Hematopoietic-substrate-1 associated protein X-1 (HAX-1) regulates liver cancer cells growth, metastasis, and angiogenesis through Akt

Abstract

The aim of this study was to investigate the effects and mechanisms of hematopoietic-substrate-1-associated protein X-1 (HAX-1) on liver cancer cells. Information on HAX-1 from liver cancer patients was analyzed by the Cancer Genome Atlas (TCGA) program. Cell migration and invasion abilities were respectively tested by scratch assay and transwell assay. Tube formation assay was applied to detect angiogenesis protein and mRNA was determined using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot. We found that the median month survival of HAX-1 overexpressing liver cancer patients was shorter than that of HAX-1 normal liver cancer patients. HAX-1 was overexpressed in liver cancer tissues and cells, and HAX-1 overexpression promoted the liver cancer cells growth, migration, and invasion, whereas silencing HAX-1 produced the opposite results. Inhibition of Akt by LY294002 reversed the migration and invasion abilities of liver cancer cells, and inhibited the ability of cells growth and angiogenesis. Silencing PIK3CA enhanced the inhibitory effects of HAX-1 silencing on the viability, migration, and invasion of liver cancer cells. HAX-1 affected liver cancer cells metastasis and angiogenesis by affecting Akt phosphorylation and FOXO3A expression.

Keywords: Akt; FOXO3A; Liver cancer; angiogenesis; hematopoietic-substrate-1 associated protein X-1.

Figure 1.

Expression characteristics of hematopoietic-substrate-1 associated protein X-1 (HAX-1) in liver cancer tissues and cells. (a) Information on HAX-1 from liver cancer patients was analyzed by The Cancer Genome Atlas (TCGA) program. (b-d) Quantitative real-time polymerase chain reaction (qRT-PCR), Western blot and streptavidin-perosidase (SP) staining were used to detect HAX-1 levels in liver cancer tissues and adjacent tissues. (e, f) QRT-PCR and Western blot were applied to tested HAX-1 mRNA and protein levels in different liver cancer cells.*P < 0.05, **P < 0.01, versus cancer tissues or human normal liver cells THLE-2 and L02.

Figure 2.

The effects of silencing or overexpressing hematopoietic-substrate-1 associated protein X-1 (HAX-1) on proto-oncogene and tumor suppressor gene. (a–o) Quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot were used to detect the expressions of proto-oncogenes and tumor suppressor genes at different levels of HAX-1 expressions in SK-Hep1 and SUN387 cells.*P < 0.05, **P < 0.01, versus control group and siNC group.

Figure 3.

The effects of silencing hematopoietic-substrate-1 associated protein X-1 (HAX-1) on SK-Hep1 cells. (a–d) The migration and invasion abilities of SK-Hep1 cells were tested by the scratch assay and Transwell assay. (e) Cell counting kit-8 (CCK-8) assay was applied to test the effects of HAX-1 low expression on SK-Hep1 cell viability.*P < 0.05, **P < 0.01, versus control group and siNC group.

Figure 4.

The effects of overexpressing hematopoietic-substrate-1 associated protein X-1 (HAX-1) on SUN387 cells. (a–d) The migration and invasion abilities of SK-Hep1 cells were tested by the scratch assay and Transwell assay. (e) Cell counting kit-8 (CCK-8) assay was applied to test the effects of HAX-1 low expression on SUN387 cell viability.*P < 0.05, **P < 0.01, versus control group and mock group.

Figure 5.

The effects of silencing or overexpressing hematopoietic-substrate-1 associated protein X-1 (HAX-1) on signaling pathways. (a–l) Western blot was applied to test JAK/STAT3 and Akt signaling pathways-related protein levels in different liver cancer cells.

Figure 6.

The effects of silencing or overexpressing hematopoietic-substrate-1 associated protein X-1 (HAX-1) and inhibiting Akt on FOXO3A. (a–b) LY294002 was used to inhibit Akt, and the expression level of FOXO3A protein was detected by Western blot. *P < 0.05, **P < 0.01, versus siNC group or mock group; ^#P < .05, ^##P < 0.01, versus siHAX-1 group or HAX-1 group; ^{^}P < 0.05, ^{^^}P < 0.01, versus siNC+ LY294002 group.

Figure 7.

The effects of silencing hematopoietic-substrate-1 associated protein X-1 (HAX-1) and inhibiting Akt on SK-Hep1cells. (a–d) The migration and invasion abilities of SK-Hep1 cells were tested by the scratch assay and Transwell assay. (e) Cell counting kit-8 (CCK-8) assay was applied to test the effects of HAX-1 low expression and LY294002 on SK-Hep1 cell viability. *P < 0.05, **P < 0.01, versus siNC group; ^#P < 0.05, ^##P < 0.01, versus siHAX-1 group; ^{^}P < 0.05, ^{^^}P < 0.01, versus siNC+ LY294002 group.

Figure 8.

The effects of overexpressing hematopoietic-substrate-1 associated protein X-1 (HAX-1) and inhibiting Akt on SUN387 cells. (a–d) The migration and invasion abilities of SUN387 cells were tested by the scratch assay and Transwell assay. (e) Cell counting kit-8 (CCK-8) assay was applied to test the effects of HAX-1 low expression and LY294002 on SUN387 cell viability. *P < 0.05, **P < 0.01, versus mock group; ^#P < 0.05, ^##P < 0.01, versus HAX-1 group; ^{^}P < 0.05, ^{^^}P < 0.01, versus mock+ LY294002 group.

Figure 9.

The effects of silencing or overexpressing hematopoietic-substrate-1 associated protein X-1 (HAX-1) and LY294002 on angiogenesis. (a–d) The tube formation assay was applied to examine the effects of silencing or overexpressing hematopoietic-substrate-1-associated protein X-1 (HAX-1) on angiogenesis. *P < 0.05, **P < 0.01, versus siNC group or mock group; ^#P < 0.05, ^##P < 0.01, versus siHAX-1 group or HAX-1 group; ^{^}P < 0.05, ^{^^}P < 0.01, versus mock+ LY294002 group.

Figure 10.

The effect of silencing or overexpressing hematopoietic-substrate-1 associated protein X-1 (HAX-1) and PIK3CA on SK-Hep1 and SUN387 cells. (a–f) The effect of silencing PIK3C on the expression of Akt, and the protein levels of PIK3CA, p-Akt and Akt in SUN387 and SK-Hep1 cells were detected by Western blot. (g–h) Cell counting kit-8 (CCK-8) assay was applied to detect the effects of HAX-1 overexpression or low expression and siPIK3CA on SK-Hep1 and SUN387 cell viability. (i–p) The migration and invasion abilities of SUN387 and SK-Hep1 cells were tested by the scratch assay and Transwell assay. *P < 0.05, **P < 0.01, versus mock group or siNC group; ^#P < 0.05, ^##P < 0.01, versus HAX-1 group or siHAX-1 group; ^{^}P < 0.05, ^{^^}P < 0.01, versus mock+siPIK3CA group or siNC+ siPIK3CA group.