DNAH17-AS1 promotes pancreatic carcinoma by increasing PPME1 expression via inhibition of miR-432-5p

Abstract

Background: The incidence and mortality rates of pancreatic carcinoma (PC) are rapidly increasing worldwide. Long noncoding RNAs (lncRNAs) play critical roles during PC initiation and progression. Since the lncRNA DNAH17-AS1 is highly expressed in PC, the regulation of DNAH17-AS1 in PC was investigated in this study.

Aim: To investigate the expression and molecular action of lncRNA DNAH17-AS1 in PC cells.

Methods: The PC expression data for the lncRNA DNAH17-AS1 was downloaded from The Cancer Genome Atlas database and used to examine its profile. Western blot and reverse transcription-quantitative PCR were employed to assess protein and mRNA expression. A subcellular fractionation assay was used to determine the location of DNAH17-AS1 in cells. In addition, the regulatory effects of DNAH17-AS1 on miR-432-5p, PPME1, and tumor activity were investigated using luciferase reporter assay, MTT viability analysis, flow cytometry, and transwell migration analysis.

Results: DNAH17-AS1 was upregulated in PC cells and was associated with aggressive tumor behavior and poor prognosis for patients. Silencing DNAH17-AS1 promoted the apoptosis and reduced the viability, invasion, and migration of PC cells. In addition, DNAH17-AS1 served as a PC oncogene by downregulating miR-432-5p which normally directly targeted PPME1 to downregulate its expression.

Conlusion: DNAH17-AS1 functions in PC as a tumor promoter by regulating the miR-432-5p/PPME1 axis. This finding may provide new insights for PC prognosis and therapy.

Keywords: DNAH17-AS1; Long noncoding RNAs; MiR-432-5p; Molecular mechanism; PPME1; Pancreatic carcinoma.

Figure 1

The expression of DNAH17-AS1 is upregulated in pancreatic carcinoma patients. A: Volcano plot of the TCGA_lncRNA profile. The top ten highly expressed lncRNAs are marked in green; B: The expression pattern of DNAH17-AS1 in pancreatic carcinoma (PC) patients and normal patients from The Cancer Genome Atlas dataset; C: DNAH17-AS1 expression in PC tissues collected by our hospital; D: DNAH17-AS1 expression in Hs766T, SW1990, and HPDE6-C7 cells; E: Dysregulation of DNAH17-AS1 is related to overall survival in PC patients; F: Dysregulation of DNAH17-AS1 is related to disease-free survival in PC patients. ^bP < 0.01. DFS: Disease-free survival; OS: Overall survival.

Figure 2

DNAH17-AS1 regulates viability, migration, and invasion of pancreatic carcinoma cells. A: DNAH17-AS1 expression in Hs766T cells with si-DNAH17-AS1 or si-NC; B and C: DNAH17-AS1 siRNA inhibited cell proliferation (B) and promoted apoptosis (C) in Hs766T cells; D and E: DNAH17-AS1 siRNA also inhibited Hs766T cell migration (D) and invasion (E). ^bP < 0.01.

Figure 3

DNAH17-AS1 acts as a sponge for miR-432-5p in pancreatic carcinoma cells. A: Subcellular fractionation assay was performed to identify the location of DNAH17-AS1 in Hs766T cells; B: The binding sites between DNAH17-AS1 and miR-432-5p; C: Luciferase activity of miR-432-5p mimic with DNAH17-AS1-WT or DNAH17-AS1-MUT; D: MiR-432-5p expression is regulated by si-DNAH17-AS1 in Hs766T cells. E-H: Cell proliferation (E), migration (F), invasion (G), and apoptosis (H) in Hs766T cells treated with miR-432-5p mimic or si-DNAH17-AS1 and miR-432-5p inhibitor. ^aP < 0.05, ^bP < 0.01.

Figure 4

MiR-432-5p regulates PPME1 expression in pancreatic carcinoma. A: Three possible mRNAs regulated by miR-432-5p were selected and are presented in a Venn diagram; B and C: The expression levels of three target mRNAs were assessed after overexpression of miR-432-5p; D and E: The protein levels of three possible proteins were assessed after overexpression of miR-432-5p; F: The binding sites between PPME1 and miR-432-5p; G: Luciferase activity of miR-432-5p mimic with PPME1-WT or PPME1-MUT; H and I: PPME1 expression regulation by miR-432-5p mimic or inhibitor in Hs766T cells; J: PPME1 expression in pancreatic carcinoma tissues; K: MiR-432-5p inversely regulates PPME1 expression. ^aP < 0.05, ^bP < 0.01.

Figure 5

DNAH17-AS1 interacts with miR-432-5p to regulate PPME1 expression. A: PPME1 is positively correlated with DNAH17-AS1 expression; B: PPME1 expression in Hs766T cells containing si-DNAH17-AS1 and PPME1 vector; C-F: Cell proliferation (C), migration (D), invasion (E), and apoptosis (F) in Hs766T cells containing si-DNAH17-AS1, miR-432-5p mimic, and PPME1 vector. ^aP < 0.05, ^bP < 0.01.