{'fraction_non_alphanumeric': 0.04537069064914101, 'fraction_numerical': 0.04784964833390983, 'mean_word_length': 4.842707982485686, 'pattern_counts': {'":': 0, '<': 18, '<?xml version=': 0, '>': 2, 'https://': 0, 'lorem ipsum': 0, 'www.': 0, 'xml': 0}, 'pii_count': 0, 'substrings_counts': 0, 'word_list_counts': {'cursed_substrings.json': 0, 'profanity_word_list.json': 0, 'sexual_word_list.json': 0, 'zh_pornsignals.json': 0}}
Molecular pathogenesis of pancreatic ductal adenocarcinoma: Impact of passenger strand of pre‐miR‐148a on gene regulation

We previously used RNA sequencing to establish the microRNA (miRNA) expression signature of pancreatic ductal adenocarcinoma (PDAC). We found that both strands of pre‐miR‐148a (miR‐148a‐5p: the passenger strand and miR‐148a‐3p: the guide strand) were downregulated in cancer tissues. Ectopic expression of miR‐148a‐5p and miR‐148a‐3p significantly inhibited cancer cell migration and invasion, indicating that both strands of pre‐miR‐148a had tumor‐suppressive roles in PDAC cells. In silico database and genome‐wide gene expression analyses identified a total of 15 genes that were putative targets regulated by these miRNAs. High expression of miR‐148a‐5p targets (PHLDA2,LPCAT2 and AP1S3) and miR‐148a‐3p targets (SMA, ENDOD1 and UHMK1) was associated with poor prognosis of patients with PDAC. Moreover, knockdown of PHLDA2 expression inhibited cancer cell aggressiveness, suggesting PHLDA2 acted as an oncogene in PDAC cells. Involvement of the passenger strand of pre‐miR‐148a (miR‐148‐5p) is a new concept in cancer research. Novel approaches that identify tumor‐suppressive miRNA regulatory networks in lethal PDAC might provide new prognostic markers and therapeutic targets for this disease.

Therefore, aberrantly expressed miRNAs can disturb entire networks of mRNAs and proteins.
Identification of dysregulated miRNAs in cancer cells is an important first step in elucidating aberrantly expressed genes and proteins in cancer. Based on this background, our groups have been comprehensively studying specimens of several cancers by genome-wide miRNA expression analyses. 6,7 Analyses of miRNA expression signatures of PDAC clinical specimens by RNA sequencing showed that some passenger strands of miRNAs (eg, miR-216a-3p, miR-216b-3p, miR-148a-5p, miR-129-1-3p and miR-129-2-3p) were significantly downregulated in cancer tissues. 8 In miRNA biogenesis, it is generally believed that 1 strand of the mature duplex miRNA (the guide strand) is incorporated into the miRNA-induced silencing complex (RISC) and its control target genes by RNA degradation and translational repression. 9 In contrast to the guide strand, the passenger strand of miRNA was previously thought to be degraded and to have no function. 10,11 Contrary to established beliefs, our recent studies indicated that several passenger strands of miRNAs acted as antitumor miRNAs in several cancers. 12,13 For example, miR-145-3p (passenger strand) was significantly downregulated in castration-resistant prostate cancer and restoration of miR-145-3p inhibited cancer cell migration and invasion through targeting of MELK, NCAPG, BUB1, and CDK1. 14 Similarly, antitumor functions have also been shown in bladder cancer and lung cancer. 15,16 Realization that the passenger strand of miRNA is involved in the regulation of RNA networks will improve our understanding of cancer cell development and tumor progression.
In the present study, our goal was to identify novel therapeutic targets in lethal PDAC. Based on our original miRNA expression signature of PDAC, we focused on pre-miR-148a (miR-148a-5p [passenger strand] and miR-148a-3p [guide strand]) and investigated the functional significance of these miRNAs and their regulatory RNA networks in PDAC cells. Elucidation of molecular networks modulated by novel antitumor miRNAs in PDAC cells may provide new insights into the pathological mechanisms underlying the disease.

| Clinical specimens and cell lines
Clinical tissue specimens (n = 30) were obtained from patients with PDAC who underwent curative surgical resection at Kagoshima University Hospital between 1997 and 2016. Normal pancreatic tissue specimens (n = 18) were obtained from non-cancerous tumoradjacent tissues. Each surgical specimen was histologically characterized according to the TNM classification system. 17 All patients in this study provided informed consent and the study protocol was approved by the Institutional Review Board of Kagoshima University.

| Plasmid construction and dual-luciferase reporter assay
The vector used for dual-luciferase reporter assay was psiCHECK-2 vector (C8021; Promega, Madison, WI, USA). The cloned sequences are listed in Figure S1. The procedure for dual-luciferase reporter assay was described previously. [18][19][20][21] 2.10 | Identification of downstream targets regulated by PHLDA2 in PDAC Genome-wide microarray analysis was applied for identification of PHLDA2 downstream targets. Expression data were deposited in the GEO database (GSE106791). Genes downregulated by PHLDA2 were assessed for PDAC prognosis using TCGA database.

| Statistical analysis
Relationships between 2 groups and expression values obtained by qRT-PCR were analyzed using the Mann-Whitney U test. Correlation between expression of miR-148a-5p, miR-148a-3p and PHLDA2 was evaluated using Spearman's rank test. Relationships among more than 3 variables and numerical values were analyzed using the Bonferroni-adjusted Mann-Whitney U test. Overall survival (OS) and DFS after surgery were gauged using Kaplan-Meier curves. Patients were divided into 2 groups based on high and low gene expression around the median, and differences in survival were estimated using the log-rank test. We used Expert StatView software (version 5.0; SAS Institute Inc., Cary, NC, USA) for these analyses. 18 Tables 1,2 . Expression levels of miR-148a-5p and miR-148a-3p were significantly lower in tumor tissues compared with normal pancreatic tissues (P < .0001 and P = .0004, respectively, Figure 1A). Spearman's rank test showed positive correlations between the expression of miR-148a-5p and miR-148a-3p (R = .669 and P < .0001; Figure 1B) (ie, neoadjuvant chemotherapy, metastasis or recurrence) and the expression of miR-148a-5p and miR-148a-3p (data not shown).
F I G U R E 1 Antitumor functions of pre-miR-148a in pancreatic ductal adenocarcinoma (PDAC) cell lines (PANC-1 and SW1990). A, Expression levels of miR-148a-5p and miR-148a-3p in PDAC clinical specimens and cell lines were determined by qRT-PCR. Data were normalized to RNU48 expression. B, Expression levels of miR-148a-3p and miR-148-5p were positively correlated (R = .699, P < .0001). C, Cell proliferation was determined by XTT assays 72 h after transfection with 10 nmol/L miR-148a-5p or miR-148a-3p, *P < .0001. D, Cell invasion activity was determined using Matrigel invasion assays, *P < .0001. E, Cell migration activity was determined by migration assays, *P < .0001 3.4 | Effects of miR-148a-5p and miR-148a-3p expression on cell growth, apoptosis, migration and invasiveness of PDAC cell lines Gain-of-function studies were carried out to investigate the functional roles of miR-148a-5p and miR-148a-3p. XTT assays showed significant inhibition of cell proliferation in PANC-1 or in SW1990 cells transfected with miR-148a-5p or miR-148a-3p in comparison with mock or control transfectants (P < .0001, Figure 1C). Apoptotic cell numbers (early apoptotic and late apoptotic cells) were significantly larger in miR-148a-5p or miR-145-3p transfectants than in mock or negative control transfectants ( Figure S4A,B). Western blot analyses showed that cleaved PARP expression was significantly increased in miR-148a-5p or miR-148a-3p transfectants compared with mock or negative control transfectants ( Figure S4C). In vitro assays showed that migration and invasion were significantly inhibited in miR-148a-5p or miR-148a-3p transfectants compared with mock or miR-control transfectants (each P < .0001, Figure 1D,E). These results suggested that pre-miR-148a could have an antitumor function in PDAC cells.

| Identification of candidate genes regulated by
miR-148a-5p and miR-148-3p in PDAC cells Next, we attempted to identify target genes coordinately regulated by miR-148-5p and miR-148a-3p. The strategies for narrowing down the genes targeted by miR-148a-5p and miR-148a-3p are shown in Figure 2. We carried out a combination of in silico analysis, GEO analysis and genome-wide gene expression analysis using PDAC cell lines (PANC-1 and SW1990) transfected with miR-148a-5p or miR-148a-3p (GEO accession number GSE93290).
In PANC-1 and SW1990 cells transfected with miR-148a-5p, a total of 1033 and 176 genes, respectively, were downregulated. Furthermore, the TargetScan database showed that 414 and 90 of those genes, respectively, had putative target sites for miR-148a-5p in their 3 0 -UTR. We found that there were 68 common genes targeted in both cell lines. Last, gene expression data showed that 6 of those genes were upregulated (fold-change log 2 > 1.0) in cancer tissues according to GEO database analyses (GEO accession number GSE15471).
In miR-148a-3p transfectants, a total of 629 and 250 genes, respectively, were downregulated in PANC-1 and SW1990. Furthermore, the TargetScan database showed that 186 and 121 genes, respectively, had putative target sites for miR-148a-3p in their 3 0 -UTR. We found that there were 59 common genes targeted in both cell lines. Last, 9 genes were upregulated (fold-change log 2 > 1.0) in cancer tissues by GEO database analyses (GEO accession number GSE15471).
We also checked the clinical significance of 6 genes targeted by miR-148a-5p (Table 3) and 9 genes targeted by miR-148a-3p (Table 4)  In the present study, we focused on the Pleckstrin Homology Like Domain Family A Member 2 (PHLDA2) because miR-148a-5p (passenger strand) achieved the greatest degree of downregulation ( Figure 4A, upper, right).

in PDAC cells
Negative correlations were detected between miR-148a-5p and PHLDA2 expression (R = À.495, P = .0007, Figure 5A). Both The putative target site in the 3 0 -UTR of PHLDA2 is shown in Figure S1. Luminescence intensity was significantly reduced by cotransfection with miR-148a-5p and the vector carrying the wildtype 3 0 -UTR (position 77-83), whereas transfection with the deletion vector (deletion of binding site) blocked the decrease in luminescence (P < .0001, Figure 5C).
A total of 30 specimens was evaluated by immunohistochemical staining. All tumors were of differentiated adenocarcinoma type (well, moderate and poor) and showed strong intracellular immunoreactivity. However, normal pancreatic tissue was not stained ( Figure 5D).   Our miRNA expression signature of PDAC showed that some passenger strands of miRNAs were significantly downregulated in PDAC. 8 We hypothesize that the data could help to identify miR-NAs important for the development and progression of PDAC. In the present study, we focused on pre-miR-148a strands: miR-148a-5p (the passenger strand) and miR-148a-3p (the guide strand). With this focus, we investigated the functional significance of these specific miRNAs and regulatory RNA networks in PDAC cells. In the human genome, the miR-148-family consists of 3 members that are located in different chromosomal regions: miR-148a is at 7p15.2, miR-148b is at 12q13.13 and miR-152 is at 17q21.32. 27,28 The mature sequences of the guide strands of these miRNAs are similar and the seed sequences (UCAGUGCA) are identical. 29 Most analyses have focused on guide strands of the miR-148-family in normal physiological conditions or in cancer cells. 30,31 A large number of previous studies showed that miR-148a-3p (the guide strand) was downregulated in several types of cancer (eg, gastric cancer, colon cancer, hepatocellular carcinoma, breast cancer and PDAC). [32][33][34][35][36] The miR-148a gene contains a large number of CpG islands in the promoter ( Figure S3A). In hepatocellular cancer, miR-148a is silenced by DNA hypermethylation. 37 In pancreatic cancer, a past study reported that aberrant hypermethylation of the miR-148a coding region progresses as pancreatic cancer malignancy increases. 38 We showed that hypermethylation suppresses miR-148a-5p and miR-148a-3p even in PDAC ( Figure S3B). Our present study of miR-148a-3p is consistent with previous reports. A new finding of the present analysis was that miR-148a-5p (the passenger strand) acted as an antitumor miRNA as did miR-148-3p (the guide strand) in PDAC cells. Involvement of the passenger strand of miRNA in cancer pathogenesis is a new concept in the field of miRNA research.

| DISCUSSION
In the present study, we identified oncogenic genes regulated by miR-148a-5p and miR-148a-3p in PDAC cells by using genome-wide gene expression and miRNA database analyses. Three genes (PHLDA2, LPCAT2 and AP1S3) were regulated by miR-148a-5p (passenger strand) and were identified as putative oncogenic genes in PDAC pathogenesis. Similarly, 3 genes (SAS, ENDOD1 and UHMK1) were found to be regulated by miR-148-3p (the guide strand). Analysis of these genes will contribute to the elucidation of the molecular mechanism of PDAC pathogenesis.
We focused on PHLDA2 and investigated its oncogenic functions because its expression in 2 pancreatic cancer cell lines showed the greatest suppression by miR-148a-5p (passenger strand). Moreover, the role of PHLDA2 has been controversial in other carcinomas (ie, its roles as an "oncogene" or "tumor suppressor" are unclear). 39,40 Clinical features in TCGA database of PDAC patients showed there were no significant relationships in T, N, and M of TMN stage (data not shown). However, high expression of PHLDA2 related to poor F I G U R E 4 Messenger RNA expression of target genes of miR-148a-5p and miR-148a-3p on clinical sample and directly regulation of mRNA expression by miR-148a-5p and miR-148a-3p transfectants in pancreatic ductal adenocarcinoma (PDAC) cell lines. A, Expression levels of miR-148a-5p target genes (PHLDA2, LPCAT2, AP1S3) in PDAC clinical specimens and cell lines were determined by qRT-PCR. GUSB was used as an internal control, data were normalized to GUSB expression (left), *P < .05. Gene expression was directly downregulated by transfection of miR-148a-5p mimic (right), *P < .0001. B, Expression levels of miR-148a-3p target genes (SMS, ENDOD1, UHMK1) in PDAC clinical specimens and cell lines were determined by qRT-PCR. GUSB was used as an internal control, data were normalized to GUSB expression (left), *P < .05. Gene expression was directly downregulated by transfection of miR-148a-3p mimic (right), *P < .0001 OS and DFS. Past studies showed that PHLDA2 acted as a growthsuppressive gene. 41 PHLDA2 is located on human chromosome 11p15.5, a location known for imprinting gene clusters. 42 It is well known that dysregulation of this gene cluster is associated with Beckwith-Wiedemann syndrome. 43 To investigate the functional significance of PHLDA2, knockout mice or transgenic mouse studies have been carried out. 44 In PHLDA2 null mice, placenta overgrowth was observed and overexpression of PHLDA2 resulted in placental stunting. 45 In humans, upregulation of PHLDA2 was observed in intrauterine growth restriction placentas. 46 However, the relationship between PHLDA2 expression and human cancers is not clear.
Our present study showed that PHLDA2 was overexpressed in PDAC clinical specimens and that its expression enhanced cancer cell migration and invasion in PDAC cells. Moreover, high expression  These results suggest that PHLDA2 might be a possible therapeutic target for PDAC patients.
The passenger strand, miR-148a-5p, had a potent antitumor function through targeting of cancer-promoting genes in PDAC cells. PHLDA2 was markedly elevated in PDAC, and it was involved in PDAC pathogenesis, suggesting PHLDA2 could be used as a therapeutic target in PDAC. The involvement of passenger strand miRNAs in the regulation of cellular processes is a novel concept in RNA research.
F I G U R E 6 PHLDA2 mRNA and PHLDA2 protein expression after si-PHLDA2 transfection and the effects of PHLDA2 knockdown in pancreatic ductal adenocarcinoma (PDAC) cell lines. A, PHLDA2 mRNA expression in PDAC cell lines was evaluated by qRT-PCR 72 h after transfection with si-PHLDA2-1 or si-PHLDA2-2. GUSB was used as an internal control, data were normalized to GUSB expression. B, PHLDA2 protein expression in PDAC cell lines was evaluated by western blot analysis 96 h after transfection with si-PHLDA2-1 and si-PHLDA2-2. GAPDH was used as a loading control. C, Cell proliferation was determined with XTT assays 72 h after transfection with 10 nmol/L si-PHLDA2-1 or si-PHLDA2-2, *P < .0001. D, Cell invasion activity was determined using Matrigel invasion assays, *P < .0001. E, Cell migration activity was determined by migration assays, *P < .0001 Rescue experiments: induction of PHLDA2 overexpression in pancreatic ductal adenocarcinoma (PDAC) cell lines with miR-148a-5p restoration. A, PHLDA2 protein overexpression in PANC-1 and SW1990 was evaluated by western blot analyses 72 h after 0.5 lg/well transfection with PHLDA2 cDNA plasmid (middle bands). PHLDA2 protein overexpression was attenuated 72 h after cotransfection with 0.5 lg/well PHLDA2 cDNA plasmid and 10 nmol/L miR-148a-5p mimic (right bands). GAPDH was used as a loading control. B, Cell migration activity was assessed 72 h after cotransfection with 0.5 lg/well PHLDA2 cDNA plasmid and 10 nmol/L miR-148a-5p mimic, *P < .0001. Cell invasion activity was determined using Matrigel invasion assays 72 h after cotransfection with 0.5 lg/well PHLDA2 cDNA plasmid and 10 nmol/L miR-148a-5p mimic, *P < .0001