**4.10 miRNA**

It is well established that non-protein coding micro (mi) RNAs play a significant role in regulation of gene expression and cellular protein levels. They are now also being increasingly recognised as major regulators of EMT and metastasis, specifically the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, miR-429 and miR-205 (Gregory et al, 2008; Park et al., 2008). Members from the miR-200 family and miR-205 are associated with increased expression of E-cadherin and decreased vimentin. In addition, these miRNAs also target the expression of ZEB1 and ZEB2, the E-cadherin transcriptional repressors. Expression levels of miR-205 and of some members of the miR-200 family were also found to vary inversely with vimentin expression in primary serous papillary carcinomas of the ovary (Park et al., 2008). In another study, EMT was induced through either TGFβ or the tyrosine phosphatase pEZ in Madin–Darby canine kidney (MDCK) cells. The levels of both miR-205 and miR-200 family members was down-regulated after EMT induction while their ectopic expression induced MET (Gregory et al., 2008).

One way that natural antisense transcripts can play a major role in EMT is by targeting the regulation of ZEB2 expression. This was documented when EMT was induced in a human colorectal cancer cell line by SNAIL. ZEB2 levels were found to be directly increased after EMT initiation which was explained as the result of the action of a natural antisense transcript that prevented the splicing of a large intron in the 5′ untranslated region (UTR) that contains an internal ribosomal entry site which lowers ZEB2 levels in epithelial cells through the inhibition of ribosome scanning. During EMT activation, the antisense transcript levels are increased. They bind to the 5'UTR and inhibit splicing, preserving the

DELTA/JAGGED acting through the NOTCH pathway are implicated in both cell fate in the normal human mammary gland (Raouf et al., 2008) and regulation of cancer stem cells (CSCs) in both ductal carcinoma *in situ* and in invasive carcinoma of the breast (Dontu et al., 2004; Stylianou et al., 2006). This pathway is known to be transcriptionally induced by TGFβ/SMAD signalling and contributes to EMT (Zavadil et al., 2004). This pathway is cell type specific and can be either oncogenic through activation of the NKFB pathway or it can be tumour suppressive. Wang et al., (2006) provided evidence demonstrating that NOTCH

The WNT signalling pathway mediates several vital processes such as cell proliferation, migration, differentiation, adhesion and death (Vincan et al., 2008). In addition, this pathway can promote migration and EMT in breast cancer cells through the stabilisation or increased expression of SNAIL1 and 2 and TWIST (Onder et al., 2008; Vogelstein et al., 2004). SNAIL has been implicated in regulatingWNT-1-induced EMT in MCF-7 cells. Furthermore, WNT signalling can also lead to the translocation of β-catenin to the nucleus where it can drive the expression of several EMT inducing transcription factors through the WNT induced inhibition of glycogen synthase kinase-3β (GSK3β)-mediated phosphorylation. However, β-catenin alone usually is not enough to induce EMT although in colorectal cancer WNT is indeed a silencer of its negative regulators SOX17 (Zhang et al., 2008), SFRPS18, 19 and DKK1 (Aguilera et al., 2006). Interestingly, both SFRP1 and DKK1

It is well established that non-protein coding micro (mi) RNAs play a significant role in regulation of gene expression and cellular protein levels. They are now also being increasingly recognised as major regulators of EMT and metastasis, specifically the miR-200 family (miR-200a, miR-200b, miR-200c, miR-141, miR-429 and miR-205 (Gregory et al, 2008; Park et al., 2008). Members from the miR-200 family and miR-205 are associated with increased expression of E-cadherin and decreased vimentin. In addition, these miRNAs also target the expression of ZEB1 and ZEB2, the E-cadherin transcriptional repressors. Expression levels of miR-205 and of some members of the miR-200 family were also found to vary inversely with vimentin expression in primary serous papillary carcinomas of the ovary (Park et al., 2008). In another study, EMT was induced through either TGFβ or the tyrosine phosphatase pEZ in Madin–Darby canine kidney (MDCK) cells. The levels of both miR-205 and miR-200 family members was down-regulated after EMT induction while their

One way that natural antisense transcripts can play a major role in EMT is by targeting the regulation of ZEB2 expression. This was documented when EMT was induced in a human colorectal cancer cell line by SNAIL. ZEB2 levels were found to be directly increased after EMT initiation which was explained as the result of the action of a natural antisense transcript that prevented the splicing of a large intron in the 5′ untranslated region (UTR) that contains an internal ribosomal entry site which lowers ZEB2 levels in epithelial cells through the inhibition of ribosome scanning. During EMT activation, the antisense transcript levels are increased. They bind to the 5'UTR and inhibit splicing, preserving the

receptor signalling regulates SNAIL 1 and 2, ZEB1 and vimentin.

are frequently silenced by methylation in breast cancer.

ectopic expression induced MET (Gregory et al., 2008).

**4.8 NOTCH** 

**4.9 WNT** 

**4.10 miRNA** 

internal ribosomal entry site sequence and thereby increasing the translational efficiency of ZEB2 which then directly inhibits E-cadherin expression, maintaining an EMT state (Beltran et al., 2008).

Although these RNA molecules are associated with the regulation of EMT and MET, other miRNAs such as miR-10b are reportedly associated with metastasis and invasion. It inhibits HOX10 translation while increasing RHOC when induced by TWIST (Ma et al., 2007). Another miRNA that seems to increase the metastatic potential of cancer cells is miR-29a; up-regulated in a mesenchymal metastatic RASXT mammary cell line compared to epithelial EpRas cells. In addition, over-expression of miR-29a suppresses expression of tristetraprolin, a regulator of epithelial polarity and metastasis, and leads to EMT and metastasis through RAS signalling. This correlates with data from breast cancer patients showing enhanced miR-29a and reduced tristetraprolin levels (Gebeshuber et al., 2009). In contrast to miR-10b, miR-335 was found to be a suppressor of invasion and metastasis through modulation of the expression of the 'six gene signature' set: COL1A1, MERTK, PLCB1, PTPRN2, TNC and SOX4 which are considered predictive markers of metastasis and invasion. miR-335 was also reported to suppress invasion and metastasis in MDAMB231, a highly metastatic and invasive ER-ve breast cancer cell line (Tavazoie et al., 2008).
