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A miRNA microarray platform (Agilent Human miRNA Microarray Kit version 14.0) covering a total of 887 human miRNAs was performed to compare miRNA profiles between 4 pairs strictly matched tumor tissues. They were strictly matched by gender, age (± 10 years), CA 19-9 (-: < 37 U/L / +: â‰¥37 U/L), TNM stage and histological differentiation. We found the majority of miRNAs, including 66 that have been reported to associate with pancreatic cancer (Table 3), were not significantly differentially expressed between these two subtypes of pancreatic cancer. The result was not surprising because all the 4 paired tissues were early stage PDACs.
Of note, we found 6 miRNAs that were significantly differentially expressed (P < 0.05) (Figure 5). There were up-regulated miR-126*, miR-455-3p and miR-375 expressions, and down-regulated miR-501-3p, miR-320d and miR-320b expressions in pancreatic body/tail cancer compared with their matched pancreatic head cancer (Table 3). If we take fold change and Rank Sum difference into consideration, miR-501-3p was the only candidate for further study with P < 0.05, fold change > 2 and largest Rank Sum difference. But we also take miR-375 into further verification because it was reported to be a potent tumor suppressor in pancreatic cancer .
Down-regulation of miR-501-3p and up-regulation of miR-375 in pancreatic body/tail cancer
A total of 15 paired tissues were used for the subsequent quantitative reverse transcription real-time polymerase chain reaction (qRT-PCR) validations. Clinical and histopathologic variables for the patients included in this study were listed in Table 4.
We found the result was consistent with the one from miRNA microarray profiles that miR-501-3p expression was significantly decreased (P = 0.004, fold change = 2.99) whereas miR-375 expression was increased (P = 0.040, fold change = 1.88) in pancreatic body/tail cancer than those in pancreatic head cancer (Figure 6).
From the up-regulation of miR-375, a tumor suppressor in pancreatic body/tail cancer, we hypothesized that pancreatic body/tail cancer might be 'less' malignant than pancreatic head cancer. However, the role of miR-501-3p in cancer is definitely unknown and should be further evaluated.
Down-regulation of miR-501-3p contributes to low-risk of tumor recurrence
There were 9 out of 30 patients (30.0%) who were found tumor recurrence during the follow up (2.3 ± 0.8 years) and the majority (8/9, 88.9%) was occurred within 1 year after operation. The recurrence rate was significantly lower in pancreatic body/tail cancer than that in pancreatic head cancer (13.3% vs. 46.7%, P = 0.046). Furthermore, from the Kaplan-Meier survival curves, we could find that both patient cumulative survival and tumor-free survival were higher in pancreatic body/tail cancer than pancreatic head cancer (Figure 7).
To determine the role of miRNAs in tumor recurrence, we then carried out COX regression analysis and found that high expression of miR-501-3p (P = 0.034; Risk Ratio = 2.322, 95%CI: 1.068-5.048) but not miR-375 was significantly associated with tumor recurrence after surgery. Patients with high expression of miR-501-3p showed a significantly lower tumor-free survival than those with low expression of miR-501-3p (P = 0.031, Figure 8A). In addition, we compared the miR-501-3p expression between patients with and without tumor recurrence and found a significantly higher expression of miR-501-3p in those with tumor recurrence (P = 0.017, Figure 8B). The data indicated that down-regulation of miR-501-3p in pancreatic body/tail cancer may contribute to low-risk of tumor recurrence.
miR-501-3p leads to the morphology change of pancreatic cancer cells
To further confirm our speculation, we performed in vitro study to evaluate the role of miR-501-3p in PDAC cells. We tranfected two frequently used pancreatic cancer cell lines (Panc-1 and colo357) with miR-501-3p Mimics and Inhibitors. We also used Mimic and Inhibitor Negative Control-transfected samples as a baseline for evaluation of the effect of the experimental miRNA Mimics and Inhibitors. Cell proliferation, viability, invasion, chemo-resistance and TRAIL-sensitivity were compared between Mimic group and its negative control, and between Inhibitor and its negative control, respectively. Each experiment was repeated at least 3 times under the same condition. Because miRNA mimics and miRNA inhibitors have different chemical structures, for example, miRNA mimics are modified double-stranded RNAs that mimic endogenous miRNAs, while miRNA inhibitors are modified single-stranded RNA molecules that can specifically bind to and inhibit endogenous miRNA. We will present the results from Mimics and Inhibitors separately.
miR-501-3p and proliferation
PDAC cells were transfected with miR-501-3p Mimics, Inhibitors and their negative controls. After tranfection for 72 hours, cells were observed under microscope. We found some cells floating in the central of well (most of them could be dead cells). Compared with their respective negative controls, Mimic group showed less dead cells but Inhibitor group had more dead cells both in Panc-1 and Colo357 (Figure 9). Therefore, in the next part, we would analyze the cell death rate between the different groups.
Then we trypsinized the cells from 6-well plates and counted them according to the 'accurate counting for 6-well' procedure. We found significantly fewer cells in the Mimics group than its negative control (P = 0.002 for Panc-1, P = 0.013 for colo357) but no significant difference was found between Inhibitor group and its negative controls (Figure 10). This may be because that we washed the cells with PBS before trypsinization, the dead cells could be removed from the following cell counting. The result indicates that miR-501-3p may reduce proliferation of PDAC cells.
miR-501-3p and cell death
Deregulation of proliferation, together with a reduction of cell death, is both necessary and sufficient for tumor development, progression, and therapy-resistance. From the observation under microscope, we suspected that miR-501-3p may lead to reduced proliferation but also decreased cell death. Therefore, we performed flow cytometry (FCM) to assess the apoptosis and death rates in PDAC cells.
After tranfection for 72 hours, Panc-1 cells were double stained with Annexin V-FITC and PI (Sigma, USA). Viable cells are Annexin V-FITC (-) and PI (-). Cells in early apoptosis (membrane integrity is present) are Annexin V-FITC (+) and PI (-), while cells in late apoptosis or already dead are FITC Annexin V (+) and PI (+). We found that significant more Annexin V-FITC (+) and PI (-) cells in the Inhibitor group than its negative control (P = 0.010), which indicates that miR-501-3p may reduce PDAC cells early apoptosis (Figure 11). However, this assay is limited by not distinguishing between cells undergoing apoptotic death versus those dying in a necrotic pathway.
Caspase activation plays a crucial role in cells for apoptosis. There are two main caspase-activated pathways for apoptosis: the intrinsic and the extrinsic. Caspase-8 is an initiator caspase in the extrinsic apoptotic pathway and can cleave its inactive pro-forms to effector caspases. It can activate downstream effector caspases independent of mitochondria, or it can cleave Bid and activate the intrinsic apoptotic pathway . Therefore, we tested the apoptosis-associated protein caspase-8 in different groups and found that the cleaved caspase-8 was weaker in Mimics group than its negative control, but much stronger in inhibitors group than its negative control, particularly in Panc-1 cells (Figure 12).
miR-501-3p and invasion
Cell invasion is crucial steps in metastasis, and the ability of cancer cells to undergo invasion allow them to enter lymphatic and blood vessels for dissemination . Tumor cell motility is the hallmark of invasion and is an initial step in metastasis, and extracellular matrix (ECM) adhesion is a key mediator in cell motility and in the process of metastasis . In the first part of our study, we found that miR-501-3p might associate with metastasis/recurrence of pancreatic cancer. Therefore, evaluating the effect of miR-501-3p on invasion ability in vitro could be a strong evidence for proving the clinical finding.
PDAC is characterized by a pronounced fibrotic reaction composed primarily of type I collagen. PDAC cells have been shown to respond to collagen I by becoming more motile and invasive although collagen I functions as a barrier to invasion . Here, we used a type I collagen coated Transwell chamber for testing the invasion ability of PDAC cells.
After tranfection for 72 hours, 5.0 Ã- 104 cells/well PDAC cells in reduced serum medium (0.5%) were seeded into the upper chamber of 24-well invading Collagen I-coated Transwell chambers. After incubation for 24 hours, the invasion ability was assessed by crystal violet assay. We found much higher invasion rate in the Mimics group (P = 0.017 for Panc1, P = 0.068 for Colo357) and remarkably less invasion rate in the Inhibitors group (P = 0.003 for Panc1, P = 0.001 for Colo357) compared with their respective negative controls, which indicates that miR-501-3p may promote invasion in PDAC cells (Figure 13).
This result was consistent with the clinical finding, which suggests that pancreatic body/tail cancer is associated with a reduced miR-501-3p expression and consequently a less invasion ability compared with pancreatic head cancer. The possible mechanism might be explained by the decreased E-Cadherin level in the Mimics group and increased E-Cadherin level in the Inhibitors group, which would be shown in the following part.
miR-501-3p and chemo-resistance
Chemo and/or radiotherapy remain the first choice of palliative treatment for PDAC, and promote cancer cell death primarily by the induction of apoptosis. Gemcitabin is now widely used as the first line chemotherapy for the end stage pancreatic cancer. To better evaluate the role of miR-501-3p in the chemo-induced apoptosis, we tested the cell viability in both chemo-resistant cell line (Panc-1) and chemo-sensitive cell line (Colo357).
PDAC cells were transfected with miR-501-3p Mimics, Inhibitors, or their negative controls for 72 hours and then seeded into the 96-well plates. Gemcitabin (1 ug/ml) was given in the treatment group when the cells had settled down. After 48 hours, the cell viability was tested by crystal violet assay (Figure 14). We found cell viability was much higher in Panc-1 cells than that in colo357 cells, which proved again that Panc-1 were gemcitabin-resistant cell line while colo357 was highly sensitive to gemcitabin treatment. However, no significant different was found between Mimics and its control or between Inhibitors and its control in both the two cell lines. It seems that miR-501-3p may be not associated with the chemo-resistance in both high and low chemo-sensitive cell lines.
miR-501-3p and TRAIL-induce apoptosis
Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a member of the tumor necrosis factor superfamily, selectively induces apoptosis in a variety of human cancer cell lines, with no toxicity against normal tissues . TRAIL induces programmed death in various cancer cells through its interaction with the death receptors, TRAIL-receptor 1 (TRAIL-R1 or DR4) and/or TRAIL-receptor 2 (TRAIL-R2 or DR5), which contain the intracellular death domain (DD) that is essential for the apoptosis signal. The decreased expression of death receptors TRAIL-R1 and TRAIL-R2 or increased expression of anti-apoptotic protein in cancer cells is involved in TRAIL resistance . However, in PDAC, death receptors such as TRAIL-R1/-R2 also show non-apoptotic functions leading to strong pro-inflammatory responses, which are related to survival, proliferation, migration and invasion . In this study, we evaluate the effect of miR-501-3p on the TRAIL-induced death and also test the expression of TRAIL-R1 and TRAIL-R2 after regulation of miR-501-3p levels.
After transfection for 72 hours, 1.0 Ã- 104 cells/well PDAC cells were seeded into 96-well plates. TRAIL (100 ng/ml) was added into the treatment group when the cells had settled down. After 24 hours, the cell viability was tested using crystal violet assay. We found no significant difference between mimic group and its control, or inhibitor group and its control in Colo357 cells. However, in Panc-1 cells, Mimic group showed high viability than its control (P = 0.027) (Figure 15).
To further study the effect of miR-501-3p on TRAIL signaling pathway, we also tested the TRAIL-R1 and TRAIL-R2 protein levels in different groups (Figure 16). Interestingly, we found decreased expression of TRAIL-R1 and TRAIL-R2 in the Mimics group compared with its negative control in Panc-1 cells but not in Colo357 cells. This result was consistent with the TRAIL treatment experiment that decreased TRAIL-sensitivity after up-regulation of miR-501-3p may due to the decreased TRAIL-R1 and TRAIL-R2 levels in Panc-1 cells.
miR-501-3p targets cell adhesion molecules
miRNA target search
The target genes for miR-501-3p have not been reported so far. Therefore, Open-sourced software using different algorithms based on sequence complementarily, such as miRBase (http://www.mirbase.org/), TargetScan (http://www.targetscan.org), PicTar (http://pictar.mdc-berlin.de/), DIANA-microT (http://diana.cslab.ece.ntua.gr/microT/) and miRanda (www.microrna.org), were used for miRNA target predictions.
MiR-501-3p has not been enrolled in PicTar database. We found 223 conserved targets from TargetScan (TargetScanHuman 6.0), 113 targets from DIANA-microT (Diana microT- v4) with a threshold of 0.5 for high precision, 1676 targets for miRanda with "good" mirSVR score (<âˆ’0.1) and seed regions. A total of 28 target genes (AFF4, ARID5B, CDH5, CIT, CPEB2, CSDE1, DCUN1D5, GAN, JHDM1D, KPNA4, NONO, NR1D2, ODZ2, PCDH9, PLD3, PPP2R5E, PPP3CA, PPP3CC, PTBP2, RCC2, RNF144A, STIM2, TARDBP, TRERF1, UBE2E2, ZFHX4, ZIC4, ZMYM4 and ZRANB2) were overlapped among all the 3 databases. Of the 28 genes, CDH5 , JHDM1D , PCDH9 , PPP3CA , PPP3CC , PTBP2 , RNF144A and STIM2 were reported to be associated with cancer (Table 5).
Differentially expressed E- and VE-Cadherin in various of PDAC cell lines
Cadherins are a class of type I transmembrane proteins. They play important roles in cell adhesion, ensuring that cells can bind together in tissues. Because three of cadherins (cadherin 5, protocadherin 9 and protocadherin 17) have been listed as the potential direct targets of miR-501-3p (http://www.targetscan.org), we paid great attention and interesting in the association between miR-501-3p and cadherins.
E-Cadherin is the most well studied member of the cadherin family. As is well known, activation of EMT is crucial for the dissemination and invasion of cancer cells. Loss of epithelial differentiation and acquisition of mesenchymal phenotype allows cancer cells to detach from the primary tumor mass and disseminate into the surrounding. The most important event of EMT is loss of E-Cadherin, which demonstrates a prerequisite for epithelial tumor cell invasion .
VE-Cadherin (CD144) is traditionally considered a strictly endothelial specific adhesion molecule located at junctions between endothelial cells. It appears to be a major adhesive protein involved in the control of endothelial cell-cell contact and is of vital important for maintaining endothelial barrier integrity and homoeostasis . The disruption of the endothelial integrity, indicated by a sharply reduced VE-Cadherin expression in the cell surface, is associated with an increased invasion of cancer cells .
Although VE-Cadherin has recently been found in some non-endothelial cells such as breast cancer cells , it is still unknown whether VE-Cadherin is expressed in PDAC cells and its function. Because CDH5 is a potential direct target gene of miR-501-3p, we will focus on its encoded protein VE-cadherin in this study.
We test the two proteins in several frequently used PDAC cell lines, including Capan-1, Capan-2, Panc-1, PancII-1, Panc89, BxPc-3 and Colo357. We found that Colo357 and Panc89 expressed VE-Cadherin, and only colo357 cells expressed high levels of VE-Cadherin protein, while the other were VE-Cadherin (-) expressed cell lines (Figure 17). According to the ultrastructural grading system , both Colo357 and Panc89 are the Grade 2 cell lines. Neither the less malignant grade 1 cell lines (Capan-1 and Capan-2) nor did the more malignant grade 3 cell lines (Panc-1, PancII-1) express VE-Cadherin. The role of VE-Cadherin in the PDAC carcinogenesis and tumor progression should be further studied.
Compared with VE-Cadherin, all studied PDAC cell lines expressed E-Cadherin. Only Panc-1 showed very weak expression but all other cells presented very high E-Cadherin protein levels (Figure 17). E-Cadherin is a marker for EMT and can function as a parameter of 'malignancy'. According to the ultrastructural grading system , higher grade of PDAC cells were associated lower expression of E-Cadherin (Figure 17). Taken together, Colo357 and Panc-1 are two special cell lines that highly express VE-Cadherin and lowly express E-cadherin, respectively, compared with other PDAC cell lines. We would use this two cell lines for the further study.
miR-501-3p targets E- and VE-Cadherin
To further verify the relation between miR-501-3p and E-Cadherin and between miR-501-3p and VE-Cadherin in pancreatic cancer, we tested the protein levels after regulation of miR-501-3p in Colo357 and Panc-1 cells. We found reduced E-Cadherin protein expression in the Mimics group and increased protein expression in the Inhibitors group compared with their respective negative controls in both the two cell lines (Figure 18). In Colo357, the only high express VE-Cadherin cells, both E-Cadherin and VE-Cadherin expressions markedly decreased after transfection with miR-501-3p Mimics but increased with miR-501-3p Inhibitors compared to the negative controls (Figure 18). It suggests that miR-501-3p targets E-Cadherin and VE-Cadherin. Because there is a direct role for E-Cadherin in the suppression of tumor invasion , the decreased E-Cadherin in the Mimics group might explain the increased invasion ability in both Colo357 and Panc-1 cells.
We further detected the miR-501-3p targeted E-Cadherin protein expression in tissue samples using by western blot (Figure 19) and found higher expressed E-Cadherin protein in pancreatic body/tail cancers compared to their paired pancreatic head cancers. However, because of the limited amount of tissue samples, we did not test the VE-Cadherin in these tissues.
Target VE-Cadherin verification
CDH5 is the leading predicted target genes of miR-501-3p. Its encoding protein VE-Cadherin were well known to be related to tumor invasion and metastasis and from the previous Western Blot analysis we have found that miR-501-3p could target VE-Cadherin. Therefore, CDH5 was chosen for the further verification (Table 6).
We cloned the 3â€²-UTR region of CDH5 (WT-3â€²-UTR) or its mutant (MUT-3â€²-UTR) downstream of the firefly luciferase reporter gene, and then co-transfected with miR-501-3p Mimics/Inhibitors or its negative control into HUVEC cells. The luciferase activity of the WT construct of CDH5 3â€²-UTR was significantly reduced/increased in the presence/absence of miR-501-3p compared with the negative control (P< 0.05). However, such an effect was not observed in the MUT construct of CDH5 3â€²-UTR (Figure 20). These data suggest that a direct and specific interaction of miR-501-3p on CDH5 3'UTR.
Pancreatic body/tail cancer is associated with better prognosis
The survival rate of pancreatic cancer is the 'worst' of all major cancers (e.g. lung cancer, liver cancer, gastric cancer and colon cancer) and usually depends on the tumor stage. Stage I-II is commonly considered as the stage during which the surgical treatment is most effective. Although there have been continuing improvements in surgery, patients with early stage pancreatic cancer still had a high recurrence rate after curative resection, as was shown by this study that a third of all patients suffered tumor recurrence during the follow up.
Besides tumor stage, the prognosis of pancreatic cancer is also associated with the tumor site. The theory of different mechanisms in carcinogenesis of tumors at different locations and the importance of subsite division have been supported by several pioneering studies. In colon cancer, a number of studies have demonstrated that right- and left-sided tumors exhibit different genetic, biological and demographical characteristics and risk factors, suggesting that the carcinogenesis and tumor progression of colon cancer may differ with tumor localization . In pancreatic serous cystic neoplasm, tumor location in the head of pancreas was independently associated with local invasiveness .
For PDAC, pancreatic body/tail cancer has been long-term considered as a 'more' malignant subtype compared with pancreatic head cancer. Because clinically, we always see patients with pancreatic body/tail cancer would more likely to be at advanced stage. The symptoms, such as jaundice and nausea, which could be a signal for early diagnosis, would rarely happen in patients with early stage pancreatic body/tail cancer. Consequently, pancreatic body/tail cancer is commonly associated with a dismal prognosis. The overall survival is much lower in pancreatic body/tail cancer than that in pancreatic head cancer.
However, recently, a large sample study from SEER registries of Unite States including information from 43,946 patients revealed that pancreatic body/tail cancer had a significant higher survival than pancreatic head cancer if both tumors were at the same local-stage. If we dig out more data, we could find that the old database of Unite States (1985-1995) also presented a higher survival rate for local-stage pancreatic tail cancer compared with local-stage pancreatic head cancer . Then we start to think whether the pancreatic body/tail cancer is 'more' malignant than pancreatic head cancer in nature. However, the data from both clinical and genetical comparison between the pancreatic head cancer and body/tail cancer is that limited. Given the complex and heterogeneous nature of PDAC, it is reasonable to hypothesize that these two subtypes maybe separated from each other and differ in phenotype and genotype. Furthermore, we suppose that the two subtypes of PDAC may be differing in malignancy.
This study performed the first comparison between pancreatic body/tail and head cancers using strictly matched early stage cancers. We found that patients with early stage pancreatic body/tail cancers presented higher survival rate than those with paired pancreatic head cancers. This finding was consistent with the previous studies mentioned before. Furthermore, we found patients with early stage pancreatic body/tail cancers had a lower tumor recurrence rate after curative resection.
Tumor recurrence is defined as the return of tumor after radical treatment and after a period of time during which the tumor cannot be detected. There are three types of tumor recurrence: local recurrence (the same tumor occurs where it started), regional recurrence (the same tumor occurs in the lymph nodes near the place it started), distant recurrence which is also called metastatic recurrence (the same tumor occurs somewhere else in the body, distance from where it started). The mechanism of tumor recurrence is still unclear. The well-known risk factors are advanced tumor stage, poor histological differentiation and high malignant tumor subtype. In addition, surgery itself maybe a facilitator of the recurrence/metastatic process . To some extent, high tumor recurrence rate could be a marker for high malignant potential, especially under the condition in this study that the same surgeon performed all operations, and tumors were at the same stage and histological differentiation. These results indicates that pancreatic body/tail cancer might be 'less' malignant than pancreatic head cancer.
Downregulation of miR-501-3p in pancreatic body/tail cancer indicates a low invasive phenotype
miRNA profiles in pancreatic cancer
miRNAs are non coding small RNAs, suppressing gene expression or inhibiting translation via binding complementary sequences in the 3'UTR of message RNA (mRNA)s. miRNAs regulate biological processes including cell proliferation, differentiation, apoptosis, development, metabolism and neoplasm transformation . It is likely that the majority of human genes are regulated by miRNAs. It has been reported that the expression pattern of miRNAs rather than mRNAs are surprisingly informative, highly accurate in reflecting the developmental lineage and differentiation state of the tumors . The discovery of miRNAs offers a new opportunity to uncover the aberrantly expressing cellular pathways during the pathognomonic carcinogenetic events for all human cancers. Comparison of miRNA expression profiles between normal and tumor tissue/fluid samples provides a novel way for the identification of tumor biomarkers, and finally clinical diagnosis of tumors.
In miRBase database, more than 700 human miRNAs have been found and more miRNAs are likely to be found. As the development of new technologies, microarray-based approaches has allowed genome-wide, high throughput screening for novel candidates involved in the pathogenesis of a various of cancers. Aberrant expression of miRNAs has been detected in many human diseases including PDAC. When searching the Pubmed using keywords 'pancreatic cancer' and 'miRNA', we found that a total of 224 literatures are involved (http://www.ncbi.nlm.nih.gov/pubmed). Among the 224 literatures, 204 and 187 were published in the past 5 and 3 years, respectively. It is becoming a very hot and promising filed for identifying the PDAC associated miRNAs expression pattern.
Current clinical screening and diagnostic management of patients with PDAC is mainly based on imaging techniques such as contrast-enhanced multi-detector CT scan and Magnetic resonance imaging. However, due to the relative low sensitivity and specificity, and the use of radiation, such techniques may only be used for the evaluation of existing of solid tumors (> 2 mm). Therefore, there is an immediate need for identification of novel biomarkers to predict the development of PDAC and early diagnosis.
During the last 5 years, a number of studies have compared the miRNA expression profiles either between PDAC and normal pancreas, or between PDAC and chronic pancreatitis, and even between PDAC and different stages of PanINs, using blood or tissue samples . Recently, considering a high volume (~1.5 L) of pancreatic juice production and excretion into the bowel, it has been hypothesized that precancerous or early cancer-related molecular changes may be detectable in feces of pancreatic cancer patients. A specific miRNA pattern has been found in stool as miRNA biomarkers for PDAC screening . Taken together, advanced technologies have helped us to find a series of frequently dysregulated miRNAs in PDAC, such as miR-21, miR-155 and miR-196a .
Different miRNA expressions between pancreatic head and body/tail cancers
Although a number of studies have identified PDAC-associated miRNA expression pattern, there is still a lack of comparison between pancreatic head and body/tail cancers. We can only find indirect evidence from the comparison between two cell lines as typical representatives originating from pancreatic head cancer (Panc-1) and body/tail cancer (MIA PaCa-2), respectively. Studies have shown specifically altered miRNAs between PDAC cell lines and human pancreatic ductal epithelium control cell lines, and between subgroups of PDAC cell lines with different invasion or metastasis properties . From these studies covering a large panel of miRNAs, we could find several differentially expressed miRNAs between Panc-1 and MIA PaCa-2, such as miR-10b, miR-15b, miR-18a, miR-21, miR-22, miR-125, miR-155, miR-181 and miR-196a .
For the first time, we performed miRNA microarray to provide direct evidence of the different miRNA patterns between pancreatic head and body/tail cancers. It is not surprising that the expressions of the majority of miRNAs in the microarray platform were similar between pancreatic body/tail cancer and head cancer because both the two subtypes are essentially PDAC. However, as expected, there were also significantly differentially expressed miRNAs between the two subtypes. Among 887 detected human miRNAs, 6 were found either remarkably up-regulated or down-regulated in pancreatic body/tail cancer compared with those in pancreatic head cancer. According to the selection criteria, the two most statistically significant miRNAs (miR-501-3p and miR-375) were further verified by qRT-PCR. We hypothesized that miR-501-3p and miR-375 may encode the diversity between pancreatic head and body/tail cancer.
miR-375 was firstly thought to be only expressed in pancreatic cells (islet Î²-cells and non-Î²-cells) , but increasing evidence shows that it could be also expressed in other tissues . miR-375 has great impact on pancreatic islet cell viability and function, and plays important role in the regulation of insulin secretion and glucose metabolism, and acts as a key determinant of blood glucose homeostasis . Furthermore, miR-375 is also closely associated with pancreatic cancer. Studies have demonstrated a down-regulation of miR-375 in pancreatic cancer . It can act as a tumor suppressor gene and targeted several oncogenes such as PDK1 (a master regulator of oncogenic phosphoinositide-3 kinase signaling), Janus kinase 2 (JAK2) and insulin-like growth factor 1 receptor (IGF1R), resulting in decreased cancer cell growth, viability and invasion . Up-regulation of miR-375 in pancreatic body/tail cancer could be an evidence for the 'less' malignancy of pancreatic body/tail cancer than pancreatic head cancer.
Unlike miR-375, miR-501-3p has definitely not reported yet in human cancers. It is also not reported to highly differentially express between PDAC and normal pancreas. Therefore, the role of miR-501-3p in pancreatic cancer needs further research.
miR-501-3p is associated with high invasiveness
Our results demonstrated that miR-501-3p was significantly associated with pancreatic cancer recurrence after surgery. High expression of miR-501-3p was a risk factor of tumor recurrence, which suggests that miR-501-3p may be a potential biomarker for tumor recurrence after radical therapy. These results hinted that pancreatic body/tail cancer could be characterized by a lower expression of miR-501-3p, which contributes to a lower recurrence risk, compared with pancreatic head cancer.
However, as mentioned before, the role of miR-501-3p in PDAC has not been reported yet and little is known about its molecular targets. We performed in vitro study to verify whether miR-501-3p could promote invasion ability in PDAC cells. In both two frequently used cell lines, the invasion rate significantly increased in the up-regulation of miR-501-3p group and remarkably decreased in the down-regulation of miR-501-3p group compared with their respective negative control groups. Moreover, because miR-501-3p targets cell adhesion molecules as has been reported in the '15th Annual Conference of Rehabilitation in Multiple Sclerosis' (http://registration.akm.ch/einsicht.php?XNABSTRACT_ID=116538&XNSPRACHE_ID=2&XNKONGRESS_ID=126&XNMASKEN_ID=900), we tested the well-known epithelial cell-cell adhesion molecular E-Cadherin among different groups to further understand the mechanism of increased invasiveness via up-regulation of miR-501-3p. The results revealed that up-regulation of miR-501-3p led to a decreased expression of E-Cadherin, while down-regulation of miR-501-3p resulted in an increased expression of E-Cadherin in both the two cell lines. E-cadherin is a transmembrane protein localized at the adherens junctions of the epithelial cell basolateral surface and plays a key role in epithelial morphology maintenance. Loss of E-cadherin expression is a well-recognized marker of EMT and promotes PDAC progression and invasion . Our results from in vitro study provided evidence for the clinical part that down-regulation of miR-501-3p in pancreatic body/tail cancer may lead to a less invasive/metastasis phenotype through up-regulation of E-Cadherin.
In addition, we have proven that CDH5 is a direct target gene of miR-501-3p. VE-cadherin, also known as Cadherin-5, is a member of cadherin superfamily, expressed by vascular endothelial cells. It has emerged as a critical player involved in maintaining endothelial barrier integrity and homoeostasis . More importantly, VE-cadherin-mediated permeability plays a critical role for carcinogenesis including tumor induced angiogenesis and inflammation. It has been demonstrated that tumor cell-secreted VEGF increased endothelial permeability in association with decreased VE-cadherin localization at the plasma membrane . Disruption of VE-cadherin and subsequently ensuing vascular permeability strongly supports tumor metastasis . PDAC cells binding can induce focal disappearance of VE-Cadherin from endothelial cell junctions and facilitate cancer invasion . In the present study, we found miR-501-3p directly targeted VE-cadherin and the down-regulation of miR-501-3p led to an increasing in VE-cadherin expression in a PDAC cell line (Colo357). We hypothesize that down-regulation of miR-501-3p in pancreatic body/tail cancer may associate with increased expression of VE-cadherin, and subsequently a better-preserved 'vascular barrier' to prevent tumor metastasis.
However, the function of VE-Cadherin in PDAC is still unclear. VE-Cadherin is traditionally been considered restrict in endothelial cells. Here, we provided the first evidence that PDAC cells (Colo357 and Panc89) could also express the vascular endothelial specific molecular. Only some of the 'moderate' malignant PDAC cells expressed the endothelial-marked molecular but neither the more nor the less 'malignant' cells expressed VE-Cadherin. The effect of VE-Cadherin on PDAC cells needs further research. For this project, the expression of VE-Cadherin in tumor tissues would be evaluated in the future. Because of the small amount of the early stage PDAC tissue samples, we are now collecting the new samples for immunostaining of VE-Cadherin in tumor tissues.
miR-501-3p reduces proliferation and apoptosis
Besides invasion, our in vitro study also demonstrated that miR-501-3p could reduce proliferation and apoptosis, although the mechanism still needs further explore. We found higher caspase-8 expression in Panc-1 cells with down-regulation of miR-501-3p than its negative control, and lower cleaved caspase-8 expression after up-regulation of miR-501-3p than its negative control. Caspase-8 is engaged in both intrinsic (mitochondria-mediated) pathway and the extrinsic (death receptor-mediated) pathway. In the extrinsic pathway, death receptors (e.g. Fas/Apo-1/CD95, TRAIL-R1, TRAIL-R2) interact with the tumor necrosis factors, leading to an activation of caspase-8, which could directly activate downstream effector caspases, causing apoptosis . In addition, caspase-8 also could cleave Bid to activate intrinsic apoptotic pathway . Our results indicated that caspase-8 might associate with miR-501-3p-induced decreased apoptosis in PDAC. Moreover, we found that miR-501-3p could reduce the expression of death receptors, TRAIL-R1 and -R2, and consequently lead to more resistant to TRAIL-induced apoptosis. That may be another explanation for the miR-501-3p-induced decreased apoptosis.
Our previous study has revealed that TRAIL-R1 and TRAIL-R2 show not only apoptotic function but also non-apoptotic functions leading to strong pro-inflammatory responses, which are related to survival, proliferation, migration and invasion . Pancreatic cancer cells can produce endogenous TRAIL, which binds to TRAIL-R1 or TRAIL-R2 and activate the non-apoptotic pathways to promote tumor progression. In another project, to find the novel non-apoptotic role of TRAIL-R1 or TRAIL-R2, we used siRNA to knockdown either TRAIL-R1 or TRAIL-R2 in Panc-1 cells. We found that down-regulation of TRAIL-R1 or TRAIL-R2 could lead to a sharp decrease of the expression of miR-370 (unpublished data), which directly targets transforming growth factor (TGF)-Î² receptor II and involves in the TGF-Î² signaling pathway . It indicates that miR-501-3p decreases the expression of TRAIL-R1 and R2, and consequently leads to an increased activation of TGF-Î² signaling pathway, which could enhance the invasion ability of PDAC cells .
So far, not enough attention has been paid to the diversity between pancreatic head and body/tail cancers. Only a few studies have focused on the difference and the overall information is limited. The current clinical data support higher incidence and easier detection of pancreatic head cancer compared with pancreatic body/tail cancer. However, for tumors at the local-stage, pancreatic head cancer has a lower survival than pancreatic body/tail cancer. Although the previous reports included large cohorts of patients, the evidence is still not so convincing. Because that there is a lack of strictly matched cohorts between the two subtypes of PDAC. The two groups of patients should be matched not only by tumor stage, but also by other well-known prognostic factors such as age, gender, race, tumor markers and histological differentiation.
The frequently used PDAC cell lines Panc-1 and MIA PaCa-2, which are matched by donor age (±10 years), tumor stage, histological differentiation and ultrastructural features, to some extent might represent pancreatic head cancer and body/tail cancer. Compared to MIA PaCa-2, Panc-1 has a greater ability of ECM adhesion and cell invasion. Furthermore, Panc-1 exhibits a 'more' oncogenic profile than MIA PaCa-2.
This study, firstly, compares pancreatic head cancer and pancreatic body/tail cancer using strictly matched early stage PDAC tissue samples. We demonstrate that miR-501-3p and miR-375 are significantly differentially expressed between pancreatic head and body/tail cancers, and may encode the diversity. Furthermore, down-regulation of miR-501-3p in pancreatic body/tail cancer contributes to a low tumor recurrence rate after radical surgery therapy compared with pancreatic head cancer. miR-501-3p could be act as a potential prognostic marker for tumor recurrence. A limitation of the clinical part is the relative small study cohort. We have collected only 15 pairs of pancreatic head and body/tail cancers during the last 3 years in one of the largest Hepatobiliary Pancreatic Surgery Department of P.R. China. Four pairs and 11 pairs were used for miRNA microarray and qRT-PCR verification, respectively. A total of 30 patients was included for survival analysis. Nevertheless, considering the relative low incidence of pancreatic body/tail cancer and more importantly, the difficulty in strictly matching of age, gender, tumor marker, tumor stage and tumor differentiation between the two groups, the study should have good value. We are collecting more matched samples for further verification.
In vitro study, we prove that miR-501-3p can promote invasion in PDAC cells, possibly via decreasing the expression of E-cadherin and VE-cadherin. Furthermore, more miR-501-3p-induced morphology change of PDAC cells has been found, such as proliferation and apoptosis. However, the detailed mechanism needs further clarify.
In conclusion, we say that diversity exists between pancreatic head and body/tail cancers, in which the pancreatic body/tail cancer might be 'less' malignant than the pancreatic head cancer. Lower levels of miR-501-3p might contribute to a less invasive phenotype and lower-risk of tumor recurrence/metastasis in pancreatic body/tail cancer than pancreatic head cancer. These findings confirm the importance of subsite division and support the development of individual treatment strategies.
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