Aberrant MicroRNAs Expression Patterns in Pancreatic Cancer and Their Clinical Translation

Biosynthesis of miRNAs

Biosynthesis of miRNAs is a multi-step process, involving both nuclear and cytoplasmic components. In brief, miRNAs are first transcribed by RNA polymerase II into large RNA precursors called pri-miRNAs, ^{12 –14} which are then processed by an RNase III enzyme (Drosha) and its cofactor (Pasha) into a 70-nucleotide pre-miRNA. ^{15 –17} This product is exported into the cytoplasm by the Exportin-5/Ran-GTP complex, ^{18 –21} where it is processed by another enzyme complex comprising Dicer, TRBP, and Argonuate 2 ^22,23 to form a miRNA duplex that is then cleaved to produce a single-stranded mature miRNA. This mature miRNA is then incorporated into an RNA-induced silencing complex (RISC), which binds the 3'-UTR of mRNA, ^25,24 leading to relatively defined outcomes based on the degree of binding complementarity. A perfect fit invariably results in target mRNA cleavage, whereas an imperfect fit usually results in translational repression or reduction in target mRNA concentration. ²⁶ The majority of interactions between miRNA and mRNA in animals are only partially complementary, so translational inhibition predominates. ²⁷ Wu et al. have shown that mRNA decay brought about by the deadenylation of target mRNA has been shown using miR-125b and let-7 as representative miRNAs. ²⁸ Interestingly, Dong et al. reported that miR-12 can up-regulate Bcl-2 via a direct interaction. ²⁹ However, in most cases, miRNAs negatively suppress their target mRNAs.

Aberrant miRNA Expression Patterns in PC

In recent years, a number of approaches have been described to quantify miRNA levels. ^{30 –37} These approaches have revealed distinct cell- and tissue-specific miRNA expression in PC specimens as compared with normal cells and tissues. The earliest report regarding pancreas was that miRNAs cloned from pancreas tissue (miR-375 and miR-376) were expressed at higher levels in mouse pancreas and pancreatic islet cells compared with mouse brain, heart, and liver tissues. ³⁸ A subsequent study found that expression of miR-376 precursor in the PC cell line Panc-1 was among the highest of the cell lines studied, while expression of miR-375 in the two PC cell lines studied did not differ from the other cell lines. ³⁹ In a recent study, the expression levels of specific miRNAs identified by microarray analyses were examined in a panel of 15 PC cell lines. Based on the microarray data, frequent and marked overexpression of miR-10a, miR-92, and miR-17-5p in PC cell lines ⁴⁰ was found.

An early reported application of real-time PCR profiled more than 200 microRNA precursors in specimens of human pancreatic adenocarcinoma, paired benign tissue, and normal pancreas. One hundred miRNA precursors were aberrantly expressed in PC or desmoplasia, including miR-155, miR-21, miR-221, miR-222, miR-376a,s and miR-301. Most of the top aberrantly expressed miRNAs displayed increased expression in tumors. Mature miRNA showed that three of the top differentially expressed miRNAs (miR-221, miR-376a, and miR-301) were localized to tumor cells and not to stroma, normal acini, or ducts. ⁴¹

Roldo et al. investigated the global miRNA expression patterns in normal pancreas, pancreatic endocrine tumors, and acinar carcinomas. Using a custom microarray, they studied 12 nontumor pancreases and 44 pancreatic primary tumors. Their data showed that a common pattern of miRNA expression distinguishes any tumor type from normal pancreas. Specifically, the expression of miR-103 and miR-107, associated with a lack of expression of miR-155, discriminates tumor from normal tissue; a set of 10 microRNAs distinguishes endocrine from acinar tumors and is possibly associated with either normal endocrine differentiation or endocrine tumorigenesis; miR-204 is primarily expressed in insulinomas and correlates with immunohistochemical detection of insulin; and the overexpression of miR-21 is strongly associated with both a high Ki67 proliferation index and the presence of liver metastasis. These results suggest that alteration in miRNA expression might prove useful in distinguishing tumors with different clinical behaviors. ⁴² A large-scale miRNA analysis of 540 samples, including lung, breast, stomach, prostate, colon, and pancreatic endocrine tumors, showed that the spectrum of miRNA expression varied among different solid tumors and normal cells (43 of 137 miRNAs, 31%). Prostate, colon, stomach, and pancreas are most similar, whereas lung and breast are represented by a fairly different signature. Overall, 55 miRNAs (miR-21, miR-17-5p, miR-191, miR-29b-2, etc) were up-regulated and 2 were down-regulated in pancreatic endocrine tumors. MiR-218-2 is consistently down-regulated in colon, stomach, prostate, and pancreas cancers, but not in lung and breast carcinomas. ⁴³ Szafranska et al. reported 377 miRNAs in tissue samples from normal pancreas, chronic pancreatitis (CP), and pancreatic ductal adenocarcinoma (PDAC), finding that the expression of some miRNAs, such as miR-29c, miR-96, miR-143, miR-148b, and miR-150, was dysregulated in both CP and PDAC samples; whereas miR-196a, miR-196b, miR-203, miR-210, miR-222, miR-216, miR-217, and miR-375 were aberrantly expressed only in the PDAC samples. The authors concluded that the aberrant expression of only two miRNAs, miR-217 and miR-196a, could distinguish PDAC from normal pancreas and pancreatitis. ⁴⁴ PDAC showed a higher expression of miR-21, miR-221, miR-155, miR-100, and miR-181b than benign lesions (intraductal papillary mucinous neoplasms and nonneoplastic tissues) by qRT-PCR. Overexpression of miR-21, miR-221, and miR-181b was confirmed by microarray analysis. ⁴⁵

In another study, Bloomston et al. demonstrated that up-regulation of miR-155, miR-181a,b,c,d, miR-21, miR-196a, and miR-221 and down-regulation of miR-148a,b, and miR-375 differentiated PC from normal pancreas and pancreatitis tissue samples. The expression of miR-196a was up-regulated in these studies, and miR-196a levels inversely correlated with survival in pancreatic adenocarcinoma patients. ^44,46 Strong expression of miR-21 and miR-200c was associated with poor survival of pancreatic adenocarcinoma patients. ^47,48 Strong miR-21 expression was predictive of poorer outcomes compared with absent or faint/focal miR-21 expression in patients with node-negative disease (median 15.2 vs. 27.7). In the high miR-200c expression group, the 5-year survival rate was 33.5%, but it was 11.2% in the low miR-200c expression group. Zhang et al. reported eight miRNAs, including miR-196a, were significantly up-regulated in most PC tissues and cell lines. The incidence of up-regulation of these eight genes between normal control subjects and tumor cells or tissues ranged from 70% to 100%. ⁴⁹

As described earlier, many miRNAs are dysregulated in PC. Therefore, it is anticipated that circulating miRNAs are also affected during PC progression. Kong et al. found that serum miR-196a could be a potential noninvasive marker for PDAC prognosis and selection for laparotomy. ⁵⁰ An investigation by Wang et al. showed that the expression levels of four miRNAs in plasma—miR-21, miR-210, miR-155, and miR-196a—were significantly higher in patients with pancreatic adenocarcinoma than in a healthy control group. ⁵¹ The miRNAs most frequently reported in the literature to exhibit aberrant expression in PC were mir-15b, miR-146a, miR-21, miR-155, miR-181b, miR-196a, miR-200, and miR-221/222. ^{39,41 –44,46,49} These miRNAs also have important functions in other cancers. Overexpression of miR-15b sensitized human gastric cancer cells to anticancer drugs by targeting Bcl-2. ⁵² MiR-146a has been reported to be lost in metastatic prostate cancer and breast cancer. It also has been shown that ectopic expression of miR-146a reduced the metastatic potential of breast cancer. ^53,54 In a comparison with normal pancreatic tissues, insulinomas and nonfunctioning endocrine tumors were shown to have lower expression of miR-155, which has been reported to be up-regulated in a variety of malignancies, including PDAC. ⁴¹ MiR-155 may be applicable to a differential diagnosis of pancreatic endocrine tumors and PDAC. Habbe et al. reported that miR-155 and miR-21 are overexpressed significantly in tissue from intraductal papillary mucinous neoplasms (IPMNs). ⁵⁵ Ectopic expression and depletion of miR-181b showed that it enhanced the activity of the matrix metallopeptidases MMP2 and MMP9 and promoted effects on hepatocellular carcinoma (HCC) cells, including growth, clonogenic survival, migration, and invasion that could be reversed by modulating TIMP3 levels. Further, depletion of miR-181b inhibited tumor growth of HCC cells in nude mice and enhanced resistance of HCC cells to the anticancer drug doxorubicin. ⁵⁶ Low expression of miR-200 family genes and high expression of their targets ZEB1 and ZEB2 are common in several malignancies, including breast and ovarian cancers. ^57,58 MiR-221 has been reported to be overexpressed in glioblastoma ⁵⁹ and in thyroid cancer. ⁶⁰ MiR-221 and miR-222 are clustered on the X chromosome, and both of them are predicted to regulate the cell cycle by targeting kit ⁵⁹ and p27Kip1. ⁶¹ PC may have a unique miRNA expression pattern at each individual basis. However, common pathways for PC pathogenesis may exist. These studies reported the aberrant expression of miRNAs in the majority of PC tissues, cell lines, and patient sera. Further investigations are required to determine their molecular functions and mechanisms.

The Function of miRNAs in PC

The mechanism of action of a specific miRNA usually involves nucleotide complementary nucleotide pairing to the 3'UTR of its specific target mRNA, where it primarily functions as a negative regulator by repressing target mRNA translation. miRNAs may directly regulate tissue or organ development and cell differentiation as well as maintain normal functions of many organ systems. The aberrant expressions of miRNAs in solid tumors are crucial in regulating key cancer genes. ^43,62 Genes targeted by miRNAs are highly enriched and play a crucial role in regulating apoptosis, proliferation, migration, and invasion of PC cells. These functions of miRNAs in PC affect the prognosis of PC patients.

MiR-34a is a significant component of the p53 transcriptional network and during DNA damage, and miR-34a is commonly deleted in human cancers such as PC. Characterization of the miR-34a primary transcript and promoter demonstrates that this miRNA is directly transactivated by p53. Expression of miR-34a causes dramatic reprogramming of gene expression and promotes apoptosis. MiR-34a-responsive genes are highly enriched for those that regulate cell-cycle progression, apoptosis, DNA repair, and angiogenesis. ⁶³ Similarly, Ji et al. reported the overexpression of miR-34 in p53-mutant human PC cell lines by using miR-34 mimics or infection with a lentiviral miR-34 construct led to an observed down-regulation of Bcl-2 and Notch1/2, which was accompanied by promotion of apoptosis. Researchers also tested the inhibitory effects of miR-34 on human PC tumor-initiating cells. Interestingly, miR-34 restoration led to an 87% reduction of the tumor-initiating cell population, and significant tumor sphere growth inhibition occurred in vitro as tumor formation inhibition occurred in vivo. ⁶⁴ Since more than 50% of primary human cancers have mutations inactivating p53 function, these findings provided impetus to explore the functional restoration of miR-34 as a novel approach to inhibit cancers with p53 loss of function.

MiR-96 is considered a potential tumor suppressor, because it directly targets and down-regulates the KRAS oncogene. In PC, miR-96 is significantly down-regulated when compared with normal pancreatic tissues. Ectopic expression of miR-96 has been seen to induce apoptosis in PC cell lines Panc-1 and Mia PaCa-2; this is mediated by the inhibition of KRAS and Akt signaling. ⁶⁵ In human clinical specimens, an inverse correlation was observed between miR-96 and KRAS expression. Thus, miR-96 may have potential therapeutic use in KRAS-driven PC.

These studies show that miRNA is involved in the regulation of apoptosis in PC cells, and it functions as a potent effector of apoptosis. Deregulation of apoptosis, however, can lead to pathological states, including uncontrolled cell proliferation, as in cancer, or cell loss as in neurodegenerative diseases. Therefore, enforced expression of specific genes that promote or inhibit apoptotic cell death can render cancer cells relatively resistant or sensitive to the cytotoxic effects of chemotherapeutic agents. ⁶⁶

MicroRNAs appear to be involved at several points along the tumor's pathway to acquisition of migratory and invasive properties. MiR-21 was found to be significantly up-regulated in PC, where it targets phosphatase and tensin homologue 2 (PTEN), programmed cell death 4 (PDCD4), trophomyosin 1 (TPM1), and tissue inhibitor of metalloproteinases 3 (TIMP3), leading to inhibition of apoptosis and consequent increased tumorigenicity. ⁶⁷ Overexpression of miR-21 and miR-221 was shown in another study to enhance the malignant phenotype of PC cells. Inhibition of these miRNAs using antisense oligonucleotides revealed decreased proliferation and increased apoptosis of PC cell lines compared with control oligonucleotides. ⁶⁸ Overexpression of miR-21 precursor in PC cells resulted in increased proliferation and invasion, compared with control cells. The reverse was observed, however, when miR-21 expression was knocked down in PC cells. Moreover, miR-21 positively correlated with the mRNA expression of invasion-related genes, matrix metalloproteinase-2 and -9, vascular endothelial growth factor (VEGF), PTEN, PDCD4, TPM1, and VEGF). ^47,69 Burk et al. reported that ZEB1 directly suppresses transcription of microRNA-200 family members miR-141 and miR-200c, which strongly activate epithelial differentiation in pancreatic, colorectal, and breast cancer cells. Notably, the epithelial-to-mesenchymal transition (EMT)-activators transforming growth factor beta2 and ZEB1 are the predominant targets that are down-regulated by these microRNAs. These results indicate that ZEB1 triggers an miRNA-mediated feed-forward loop that stabilizes EMT and promotes invasion by cancer cells. ⁷⁰ MiR-520h displays an inhibitory effect on PC cell migration and invasion. This effect may be mediated through direct target inhibition of ATP-binding cassette subfamily G member 2 (ABCG2) gene expression, which is also known as breast cancer resistance protein. ⁷¹ MiR-20a, a member of the miR-17-92 family, regulates Stat3 at the post-transcriptional level, with consequent inhibition of cell proliferation and invasion of PC. The PC cell lines (Panc-1 and BxPC-3) stably overexpressing microRNA-20a showed reduced proliferation and invasion capacity in vitro and in vivo. ⁷² Re-expression of miR-146a inhibited the invasive capacity of PC cells with concomitant down-regulation of EGFR and the NF-kappaB regulatory kinase interleukin 1 receptor-associated kinase 1 (IRAK-1). ⁷³ Furthermore, a similar study showed that miR-146b-5p may be involved in PC cell migration and invasion by targeting MMP16. ⁷⁴ In a recent study, manipulation of miR-31 expression led to reduced cell migration and invasion in PC. ⁷⁵ MiR-10a was overexpressed in PC cells isolated from a subset of primary tumors (12 of 20, 60%) compared with precursor lesions and normal ducts by microdissection analysis. In vitro experiments revealed that miR-10a inhibitors decreased the invasiveness of PC cells, but had no effect on their proliferation. Inhibition of HOXA1, a target of miR-10a, promoted the invasiveness of PC cells. ⁴⁰ These studies have shown that a variety of miRNAs are relevant to PC cell invasion, and they play an important role in PC metastasis, suggesting the possibility that the engineered expression of these miRNAs may prove useful in treating and improving the prognosis for human PC. Nevertheless, further investigation is needed in order to confirm these observations as a general rule for PC.

Tumor cell sensitivity to radiotherapy or chemotherapy is an important prognostic factor in patients with cancer. A variety of miRNAs have been shown to induce changes in the chemosensitivity or radiosensitivity of PC cells. PC cells overexpressing miR-21 precursor showed increased chemoresistance to gemcitabine compared with control cells. ⁴⁷ Further, miR-21 also appears to induce chemoresistance to gemcitabine in PC cell lines. For example, when PC cell lines Panc-1, LPc111, and LPc006 were transfected with the miR-21 precursor, those cells were resistant to gemcitabine treatment. ^69,76 Hwang et al. demonstrated that low miR-21 expression was associated with benefit from adjuvant treatment in two independent cohorts of PDAC cases, and anti-miR-21 increased 5-fluorouracil (5-FU) activity in vitro. Therefore, miR-21 might be a useful biomarker for chemoresistance prediction. ⁷⁷ As previously mentioned, Bcl-2 protein is directly regulated by miR-34. He et al. showed that ectopic expression of miR-34 induces cell cycle arrest in both primary and tumor-derived cell lines; this finding was consistent with the ability of miR-34 to down-regulate a program of genes promoting cell-cycle progression. ⁷⁸ Loss of miR-34 has been linked to chemoresistance in chronic lymphocytic leukemia. ⁷⁹ MiR-34 also appears to sensitize PC cells to radiotherapy. Ji et al. showed that miR-34 potently inhibits Bcl-2 expression and cell growth and increases cell death and response to radiotherapy in the overall population of MIA-PaCa-2 cells. ⁶⁴ MiR-200 may also be involved in chemoresistance. For the first time, Ali et al. reported that treatment with curcumin, a major chemical component in turmeric, up-regulated miRNA-200b and miRNA-200c while it down-regulated miR-21 in both gemcitabine-sensitive (BXPC-3) and gemcitabine-resistant (MIA-PaCa-E and MIA-PaCa-M) cell lines, which were associated with induction of apoptosis. ⁸⁰ A deeper understanding of the relationship between miRNA and chemo- or radio sensitivity of PC could be exploited for designing novel strategies for the prevention of tumor progression and/or treatment of PDAC using the combination of regulating miRNAs and gemcitabine in the future.

As discussed earlier, miRNAs affect PC development, apoptosis, invasion, prognosis, and treatment by regulating tumor-related gene expression. MiR-200 affects the expression of PTEN and MT1-MMP. ⁸¹ MiR-21 targets the inhibition of PTEN, PDCD4, TPM1, and TIMP3; tumor-initiating cells show high expression levels of Notch and Bcl-2 with loss of miR-34 expression and miR-34 affecting the expression of P53; miR-196a inhibits the expression of HOXB8, ANXA1, and HMGA2; and miR-18a regulates expression of the pancreatic transcription factor Ptf1a (Fig. 1). The discovery of miRNAs provides a new opportunity for studying the tumorigenesis and metastatic mechanisms of PC. Increasing an understanding of these processes may be expected to contribute to the development of systemic delivery systems for miRNAs to treat metastatic PC.

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FIG. 1.

Important miRNAs that are aberrantly expressed in pancreatic cancer. MiRNAs that are up-regulated and down-regulated in pancreatic cancer are depicted in red (left) and green (right), respectively, and these miRNAs target genes (PTEN phosphatase and tensin homolog, PDCD4 programmed cell death 4, TPM1 tropomyosin 1, TIMP3 tissue inhibitor of metalloproteinases 3, TP53INP1 tumor protein 53-induced nuclear protein 1, HOXB8 Homeobox B8, ANXA1 annexin A1, HMGA2 high-mobility group AT-hook 2, HOXA1 Homeobox A1, FGFRL1 fibroblast growth factor receptor-like 1, HOXA9 Homeobox A9, ZEB1 zinc finger E-box-binding homeobox 1, ZEB2 zinc finger E-box-binding Homeobox 2, CDKN1B (p27) cyclin-dependent kinase inhibitor 1B, PUMA p53 up-regulated modulator of apoptosis, CCNE1 cyclin E1, TCL1 T cell leukemia/lymphoma 1, Plk1 polo-like kinase 1, FRAP1/mTOR FK506 binding protein 12-rapamycin-associated protein 1/mammalian target of rapamycin, DNMT3b DNA methyltransferase 3b, Mitf microphthalmia-associated transcription factor, TRAF6 TNF receptor-associated factor 6, IRAK1 interleukin-1 receptor-associated kinase 1, Stat1 signal transducer and activator of transcription 1, Stat3 signal transducer and activator of transcription 3, and PDK1 3-phosphoinositide-dependent protein kinase-1).

Biomarkers for Early Diagnosis of PC

It is generally recognized that PC is an insidious disease with no specific early clinical symptoms, except when the primary tumor is located in the head of the pancreas (obstructive jaundice). The most commonly reported symptoms include abdominal pain, unusual bloating, belching, heartburn, altered bowel habits (either constipation or diarrhea), symptoms of biliary obstruction (jaundice, pale stools, and pruritus), and general constitutional symptoms (fatigue, inability to sleep, and weight loss). ⁸² A longer interval between the onset of symptoms and the initial diagnosis of PC is associated with the disease being first identified at a more advanced stage with a very poor prognosis. At the time of diagnosis, less than 15% of patients have surgically resectable disease. The median survival of unresectable PC is 4–6 months. Although the overall 5-year survival of large resected PC (median size 3 cm) is only 10%–20%, it is 30%–60% after resection of small PC (tumor size ≤2 cm), and exceeds 75% when minute PC (≤10 mm in size) is resected. ⁸³

Early detection of PC would logically require the detection of small lesions. Current noninvasive imaging techniques such as ultrasound, contrast-enhanced multidetector computed tomography, magnetic resonance imaging, and integrated positron emission tomography are inadequate for the detection of PC at an early stage, because they do not reliably detect tumors <1–2 cm in size. ⁸⁴ Even invasive techniques, such as endoscopic retrograde cholangiopancreatography and endoscopic-ultrasound (EUS) guided fine-needle aspiration (FNA), which are used to distinguish foci of malignant change in the background of CP, present difficulties. In a retrospective study of more than 1000 FNAs conducted at a single institution, it was reported that EUS-guided FNA sensitivity, specificity, and accuracy were 86.5%, 100%, and 91.9%, respectively, in detecting intra-pancreatic lesions which were less than 3 cm in diameter. ⁸⁵ However, EUS-FNA should not be performed in those patients with risk factors for PC without an apparent mass, due to the risk of procedure-related complications (pancreatitis, perforation, and bleeding).

Serum and plasma remain the most easily accessible tissues for diagnostic testing and, hence, are an attractive medium for biomarker testing to screen for early-stage disease. To date, the clinical role of PC markers for diagnosis has been limited; meanwhile, the development of minimally invasive biomarker assays for early detection and effective clinical management is urgently needed. Several reports indicate that miRNA expression profiles could be useful in diagnosis of specific cancer types. ^{86 –98} For example, Lawrie et al. found circulating miR-21, in sera from diffuse large B-cell lymphoma patients. High expression levels of miR-21 were found to be associated with improved relapse-free survival times.

Roldo et al. reported that increased expression of miR-103 and miR-107 along with decreased expression of miR-155 was useful to discriminate tumors from normal pancreas. ⁴² Habbe et al. found that miR-155 and miR-21 were overexpressed significantly in tissue from IPMNs. ⁵⁵ Elevated levels of miR-155, miR-203, miR-210, and miR-222 expression in PDCA were significantly associated with increased risk (6.2-fold) of death compared to patients with tumors having reduced expression of these miRNAs. ⁹⁹ Similarly, it has been reported that miR-21, miR-221, miR-222, miR-181a, miR-181b, miR-181d, and miR-155 are overexpressed in PC samples compared with benign pancreatic tissue. ⁴⁶ Combining expression data for miR-196a and miR-217 further improved the ability to distinguish between healthy tissue, PDAC, and CP, as well as to segregate PDAC FNA samples from other FNA samples. ⁹¹ Hypermethylation of the DNA region encoding miR-148a led to the inhibition of its gene expression in preneoplastic pancreatic intraepithelial neoplasia (PanIN). ¹⁰⁰ Importantly, this phenomenon of hypermethylation can differentiate PDAC from CP.

So far, no PC marker has been shown to be useful in the early diagnosis of an asymptomatic population. Early diagnosis for PC requires markers with high sensitivity and specificity. The standard serum marker, salivated Lewis blood group antigen CA19-9, is widely used, but its use is limited to monitoring responses to therapy, not as a diagnostic marker. ^101,102 Recent study results from our group indicate that sera or plasma from patients with PDAC have a unique miRNA expression pattern compared with normal as well as CP. ^103,104 The combination of miR-16, miR-196a, and CA19-9 was more effective for discriminating PC from non-PC (normal and CP) with a sensitivity of 92.0% and a specificity of 95.6%. These studies suggest that the amount of miRNAs in serum have potential as diagnostic and prognostic biomarkers for PDAC. ⁵⁰ MiR-210 also has been detected in the serum of PC patients, where it was expressed at levels that were fourfold higher than in normal controls. ¹⁰⁵ In conclusion, serum and plasma miR-21, miR-155, miR-210, and miR-196a are promising biomarkers for early diagnosis of PC.

Clinical Therapeutic Implications in PC

Many miRNAs down-regulate genes that are highly relevant to PC and contribute to disease progression; thus, chemically modified antisense oligonucleotides or ectopic overexpression of miRNAs might be considered for therapy. Since a single miRNA could potentially affect several clinically relevant targets, artificially increasing or decreasing the expression level of a given miRNA offers interesting therapeutic possibilities.

RNAi was identified in C. elegans in 1998 ¹⁰⁶ and in mammalian cells, in 2001. ¹⁰⁷ Since then, RNAi has generated increasing interest and publications in diverse research areas. The main problem faced in inRNAi-based gene therapy is the delivery of the effector molecule, which should preferably be controllable, sustained, and tissue specific. Several groups have opted for nonviral delivery of synthetic miRNA molecules. MiRNA mimics or miRNA antagomirs can be repeatedly delivered locally or systemically, causing transient suppression of target gene expression. ¹⁰⁸ Morrissey et al. intravenously injected mice carrying replicating HBV with a stabilized siRNA targeted to the HBV RNA that had been incorporated into a specialized liposome to form a stable nucleic-acid-lipid particle (SNALP). The improved efficacy of siRNA-SNALP compared with unformulated siRNA correlates with a longer half-life in plasma and liver. RNAi incorporated into SNALPs will protect them from degradation, prevent immunostimulation, and facilitate their uptake in endosomes. ¹⁰⁹ In addition, 2'O-methyl modifications increase the stability of synthetic molecules, preventing off-targeting. ¹¹⁰ Aberrant miRNA expression in PC oncogenically affects cancer suppressor genes, causing subsequent effects on PC cell proliferation, apoptosis, and metastasis. For example, transfection of the synthetic miRNA (Gli-1-miRNA-3548) and its corresponding duplex (Duplex-3548) significantly inhibits Gli-1, leading to the inhibition of the proliferation, delayed cell division, and activation of late apoptosis in MIA-PaCa-2 cancer cells. ^111,112 As already mentioned, miR-96 directly targets the KRAS oncogene, and ectopic expression of miR-96 can reduce pancreatic cell proliferation, migration, and invasion, indicating its potential therapeutic role in PC. ⁶⁵ Other miRNAs with oncogenic or tumor suppressor functions, including let-7, miR-21, miR-27a, miR-31, miR-200, and miR-221, could be used as novel therapeutic agents for PC. ^{68,69,72,75,113 –115} Antisense to miR-21 and miR-221 sensitized the effects of gemcitabine, and the antisense-gemcitabine combinations were synergistic at the high fractions affected. Antisense to miR-21 and miR-221 results in significant cell killing under various conditions. ^68,69 MicroRNA-20a regulates Stat3 at the post-transcriptional level, resulting in inhibition of cell proliferation and invasion of pancreatic carcinoma. ⁷² Both the inhibition of miR-31 in AsPC-1 and HPAF-II PC cells with high endogenous expression and forced expression of miR-31 in MIA PaCa-2 with low endogenous levels led to reduced cell proliferation, migration, and invasion. More importantly, in AsPC-1 cells, further enhancement of miR-31 also resulted in reduced cell migration and invasion, implicating that the level of miR-31 is critical for these phenotypes. ⁷⁵ MiR-27a plays an oncogenic role by targeting Spry2 and modulating the malignant, biological behavior of PC cells. ¹¹³ The microRNA-200 family regulates epithelial to mesenchymal transition (EMT). ¹¹⁴ Let-7 expression was repressed in patients with PDAC who were not eligible for surgery. Restoring let-7 levels in cancer-derived cell lines strongly inhibits cell proliferation, K-ras expression, and mitogen-activated protein kinase activation, but fails to impede tumor growth progression after intratumoral gene transfer or after implantation of Capan-1 cells stably overexpressing let-7 microRNA. ¹¹⁵ These studies opened a new perspective and provided early steps for miRNA replacement therapy for PC.

Conclusions and Perspectives

It is now acknowledged that miRNAs are key players in a wide variety of biological processes, including development, cellular proliferation, invasion, apoptosis, and prognosis. In PC, miRNAs have aberrant processing and expression profiles. In addition, the profile of circulating miRNAs is affected, rendering them potential biomarkers with possible applications in diagnosis and prognosis of PC, especially for early, presymptomatic disease. Although some miRNAs have been found to be associated with proliferation, invasion, and prognosis of PC, the precise mechanisms controlling the processes mentioned earlier remain unclear. The discovery of miRNA-mediated gene regulation as a fundamental post-transcriptional mechanism increases the complexity of cancer genetics; however, understanding the molecular mechanisms by which miRNAs regulate development and tumorigenesis may lead to novel concepts in the early diagnosis and treatment of PC. Further investigation is needed to evaluate and possibly realize the potential of exploiting the regulatory mechanisms of miRNAs in PC. To elucidate early diagnostic strategies, all identified miRNA biomarkers should be tested in more clinical serum samples, including those from patients with PC, those with pancreatitis, and normal controls.