HIPK3 Promotes Growth and Metastasis of Esophageal Squamous Cell Carcinoma via Regulation of miR-599/c-MYC Axis

Abstract

Background/aims: this experimental design was based on HIPK3 to explore the pathogenesis of ESCC.

Methods: RT-qPCR was used to detect the expression of CircHIPK3 and miR-599 in ESCC tissues and cell lines.CCK-8, colony formation, flow cytometry and transwell assay were used to detect the effects of CircHIPK3 and miR-599 on tumor cell proliferation, apoptosis and migration and invasion. Target gene prediction and screening, luciferase reporter assays were used to validate downstream target genes of CircHIPK3 and miR-599.mRNA and protein expression of c-MYC were detected by RT-qPCR and Western blotting. The tumor changes in mice were detected by in vivo experiments in nude mice.

Results: HIPK3 was highly expressed in ESCC tissues and cell lines. In addition, HIPK3 expression levels were associated with advanced TNM stage, lymph node metastasis and tumor size. Moreover, HIPK3 was significantly promoted cell proliferation and migration of ESCC cells. In addition, HIPK3 was able to inhibit miRNA-599 expression and up-regulate the expression level of c-MYC. Finally, the results of in vivo animal models confirmed that HIPK3 promoted ESCC progression by modulating the miR-599/c-MYC axis.

Conclusion: HIPK3 can regulate the proliferation of esophageal squamous cell carcinoma cells by regulating miR-599/c-MYC axis, thereby inhibiting the occurrence and development of esophageal squamous cell carcinoma.

Keywords: HIPK3; c-MYC; esophageal squamous cell carcinoma; miR-599; proliferation.

Figure 1

HIPK3 was upregulated in ESCC tissues and cell lines. (A) The expression of HIPK3 in 42 pairsof ESCC tissues and in paired normal tissues. (B) The expression of HIPK3 in ESCC cell lines and NE1 cells. ** p <0.01, *** p <0.001.

Figure 2

CircHIPK3 promoted proliferation and metastasis of ESCC cells. (A) HIPK3 levels in si-HIPK3 or LV-HIPK3 transfected cells. (B) Effect of HIPK3 on cell viability. (C) Effect of HIPK3 on cell proliferation. (D) Effect of HIPK3 on apoptosis. (E, F) Effect of HIPK3 on cell migration and invasion.*p <0.05, ** p <0.01, *** p <0.001.

Figure 3

HIPK3 directly targeted miR-599 in ESCC. (A) Bioinformatics analysis revealed predicted binding sites between HIPK3 and miR-599. (B) Relative luciferase activity in TE-13 cells co-transfected with wild-type (HIPK3-WT) or mutant reporter plasmid (HIPK3-mut) and miR-599 mimic or negative control. (C) Cellular lysates from ESCC cells were used for RIP with an Ago2 antibody. The Ago2 protein level was detected by Western blot, (D) and the relative expression of HIPK3 and miR-599 in the immunoprecipitate was measured by RT-PCR. (E) The miR-599 expression in ESCC tissues and matched normal tissues. (F) Expression of miR-599 in the presence of si-HIPK3 or LV-HIPK3. (G) The relationship between HIPK3 and miR-599 in the ESCC organization.(** P <0.01).

Figure 4

(A) HIPK3 promoted cell growth and metastasis by inhibiting miR-599. (B) Cell viability assay. (C) Cell proliferation assay. (D) Apoptosis assay. (E, F) Cell migration and invasion assays.*p <0.05, ** p <0.01.

Figure 5

HIPK3 increased c-MYC expression levels by inhibiting miR-599. (A) Bioinformatics analysis revealed predicted binding sites between c-MYC and miR-599. (B) Luciferase activity was detected in cells co-transfected with miR-599 and a luciferase reporter containing c-MYC or mutant forms. (C) c-MYC mRNA expression levels in TC-13 cells. (D) c-MYC protein expression levels in TC-13 cells. (E, F) Changes in expression of c-MYC protein in TE-13 cells. (G) A positive correlation between HIPK3 and c-MYC expression in ESCC tissues. (H) Negative correlation between c-MYC and miR-599 in ESCC organization.(#P <0.05, **P <0.01).

Figure 6

HIPK3 promoted ESCC progression in vivo by modulating the miR-599/c-MYC axis. (AC) Tumor size and tumor weight. (D) Expression changes of c-MYC protein in each group. (#P <0.05, **P <0.01).