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MicroRNA:New Diagnostic&Therapeutic target
Introduction

MicroRNAs (miRNAs) are ~22 nucleotide long, endogenous non-protein-coding RNA molecules which show their functions as an important regulatory molecules which can base pair with messenger RNAs (mRNAs) and thereby regulate gene and protein expression through RNA interference (RNAi) machinery. MiRNAs have been implicated in the control of all biological, developmental, physiological, cellular processes, including the biology of stem cells[5].

In eukaryotic cells, miRNAs play a important roles in regulating biological processes such as differentiation,proliferation,apoptosis and stem cell self-renewal. The human genome contain approximately 1,000 miRNAs, and more than 700 have been identified[5].

MicroRNA Biogenesis: First miRNA are transcribed by RNA polymerase II resulting in formation of long primary miRNA,these primary miRNA contain a 5’ cap and 3’ polyadenylated tail which show the similarity to mRNAs.Futher this primary miRNA having a hairpin shaped stem loop structure which are cleaved by Rnase III endonuclease Drosha and DGCR8 protein which result in the formation of pre-miRNA which is transported from nucleus to cytoplasm by Exportin-5 and its cofactor Ran GTP[6].

Futher, in the cytoplasm pre-miRNA is cleaved by Rnase III endonulease Dicer-1 and TRBP protein which result in the formation of short imperfect double-stranded miRNA duplex5. Unwinding of this duplex into mature miRNA is take place by the action of Helicase,after that TRBP recruits the catalytic Argonaute 2 to the Dicer complex with mature miRNA result in forming of RNA-induced silencing complex(RISC).

Then this RISC complex regulates gene expression by mRNAs degradation or by translational repression, Therefore miRNA negatively regulate the gene and protein expression through the RNA interference pathway[6].

'''Micro-RNA in different process of devlopment ''' Micro-RNA in regulating and maintaining the cancer stem cell population[4]: Cancer stem cell have an abnormal levels of specific miRNA,which result in dysregulation of self renewal properties of cancer stem cell through the signaling pathways.miRNA in cancer cell acts as both tumor suppressors and oncogenic4. miR-21,

miR-17-92 cluster, miR-135 acts as a oncogenic miRNAs[5]. miR-21: function as an oncogene in breast cancer,and over expression of miR-21 leads to dysregulation of Bcl-2 pathway which result in increased tumor growth and decreased cell apoptosis. miR-17-92 cluster: it over expressed in lung cancer and induced tumor and activates the Hedgehog signaling pathway which result in increased the cancer stem cell self renewal properties. miR-135: it down-regulate the APC gene(adenomatous polyposis) expression, which is the part of Wnt signaling pathway.if this gene is not expressed correct then, β-catenin will accumulate which leads to activations of self renewal gene this result in dysregulation of cancer stem cells.

Now miR-199b-5p, Let-7, miR-34 acts as a tumor suppressor miRNAs[5].

miR-199b-5p: it down-regulate the expression of transcription factorin Notch signaling pathway which blocked the Notch pathway thus decrease the self renewal properties of cancer stem cell.

Let-7: it down-regulate the Lin 28, Myc expression,thus it negatively regulates the targets in different pathways that result in dysregulatation of self-renewal properties of cancer stem cell[5].

miR-34: it down-regulate of Bcl-2 and Notch pathway thus acts tumor suppressor miRNA of cancer stem cells by regulating both apoptosis and self-renewal properties of cancer stem cell.

'''Micro-RNA play an essential role in stem cell self-renewal and differentiation: ''' Antagonism network: It implies that miRNA can regulate other miRNAs which initiating dysregulation of cancer stem cell self-renewal properties[5]. Example: let-7 and the embryonic stem cells cell cycle regulating (ESCC) miRNAs like miR-294 ,they both have opposing effects of embryonic stem cell self-renewal and differentiation properties.

• Self-renewing state: miRNA-294 indirectly increase expression of Lin28 and c-Myc(highly conserved RNA binding protein) thus Lin 28 block the functions of let-7and c-Myc bind and activates ESCC miRNA expression.

This keep the cell in self renewal state,it show that oncogenic miRNA regulate and block the expression of tumor suppressor miRNA(Let-7) which result in stem cell dysregulation5.

• Differentiate state: In this state c-Myc expression are down-regulated which result in loss of Lin-28 expression, which means that Let-7 expression increased.This lead to loss of self-renewal propertie of stem cell and prevent expression of oncogenic miRNA. This shows that tumor suppressor miRNA regulate and prevent the expression of oncogenic miRNA[5].

Thus, the antagonism network of miRNA stabilizes the switch between self-renewal and differentiation of cancer stem cell.

'''Micro-RNAs in cancer diagnostics ''' Cancer is the second leading cause of death. Most of the cancers are diagnosed in advanced stages, which lead to poor outcomes. Todays research is focused on seeking specific molecular changes that are able to identify patients with early cancer. Biological samples such as blood, serum, stool, pancreatic juice or urine, as well as both DNA and RNA, have been analysed for tumour-specific changes. Additionally genomic DNA alterations, various mutations such as KRAS either in serum, blood or circulating cancer cells in blood samples have been evaluated to allow the early diagnosis of cancer patients[7].

• MicroRNAs can function as tumour suppressor genes or oncogenes. • MicroRNAs display tissue and cell lineage specificity. • Their small size and resistance to RNase degradation render them superior to mRNAs as molecular markers. • MicroRNA expression patterns provide more pathognomonic information as compared to mRNA expression[7].

The unique patterns of miRNA expression in each type of cancer, and their stability in serum and their role as biomarkers of disease risk suggest that miRNAs may potentially serve as novel molecular biomarkers for clinical cancer diagnosis[8].

'''Therapeutic applications of microRNA ''' MicroRNAs can be used in therapeutic applications by targeting the different oncogene and signaling pathway.It work as a tumor suppressor gene in pancreatic cancer,and dysregulation of these miRNAs may result in abnormal expression of their target which leads to tumorigenesis.

Here, miRNA-96 directly target the KRAS oncogene and Akt signaling pathway which leads to suppression of tumor gene in pancreatic cancer[9]. KRAS is the member of RAS oncogenic family which encode small GTPase that involve in cellular transduction.Activation of KRAS oncogene promotes pancreatic tumor through activating different downstearm pathways, which include phosphatidylinositol 3-kinase (PI3K)/Akt, extracellular signal regulated kinase (ERK), Bad[9].

KRAS downregulation may leads to reduce in tumor growth and enhance gemcitabine chemotherapy efficacy for pancreatic cancer treatment[10]. Thus the downregulation or silencing would be efficient therapeutic strategy in pancreatic cancer.

Micro-RNA-96 play a important role in tumor suppressor in human pancreatic cancer progression,by direct targeting the KRAS oncogene,Akt signaling pathway and inhibits cell proliferations,migration, invasion which effect the tumor cell growth.

'''Overexpression of miR-96 in pancreatic cancer: '''

KRAS play a important role in cell proliferation and regulation of the cell cycle[9]. Here, the two pancreatic cancer cell line would be taken which are MLA Paca-2 and PANC-1. When both the cell line are transfected with pre-miRNA-96,scramble or untreated. So, pre-miRNA-96 treated cell line show the reduction in growth of tumor cell as compared to scramble and untreated cell line.

Futher, miRNA-96 regulate the cell cycle of MLA Paca-2 and PANC-1 cell line,it reduceses the number of cell in S-phase of the cell cycle. On the other hand, miRNA-96 expression on these two cell line effects on the migratory and invasive property of the cell, it inhibite the invasive capacity of MLA Paca-2 and PANC-1 cell line and significantly reduce the cell migration[9].

'''Micro-RNA-96 affects tumor cell growth in vitro and in vivo: ''' in vitro: first to evaluate the growth capacity of pre-miR-96, scramble-transfected or non transfected pancreatic cancer cell line[9]. When the MLA Paca-2 and PANC-1 cell are transfected with pre-miR-96, it show fewer and smaller colonies as compared with scramble-transfected and non-transfected cell.

This show the growth inhibitory role of miR-96 In vitro.

In vivo:The MLA Paca-2 cell are transfected with pre-miR-96, scramble, untreated were injected into the posterior flank of nude mice. After seven week the growth of tumor are less in miR-96 transfected mice as compared to scramble transfected or untreated[9]. The volume of tumor induced by pre-miR-96 transfected cell were less as compared to the volume of scramble transfected or untreated.

Thus, it indicates that the introduction of miR-96 in the nude mouse inhibits the tumorigenicity of MLA Paca-2 cell In vivo[9].

Reference

1.Qing, J. I., Karnak, D., Hao, P., et al. “No small matter: microRNAs - key regulators of cancer stem cells,” Int J Clin Exp Med., vol.3, pp. 84-87, 2010.

2.Gangaraj, K., Lin, H., “MicroRNAs: key regulators of stem cells,” Nature., vol.10, pp.116-126, 2009.

3.Yang, C., Nakashima, S., Rana, T., et al. “Small RNA-mediated regulation of iPS cell generation,” EMBO., vol.30, pp. 823-834, 2011.

4.Mallanna, S., Rizzino, A., “Emerging roles of microRNAs in the control of embryonic stem cells and the generation of induced pluripotent stem cells,” Dw. Biol., vol.344, pp. 16-25, 2010.

5.Jeffrey, T., DeSano., Liang, X., et al. “MicroRNA Regulation of Cancer Stem Cells and Therapeutic Implications,” AAPS., vol.11, pp. 682-692, 2009.

6.Chunsheng, l., Feng, Y., Coukos, G., et al. “Therapeutic MicroRNA Strategies in Human Cancer,” AAPS., vol.02, pp. 747-757, 2009.

7.Sempere, F., Kauppinen, S., “Translational Implications of MicroRNAs in Clinical Diagnostics and Therapeutics,” Handbook of Cell Signaling., vol.03, pp. 2965-2981, 2010.

8.James, V., James, W., Jacobson., et al. “MicroRNA: Potential for Cancer Detection, Diagnosis, and Prognosis,” Cancer Res., vol.67, pp. 4553-4555, 2007.

9.Shuangni, Y., Zhaohui, L., Changzheng, L., et al. “miRNA-96 Suppresses KRAS and Functions as a Tumor Suppressor Gene in Pancreatic Cancer,” Cancer Res., vol.70, pp. 6015-6025, 2010.

10.Wang, J., Sen, S., “MicroRNA functional network in pancreatic cancer: From biology to biomarkers of disease,” J. Biosci., vol.36, pp. 481-491, 2011.