Biochemistry of hnRNA C and hRALY in cancer and normal cells

Biochemistry of hnRNA C. And hRALY in Cancer and Normal Cells using Northern Blots Analysis

Department of Chemistry

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two groups working independently During the mid-1990s discovered hRaly, which is a protein that shares a great deal of primary sequence homology with the heterogeneous nuclear ribonucleoproteins C1 and C2 (hnRNP C). These two proteins have been posited by other researchers to play a number of important roles in pre-mRNA biogenesis as well as mRNA metabolism. To this end, this study screened 24 paired tissue samples from normal and tumor cells for hraly and hnRNP C. expression. Based on an analysis of the resulting data, the results of this study showed that hnRNP C. And hRaly may be capable of providing the same types of analytical outcomes for uterine cancer detection purposes. In addition, this study determined that the RNA binding properties of hnRNP C. And indistinguishable from the published evidence for hRaly. This study also showed that hRaly and hnRNP C. interact with a common set of proteins as defined by a Yeast Two Hybrid Assay. Finally, the results of this study also determined that there was a significant difference in the expression of some proteins in some cancer cells than the normal cells. The findings from this and previous studies can be used to help identify valuable cancer biomarkers that can help diagnose or even detect cancer before it is too late to use existing treatment regimens.

Table of Contents

Chapter One -- Introduction

Chapter Two -- Literature Review

Chapter Three -- Methods

Chapter Four -- Results and Data Analysis

Chapter Five -- Discussion

Chapter Six -- Conclusions

Biochemistry of hnRNA C. And hRALY in Cancer and Normal Cells using Northern Blots Analysis

Chapter One: Introduction

Structure and Function of hnRNP Proteins

The structure and function of heterogeneous nuclear ribonucleoproteins (hnRNP) provide the basis for a growing body of research concerning ways to detect cancer early on so that existing treatments will be more effective and therefore improve clinical outcomes. There has been increasing interest in the scientific community concerning hnRNP proteins for this purpose; these are highly abundant proteins that bind to pre-mRNA in the nucleus. In this regard, Dreyfuss, Matunis, Pinol-Roma and Burd (1993) report that, "Messenger RNAs (mRNAs) are formed in the nuclei of eukaryotic cells extensive posttranscriptional processing of primary transcripts of protein-coding genes. These transcripts are produced by RNA polymerase II and are termed heterogeneous nuclear RNAs (hnRNAs), a historical term that describes their size heterogeneity and cellular location" (p. 290). To date, researchers have identified more than 20 different hnRNP proteins which have been designated using the letters A through U. based upon the extent of their migration on SDS-PAGE gels (Dreyfull et al., 1993). Likewise, Ford, Wright and Shay (2002) note that, "The heterogeneous nuclear ribonucleoproteins (hnRNPs) are a large family of nucleic acid binding proteins that are often found in, but not restricted to, the 40S-ribonucleoprotein particle" (p. 580). Therefore, the ribonucleoprotein complex provides the framework in which the pre-mRNA is processed into mature messenger RNA (Ford et al., 2002).

The importance of RNA in protein synthesis relates to its potential to provide improved methods that can be used for the early identification of several types of cancer by measuring RNA amount of hnRNP C. And hRALY in both cancer and normal cells. In this regard, the functions of hnRNP C. include:

1. Pre-mRNA Packaging

2. Pre-mRNA splicing

3. Pre-mRNA transport

4. Regulate mRNA stability

5. Component of Telomerase

6. Regulation of IRES mediated Translation

7. Cancer Biology and hnRNP Proteins

8. hnRNP A and B. Proteins and Lung Cancer

It has been suggested that the distribution of hnRNPs on pre-mRNA results from their different binding specificities. However, the high concentration of the individual hnRNPs in the nucleus disputes this observation and suggests that the proteins are organized randomly based upon non-specific binding. Though their distribution on pre-mRNA remains a mystery it is clear that many of the hnRNP proteins are associated with pre-mRNA biogenesis. However, multiple functions have been proposed for most the hnRNPs and many of these remains controversial in the scientific community. For example hnRNP C, has been proposed to be involved in RNA splicing, RNA polyadneylation, regulation of mRNA stability, internal ribosome entry site mediated translation, and also has been shown to be a component of the telomerase holoenzyme. Likewise the hnRNP A and B. proteins have been shown to regulate alternative splicing and to be associated with mRNA transport from the nucleus, as well as regulating RNA stability. Several of the hnRNP proteins have been found to be integral components of telomers. In general a plethora of functions have been proposed for each of the individual hnRNP, too many to address in this thesis. According to Torosyan, Dobi, Glasman, Mezhevaya, Naga, Huang, Paweletz, Leighton, Pollard and Srivastava (2010), though, "Representing the most abundant nuclear proteins that are implicated in the spliceosome packaging of excised intron sequences, hnRNPs typically bind to exonic/intronic splicing silencers, thereby repressing splicing" (p. 2460). These researchers are quick to point out, though, that, "hnRNPs can hinder communication between factors bound to different splice-sites, thereby having a positive role in RNA splicing. Most importantly, the concentration of splicing factors can alter the kinetic equilibrium in splicing, resulting in changes in splice-site selection" (Torosyan et al., p. 2460). Other researchers have also identified these mechanisms. For instance, according to Rajan, Dalgliesh, Bourgeois, Heiner, Emami, Clark, Bindereif, Stevenin, Robson, Leung and Elliott (2009), "Active pre-mRNA splicing occurs co-transcriptionally, and takes place throughout the nucleoplasm of eukaryotic cells. Splicing decisions are controlled by networks of nuclear RNA binding proteins and their target sequences, sometimes in response to signaling pathways" (p. 1).

Associations between these proteins have also been investigated. For example, Mili, Shu, Zhao and Pinol-Roma (2001) report that, "In the cytoplasm, A1 is associated with its nuclear import receptor (transportin), the cytoplasmic poly (A)-binding protein, and mRNA. In the nucleus, A1 is found in two distinct types of complexes that are differently associated with nuclear structures" (p. 7307). The first class of the cytoplasm A1 has the pre-mRNA and mRNA, and is the same as hnRNP complexes that have been described in the research in the past; however, the other class of the cytoplasm A1 functions as freely diffusible nuclear mRNPs (nmRNPs) during the late nuclear maturation stages and the potential exists that it is linked with nuclear mRNA export (Mili et al., 2001).

These nmRNPs are distinguishable from hnRNPs because although they contain shuttling hnRNP proteins, the mRNA export factor REF, as well as mRNA, they do not contain pre-mRNA or nonshuttling hnRNP proteins (Mili et al., 2001). Significantly, nmRNPs also contain proteins that are not found in hnRNP complexes as well, including the alternatively spliced isoforms D01 and D02 of the hnRNP D. proteins, the E0 isoform of the hnRNP E. proteins, and LRP130; the latter isoform has also been described previously in the research and is a protein whose function remains unknown but seems to possess a unique sort of RNA-binding domain (Mili et al., 2001). According to these researches, "The characteristics of these complexes indicate that they result from RNP remodeling associated with mRNA maturation and delineate specific changes in RNP protein composition during formation and transport of mRNA in vivo" (Mili et al., 2001, p. 7307).

Studies have also shown that hnRNP proteins are concentrated in the nucleus when growth conditions are normal growth at which point they seem to be excluded from the nucleolus (Mili et al., 2001). In addition, it is known that a subset of the hnRNP proteins, hnRNPs A1 and K, shuttle between the cytoplasm and the nucleus constantly while others such as hnRNP C1/C2 and hnRNP U. do not exhibit this shuttling process and remain in the nucleus (Mili et al., 2001). Mediation of the nuclear export of hnRNP A1 is achieved through the function of a specific amino acid sequence known as M9; this amino acid satisfies the criteria for an authentic nuclear export signal (NES); in addition, M9 operates as the hnRNP A1 nuclear location signal via mediating binding of its nuclear import receptor, transportin (Mili et al., 2001). It is also known that hnRNP A1 keeps the capability to bind mRNA, temporarily, in the cytoplasm and most likely as its passes through the nuclear pore complex (NPC) as well (Mili et al., 2001). By contrast, hnRNP A1, hnRNP C1 and hnRNP C2 remain in the nucleus and the process for retention is mediated via another specific amino acid sequence contained in C. proteins that operates as a nuclear retention sequence (NRS) (Mili et al., 2001). Significant as well is the fact that this NRS is capable of taking precedence over NESs; consequently, it is thought that the removal of hnRNP proteins that contain NRS from mRNA is required in order for the nuclear export of mRNA to proceed (Mili et al., 2001).

Not surprisingly, other researchers have also investigated the recent trends in this area. In this regard, Martinez-Contreras, Cloutier, Shkreta, Fisette, Revil & Chabot report that, "Proteins of the heterogeneous nuclear ribonucleoparticles (hnRNP) family form a structurally diverse group of RNA binding proteins implicated in various functions in metazoans" (2007, p. 123). In this study, Martinez-Contreras and her associates report the results of recent research that has provided additional evidence concerning the function of these proteins in precursor-messenger RNA (pre-mRNA) splicing (2007).

The splicing repression can function in two discrete ways in heterogeneous nuclear RNP proteins; the first way is by antagonizing the recognition of splice sites directly and the second way is through interference with the binding of proteins that are bound to enhancers (Martinez-Contreras et al., 2007). A growing body of research concerning the role of hnRNP proteins has determined that these proteins can restrict communication between factors bound to different splice sites; by contrast, a number of studies have identified a positive role for some hnRNP proteins in pre-mRNA splicing (Martinez-Contreras et al., 2007). Moreover, the research to date suggests cooperative interactions between bound hnRNP proteins that may facilitate splicing between specific pairs of splice sites while concomitantly repressing other combinations (Martinez-Contreras et al., 2007). Therefore, it has become increasingly apparent that hnRNP proteins employ a wide range of methods to control splice site selection in a fashion that is important for alternative as well as constitutive pre-mRNA splicing (Martinez-Contreras et al., 2007).

The functioning of hnRNPs has also been investigated by Prahl, Vilborg, Palmberg, Jornvall, Asker and Wiman (2008) who report that hnRNPs take part in the maintenance of telomere length, transcriptional regulation, alternative pre-mRNA splicing and pre-mRNA 30 end processing within the nucleus. According to these researchers, "In the cytoplasm, hnRNPs can regulate mRNA localization, translation and turnover. hnRNP A2 (36 kDa) and B1 (38 kDa) are isoforms derived from the same gene and differ by only 12 amino acids, due to the presence of exon 2 in the B1 transcript" (Prahl et al., 2008, p. 2173). In transformed cells, the targeting of hnRNP A2/B1 facilitates the death of cells; however, targeting hnRNP A2/B1 in primary cells does not promote their death (Prahl et al., 2008). In addition, the suppression of hnRNP A2 results in a non-apoptotic inhibition of cell proliferation (Prahl et al. 2008).

A study by David, Chen, Assanah, Canoll and Manley (2010) notes that there are three heterogeneous nuclear ribonucleoprotein (hnRNP) proteins: (a) polypyrimidine tract binding protein (PTB, also known as hnRNPI); (b) hnRNPA1 and (c) hnRNPA2; all three of these hnRNP proteins repressively bind to sequences that are adjacent to exon 9 causing exon 10 inclusion. In their study, these researchers also demonstrate that the oncogenic transcription factor c-Myc upregulates the transcription of hnRNPA1, hnRNPA2 as well as PTB thereby assuring an elevated ratio for PKM2/PKM1 (David et al., 2010). Establishing a relevance to the function of these proteins with regards to cancer, these researchers also determined that the overexpression of human gliomas PTB, hnRNPA1, c-Myc, and hnRNPA2 are congruent with the same type of overexpression that occurs in PKM2 (David et al., 2010). Based on these results, these researchers concluded that these findings define a pathway that regulates an alternative splicing event that is implicated in tumor cell proliferation (David et al., 2010).

The role of PKM2 proteins in cancer proliferation was also the subject of a study by Clower, Chatterjee, Wang, Cantley, Vander Heiden and Krainer (2010), who report, "Cancer cells exhibit a metabolic phenotype characterized by increased glycolysis with lactate generation, regardless of oxygen availability -- a phenomenon termed the Warburg effect" (p. 1894). In addition, Clower et al. point out that recent research has shown that the expression of the type II isoform of the pyruvate-kinase-M gene (PKM2, referred to as PK-M by these researchers) is "a critical determinant of this metabolic phenotype, and confers a selective proliferative advantage to tumor cells in vivo" (2010, p. 1894). The significance of this finding is summarized by these researchers thusly: "This finding adds to the growing body of evidence that alterations in alternative pre-mRNA splicing play important roles in different aspects of cancer progression" (Clower et al., 2010), p. 1894).

Clearly, the expression of pre-mRNA plays an important role in the cancer-development process, and measuring the constituent elements of the process may help researchers develop superior early detection mechanisms that can provide earlier treatment and improved clinical outcomes. For instance, Clower et al. add that, "Proliferating cells and cancer cells preferentially express PK-M2 over PK-M1 at the protein level" (2010, p. 1895). Taken together, the foregoing suggests that additional research into hnRNP proteins is warranted, and the research objective of this study is discussed further below.

Research Objective

Work in our laboratory has previously shown that all vertebrate cells have very high concentrations of a protein called hnRNP C. In their nuclei. The function of this protein has remained elusive. It has been proposed to be involved in many cellular activities. The research objective of this study was to investigate the expression of hRaly and hnRNP C, which have been found to be ubiquitous in all tissues examined in previous studies. To this end, the study collected 24 paired tissue samples from normal and tumor cells which were then screened for hRALY and hnRNP C. expression. Another protein, though, hRaly, has been discovered that is virtually identical to hnRNP C; this protein shares fully 43% primary sequence homology with hnRNP C1 and C2. Therefore, identifying techniques that can measure RNA amounts of hnRNP C. And hRALY in both cancer and normal cells represents a timely and valuable enterprise as discussed further below.

Chapter Two: Literature Review

Background and Overview

The need for reliable biomarkers for use in the early detection of cancer has been well established, but there remains a fundamental lack of specific biomarkers that are capable of assessing the effects of long-term exposure to various environmental conditions, even over the course of several decades (Brody & Rudel, 2003). Further, women in particular at are risk for a number of diseases that may be affected by environment conditions, including breast cancer (the most common type) (Brody & Rudel, 2003), as well as uterine cancer (the fourth most common with about 40,000 new cases in the United States each year) (Identifying uterine cancer, 2006). Biomarkers may therefore help improve the assessment of various environmental exposures on the incidence of cancer in general and in women in particular (Heck, Andrew, Onega, Rigas, Jackson, Karagas & Duell, 2009). In this regard, Boukakis, Patrinou-Georgoula, Lekarakou, Valavanis and Guialis (2010) note that, The biogenesis of mRNA in higher eukaryotes is largely based on the interplay of a large number of RNA-binding proteins (RBPs). Heterogeneous nuclear ribonucleoproteins (hnRNPs) are RBPs that are essential players in mRNA metabolism, acting as coordinators of post-transcriptional events (splicing, transport, cellular localisation, decay and translation of mRNA) by participating in an extensive network of RNA-RBP interactions" (p. 434). Likewise, according to Sun, Xu, Poon, Day and Luk (2010), early cancer detection using mRNA-level biomarkers can also help improve clinical outcomes for liver cancer patients. Similarly, Katsimpoula, Patronou-Georgoula, Makkrilia, Dimakou, Guialis, Orfandious and Syrigo (2009), the overexpression of hnRNP A2/B1 has increasingly been cited as representing a potential useful marker for early detection of lung cancer.

In addition, individual hnRNPs function in several other cellular processes are of great interest to researchers, including transcription, DNA repair, telomere biogenesis and cell signaling; consequently, any malfunctioning in the various regulatory roles gene expression in these proteins, particularly with regards to their deregulated expression in cancer, is believed to affect the physiological network of RNA-RBP interactions (Boukakis et al., 2010). Recent research in this area is discussed further below.

Recent Clinical Research

According to Sueoka, Goto, Sueoka, Kai, Kozu and Fujiki (1999), heterogeneous nuclear ribonucleoprotein (hnRNP) A2/B1 is an RNA binding protein and this binding protein is an essential component needed for the successful maturation of the mRNA precursor. These researchers add that, "hnRNP A2/B1 protein is a major component of the hnRNP core complex in mammalian cell nuclei. Although the function of hnRNP A2/B1 has not yet been fully elucidated, recent studies have revealed that hnRNP A2/B1 is involved in RNA splicing in nuclei as well as in mRNA transport from nucleus to cytoplasm" (Sueoka et al., 1999, p. 1404).

When hnRNP A2/B1 mRNA and hnRNP B1 mRNA were investigated separately, these researchers developed original evidence that hnRNP B1 mRNA, which is a splicing variant of hnRNP A2 mRNA, was significantly increased in lung cancer tissues compared to hnRNP A2/B1 mRNA (Sueoka et al., 1999). Further, Sueoka et al. note that the hnRNP B1-specific polyclonal antibody used in their study specifically recognized hnRNP B1 protein as a Mr. 37,000 nuclear protein through the use of Western blotting techniques; however, it did not recognize hnRNP A2 protein. As to the procedures, Sueoka et al. report that, "Immunohistochemical staining with the hnRNP B1 antibody revealed that hnRNP B1 protein was specifically stained in the nuclei of human cancer cells, and in squamous cell carcinomas in particular, but not in those of normal adjacent lung epithelial cells" (1999, p. 1404). Based on these findings, these researchers concluded that hnRNP B1 protein of Mr. 37,000, rather than hnRNP A2, represent a better biomarker for the detection of human lung cancer (Sueoka et al., 1999). Likewise, a more recent follow-up study by Sueoka, Sueoka, Goto, Matsuyama, Nishimura, Sato, Fujimura, Chiba and Fujiki (2001) notes that, "Heterogeneous nuclear ribonucleoprotein (hnRNP) B1 is a RNA binding protein of Mr. 37,000. hnRNP B1 was specifically overexpressed in the nuclei of human lung cancer cells, particularly in squamous cell carcinoma" (p. 1896).

An early study by Rhodes, Valbracht, Nguyen and Vaughanf (1997) investigated the role of the p542 protein in the Raly gene. These researchers report that anti-EBNA-1 antibodies are produced in infectious mononucleosis (IM), which then cross-reacts with numerous human proteins that are normal. According to these researchers, "The cross-reactions can be inhibited with synthetic peptides representing the glycine/alanine repeat in EBNA-1, which implies that the cross-reactivity is due to anti-gly/ala antibodies that cross-react with host proteins containing configurations like those in the EBNA-1 repeat" (p. 447). In this study, Rhodes et al. (1997) report the isolation of five gene fragments from a Raji B. lymphocyte cDNA library encoding peptides reactive with autoantibodies in an IM serum; of these five isolated gene fragments, p542 was determined to encode a glycine rich 28-mer that comprises its cross-reactive epitope. These researchers found that p542 was discernible in three B. lymphocyte lines using Northern blots (Rhodes et al., 1997). Following an analysis of protein sequence characteristics comparable to P542 in the GenBank, Rhodes et al. (1997) identified a high degree of consistency with the mid- and 3? terminal regions of the recently published mouse gene, Raly, which has been shown to encode a protein that has the structure of an hnRNP. These researchers also confirmed their guiding presumption that the 5? sequences of p542 have a high level of identity with Raly as well, including the presence of RNA binding motifs that distinguish hnRNPs. (Rhodes et al., 1997). In addition, they determined that there was a sequence homology with human hnRNP C2 (Rhodes et al., 1997). Based on these findings and the results from previous on-point studies, these researchers concluded that the autoantigen for one of the cross-reactive autoantibodies created during human immune responses to the Epstein Barr virus, anti-p542, is also probably an hnRNP (Rhodes et al., 1997).

Likewise, Ji, Chen, Xie, Wang, Qian, Zhao, Jin, Wu, Xu, Ying and Mao (2003) investigated the role of the p542 and report, "High throughput cDNA sequencing and 5'-rapid amplification of cDNA ends (5'RACE) isolated two cDNAs that shared the same open reading fragment (ORF)" (p. 61). Like Rhodes and his colleagues, Ji et al. (2003) also used Northern blot analysis with the fetal brain mRNA blots which identified two transcripts with the respective lengths of 3.2 kb and 2.2 kb. The ORF is known to encode a 291 residues putative protein that is highly homologous with hRALY and hnRNPC; consequently, like hRALY, this protein was named hRALYL (Ji et al., 2003). According to Ji and his associates, "Expression pattern was detected by multiple-issue cDNA (MTC) panel-based RT-PCR. It revealed that the transcripts of hRALYL were expressed ubiquitously in human tissues with different intensities" (p. 61). These researchers determined that the highest transcript was situated in the brain, and blast analysis showed that the cDNA corresponding to a contig NT_008292 found that the gene housed at least 9 exons; the gene in question was situated on human chromosome 8q21.13-8q21.2 and consequently, these researchers posit that hRALYL could therefore be a member of the hnRNPC subfamily (Ji et al., 2003).

Other hnRNA Proteins Related to Other Cancers

A study by Sueoka, Sueokaa, Iwanagaa, Satoa, Sugab, Hayashia, Kagasawaa and Nakachic (2005) notes that one of the diagnostic tools that has been used for cancer detection is circulating cell-free nucleic acids. For this purpose, Sueoka et al. (2005) report that, "Heterogeneous nuclear ribonucleoprotein (hnRNP) B1, an RNA binding protein, has been found overexpressed in the early stage of lung cancer, including bronchial dysplasia, a premalignant lesion of lung squamous cell carcinoma" (p. 77). In order to assess the efficacy of plasma hnRNP B1 RNA as a detection marker for lung cancer, Sueka et al. (2005) analyzed plasma hnRNP B1 mRNA of lung cancer patients by real-time RT-PCR; for this purpose, 100 subjects (44 lung cancer patients, 7 lung neoplasm patients, 24 benign lung diseases and 25 healthy volunteers) had plasma RNA extracted from their lugs (Sueoka et al., 2005). The analysis by Sueoka et al. identified a mean concentration of plasma hnRNP B1 mRNA of 0.99pg/?g RNA in the study's subjects (n=44 lung cancer patients) versus respective rates of 0.23pg/?g RNA in the control group of 25 healthy volunteers and 0.30pg/?g RNA in the subjects who had benign lung disease (n=24 patients) (Suekoa et al., 2005). These researchers add that, "Twenty of 44 (45.5%) lung cancer patients showed more than 0.70pg/?g RNA of plasma hnRNP B1 mRNA, compared with only 3 of 25 (12.0%) healthy volunteers. Looking at histological subtype, squamous cell carcinoma patients showed higher hnRNP B1 mRNA in the plasma than did adenocarcinoma patients, which is consistent with our previous immunohistochemistry results" (p. 77). Based on these findings, Suekoa et al. (2005) conclude that plasma hnRNP B1 mRNA represents a useful non-invasive markers for detection of lung cancer.

The need for such early detection tools is paramount and its urgency increases daily. In this regard, Fielding, Turnbull, Prime, Walshaw and Field (1999) report that, "Clinical detection of lung cancer usually occurs late in the disease, when it is often beyond effective treatment; consequently, there is a high mortality rate. Detection at the earlier stage would influence both the mortality and morbidity rates" (p. 4051). Pathologically, Fielding et al. report that hnRNP3 has been characterized as a Mr. 31,000 protein that is responsible for posttranscriptional regulation of gene expression via capping, splicing, polyadenylation, and the cytoplasmic transport of mRNAs. According to Fielding et al., "Overexpression of hnRNPA2/B1 is critical in certain stages of mammalian lung development [and] the tumor-associated monoclonal antibody 703D4 may be used as an early detection marker for lung cancer" (p. 4048). Based on their findings, Fielding and his associates conclude that, "The results of this study further support the hypothesis that hnRNP overexpression may be considered a potential early detection marker" (1999, p. 4051).

In addition, He, Brown, Rothnagel, Saunders and Smith (2005) confirm that, "Overexpression of heterogeneous nuclear ribonucleoproteins (hnRNPs) A2 and B1 has been observed in a variety of tumour types, however, it is unknown whether this dysregulation is a consequence of, or a driving force for, unregulated cell proliferation" (p. 3173). In this study, He et al. (2005) demonstrated that the levels of hnRNPs A1, A2 and B1, are modulated during the cell cycle of Colo16 squamous carcinoma cells but not A3; in addition, the researchers showed that HaCaT immortalized keratinocytes indicating that A1, A2 and B1 are prerequisites at specific cell cycle stages. The levels of hnRNP A1, A2 and B1 mRNAs, though, were found to be constant, suggesting that regulation of protein levels was controlled at the level of translation (He et al., 2005). According to these authorities, "RNAi suppression of hnRNP A1 or A3 alone did not affect the proliferation of Colo16 cells but the proliferation rate was significantly reduced when both were suppressed simultaneously, or when either was suppressed together with hnRNP A2" (He et al., p. 3173). The reduction of hnRNP A2 expression in Colo16 and HaCaT cells by RNAi resulted in a non-apoptotic-related decrease in cell proliferation, lending further support to the notion that this protein is required for cell proliferation (He et al., 2005). Other relevant findings that emerged from the He et al. study (2005) included the fact that suppression of hnRNP A2 in Colo16 cells was found to be associated with increased p21 levels; however, p53 levels were unaffected. Moreover, the expression of BRCA1 was downregulated, at both protein and mRNA levels. Based on these results, these researchers concluded that, "The observed effects of hnRNP A2 and its isoforms on cell proliferation and their correlation with BRCA1 and p21 expression suggest that these hnRNP proteins play a role in cell proliferation" (He et al., 2005, p. 3173).

A study by Peebles, Dwyer-Nield and Malkinson (2007) extended the findings from a previous proteomic investigation of lung neoplasia in vitro study by these researchers that found a high concentration of the lung cancer biomarker and splicing factor, hnRNP A2/B1, in the transformed mouse lung epithelial cell line, E9. Noting that because alterations in pre-mRNA splicing have an significant effect on neoplastic progression, Peebles et al. investigated hnRNP A2/B1 expression in chemically induced primary mouse lung tumors which was used to model an in vivo pulmonary adencocarcinoma. According to Peebles et al., "Tumor hnRNP A2/B1 content and spatial distribution assessed by immunohistochemistry varied with stage of progression, genetic background, and whether tumors were induced by a single agent (urethane) or by 2-stage initiation/promotion (3-methylcholanthrene/butylated hydroxytoluene) carcinogenesis" (2007, p. 887). This follow-up study by Peebles et al. (2007) also used in vitro models in order to address mechanisms governing hnRNP A2/B1 expression changes. These researchers found that hnRNP A2/B1 protein was overexpressed in E9, the spontaneous tranformant of immortalized but non-neoplastic E10 cells; however, they also determined that expression was not solely a function of enhanced proliferative rate in neoplastic cells (Peebles et al., 2007).

Increased mRNA content was shown to be positively correlated with cell division in both E10 and E9; however, hnRNP A2/B1 protein levels declined in proliferating E10 cells (Peebles et al., 2007). Enhanced mRNA stability was produced by the elevated mRNA levels as demonstrated through time-dependent mRNA decay following inhibiting transcription (Peebles et al., 2007). Based on their findings, these researchers concluded that, "Dysregulation of hnRNP A2/B1 expression during lung neoplasia in vivo thus depends on complex gene-environmental interactions that affect cell type-specific changes in mRNA processing and, most probably, the rates of translation and/or protein degradation" (Peebles et al., 2007, p. 887).

Finally, the implications of the foregoing research have caused other researchers to investigate the role played by these biomarker proteins in detecting colorectal cancer. Noting that colorectal cancer (CRC) is the third most common cancer worldwide and has poor prognosis, the objective of a study by Ma, Peng, Zhang, Huang, Liu, Shen, Chen, Zhou, Zhang, Chu and Qin (2009) was to identify the proteins that are implicated in colorectal carcinogenesis. To this end, Ma et al. (2009) employed a 2-DE and MALDI-TOF/TOF-based proteomics approach to investigate the differentially expressed proteins in tumor and adjacent nontumor tissue samples. According to these researchers, "Samples from 10 colorectal patients were analyzed. Of the 7 significantly and consistently altered proteins identified, hnRNP A1 was one of the most significantly altered proteins and its overexpression was confirmed using RT-PCR and Western blot analyses" (Ma et al., 2009, p. 4525). An immunohistochemical analysis found that that the enhanced expression of hnRNP A1 was associated with the increased severity of colorectal tissue as well as the progression of the colorectal cancer, as well as International Union against Cancer staging, histo-differentiation, recurrence and diminished survival rates (Ma et al., 2009). These researchers suggest that the development of a highly sensitive immunoassay could detect hnRNP in human serum, given that this level was substantially increased in colorectal cancer patients vs. The levels found in healthy volunteers (Ma et al., 2009). These researchers suggest that hnRNP A1 could be regarded as being a novel serum tumor marker for colorectal cancer that has been potential for improved detection and management of patients with this disease. Based on their findings, these researchers concluded that, "These data suggested that hnRNP A1 may be a potential biomarker for early diagnosis, prognosis, and monitoring in the therapy of colorectal cancer" (p. 4525). Like the other researchers reviewed herein, Ma et al. (2009) cite the need for additional research in this area to better assess the potential clinical value of this biomarker candidate.

Chapter Three: Methods

In this study, 24 paired tissue samples from normal and tumor cells were screened for hraly and hnRNP C. expression using the methods described below.

1.

Chemicals, and other Biological Kits Reagents

Chemiluminescent detection systems have emerged as the best all-around detection method for use with western blots and ELISA. These detection assays eliminate the hazards associated with radioactive materials and toxic chromogenic substrates. The speed and sensitivity of these methods are unequalled by traditional alternatives. Streptavidin-HRP is used with biotinylated proteins and specific chemiluminescent substrates to generate light signal. Streptavidin-HRP conjugates have a very high turnover rate, coupling high sensitivity with short reaction times (Streptavidin-HRP, 2010).

In this study, North2South Chemiluminescent Hybridization and Detection Kit were used from Thermo Scientific, product number 17097 following the manufacturer-supplied directions at http://www.piercenet.com/products/browse.cfm?fldID=06010501 and http://www.piercenet.com/files/2161758.pdf. The kit contains sufficient reagents for 1,000 cm2 of membrane. Kit Contents are: Stabilized Streptavidin-Horseradish Peroxidase Conjugate, 1.5 ml, Chemiluminescent Substrate, stable for six months at room temperature or one year at 4°C, Luminol/Enhancer Solution, 80 ml, Stable Peroxide Solution, 80 ml, Blocking Buffer, 500 ml, 4X Wash Buffer, 500 ml, Substrate Equilibration Buffer, 500 ml, store at room temperature or 4°C, North2South® Hybridization Buffer, 125 ml, North2South® Hybridization Stringency Wash Buffer (2X), 375 ml. Total RNA Array was purchased from www.biochain.com Catalog No.: H1235706 Lot No.

Additional information concerning the other materials used in this study can be found in the following publications:

1. http://www.biochain.com/biochain/datasheet/H1235706-A307054.pdf

2. http://www.biochain.com/biochain/coa/total_RNA_Array_CoA.pdf

3. http://www.biochain.com/biochain/details/H1235706Detail.htm

4. http://www.biochain.com/biochain/ProtocolManuals/NothernBlotManual.pdf

5. http://www.biochain.com/biochain/Products%20and%20Services/Total_RNA.htm

6. http://www.piercenet.com/products/browse.cfm?fldID=06010501

7. http://www.piercenet.com/files/2161758.pdf

2.

Description of RNA Array Used in This Study

Total RNA samples are spotted on a nylon membrane. Each array includes one human genomic DNA (50 ng) as positive control, and one pBg18 plasmid DNA (250 pg) as negative control. Each RNA spot contains 5 ?g of total RNA. The total RNA samples have the same high quality as used in our total RNA Northern Blots. Total RNA Dot Blot allows a RNA population to be screened for the presence of a specific RNA. These premade blots enable researchers to study gene expressions in normal, fetal, and tumor samples without spending time on the difficult process of acquiring samples, isolating RNA, and making RNA blots. The left upper corner of membrane has been cut to provide orientation. TD1 was labeled at bottom right corner of each blot as shown in Table 1 below.

Table 1

RNA Array key

Tumor

Normal

Tumor

Normal

Tumor

Normal

Brain

Brain

Liver

Liver

Ovary

Ovary

Lung

Lung

Gall Bladder

Gall Bladder

Breast

Breast

Esophagus

Esophagus

Parotid

Parotid

Fallopian

Fallopian

Stomach

Stomach

Kidney

Kidney

Uterus

Uterus

Duodenum

Duodenum

Uterus

Uterus

Thyroid

Thyroid

Small Intes

Small Intes

Bladder

Bladder

Adrenal

Adrenal

Colon

Colon

Prostate

Prostate

Thymus

Thymus

Rectum

Rectum

Testis

Testis

Lymphoma

Normal Lymph

Stripping and Reprobing Northern blots

Prior to commencing the experiment, the RNA array must be stripped off in order to remove any previous probe and detecting reagents so that it will be possible to reprobe for the same or different target. Therefore, the RNA array used in this study was washed with target side up and handled only at the corners. The product manufacturers caution, though, that, "Any stripping procedure has the potential to denature sensitive nucleic acids, rendering them unrecognizable by the probe. Should this occur, reprobing will not be possible, and a new blot will have to be prepared for each probing experiment. Furthermore, any physical defects of a membrane will be exaggerated when stripped and reprobed" (Strip Northern and Southern blots, 2008, p. 1). Therefore, the RNA array in this study was carefully stripped with boiling 0.1% SDS at 80°C for 15 minutes. Following this step, the array was rinsed with clean water and was then ready for reprobing. The array was kept wet between hybridization and stripping. Best practices show that it is always better to test the efficiency of the stripping by exposing the array to a film for at least 30 minutes. Following this exposure, if no signal was detected, this indicates that the stripping was successful. The next step was to keep the blots wet after stripping and store them in 0.1% SDS or 1X TE at 4°C.

Pre-Hybridization and Hybridization

Equilibrate kit buffers to room temperature before use. If there is a precipitate in any of the kit buffers, heat buffer in a 37oC water bath until precipitate disappears. Heat incubator to 55°C for DNA hybrids. Quantify probe using a spectrophotometer. Equilibrate the North2South® Hybridization Buffer to room temperature (RT). Place blot in a container such as a 50 ml centrifuge tube and add sufficient Hybridization Buffer to completely cover the blot. Use at least 0.1 ml per cm2 of membrane. Seal the container and pre-hybridize the membrane with shaking or rotating for at least 30 minutes. For DNA hybrids incubate at 55°C. While pre-hybridizing, denature the biotinylated DNA probe. Heat DNA probe (we did not use RNA probe), at 100°C for 10 minutes and place on ice for 5 minutes. After pre-hybridization, add the denatured biotinylated probe. For biotinylated DNA probes (we did not use RNA probe), it is necessary to add ~30 ng of probe per milliliter of hybridization buffer. Following this step, incubation overnight with shaking or rotating at 55°C for DNA hybrids was performed.

Stringency Washes

On the next day, the North2South® Hybridization Stringency Wash Buffer (2X) was equilibrated to RT. Once the Wash Buffer was fully in solution, an equal volume of sterile ultrapure water was added. Therefore, the resulting 1X buffer contains 2X SSC/0.1% SDS. The next step involved washing the blot three times for 15-20 minutes per wash with agitation. For this purpose, 0.2 ml of 1X Stringency Wash Buffer per cm2 of membrane was used and washes were performed at 55C for DNA hybrids or 65C for RNA:RNA hybrids.

Probe Detection

A probe is a nucleotide sequence that is used to identify a specific RNA within a population of RNA. For this purpose there are two types of probe to be considered, a DNA probe (which is used analyzing RNA population for the presence of a gene similar to the gene of interest) and an RNA probe (which is used analyzing RNA population for the presence of a particular gene using the genetic transcript). The reagent volumes indicated are for a 10 x 10 cm membrane. If larger or smaller membranes are used, it is necessary to adjust volumes accordingly. The performance of all blocking and detection incubations in clean trays or in plastic weigh boats on an orbital shaker was the next step. Thereafter, the Blocking Buffer was warmed slowly with the 4X Wash Buffer to 37-50°C in a water bath until all particulates were dissolved. These buffers may be used between room temperature and 50°C provided all particulate remains in solution. The Substrate Equilibration Buffer may be used between 4°C and room temperature.

To block membrane, 16 ml Blocking Buffer were added and incubated for 15 minutes with gentle shaking. The next step required the preparation of the conjugate/blocking buffer solution by adding 50 ?l of the Stabilized Streptavidin-Horseradish Peroxidase Conjugate to 16 ml Blocking Buffer (1:300 dilutions). The blocking buffer was decanted from the membrane and 16 ml of the conjugate/blocking solution was added. The membrane was then incubated in the conjugate/blocking buffer solution for 15 minutes with gentle shaking. A preparation of 1X wash solution was made by adding 40 ml of 4X Wash Buffer to 120 ml ultrapure water. The membrane was transferred to a new container and rinsed briefly with 20 ml of 1X wash solution. The membrane was then washed four times for 5 minutes each in 20 ml of 1X wash solution with gentle shaking. The membrane was once again transferred to a new container and 30 ml of Substrate Equilibration Buffer was added and the membrane was then incubated for 5 minutes with gentle shaking.