**2. MicroRNA strategy**

MicroRNAs (miRNAs) are a class of naturally occurring small non-coding RNAs, approximately 22 nucleotides in length, that target protein-coding mRNAs at the posttranscriptional level [4]. Generally, mature miRNAs are integrated into a protein–RNA complex called a microRNA RNPs (similar to the RISCs (RNA-induced silencing complexes) for siRNA). miRNAs bind through partial sequence homology to the 3\_-untranslated regions (3'-UTRs) of target genes. Because of this unique feature, a single miRNA has

PAMAM Dendrimer as Potential Delivery System for

grade III gliomas (anaplastic gliomas, AAs) and GBMs.

Combined Chemotherapeutic and MicroRNA-21 Gene Therapy 501

in-situ hybridization (ISH) of surgery resected glioma samples proved that miR-21 displayed varying degrees of intensity in glioma with different grades and the positive rate increased with the ascending order of the glioma WHO grade. In hence, it was important to note that miR-21 ISH was conducted at both the tissue level and the cellular level to indicate that miR-21 disregulation could be a marker to predict the outcome of glioma patients. To identify miR-21 that was abnormal upregulated in high-grade gliomas, we used ISH to test miR-21 insitu expression in human non-neoplastic brain tissues, I~II grade gliomas,

Our group showed that miR-21 was over expressed in 57 of 60 glioma samples and miR-21 was detected in the cytoplasm of the neoplastic cells of all the positive cases. MiR-21 displayed varying degrees of intensity in glioma with different grades and the positive rate increased with the ascending order of the WHO grade. There were 27 of 30 (90%) in WHO I and II gliomas, 15 of 15 (100%) in AAs and GBMs, whereas miR-21 was rarely detected in control brain tissues. The first indication of miR-21's aberrant expression came from the miRNA profiling of human glioblastoma. Compared to normal brain tissue, miR-21 relative expression was seven to eleven folds in low-grade astrocytomas, AAs and GBMs. Besides providing the consistent data to the previous study, it is important to note that miR-21 ISH was conducted at both the tissue level and the cellular level to indicate that miR-21

To evaluate the significance of miR-21 overexpression in glioma cells, we used a loss-offunction antisense approach. An As-miR-21 oligonucleotide (ODN) was used to knock down miR-21 expression in U251 and LN229 cells. RT-realtime PCR results determined that the relative expression level of miR-21 in As-miR-21 ODN-treated U251 cell was 6.25% (P<0.01) and 12.5% for LN229 cells (P<0.01) compared with their control cells, respectively. In addition, LNA-based in situ hybridization showed that transfection of a scrambled ODN had no effect on miR-21 expression. In contrast, the cy3 red fluorescence signal in As-miR-21-transfected U251 cells was lower (Figure 1B). These data suggested that As-miR-21 can specifically inhibit

The GBM cell growth inihibitory effect (MTT assay) of decreased miR-21 reached maximum three days post transfection. G1 phase blockage was observed to indicate cell cycle distribution changed significantly after miR-21 inhibitory. Additionally, the Annexin V positive early phase apoptotic cells were significantly increased in cells transfected with AS-

The in vitro experiments suggest that miR-21 is a potential target for therapy in GBM. To further confirm this, we performed a proof-of-principle experiment using a U251 glioma xenograft model and a lipofectamine-mediated gene therapy approach. The xenograft tumors volume suppression indicated that miR-21 contributed a lot to U251 GBM cell proliferation in vivo. Pathological examination found micro-vessel density indicated that was and evaluated

Despite the apparently predominance of microRNA in cancer therapy, several problems have to be overcome for successful clinical application. They show a poor stability towards nuclease activity, low intracellular penetration and low bioavailability. Although chemical modifications were brought to the basic microRNA, their sensitivity to degradation and poor intracellular penetration is still hampering their widespread clinical applications. In fact, the major bottleneck in the development of miRNA strategy is the delivery of these

miR-21 as compared to that in parental cells and cells treated with scrambled ODN.

miR-21 expression by in situ hybridization and cellular apoptosis by TUNEL assay.

disregulation could be a marker to predict the outcome of glioma patients.

**2.3 miR-21 regulated GBM cell growth in vitro and in vivo** 

the endogenous miR-21 expression in U251 and LN229 cells.

multiple targets. It is thought that more than 30% of human genes are posttranscriptionally regulated by miRNAs [5]. miRNAs have diverse functions in biological processes, including the regulation of cellular proliferation, differentiation, and cell death. As dysregulation of these biological processes frequently occur in human cancer, miRNAs may, therefore, play a critical role in the process of tumorigenesis.

This regulatory mechanism was first shown in the developmental processes in worms, flies, and plants [6]. Subsequently, miRNAs have been shown to have important roles in many physiological processes of mammalian systems by influencing cell apoptosis, development, and metabolism through regulation of critical signaling molecules including cytokines, growth factors, transcription factors, and pro-apoptotic and anti-apoptotic proteins. Increasing number of miRNAs have been identified in the human genome and they are collectively called the miRNome [7]. Accumulating evidence shows the potential involvement of altered regulation of miRNAs in initiation and progression in a wide range of human cancers. Altered expression profiles of miRNAs are associated with genetic and epigenetic alterations including deletion, amplification, point mutation, and aberrant DNA methylation.
