Depletion of DNTTIP2 Induces Cell Cycle Arrest in Pancreatic Cancer Cells

Abstract

Background/aim: Pancreatic cancer is one of the most lethal malignant cancers worldwide and the seventh most common cause of cancer-related death in both sexes. Herein, we analyzed open access data and discovered that expression of a gene called deoxynucleotidyltransferase terminal-interacting protein 2 (DNTTIP2) is linked to prognosis of pancreatic ductal adenocarcinoma (PDAC). We then elucidated the role of DNTTIP2 in the proliferation of pancreatic cancer cells in vitro.

Materials and methods: A WST-8 assay, cell cycle analysis, Annexin-V staining, quantitative reverse transcription-PCR, and western blot analysis were conducted to assess cell proliferation, cell cycle, apoptosis, and expression of DNTTIP2 mRNA and protein, respectively, in DNTTIP2-depleteted MIA-PaCa-2 and PK-1 cells.

Results: Depletion of DNTTIP2 induced G₁ arrest in MIA-PaCa-2 cells by decreasing expression of special AT-rich sequence binding protein 1 (SATB1) and cyclin-dependent kinase 6 (CDK6). In addition, depletion of DNTTIP2 induced G₂ arrest in PK-1 cells by decreasing expression of CDK1. Depletion of DNTTIP2 did not induce apoptosis in MIA-PaCa-2 or PK-1 cells.

Conclusion: DNTTIP2 is involved in proliferation of pancreatic cancer cells. Thus, DNTTIP2 is a potential target for inhibiting progression of pancreatic cancers.

Keywords: CDK1; CDK6; DNTTIP2; Pancreatic cancer; SATB1; cells.

Figure 1. Study flow chart. We used open access databases to identify DNTTIP2 as a molecule that plays a significant role in the proliferation of pancreatic cancer cells. We performed the experimental validation to reveal the role of DNTTIP2 gene in the proliferation of pancreatic cancer cells.

Figure 2. Effect of DNTTIP2 on survival of patients with PDAC, and the effects of depleting DNTTIP2 on proliferation of pancreatic cancer cells. (A) Survival of the DNTTIP2 high expression (N=89) and low expression (N=88) groups of PDAC patients. OS was assessed using the Kaplan-Meier method, and data were compared using the log-rank test (p=0.0004). (B) Expression of DNTTIP2 mRNA in MIA-PaCa-2 cells treated with DNTTIP2 siRNA s26947 (siDNTTIP2 #1) and DNTTIP2 siRNA s26949 (siDNTTIP2 #2) for 48 h. Data are presented as the mean+standard error of the mean (SEM) of three independent biological replicates, each with three technical replicates. ***p<0.001 [vs. negative control siRNA (siCtrl)]. (C, D) Expression of DNTTIP2 mRNA transcripts in PK-1 cells treated with siDNTTIP2 #1 and #2 for 48 and 72 h. Data are presented as the mean+SEM of three independent biological replicates, each with three technical replicates. ***p<0.001 (vs. siCtrl). (E, F) Expression of DNTTIP2 protein by MIA-PaCa-2 cells treated with siDNTTIP2 #1 and #2 for 48 h, and by PK-1 cells treated with siDNTTIP2 #1 and #2 for 72 h. Data of three independent biological replicates, each with one technical replicate, are shown. (G, H) Proliferation of MIAPaCa-2 and PK-1 cells treated with siDNTTIP2 #1 and #2 for 72 h. Data are presented as the mean+SEM of three independent biological replicates, each with four technical replicates. ***p<0.001 (vs. siCtrl).

Figure 3. Cell cycle and apoptosis analysis after depletion of DNTTIP2 from pancreatic cancer cells. (A) Cell cycle analysis of MIA-PaCa-2 cells treated with siDNTTIP2 #1 and #2 for 48 h. The data in histograms are representative of three independent experiments. Data in the bar graphs are expressed as the mean+standard deviation (SD) of three independent biological replicates, each with one technical replicate. *p<0.05; **p<0.01 (vs. siCtrl). (B) Cell cycle analysis of PK-1 cells treated with siDNTTIP2 #1 and #2 for 72 h. The data in the histograms are representative of three independent experiments. Data in the bar graphs are presented as the mean+SD of three independent biological replicates, each with one technical replicate. *p<0.05 (vs. siCtrl). (C, D) Apoptosis of MIA-PaCa-2 and PK-1 cells treated with siDNTTIP2 #1 and #2 for 72 h. Data in the dot plots are representative of three independent experiments. Data in the bar graphs are presented as the mean+SD of three independent biological replicates, each with one technical replicate. NS: Not significant; *p<0.05; **p<0.01 (vs. siCtrl).

Figure 4. mRNA and protein levels of cell cycle-regulating molecules in pancreatic cancer cells depleted of DNTTIP2. Evaluation of (A) CDK1, (B) CDK6, and (C) CDK4 mRNA by qRT-PCR. MIA-PaCa-2 cells were treated with siDNTTIP2 #1 and #2 for 48 h, and PK-1 cells were treated with siDNTTIP2 #1 and #2 for 72 h. Data are presented as the mean+SEM of three independent biological replicates, each with three technical replicates. NS: Not significant; *p<0.05; **p<0.01; ***p<0.001 (vs. siCtrl). (D) CDK1 and CDK6 protein expression in MIAPaCa-2 cells treated with siDNTTIP2 #1 and #2 for 48 h. Data of three independent biological replicates, each with one technical replicate, are shown. (E) CDK1 and CDK6 protein expression in PK-1 cells treated with siDNTTIP2 #1 and #2 for 72 h. Data of three independent biological replicates, each with one technical replicate, are shown.

Figure 5. Relationship between DNTTIP2 and SATB1. (A, B) Expression of SATB1 mRNA, as assessed by qRT-PCR. MIA-PaCa-2 and PK-1 cells were treated with siDNTTIP2 #1 and #2 for 48 h. Data are presented as the mean+SEM of three independent biological replicates, each with three technical replicates. ***p<0.001 (vs. siCtrl). MIA-PaCa-2 cells were treated with SATB1 siRNA s12480 (siSATB1 #1) and SATB1 siRNA s12481 (siSATB1 #2) for 48 h, and expression of SATB1 (C), CDK6 (D), and CDK1 (E) mRNA was assessed by qRT-PCR. Data are presented as the mean+SEM of three independent biological replicates, each with three technical replicates. *p<0.05; ***p<0.001 (vs. siCtrl). PK-1 cells were treated with siSATB1 #1 and #2 for 72 h, and expression of SATB1 (F), CDK6 (G), and CDK1 (H) mRNA was evaluated by qRT-PCR. Data are presented as the mean+SEM of three independent biological replicates, each with three technical replicates. NS: Not significant; *p<0.05; **p<0.01; ***p<0.001 (vs. siCtrl).

Figure 6. Proposed mechanism by which DNTTIP2 regulates CDK1 and CDK6. DNTTIP2 regulates CDK6 through SATB1 and regulates CDK1 directly. DNTTIP2 also regulates SATB1. CCNA: Cyclin A; CCNB: cyclin B; CCND: cyclin D; CDK1: cyclin-dependent kinase 1; CDK4: cyclindependent kinase 4; CDK6: cyclin-dependent kinase 6; DNTTIP2: deoxynucleotidyltransferase terminal-interacting protein 2; OS: overall survival; PDAC: pancreatic ductal adenocarcinoma; qRT-PCR: quantitative reverse transcription-PCR; RNAi: RNA interference; SATB1: special AT-rich sequence binding protein 1; TCGA: the cancer genome atlas.