Cancer/Testis Antigens (CTA)

Cancer/Testis Antigens (CTA)

Cancer/Testis Antigens identified several proteins expressed by germ cells, tumors and trophoblasts and the apparent of the genes expressed present in the germline in cancerous cells and range of tumors. However, CTAs normally expressed in the restricted in ESCs and the germline in the testis. CTAs antigens are majorly expressed during the spermatogenesis process even though their role are not yet identified (Cheng, Wong & Cheng 2011). The findings in this research classifies the large group of the germline genes as the Cancer/testis genes. These genes play a vital role in encoding the proteins that are normal in the testis of healthy adults known as the Cancer/testis antigens. Nonetheless, these antigens can also be found in the various cancer types of a wider range. Currently, little is known about the functions of these antigens in the testis but most of the evidence supports the transition from soma-germline cells. For instance, processes include invasiveness and cellular mitotic fidelity regulation (Srivastava et al 2016). Most importantly, the findings discussed above give newer avenues for the hindrance of oncogenic Cancer/testis antigens through therapeutics (Fratta et al 2011).

Testis Expressed 19 (TEX19) as a marker of pluripotency

This dissertation illustrates that TEX19 is a gene that is specific to mammals in which the rodents have a pair of paralogues while the humans have only a single gene (Macclennan et al 2017). Chromosome 11 is the location of both Tex19.1 and Tex19.2 of the mice while the humans have their TEX19 located in the chromosome 17. The human TEX19 is similar to the TEX19.1 of the rodents and has expressions that are specific in the embryonic stem cells (ESCs) and the germline tissues of the testes. Expression of TEX19 in humans is common in the testis and placental cells of the adults.

 TEX19 in the cancer cells

TEX19 is cancer/testis (CT) gene and its first detection in the ovary and testis was found out as a murine orthologue.TEX19, therefore, manifests in the human testis and also in the mouse and human embryonic stem cells. TEX19 has recently been identified that is vital in the expression of transposable elements regulation (McFarlane and Wakeman 2017). Transposable elements are sequences of DNA are found within the genome and they change their position, therefore, resulting to chromosome rearrangements, disruption of gene and deleterious mutations that may lead to cancer. Even though the TEX19 is found in the normal human spermatogonial, this is also found in the cancer tissues in a wide range. Notably, it is a shuttling protein between the cytoplasm and nucleus cancer cells. Additionally, TEX19 suppresses quiescent-like state and enables both in vitro and in vivo proliferation in colorectal cancer cells (McFarlane and Wakeman 2017). Studies indicate TEX19 depletion the cells defect in the S-phase in the cell cycle specifically during conditions of TEX19 knockdown which also indicating that TEX19 is required for cancer cells self/renewal and proliferation. However, TEX19 depletion results in subsets of the genes expression alteration. In this sense, the alterations are linked to the regulation and proliferation of cell cycle (McFarlane and Wakeman 2017).

Histone H3 modification in normal cells

Histone H3 is the majorly modified histone which undergone post-translational modifications are known as marks and its role in gene expression regulation is to organize active regions of the genome of the chromatin (Swammle et al 2016). In addition to this, the heterochromatin regions that are inactive in nature whereby DNA compaction is increased and is made less accessible for the transcription process. Histone H3 modifications predict chromatin type (Euchromatin and Heterochromatin), distinguishes the elements that are functional to a genome enhancers, and promoters and also categorizes the outlined elements into the repressed and active states.

Histone H3 modification cancer cells

The research illustrate the epigenetic changes of Total Histone H3,H3K9 acetylation and H3K4 Tri-methylation.H3 Histone being the most modified type of Histone implicated that repression and activation of DNA during transcription on the basis of the methylation site (Yang et al 2015).In regard to this, H3k4 is majorly found in the gene promoters and is the activation marks. Histone 3 acetylation is therefore associated with the opening of chromatin that enhances the transcription process.H3K9 is associated with the promoters and enhancers that initiate activation of genes.

 Histone methylation​​

H3 methylation of Lysine is implicated in both repression and transcriptional activation on the basis of the methylation site while promotion of transcriptional activation is made possible by the arginine methylation as discussed in this research. Lysines can either be tri-, di-, and mono-methylated therefore giving diversity in the functions to each methylation site. For example, both mono- or tri-methylation of K4 (H3K4me1 and H3K4me3) are the marks that are active but the transcriptional enhancers have H3K4me1 (Messier et al 2016). H3K4me3 is majorly found within the site of the gene promoters (Judes et al 2016). Most importantly, Tri-methylation of K9 on H3 histone serves as a signal for repression activity. Conversely, H3K9me3 offers a signal that is a permanent signal for the formation of heterochromatin chromosomal regions in line with the structures that are repetitive. Methylation of DNA, therefore, leads to silencing of the gene expression that leads to catalysis by DNA methyltransferases. In most cases, the CpG islands majorly become demethylated in the cancer cells. Cancer/testis genes are therefore activated through hypomethylation.

Histone acetylation

This characteristic is often associated with the chromatin structure that is open. In this sense, the transcription factors are made accessible and gene expression is consequently accelerated. Majorly, acetylation of histone largely targets the promoter regions within the cell lines. For instance, acetylation of K27 and K9 on the H3 Histones closely associated with the promoters and enhancers of the genes that are active. Too important to note is that the lowest levels of acetylation have an unidentified function and are found throughout the genes that are transcribed (Su, Wellen & Rabinowitz 2016). Most importantly, the enzymes that are responsible for erasing and writing the histone tails acetylation are the deacetylases and Histones acetyltransferases (Zanhow et al 2016).

Testis-Specific Histone Variant H3t Gene

Testis histone tH3 plays a significant role in spermatogenesis and majorly results to azoospermia. The expression of tH3 is important in the differentiation of spermatogonial but in most cases gets lost. However, the stem cells are committed to undergo chromatin and epigenetic rearrangements. Consequently, the genomic structures ensure stability of H3t variant gene. The reports suggest that modifications of histones Ht3 play critical role in epigenetic gene regulation and majorly affects the nucleosome structure (Maehara et al 2015).

The variants histones, therefore, play a significant role in differentiating nucleosomes and are the key regulators of the function and structure of the chromatin. In this sense, the studies on proteomics are valuable histone-variants sources. In particular, Histone H3 variants such as the testis-specific variants are majorly found in the somatic mammalian cells.

RAD51 genes

This is a protein that is found in the pathway of combinational DNA repair that plays a critical role in DNA repair. Notably, this is a critical step for the mitotic recombination and is the major strand-transferase. This gene contributes to instability in the genomics consequently leading to the development of a tumor (Fratta et al 2011).In this sense, the genomic instability slows the growth of the cells. Sometimes, RAD51 in many tumors is overexpressed and therefore the complementing ability of the increased levels of this gene results in limiting of the genomic instability during the progression of cancer. Most importantly, the wild-type p53 is majorly seen to repress the expression of RAD51

Krüppel-like factor 4 (KLF4) genes

Krüppel-like factor 4 is majorly expressed in the tissues of humans such as the skin and the intestine and serves a critical role in the physiologic processes such as maintenance of homeostasis that is normal to tissues, differentiation, and development. Notably, this factor is a transcriptional factor that is bi-functional and can either repress or activate transcription while utilizing various mechanisms depending on the target gene (Fadous-Khalife et al 2016). Additionally, Krüppel-like factor 4 gene functions as a tumor suppressor or oncogene depending on the cancer type that is involved (Lin et al 2016). Together with three other transcriptional factors, this gene can reprogram the fibroblasts through differentiation into the state where it looks like the embryonic stem cells.

KLF4 a as a tumor suppressor and as an oncogene

Majorly, expression of KLF4 gene is downregulated by both losses of heterozygosity and hypermethylation in the human colorectal cancer as cited by Filarksy et al (2016). However, no evidence has been found to correlate the tumor staging and downregulation of the patients with metastatic cancer. This, therefore, suggests that an earlier detectable event could be the loss of Krüppel-like factor 4 gene. In the mice studies, KLF4 examination of colorectal cancer in the mouse models have yielded the same results as cited by Sun et al (2015). However, the colon carcinoma cell lines in humans have a good number of point mutations that have been found within the Krüppel-like factor 4 gene. Majorly, one mutation has an effect on the activation of p21Cip1/WAF1 in NIH3T3 cells. Notably, in the HCT116 colorectal carcinoma cell line, prevention of the amplification of centrosome after the irradiation from gamma rays is made possible by the KLF4 gene. In addition to this, chromosomal instability is influenced by the loss of this factor. To explain this, the expression of ornithine decarboxylase proto-oncogene is repressed by KLF4 that takes part in the transformation of NIH3T3 cells. This, therefore, outlines Krüppel-like factor 4 serves as a tumor repressor.

As an oncogene, Krüppel-like factor 4 gene overrides RasV12   through the induced transformation and senescence in the fibroblasts. The studies outlined that overexpression of KLF4 gene resulted in the arrest of the cell cycle (Xie et al 2018). However, the addition of RasV12 led to p21Cip1/WAF1 inhibition thus allowing repression of p53 by KLF4. Notably, upon repression of p53, apoptosis is blocked and this leads to transformation.

Sw480 colon cancer

According to Bundscherer et al (2017), a unique feature of colon cancer cell lines (SW480) is that they are important in checking the colon carcinoma progression. The cells of SW480 migrate faster across the HMEC monolayers due to its high ability to come up with metastasis and its locomotive ability. Notably, the overexpression of HSP27 correlates with the unique human colorectal cancer cells behavior on the expression of Cx43. These genes are therefore released by SW480 that play a vital role in the phosphorylation of the endothelial cells. Activation of the Cx43 by SW480, therefore, is important in illustrating the tumor cells heterogeneity (Kuchta-Noctor et al 2016).

Method

 Cell Lines

The Human colon cancer cell lines which were obtained from ATCC were used in the experiment .Notably, the 1640 Eagle’s Minimum Essential Medium (EMEM) which was supplemented with 10 𝜇g/mL streptomycin, 100 IU/mL penicillin and 50 𝜇g/mL neomycin were used to culture the selected cells. Most importantly, the fetal bovine serum which in 10% heat inactivated is important in culturing of colorectal cancer cell lines. Additionally, the conditions that were necessary for incubation of these cells were: change of medium after every 48 hours, 37∘ C Hummified atmosphere, 5% Co2 and the initial cell density of 2.5 × 104 cells/flask.

RNA Isolation

The storage of tissues was done -80∘ C in RNAlater Solution for the purpose of desfrostation.The residual RNAlater was thereafter removed through rinsing the tissues in 1 minute with PBS biuffer.Homogenization of the samples was done with 600mL RLT buffer using the Ultra-Turrax T-10 basic dispersing tool in a period of 5 minutes at 30,000 rpm/it.Additionally, RNeasy Fibrous Tissue Mini Kit  was used to extract the total RNA.The purity and concentration of RNA were detected by spectrophotometer through measuring absorbance. The next protocol involved reverse transcribing every RNA sample into the Cdna using the Revert Aid First Strand cDNA Synthesis

siRNA transfection

TEX19 siRNA A (5′-AGGA TTCACCATAGTCTCTTA-3′) and B (5′-TTC AACATGGAGATCAGCTAA-3′) were used in this study alongside the control that was negative.HiPerFect was therefore used to carry out siRNA transfection together with following the instruction from the manufacturer. Notably, 6 μl of HiPerFect together with100 μl of cell medium was mixed with 150 ng of siRNA.Incubation for 15 minutes at room temperature of the mixture was thereafter done to allow formation of the complexes of transfection and this was added to the cells dropwise. This experiment was verified by western blotting and Reverse-Transcriptase-qPCR.

Real-Time Quantitative Reverse Transcription PCR (RQPCR)

Real-time RT-PCR was used in the MRNA levels quantitative assessment on the ABI 7500 Fast instrument with Power SYBR Green PCR Master Mix reagent. The conditions were as outlined: 60∘ C (1 min), 40 cycles at 95∘ C (15 sec) and 95∘ C (15 sec).In this sense, only one product of PCR was amplified under the above conditions according to analysis of melting points.Notably,the endogenous 𝛽-2 microglobulin control and 𝛽-actin were used as the calibrators and the quantity of the target was therefore normalized by this. This was then calculated as fold different prior processing through statistical analysis.

 Statistical Analysis

The Wilcoxon signed-rank test in the CTA expression was used to compare samples from normal colonic mucosa and colon cancer. These data was thereafter analysed as cancer tissue relative expression to normal tissue and the absolute cancer tissue expression in the selected samples and calculated as the ratios. 𝑝 < 0.05 was therefore considered to be statistically significant.

 

 

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