SP1-induced HOXD-AS1 promotes malignant progression of cholangiocarcinoma by regulating miR-520c-3p/MYCN

Abstract

The purpose of this article is to explore the function and mechanism of HOXD-AS1 in cholangiocarcinoma. TCGA, StarBase and JASPAR were applied to predict the differential expression and molecular mechanism. The qRT-PCR was conducted to detect molecular expression. The effect of HOXD-AS1 on tumor proliferation, metastasis and stemness was measured through corresponding experiments. ChIP, luciferase reporter and RIP assays were implemented to explore the regulatory mechanism of HOXD-AS1 in CCA. In this study, HOXD-AS1 expression was significantly upregulated in CCA tissues and cells compared with control groups, respectively. Increased HOXD-AS1 was markedly correlated with lymph node invasion, advanced TNM stage and poor survival of CCA patients. Moreover, HOXD-AS1 was confirmed to be an unfavorable independent prognostic factor for CCA patients. Functionally, gain- and loss-of-function experiments demonstrated that HOXD-AS1 facilitated tumor proliferation, migration, invasion, EMT, stemness and drug resistance in vitro and in vivo. For the mechanism, transcription factor SP1-induced HOXD-AS1 upregulated oncogene MYCN through competitively binding to miR-520c-3p. Furthermore, HOXD-AS1-induced malignant phenotypes were rescued by interfering miR-520c-3p and MYCN, respectively. SP1/HOXD-AS1/miR-520c-3p/MYCN plays a vital role in initiation and progression of CCA, and HOXD-AS1 is expected to be an efficient biomarker and therapeutic target.

Keywords: HOXD-AS1; MYCN; cholangiocarcinoma; miR-520c-3p; prognosis.

Figure 1

The expression of HOXD-AS1 and its correlation with clinicopathological characteristics and prognosis. (A) HOXD-AS1 expression in CCA tissues and paired adjacent nontumor bile duct tissues was detected by qRT-PCR. (B) HOXD-AS1 expression in tissues at TNM I+II stage and TNM III+IV stage was detected by qRT-PCR. (C) HOXD-AS1 expression in tissues with lymph node invasion and without lymph node invasion was detected by qRT-PCR. (D) CCA patients were divided into two groups according to average value of HOXD-AS1 expression. Overall survival was evaluated between high and low HOXD-AS1 expression groups by using Kaplan-Meier method and log-rank test. (E) The correlation between relative HOXD-AS1 expression and survival time of CCA patients was assessed by Pearson correlation analysis. (F) The sensitivity and specificity of HOXD-AS1 as a prognostic marker were analyzed by ROC curve. ^**P < 0.01, ^***P < 0.001.

Figure 2

Upregulated HOXD-AS1 facilitated the viability, migration and invasion of CCA cells. (A) The expression of HOXD-AS1 in CCLP-1, QBC939, TFK-1, RBE cells and normal HIBEC. (B) CCK-8 proliferation curves were drawn to show the impact of HOXD-AS1 on cellular proliferation. (C) EdU assay was performed to detect the effect of HOXD-AS1 on cellular proliferation. (D) The effect of HOXD-AS1 on cell colonies was revealed by colony formation assays. (E) The wound closure of CCA cells was evaluated by wound healing assays. (F) Transwell assays displayed that migrating cells were decreased in si-HOXD-AS1 cells and increased in QBC939 cells transfected with HOXD-AS1 plasmid. (G) The invasive ability of CCA cells was confirmed by transwell assay. (H) EMT-related proteins including epithelial marker (E-cadherin) and mesenchymal markers (snail and vimentin) were measured via western blot. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 3

HOXD-AS1 promoted stemness maintenance and chemo-resistance in CCA. (A) Spheroid forming abilities of transfected QBC939 and CCLP-1 cells were evaluated by spheroid formation assay. (B) The effect of HOXD-AS1 on stem markers (SOX2, OCT4, Nanog) was detected by western blot. (C) The effect of HOXD-AS1 on the treatment of cisplatin and gemcitabine was measured by CCK-8 assay. (D) Mice were subcutaneously injected with QBC939 cells transfected with HOXD-AS1 plasmid and intraperitoneally injected with PBS, cisplatin or gemcitabine. (E) The stem markers (SOX2, OCT4, Nanog) in xenograft tumors were measured by western blot. ^*P < 0.05, ^**P < 0.01.

Figure 4

HOXD-AS1 was induced by transcription factor SP1. (A) SP1 sequence and binding sites (E1 and E2) to HOXD-AS1 promoter region were predicted by using JASPAR database (http://jaspar.genereg.net/). (B) The knockdown efficiency and amplification efficiency of SP1 in CCLP-1 and QBC939 were detected by qRT-PCR. (C) Upregulated SP1 facilitated HOXD-AS1 expression and decreased SP1 restrained HOXD-AS1 expression in QBC939 and CCLP-1 cells. (D) ChIP assays were performed to confirm the direct binding of SP1 to HOXD-AS1 promoter in QBC939 and CCLP-1 cells. (E) Luciferase reporter assay showed that SP1 bound to E1 rather than E2. (F) Luciferase reporter assay displayed that SP1 bound to E1 wild type rather than mutant type. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 5

HOXD-AS1 functioned as a sponge for miR-520c-3p in CCA cells. (A) Subcellular localization of HOXD-AS1 was tested by subcellular fractionation assays. GAPDH and U6 were used as endogenous controls for cytoplasm and nucleus, respectively. (B) The expression levels of predicted miRNAs were detected after knocking down or amplifying HOXD-AS1 in CCLP-1 and QBC939 cells, respectively. (C) The expression of miR-520c-3p in CCA tissues and paired adjacent nontumor bile duct tissues. (D) The correlation between relative HOXD-AS1 expression and relative miR-520c-3p expression in CCA tissues. (E) The miR-520c-3p expression in CCA cells (CCLP-1, QBC939, TFK-1, RBE) and normal HIBEC. (F) Luciferase reporter plasmids were constructed with miR-520c-3p-binding site region of HOXD-AS1 sequence, including wild type and mutant type. (G) Luciferase reporter assays showed that cotransfected miR-520c-3p mimics significantly inhibited luciferase activity of HOXD-AS1 wild type. (H) AGO2 RIP assays further suggested the binding of miR-520c-3p to HOXD-AS1. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 6

MiR-520c-3p was a direct regulator of MYCN in CCA cells. (A) MiR-520c-3p restrained MYCN mRNA expression certified by qRT-PCR. (B) MiR-520c-3p refrained MYCN protein expression testified via western blot. (C) The expression of MYCN mRNA in CCA tissues and paired adjacent nontumor bile duct tissues. (D) The correlation between relative MYCN mRNA expression and relative miR-520c-3p expression in CCA tissues. (E) The MYCN mRNA expression in CCLP-1, QBC939, TFK-1, RBE and normal HIBEC. (F) The MYCN protein expression in CCA cells (CCLP-1, QBC939, TFK-1, RBE) and normal HIBEC. (G) Luciferase reporter plasmids were constructed with miR-520c-3p-binding site region of MYCN sequence, including wild type and mutant type. (H) The luciferase activity of MYCN wild type was observably inhibited by miR-520c-3p mimics cotransfection. (I) AGO2 RIP assays were conducted to further demonstrate the binding of miR-520c-3p to 3’UTR of MYCN. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 7

HOXD-AS1 boosted malignant progression of CCA through mediating miR-520c-3p/MYCN. (A) MYCN downexpression caused by HOXD-AS1 knockdown was saved by silencing miR-520c-3p. (B) MYCN overexpression caused by HOXD-AS1 amplification was saved by increasing miR-520c-3p. (C–G) Rescue assays of EdU, transwell, spheroid formation, stem marker analysis and chemo-resistance certified that inhibition of proliferation, invasion, stemness maintenance and chemo-resistance induced by knocking down HOXD-AS1 was retrieved through silencing miR-520c-3p in CCLP-1 cells, respectively. (H–L) Restoration of MYCN expression rescued the promotion of proliferation, invasion, stemness maintenance and chemo-resistance generated through HOXD-AS1 amplification in EdU, transwell, spheroid formation, stem marker analysis and chemo-resistance assays, respectively. ^*P < 0.05, ^**P < 0.01.

Figure 8

HOXD-AS1/miR-520c-3p/MYCN contributed to CCA tumorigenesis in vivo. (A) CCLP-1 cells cotransfected with sh-HOXD-AS1 and antagomir-520c-3p were subcutaneously injected into the posterior flanks of mice, and xenograft tumors were dissected on the 21^st day after injection. (B) Tumor volumes were calculated every 3 days throughout the course of tumor growth. (C) Tumor weights were measured after excision. (D) MYCN mRNA expression in xenograft tumors of the three groups (sh-NC/antagomir-NC, sh-HOXD-AS1/antagomir-NC, sh-HOXD-AS1/antagomir-520c-3p). ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.