**2.1 miR-21 general aspects**

MiR-21 has been identified as the best hit in a number of miRNA profiling studies designed for the detection of miRNAs dysregulated in human cancer [8]. MiR-21 could suppress several tumor suppressor proteins translation, including PTEN, PDCD4, TMP1 and p53, to mediated cancer cell malignant phenotype alternation [9-10]. MiR-21 was emerging as key regulators of multiple pathways involved in tumorgenesis and may become the next targeted therapies in human cancers [11]. Previously we identified miR-21's aberrant expression in glioblastoma(GBM), and we focused on in what way miR-21 regulated GBM development and progression. Thus, in the current chapter, we manage to elucidate the view point that miR-21 was an effective molecule with great potential of human cancer gene therapy; of course, we will take miR-21's biological role to GBM as an example.

Recent reports suggested that miR-21 functions as an oncogene in human cancers. Ciafre` et alprofiled the expression of 245 miRNAs in 10 glioblastoma (GBM) cell lines and nine freshly resected GBM samples and observed that miR-21 was overexpressed in human brain tumors [12]. It was shown that when miR-21 was suppressed, cell growth inhibition and caspase-dependent apoptosis were observed in A172, U87, LN229, and LN308 cells. It has been shown that miR-21 modulates breast cancer cell anchorage-independent growth through suppressing TMP1 expression. In human colorectal, breast cancer, and renal cell carcinoma, miR-21 contributes to invasion and metastasis cell by inhibiting Pdcd4 mRNA at the post-transcription level. A recent study showed that miR-21 targets PTEN gene through a binding site on the 3'-UTR in hepatocellular carcinoma [13]. PTEN has been shown to be a critical tumor suppressor gene that is commonly inactivated in GBM by deletion, mutation, or attenuated expression. Thus, increased expression of miR-21 may contribute to the attenuated expression of PTEN in GBM.

### **2.2 miR-21 was up regulated in GBM cell lines and tissue samples**

Microarray assay was used to screen the miRNA expression status in GBM cell lines. Data showed miR-21 exhibited a 7.0-fold increase relative to normal brain tissue [14]. In addition,

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

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

MiR-21 has been identified as the best hit in a number of miRNA profiling studies designed for the detection of miRNAs dysregulated in human cancer [8]. MiR-21 could suppress several tumor suppressor proteins translation, including PTEN, PDCD4, TMP1 and p53, to mediated cancer cell malignant phenotype alternation [9-10]. MiR-21 was emerging as key regulators of multiple pathways involved in tumorgenesis and may become the next targeted therapies in human cancers [11]. Previously we identified miR-21's aberrant expression in glioblastoma(GBM), and we focused on in what way miR-21 regulated GBM development and progression. Thus, in the current chapter, we manage to elucidate the view point that miR-21 was an effective molecule with great potential of human cancer gene therapy; of course, we will take miR-21's biological role to GBM as an

Recent reports suggested that miR-21 functions as an oncogene in human cancers. Ciafre` et alprofiled the expression of 245 miRNAs in 10 glioblastoma (GBM) cell lines and nine freshly resected GBM samples and observed that miR-21 was overexpressed in human brain tumors [12]. It was shown that when miR-21 was suppressed, cell growth inhibition and caspase-dependent apoptosis were observed in A172, U87, LN229, and LN308 cells. It has been shown that miR-21 modulates breast cancer cell anchorage-independent growth through suppressing TMP1 expression. In human colorectal, breast cancer, and renal cell carcinoma, miR-21 contributes to invasion and metastasis cell by inhibiting Pdcd4 mRNA at the post-transcription level. A recent study showed that miR-21 targets PTEN gene through a binding site on the 3'-UTR in hepatocellular carcinoma [13]. PTEN has been shown to be a critical tumor suppressor gene that is commonly inactivated in GBM by deletion, mutation, or attenuated expression. Thus, increased expression of miR-21 may contribute to the

Microarray assay was used to screen the miRNA expression status in GBM cell lines. Data showed miR-21 exhibited a 7.0-fold increase relative to normal brain tissue [14]. In addition,

critical role in the process of tumorigenesis.

methylation.

example.

**2.1 miR-21 general aspects** 

attenuated expression of PTEN in GBM.

**2.2 miR-21 was up regulated in GBM cell lines and tissue samples** 

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

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