miR-216 and miR-217 expression is reduced in transgenic mouse models of pancreatic adenocarcinoma, knockout of miR-216/miR-217 host gene is embryonic lethal.

Mice harboring a G12D activating Kras mutation are among the most heavily studied models in the field of pancreatic adenocarcinoma (PDAC) research. miRNAs are differentially expressed in PDAC from patients and mouse models of PDAC. To better understand the relationship that Kras activation has on miRNA expression, we profiled the expression of 629 miRNAs in RNA isolated from the pancreas of control, young, and old P48+/Cre;LSL-KRASG12D as well as PDX-1-Cre;LSL-KRASG12D mice. One hundred of the differentially expressed miRNAs had increased expression in the advanced disease (old) P48+/Cre;LSL-KRASG12D compared to wild-type mice. Interestingly, the expression of three miRNAs, miR-216a, miR-216b, and miR-217, located within a ∼30-kbp region on 11qA3.3, decreased with age (and phenotype severity) in these mice. miR-216/-217 expression was also evaluated in another acinar-specific ELa-KrasG12D mouse model and was downregulated as well. As miR-216/-217 are acinar enriched, reduced in human PDAC and target KRAS, we hypothesized that they may maintain acinar differentiation or represent tumor suppressive miRNAs. To test this hypothesis, we deleted a 27.9-kbp region of 11qA3.3 containing the miR-216/-217 host gene in the mouse's germ line. We report that germ line deletion of this cluster is embryonic lethal in the mouse. We estimate that lethality occurs shortly after E9.5. qPCR analysis of the miR-216b and miR-217 expression in the heterozygous animals showed no difference in expression, suggesting haplosufficiency by some type of compensatory mechanism. We present the differential miRNA expression in KrasG12D transgenic mice and report lethality from deletion of the miR-216/-217 host gene in the mouse's germ line.

MicroRNA replacement therapy for cancer.

MicroRNA are small noncoding RNAs that translationally repress their target messenger RNAs. Many microRNAs are expressed at reduced levels in tumors. microRNAs with reduced expression in cancer often regulate oncogenes, resulting in enhanced tumor growth. One therapeutic option is to restore microRNA levels in the tumor to that of the non-diseased tissue. This is possible by delivering microRNA to the tumor in the form of an oligonucleotide mimic or by expressing the microRNA in the cancer using a gene vector. This article surveys the field of oligonucleotide mimics and gene vector approaches to restore microRNA levels in tumors and reviews the literature on experimental and pre-clinical studies that have used these approaches to treat cancer.

Expression Profiling Identifies the Noncoding Processed Transcript of HNRNPU with Proliferative Properties in Pancreatic Ductal Adenocarcinoma.

A gene array was used to profile the expression of 22,875 long non-coding RNAs (lncRNAs) and a large number of protein coding genes in 47 specimens of pancreatic ductal adenocarcinoma (PDAC), adjacent benign pancreas and the pancreas from patients without pancreatic disease. Of the lncRNAs profiled, the expression of 126 were significantly increased and 260 were decreased in the tumors (p < 0.05, 2-fold). The expression of one lncRNA in particular, heterogeneous nuclear ribonucleoprotein U (HNRNPU) processed transcript (also known as ncRNA00201) was among the most significantly deregulated (increased four-fold) in the tumors compared to normal/adjacent benign tissues. Increased expression of HNRNPU processed transcript was associated with poor prognosis for patients with PDAC. The expression of HNRNPU processed transcript was increased in PDAC cell lines compared to noncancerous pancreatic cell lines. LNATM gapmer mediated inhibition of HNRNPU processed transcript reduced cell proliferation in Patu-T and PL45 pancreatic cancer cell lines. Reduced invasion and migration was reported upon HNRNPU processed transcript knockdown in Patu-T cells. Small interfering RNA (siRNA) knockdown of the HNRNPU protein coding gene correlated with a 55% reduction in the HNRNPU processed transcript expression and a corresponding reduction in proliferation of Patu-T and PL45 cells. However, gapmer inhibition of HNRNPU processed transcript did not affect HNRNPU mRNA levels. The lncRNA HNRNPU processed transcript expression is increased in both PDAC tissues and cell lines; knockdown of this lncRNA further reduces proliferation and invasion/migration of pancreatic carcinoma cells.

Low active loading of cargo into engineered extracellular vesicles results in inefficient miRNA mimic delivery.

Extracellular vesicles (EVs) hold great potential as novel systems for nucleic acid delivery due to their natural composition. Our goal was to load EVs with microRNA that are synthesized by the cells that produce the EVs. HEK293T cells were engineered to produce EVs expressing a lysosomal associated membrane, Lamp2a fusion protein. The gene encoding pre-miR-199a was inserted into an artificial intron of the Lamp2a fusion protein. The TAT peptide/HIV-1 transactivation response (TAR) RNA interacting peptide was exploited to enhance the EV loading of the pre-miR-199a containing a modified TAR RNA loop. Computational modeling demonstrated a stable interaction between the modified pre-miR-199a loop and TAT peptide. EMSA gel shift, recombinant Dicer processing and luciferase binding assays confirmed the binding, processing and functionality of the modified pre-miR-199a. The TAT-TAR interaction enhanced the loading of the miR-199a into EVs by 65-fold. Endogenously loaded EVs were ineffective at delivering active miR-199a-3p therapeutic to recipient SK-Hep1 cells. While the low degree of miRNA loading into EVs through this approach resulted in inefficient distribution of RNA cargo into recipient cells, the TAT TAR strategy to load miRNA into EVs may be valuable in other drug delivery approaches involving miRNA mimics or other hairpin containing RNAs.

Globally increased ultraconserved noncoding RNA expression in pancreatic adenocarcinoma.

Transcribed ultraconserved regions (T-UCRs) are a class of non-coding RNAs with 100% sequence conservation among human, rat and mouse genomes. T-UCRs are differentially expressed in several cancers, however their expression in pancreatic adenocarcinoma (PDAC) has not been studied. We used a qPCR array to profile all 481 T-UCRs in pancreatic cancer specimens, pancreatic cancer cell lines, during experimental pancreatic desmoplasia and in the pancreases of P48Cre/wt; KrasLSL-G12D/wt mice. Fourteen, 57 and 29% of the detectable T-UCRs were differentially expressed in the cell lines, human tumors and transgenic mouse pancreases, respectively. The vast majority of the differentially expressed T-UCRs had increased expression in the cancer. T-UCRs were monitored using an in vitro model of the desmoplastic reaction. Twenty-five % of the expressed T-UCRs were increased in the HPDE cells cultured on PANC-1 cellular matrix. UC.190, UC.233 and UC.270 were increased in all three human data sets. siRNA knockdown of each of these three T-UCRs reduced the proliferation of MIA PaCa-2 cells up to 60%. The expression pattern among many T-UCRs in the human and mouse pancreases closely correlated with one another, suggesting that groups of T-UCRs are co-activated in PDAC. Successful knockout of the transcription factor EGR1 in PANC-1 cells caused a reduction in the expression of a subset of T-UCRs suggesting that EGR1 may control T-UCR expression in PDAC. We report a global increase in expression of T-UCRs in both human and mouse PDAC. Commonalties in their expression pattern suggest a similar mechanism of transcriptional upregulation for T-UCRs in PDAC.

RNA isolation from mouse pancreas: a ribonuclease-rich tissue.

Isolation of high-quality RNA from ribonuclease-rich tissue such as mouse pancreas presents a challenge. As a primary function of the pancreas is to aid in digestion, mouse pancreas may contain as much a 75 mg of ribonuclease. We report modifications of standard phenol/guanidine thiocyanate lysis reagent protocols to isolate RNA from mouse pancreas. Guanidine thiocyanate is a strong protein denaturant and will effectively disrupt the activity of ribonuclease under most conditions. However, critical modifications to standard protocols are necessary to successfully isolate RNA from ribonuclease-rich tissues. Key steps include a high lysis reagent to tissue ratio, removal of undigested tissue prior to phase separation and inclusion of a ribonuclease inhibitor to the RNA solution. Using these and other modifications, we routinely isolate RNA with RNA Integrity Number (RIN) greater than 7. The isolated RNA is of suitable quality for routine gene expression analysis. Adaptation of this protocol to isolate RNA from ribonuclease rich tissues besides the pancreas should be readily achievable.

Tumor suppressive function of mir-205 in breast cancer is linked to HMGB3 regulation.

Identifying targets of dysregulated microRNAs (miRNAs) will enhance our understanding of how altered miRNA expression contributes to the malignant phenotype of breast cancer. The expression of miR-205 was reduced in four breast cancer cell lines compared to the normal-like epithelial cell line MCF10A and in tumor and metastatic tissues compared to adjacent benign breast tissue. Two predicted binding sites for miR-205 were identified in the 3' untranslated region of the high mobility group box 3 gene, HMGB3. Both dual-luciferase reporter assay and Western blotting confirmed that miR-205 binds to and regulates HMGB3. To further explore miR-205 targeting of HMGB3, WST-1 proliferation and in vitro invasion assays were performed in MDA-MB-231 and BT549 cells transiently transfected with precursor miR-205 oligonucleotide or HMGB3 small interfering RNA (siRNA). Both treatments reduced the proliferation and invasion of the cancer cells. The mRNA and protein levels of HMGB3 were higher in the tumor compared to adjacent benign specimens and there was an indirect correlation between the expression of HMGB3 mRNA and patient survival. Treatment of breast cancer cells with 5-Aza/TSA derepressed miR-205 and reduced HMGB3 mRNA while knockdown of the transcriptional repressor NRSF/REST, reduced miR-205 and increased HMGB3. In conclusion, regulation of HMGB3 by miR-205 reduced both proliferation and invasion of breast cancer cells. Our findings suggest that modulating miR-205 and/or targeting HMGB3 are potential therapies for advanced breast cancer.