The importance of microRNA expression in maintaining normal cellular physiology has been well documented 
. Inhibition of microRNA processing has been shown to enhance cellular transformation 
. The incomplete knockdown of Dicer 1 in mouse lung adenocarcinoma cells resulted in increased cellular proliferation and invasiveness as well as enhanced in vivo
tumor formation 
. Furthermore, decreased microRNA expression in mouse embryonic fibroblasts resulted in incomplete transformation of the cells 
. The mechanisms by which microRNA expression decreases in the malignant cells as opposed to the normal cells is not clear. Recently, epigenetic regulation of microRNA expression has been demonstrated in a variety of tumors 
. MicroRNA genes were silenced in human tumors by aberrant hypermethylation of CpG islands adjacent to and surrounding the microRNA genes and by acetylation of the microRNA gene associated histones 
. It has been found that a higher proportion of known microRNA genes have been found to be epigenetically regulated through methylation (11.6%) than that found in the protein-encoding genes (1–2%) 
In the present publication, we have demonstrated that 3-Cl-AHPC modulates expression of a number of microRNAs that regulate the translation and degradation of mRNA. These mRNA encode the expression of proteins previously demonstrated to have important physiological roles in cellular proliferation and death. We have found that 3-Cl-AHPC exposure resulted in the up-regulation of a number of microRNAs including miR-150* and miR-630. The significance of modulating of these microRNAs was demonstrated by observing subsequent modulation of their predicted target genes- including EGFR, c-Myb, SMAD1, ABCC1, CPEB3, IGF-1R and CDK6.
3-Cl-AHPC up-regulation of miR-150* resulted in the decreased expression of c-Myb and IGF-1R. Both proteins play critical roles in enhancing cellular proliferation. Over-expression of IGF-1R has been found in a variety of malignancies including pancreatic cancer 
. Inhibition of IGF-1R utilizing small molecule kinase inhibitors has resulted in reduced pancreatic cancer growth 
. We have previously shown that knock down of IGF-1R expression reduced pancreatic cancer cell proliferation while also enhancing 3-Cl-AHPC-mediated apoptosis 
. We have recently demonstrated that the expression of the microRNA miR-223 also suppresses cell proliferation by targeting IGF-1R. Enhanced miR-223 resulted in a decrease in IGF-1R expression as well as inhibition of the downstream Akt/mTOR/p70S6K pathway.
3-Cl-AHPC-mediated 7-fold increase in miR-150* decreased IGF-1R and c-Myb expression. In order to further demonstrate miR-150* modulation of c-Myb and IGF-1R in the PANC-1 cells, we expressed pre-miR-150* in the cells and this resulted in a marked inhibition of IGF-1R and c-Myb mRNA and protein levels (). Futhermore, addition of the miRNA inhibitor OME-150 with 3-Cl-AHPC blocked 3-CL-AHPC increase in miR-150* levels and 3-Cl-AHPC mediated decrease in c-Myb protein. These results strongly suggest that the 3-Cl-AHPC-mediated decrease c-Myb levels was due to the 3-Cl-AHPC increase in pre-miR-150* expression. Moreover, enhanced expression of miR-150* in the PANC1 cells resulted in enhanced apoptosis in the cells both in the presence and absence of 3-Cl-AHPC. Thus enhanced mir-150* expression and its down-regulation of c-Myb and IGF-1R have a significant effect on cell survival. Bcl-2 has been found to function as a negative regulator of apoptosis and has been found to play an important role in tumor cell survival 
. Bcl-2 down-regulation has been shown to be important in the induction of tumor cell death 
. We found that 3-Cl-AHPC exposure resulted in down-regulation of Bcl-2 expression by decreasing c-Myb expression and binding to the Bcl-2 promoter.
Aberrant expression of miR-150 has been reported in a number of malignant cells 
. miR-150 was found to be down-regulated in lymphoma and leukemia while its expression has been found to be increased in gastric and colorectal carcinomas. miR-150 appears, however, to have pleiotropic effects on malignant cells. Enhanced miR-150 expression promotes gastric carcinoma cell proliferation while it functions as a tumor suppressor in malignant lymphoma 
. Recently, it has been found that miR-150 directly targets the Muc4 3′mRNA 3′-UTR 
. Binding of miR-150 to this region resulted in a marked decrease expression of MUC4 mRNA and protein. This decreased expression was shown to have a marked inhibitory effect on the pancreatic cell growth, migration, invasion and clonogenicity 
EGFR plays an important role in pancreatic cancer cellular proliferation, metastatic spread, and apoptosis 
. Elevated expression of EGFR has been found to be an independent poor prognostic factor in patients with pancreatic cancer 
. EGFR is targeted by miR-133, decreasing expression of EGFR mRNA and protein in NSCLC cells 
. We have found that exposure of the pancreatic cancer cells to 3-Cl-AHPC resulted in 4-fold increase in miR-134 levels with an associated marked inhibition of EGFR mRNA and protein levels. Previous research has shown that cisplatin (CDDP) induced a 5-fold increased expression of miR-630 at 12 h 
. pre-miR-630 arrested cell growth at G0-G1 in lung cancer NSCLC A549 cells. We have found that 3-Cl-AHPC up-regulates of miR-630 expression. pre-miR-630 reduced target protein IGF-1R levels and induced growth inhibition and apoptosis in PANC-1 cells. These results suggested that IGF-1R is the target gene of miR-630.
Modulation of miRNA levels by a number of agents has been demonstrated to function as cancer chemopreventive agents 
. Epigallocatechin gallate an important cancer chemopreventative agent of green tea has been shown to upregulate miR-16 with subsequent down-regulation of Bcl-2 in hepatocarcinoma cells 
. Sun et al.
have recently shown that the antitumor agent curcumin which has been shown to inhibit the proliferation and induce apoptosis of a variety of tumor types may mediate the death of pancreatic cancer cells through its upregulation of the expression of miR-22 and the subsequent targeting of the transcription factor SP1 and estrogen receptor 1 
. Similarly, the anti-tumor effects of the natural antioxidant revesterol and the isoflavone genistein have been partially or wholly attributed to their ability the levels of a variety of miR levels 
. More direct approaches have been utilized to modulate miRNA levels both in vitro
and in vivo
. These approaches have included the use of anti miRNA oligonucleotides (AMO) miRNA sponges – that have multiple miRNA binding sites thus possessing the capacity to sequester multiple miRNA - and miRNA masks - that are complementary to the 3′-untranslated region of the target miRNA and result in the competitive inhibition of miRNA modulation of its target genes 
A number of microRNA delivery systems have been developed and have shown promise both in vitro and in vivo in their ability to enhance specific miRNA expression in the malignant cells resulting in the inhibition of tumor growth 
In this report we show that 3-Cl-AHPC modulated exposure to PANC-1 cells modulate miRNAs that appear to play key roles in growth inhibition and apoptosis induction in pancreatic cancer cells. Specifically, we've shown that up-regulation of miR-150* and miR-630 is important to the inhibition of IGF-1R, which inhibits growth and induces apoptosis in pancreatic cancer cells. This study broadly supports the framework that microRNAs are essential for apoptosis induction and growth inhibition in pancreatic cancer. Further understanding microRNAs regulation may have promising clinical implications for the treatment of cancer.