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miR-4653-3p overexpression is associated with a poor prognosis of pancreatic ductal adenocarcinoma via HIPK2 downregulation

Pancreatic ductal adenocarcinoma (PDAC) is a lethal malignant tumor. Several upregulated and downregulated microRNAs (miRNAs) are associated with invasiveness, tumorigenesis, and prognosis of PDAC. Herein, using in situ hybridization, we evaluated miR-4653-3p expression and pancreatic intraepithelial neoplasia (PanIN) and the association between miR-4653-3p expression and clinicopathological factors in PDAC patients. The miR-4653-3p target was also identified. Ninety PDAC cases, including 30 each with normal pancreatic ducts, low-grade PanINs, and high-grade PanINs, were evaluated. miR-4653-3p expression increased in the order—normal pancreatic duct, low-grade PanIN, high-grade PanIN, and PDAC—with no expression detected in normal pancreatic duct. High expression significantly correlated with advanced pathological T stage, lymph node metastasis, advanced Union for International Cancer Control stage, perineural invasion, venous involvement, and shorter overall and disease-specific survival. Homeodomain Interacting Protein Kinase 2 (HIPK2) was identified as a miR-4653-3p target based on mRNA microarray analysis and database screening. In MIA PaCa-2 cells, miR-4653-3p significantly downregulated HIPK2 expression. HIPK2 expression, unlike that of miR-4653-3p, decreased in the order—normal pancreatic duct, low-grade PanIN, high-grade PanIN, and PDAC. Low HIPK2 expression was associated with shorter overall and disease-specific survival in PDAC patients. Thus, miR-4653-3p associates with tumorigenesis and worse prognosis, partly by reducing HIPK2 expression.

upregulated in tumor cells and are classified as potent oncogenes, while others are downregulated and classified as tumor suppressors 7 . For instance, miR-21 is classified as an oncogene because its overexpression increases the proliferation, invasion, and chemoresistance of pancreatic cancer cells to gemcitabine 8 . In contrast, miR-96 is classified as a tumor suppressor because it decreases invasion and migration of cancer cells, delaying tumor growth through the downregulation of KRAS 9 . Both upregulation and downregulation of miRNAs have also been reported in PanIN. For instance, miR-148a, miR-217, and miR-145 are downregulated, whereas miR-10b and miR-196 are upregulated in PanIN II-III compared with that in normal pancreas 6,10,11 . miR-4653-3p is a mature miRNA. Its upregulation has been reported in xanthohumol-treated glioblastoma cells 12 , in glioma stem cells compared with that in neural stem cells 13 , and in breast cancer MCF-7 cells overexpressing the HER-2 gene 14 . Moreover, miR-4653-3p downregulation has been reported in paclitaxel-resistant nasopharyngeal carcinoma CNE-1/Taxol cells compared with that in parental CNE-1 cells 15 and in recurrence/ metastatic lesions compared with that in primary lesions in patients with breast cancer who relapsed after tamoxifen treatment 16 . However, to the best of our knowledge, the expression of miR-4653-3p in PDAC patients has not been investigated, as of date.
In this study, we investigated the association between miR-4653-3p expression, as determined via in situ hybridization (ISH), and clinicopathological factors and assessed the impact of miR-4653-3p expression on survival outcomes in patients with PDAC. Furthermore, we identified the target of miR-4653-3p, which is associated with survival outcomes, and found that miR-4653-3p downregulates HIPK2 and that it may be associated with metastasis, shorter survival, and tumorigenesis in PDAC.
Luciferase reporter assay, reverse transcription-quantitative polymerase chain reaction (RT-qPCR), and western blot analysis. To determine whether miR-4653-3p could downregulate HIPK2 expression, we performed real-time RT-qPCR, western blot, and luciferase assays. The miR-4653-3p mimic downregulated luciferase activity to 50.0% and 43.2% at miR-4653-3p target position 929-935 (target 1) and 7620-7627 (target 3) compared with that in the negative control (NC) mimic, but not at target position 5947-5953 (target 2; p = 0.500). Means ± (standard deviation) SD from four independent experiments are shown in Fig. 4a. In real-time RT-qPCR, HIPK2 mRNA expression in PDAC cells transfected with miR-4653-3p mimic was downregulated to 16.6% compared with that in NC cells (p < 0.001). In contrast, there was no significant difference in the expression of HIPK2 mRNA between PDAC cells transfected with miR-4653-3p inhibitor and NC cells (p = 0.683, Fig. 4b). In the western blot assay, the expression of HIPK2 in PDAC cells transfected with miR-4653-3p mimic was downregulated to 17.3% compared with that in NC cells (p < 0.001). In contrast, there was no significant difference in the expression of HIPK2 mRNA between PDAC cells transfected with miR-4653-3p inhibitor and NC cells (p = 0.935; Fig. 4c, d). Taken together, these results show that HIPK2 is the target of miR-4653-3p and that miR-4653-3p downregulates HIPK2 in PDAC cell lines. was expressed in the nuclei of normal pancreatic ducts, normal acinar cells, normal islet cells, PanIN cells, and PDAC cells (Fig. 5). The mean H-score for HIPK2 was 282.3 (240-300) in patients with normal pancreatic ducts, 267 (150-300) in patients with low-grade PanIN, 240.5 (100-300) in patients with high-grade PanIN, and 223.1 (110-300) in patients with PDAC ( Fig. 2b). Significant differences were observed in the mean HIPK2 H-scores between normal pancreatic duct and PDAC (p < 0.001), low-grade PanIN and PDAC (p < 0.001), and normal pancreatic duct and high-grade PanIN (p = 0.003). However, no significant differences in the mean H-scores of HIPK2 between low-grade PanIN and high-grade PanIN (p = 0.077), high-grade PanIN and PDAC (p = 0.213), and normal pancreatic duct and low-grade PanIN (p = 0.064) were observed. Among the patients with PDAC, 18 (20%) had low HIPK2 expression, while 72 (80%) had high HIPK2 expression. There was a weak inverse correlation between the H-score of immunohistochemistry of HIPK2 and the H-score of ISH of miR-4653-3p in PDAC (r = − 0.218, p = 0.039) (Fig. 6). Low HIPK2 expression was associated with shorter overall and disease-specific survival in the log-rank test.  Fig. 3d). However, multivariate analysis showed that miR-HIPK2 was not an independent prognostic factor (Supplementary Table 1b). There was no significant difference in progression-free survival between low and high HIPK2 expression groups (log-rank test: p = 0.687, Fig. 3f). There were no associations with other clinicopathological factors, except for overall and disease-specific survival (Supplementary Table 3).

Discussion
In this study, we used ISH to evaluate the miR-4653-3p expression in patients with PDAC and its correlation with clinicopathological factors. High miR-4653-3p expression significantly correlated with advanced pT, lymph node metastasis, advanced UICC stage, perineural invasion, venous involvement, and shorter overall and diseasespecific survival. In addition, miR-4653-3p was not expressed in normal pancreatic ducts, and its expression increased in the order-normal pancreatic duct < low-grade PanIN < high-grade PanIN < PDAC. These results suggest that miR-4653-3p is involved in the early stages of PDAC tumorigenesis in the normal pancreatic duct, low-grade PanIN, and high-grade PanIN. Furthermore, miR-4653-3p reduced HIPK2 expression in vitro, and miR-4653-3p expression inversely correlated with HIPK2 expression in vivo. Unlike that of miR-4653-3p, HIPK2 expression decreased gradually in the order-normal pancreatic duct, low-grade PanIN, high-grade PanIN, and www.nature.com/scientificreports/ PDAC. In addition, low HIPK2 expression was associated with shorter and disease-specific survival in patients with PDAC. These results indicate that miR-4653-3p is associated with tumorigenesis and a shorter prognosis, partly by reducing HIPK2 expression. HIPK2 is a nuclear serine/threonine kinase that represses the transcription of homeodomain-containing transcription factors 19 . Its expression and role have been reported in a variety of tumors. HIPK2 is downregulated in tumor cells compared with normal tissues in hepatocellular carcinoma 20 , esophageal squamous cell carcinoma 21 , urinary bladder cancer 22 , colorectal cancer 23 , and pancreatic cancer 24 . In these tumors, the downregulation of HIPK2 was associated with poor prognosis, promoted migration, tumor growth, metastasis, or increased cell viability [20][21][22][23][24][25][26] . In contrast, HIPK2 expression was upregulated in tumor cells in tonsillar squamous cell carcinoma 27 and cervical cancer 28,29 . In tonsillar squamous cell carcinoma, HIPK2 overexpression was associated with shorter overall survival and disease-free survival 27 . The expression and role of HIPK2 may differ depending on tumor site and histological type.
The expression of HIPK2 in pancreatic tissues, PanIN, and PDAC has been reported in several studies. Qin et al. reported higher HIPK2 expression in adjacent normal pancreatic tissue compared with that in pancreatic cancer tissue, and low HIPK2 expression was associated with poor prognosis 23 . Valente et al. reported that HIPK2 expression was significantly lower in PanIN-3 (currently classified as high-grade PanIN) and PDAC than in normal tissues 30 . These reports and our results of HIPK2 expression support the notion that HIPK2 downregulation may be associated with the tumorigenesis of PDAC.
We hypothesize that the association between HIPK2 and p53 may also play an important role in the prognosis of pancreatic cancer. The tumor suppressor p53 interacts with HIPK2. HIPK2-mediated phosphorylation of p53 at Ser46 enhances p53-induced apoptosis [31][32][33] . HIPK2 may stabilize p53 by downregulating p53-induced Mdm2 protein. p53 is one of the major genes associated with tumorigenesis and prognosis of PDAC 34 . Therefore, downregulation of HIPK2 in pancreatic cancer leads to the downregulation of p53-associated tumor suppressors, resulting in tumorigenesis and poor prognosis. The inactivation of p53 occurs late in the development of pancreatic neoplasia, for example, in high-grade PanIN and PDAC, but not in low-grade PanIN 35 . Based on our results and the HIPK2 expression pattern reported previously, we hypothesized that HIPK2 might be associated with the late stage of PDAC tumorigenesis, which involves p53. In contrast, miR-4653-3p expression was significantly higher in the normal pancreatic duct than in low-grade PanIN. These results indicate that other molecules, besides HIPK2, are controlled by miR-4653-3p and are associated with PDAC tumorigenesis. was not expressed in normal pancreatic ducts, and its expression increased in the order normal pancreatic duct < low-grade PanIN < high-grade PanIN < PDAC (a). In contrast, HIPK2 expression decreased gradually, in the order normal pancreatic duct, low-grade PanIN, high-grade PanIN, and PDAC (b). *represents significant differences in miR-4653-3p expression and HIPK2 H-scores (p < 0.05). www.nature.com/scientificreports/

Scientific Reports
In conclusion, this study shows that miR-4653-3p might promote the tumorigenesis and invasiveness of PDAC, in part by inhibiting the expression of HIPK2. ISH of miR-4653-3p may prove useful in predicting tumor invasiveness and prognosis of PDAC. However, more complex molecular mechanisms and other target molecules may also be associated with PDAC tumorigenesis. Thus, further studies are warranted to clarify the association between target molecules and miR-4653-3p in PDAC tumorigenesis. High miR-4653-3p expression was associated with shorter overall (a) and diseasespecific survival (c). In contrast, low HIPK2 expression was associated with shorter overall (b) and diseasespecific survival (d). There was no significant difference in the association between progression-free survival and the expression of miR-4653-3p (e) and HIPK2 (f).    Western blot analysis. Western blot analysis was performed using a simple western blot assay (Protein-Simple, Santa Clara, CA, USA). Cultured cells were lysed in cell lysis buffer containing protease inhibitors before sonication. The cell lysates were then centrifuged at 10,000 rpm for 3 min to remove the soluble material. The protein concentration of cell lysates was measured using a DC protein assay (Bio-Rad, Hercules, CA, USA). For the simple western blot assay, cell lysates were diluted to 0.4 μg/μL using 0.1 × sample buffer and 5 × Fluorescent Master Mix. The primary antibodies used were as follows: HIPK2 (ab28507, Abcam, Cambridge, United Kingdom) at 1:100 dilution and β-actin monoclonal antibody (A5441, clone AC-15, Sigma-Aldrich, St. Louis, MO, USA) at 1:4,000 as the internal control. Secondary antibodies were prepared by the manufacturer. The assay was conducted according to the manufacturer's instructions. The assay plate and capillary cartridge were placed into a simple western machine (WES; Protein Simple). All subsequent separation, immunodetection, and analysis steps were performed automatically on the WES. The Compass software (ProteinSimple) was used to visualize the chemiluminescent image of the capillary, analyze the size or charge data, and process the results. The relative HIPK2 expression peak area was normalized to that of β-actin (n = 3 in each experimental group).
Fisher's exact test and Pearson's χ 2 test were used to analyze the relationship between clinicopathological factors and the expression of miR-4653-3p or HIPK2. Two-sample Student's t-test or Welch's t-test was used to compare the expression of HIPK2 or miR-4653-3p as determined with RT-qPCR, western blot, immunohistochemistry, and ISH analyses. The correlation between the expression of miR-4653-3p and HIPK2 was evaluated using Spearman correlation analysis. Survival curves were plotted using the Kaplan-Meier method and compared using a log-rank test. Survival was also evaluated in univariate and multivariate analyses using Cox proportional hazard regression models to identify independent prognostic factors. First, a univariate analysis of overall survival was performed for each clinicopathological factor. Statistically significant factors in the univariate analysis were then included in the multivariable analysis using the forward selection method (likelihood ratio); p < 0.05 was considered statistically significant.
Ethics approval and consent to participate. This study was approved by the Research Ethics Committee of Tokai University School of Medicine (No. 17R275) and was performed according to the Declaration of Helsinki. The informed consent was obtained from all subjects through an opt-out methodology.

Data availability
All data analyzed in this study are included in this published article and its supplementary information files. The datasets of gene expression generated and/or analyzed during the current study are available in the ArrayExpress repository, ArrayExpress accession E-MTAB-11813.