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Skin is an important barrier between the body and the environment, and also serves many other roles such as thermoregulation, sensation (touch, heat, pressure, pain). The skin is an impermeable barrier preventing water loss by evaporation, protects organism from some effects of impact or friction injuries (trauma), also it contains melanin which is a pigment is produced by melanocytes to further protect against UV radiation (Luiz Carlos Junqueira, 2005).
The structure of the skin is categorized into three categories; the Epidermis, Dermis and Subcutaneous tissue.
This is a superficial, protective layer in contact with the external environment. This layer contains five sub-layers called strata(s) (Luiz Carlos Junqueira, 2005) The stratum basale is the deepest layer where most keratinocytes are produced, this layer also contains tonofilaments. These filaments help hold cells together in the epidermis and eventually the protein is transformed into keratin. In this layer stem cells are found which reflects the constant cell division taking place in this layer. (Gerard j. Tortora, 2009) In the Stratum spinosum you will find many-sided keratinocyte are, these contain bundles of tonofilaments. The third layer is the stratum granulosum and contains flattened keratinocytes which are beginning to degenerate. The cells here contain a protein called keratohyalin which converts the tonofilaments into keratin. The stratum lucidium is the fourth layer, but is only present in areas of the body where thick skin is needed. The last strata of the epidermis is the stratum corneum and has about 25 to 30 rows of dead, flat keratinocytes containing high levels of keratin protein. (Gerard j. Tortora, 2009)
Mostly made of connective tissue, and connects the epidermis and subcutaneous tissue (hypodermis). There are two layers in the dermis; the papilallary layer which supports and nourishes the epidermis through the network of capillaries and nerves. The reticular layer is thicker and most made of type 1 collagen, this region provides most of the elastic property of skin. (Martini, 2010)
1.4 Subcutaneous Tissue
Loose connective tissue binds skin loosely to surrounding lower organs. This allows the skin to slide over the organs. Adipose tissues are common in this layer, the number of tissues found depends on the area of the body and the size is a reflection of the nutritional state.(Luiz Carlos Junqueira, 2005)
1.5 Skin Cancer
Cancer is caused by changes in the regulation of tissue growth. The difference between a normal cell and a cancerous cell is changes in the genes that regulate cell growth and differentiation. These are changes in oncogenes, tumour-suppressor genes and microRNA genes(Croce, 2008). There are different types of neoplasmââ‚¬â„¢s that can develop on the skin (Owens and Watt, 2003) most common neoplasias are basal cell carcinoma (BCC), squamous cell carcinoma (SCC) and malignant melanoma (MM) (Miller and Weinstock, 1994).
Basal cell carcinomas have sometimes been considered the most common type of cancer in humans(Telfer et al., 1999). This form of non-melanoma cancer is associated with difference in skin colour and the response of the skin to sunlight (constitutional factors) and this is evident in some Caucasians with relatively lighter features such as blue eyes and blonde or red hair where they suffer from sun burn rather than produce a tan when their skin is exposed to sunlight (Gallagher et al., 1995). The big environmental cause of BCC and SCC is exposure to sunlight especially UVB in sunlight (Situm et al., 2008; Welsh et al., 2008).
The body has various important DNA repair mechanisms to avoid tumours including formation of BCCs and SCCs. The p53 gene plays an important role in these mechanisms (Levine, 1997). However there can be mutations in p53 itself and these mutations in the p53 gene have been detected in about 56% of BCC (Soehnge et al., 1997) and a higher value of above 90% in SCC (Ziegler et al., 1994). The p53 gene can be activated by many different stresses such as oxidative stress, DNA-damaged agents and radiation (Vousden and Lane, 2007). A fully functioning p53 protein can promote apoptosis if it is required due to the extent of the DNA damage and p53 also influences cell cycle arrest. In cell cycle arrest cellular processes such as P21 (G1 arrest) and 14-3-3ÃÆ’ (G2 arrest) are manipulated by the p53 protein (Fei et al., 2002; Hermeking et al., 1997; Yu et al., 2001). Recently with ever ongoing research into formation of tumourigenesis, the interactions of p53 and microRNA have been investigated. Some studies have shown MicroRNA that have targeted p53, and others have seen direct transactivation by p53. (Takwi and Li, 2009)
BCC formation has been linked to mutation in the ptch1 gene (Hahn et al., 1996; Johnson et al., 1996). The ptch1 protein is a receptor for the Hedgehog (HH) family of extracellular ligands. When bound this reduces the inhibition that the receptor has on other cellular components specifically the smoothened protein (SMO). This causes numerous signals to be sent including to the interacting protein, Suppressor of fused (SUFU) and this eventually causes the Gli family of transcription factor family to down-regulate. (Kinzler et al., 1987; Lum and Beachy, 2004; Rohatgi and Scott, 2007; Varjosalo and Taipale, 2007). BCC formation has been linked with over expression of Gli-2 in keratinocytes (Grachtchouk et al., 2000)
Micro RNA is a class of small non-coding RNAs, first described by Victor Ambros and Gary Ruvkin when they discovered Lin-4 (Wightman et al., 1993) (Lee et al., 1993). Research only really began in these classes of miRNA in the 2000 (Mueller and Bosserhoff, 2009). The formation of miRNA begins when RNA polymerase II transcribes the miRNA genes, which forms primary transcripts called pri-miRNA that are often several kilobases in length (Filipowicz et al., 2008). In the nucleus the pre-mir is subjected to a mini-processor complex which is essentially RNAs III (Drosher) enzyme and its co-factor, DiGeorge syndrome critical region 8(DGCR8). This creates a sequence about 60-110 nucleotide long in the shape of a stem-loop containing mature miRNA sequence. Following this Ran-GTP-dependent nuclear export factor called exportin-5 quickly translocates the newly formed pre-miRNA (pre-mir) to the cytoplasm. (Mueller and Bosserhoff, 2009)
Another RNAs III enzyme called Dicer along with its co-factor called TRBP cuts about a 18-24 nucleotide double strand. This forms an RNA duplex, which acts through miRNA-induced-silencing complex (miRISC), where only the stable strand acts as miRNA and forms post-transcriptional regulation (silencing) in target genes(Mueller and Bosserhoff, 2009). Fig 1 illustrates some key aspects of microRNA biogenesis.
Fig-1 A representation of biogenesis reproduced from the review on MicroRNA and the Skin (Sand et al., 2009)
There are two methods by which miRNA inhibit protein expression, one method is mature miRNA to act through the RNA-induced silencing complex (RISC) to target and cleave mRNA (Hammond et al., 2000) miRNA can cleave their target mRNA without perfect complementarity (Hutvagner and Zamore, 2002).
The second method is to inhibit translocation in mRNA which occurs when miRNA does not align fully with its mRNA target (Zeng et al., 2003). An example of this was shown by Olsen and Ambros (1999) where protein translocation was inhibited by miRNA when they isolated lin-4 miRNA and Lin-14 mRNA from cytoplasmic ribosomal complexes (Olsen and Ambros, 1999)
The miRNA are associated and known to regulate several cellular processes, such as proliferation, apoptosis, cell cycle regulation and differentiation. Thus if there is irregular miRNA activity this has been shown to contribute in the formation and growth of cancer (Mirnezami et al., 2009; Visone and Croce, 2009).
A loss of function in the miRNA can be due to similar reasons as a protein-coding gene such as genomic deletion, mutation, epigenetic silencing and maybe also miRNA processing alteration. Such changes could contribute to the formation of malignant cells (Calin et al., 2002; Calin et al., 2005; Nakamura et al., 2007; Saito et al., 2006).
The miRNA, Mir-15a and miR16-1 were the first to be associated with human cancer and came from studies following the 13q14 deletion in human chronic lymphocyte leukaemia (Calin et al., 2002).
With different miRNA there are different levels of effects following their activity in malignant cells, for example miR-143 and miR-145 are found to be down regulated in colon carcinomas, and the mir17-92 cluster is up-regulated in B-cell lymphomas and lung cancers (Johnson et al., 2005)
There have not been many studies in the expression and function of miR-214, however recently more and more information is underway. In a 2008 paper abnormal levels of miR-214 alongside other miRNA was detected in ovarian cancers, especially in high grade and late stage tumours. The study went further by identifying miR-214 as a negative regulator of phosphatase and tensin homolog (PTEN) by binding to the 3ââ‚¬â„¢UTR. This causes inhibition in PTEN and small molecule AKT (protein kinase B) inhibitors which contributed to cell death, to override cell survival and reduces cisplatin resistance. However due to the level of miR-214 this does not take place and contributes to the survival of malignant cells. (Yang et al., 2008)
In this study the goal is to detect if miR-214 is expressed in the skin and if its expression is changed during progression of tumour development in mouse skin. This choice was used to avert the problems faced with studying biological mechanisms and agents in vivo cells that have carcinogenesis induced. An example of a common procedure to produce these artificial cells, is administrating the polycyclic aromatic hydrocarbon 7,12 ââ‚¬"dimethyl-benz [a] anthracene (DMBA). Then follows several weeks of weekly administering of phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA). The result is formation of several benign papillomas of which some become malignant squamous cell carcinomas (Zoumpourlis et al., 2003). This model is used to study alterations in signal transduction pathways implicated in tumour progression during multistage mouse skin carcinogenesis (Zoumpourlis et al). This is a common model used by many scientists in such studies, and it is therefore thought suitable for this project.