell assay. The similar results was obtain in Hep3B cells.

Figure 5

TLX3 sensitized HCC cells to 5-FU. (A) MTT assay showed that 20μm was the lowest concentration at which 5-FU treatment caused the significantly repression of MHCC97h cell viability; TLX3 over-expression was found to strengthen the pro-apoptotic function of 5-FU treatment on MHCC97h cell by both annexin V/PI flow cytometry assay(B) and Caspase 3/7 activity assay (C); Consistently, TLX3 over-expression was confirmed to enhance the inhibitory effect of 5-FU treatment on MHCC97h cell viability by MTT assay (D); Orthotopic HCC models established by portal vein implantation of MHCC97h showed that TLX3 over-expression enhanced the anti-HCC activity of 5-FU treatment dramatically in vivo.

Figure 6

TLX3 blocked IL-6-induced phosphorylation of STAT3 through binding with STAT3 protein directly and then restrained SNAI1 expression. (A) over-expression of TLX3 weakened phosphorylation of STAT3 induced by IL-6 treatment, while silencing TLX3 strengthened the positive regulatory effect of IL-6 treatment on STAT3 phosphorylation notably; (B) CO-IP assay confirmed that TLX3 protein was bound with STAT3 protein directly in both MHCC97h and Huh7 cells; (C) After searching UCSC Genome Browser, 11 potential p-STAT3 DNA binding sites was found in the promoter of SNAI1; (D) ChIP assay verified that p-STAT3 was bound directly with the -1301~-846 bp of SNAI1 promoter fragment.