RESULTS
Histone H3 modifications of Cancer/Testis Antigens in the colorectal cancer cell lines
As evident in Figure 1 below, the cancer/Testis Antigens are epigenetically modified through the processes of Total histone 3 modification, H3K4 tri methylation and H3K9 Acetylation. The expression of CTAs is regulated and controlled through DNA methylation at the promoter level. The three cell lines under study were therefore observed that sub toxic dose was used to express CTAs.Additionally, the CRC cell lines did not equally respond to the Demethylating DNA drug. There was significant increase in the HTB-39 and HTB-37 cell lines and other several Cancer/Testis genes de novo. Most importantly, the modifications of Histone H3 results to expression of Cancer/Testis Antigens in the colorectal cancer cell lines.
Total histone 3
H3K4 tri methylation
H3K9 Acetylation
Expression of CTAs in Colon Cancer Cell Lines.
A panel of the CTA individuals that express colon cancer in this study included MAGEA1, A2, and A3; OIP5; PLAC1; CAGE; SSX4; FBXO39; NY-ESO-1; and HAGE.The expression of these Cancer/Testis Antigens in the adenocarcinoma cell lines of colon cancer such as CL-188, HTB39, and HTB-37.In this sense, HTB39 and HTB-37 were of a male origin while CL-188 was female’s. Additionally, isolation of HTB-37 was from primary tumours while HTB-39 were from site of metastatis.Most importantly, fold difference was used to measure Cancer/Testis expression and this was in comparison to the normal colon.Notably,there was CTAs variability in the cell lines tested. The highest CTA-expressing cell line is CL-188 as revealed by RQ-PCR that gave 50% of the Cancer/Testis Antigens were overexpressed. In addition to this, HTB-39 cell line came second and showed 38% of the overexpressed CTAs.Lastly, HTB37 gave the least percentage of 33% of the overexpressed Cancer/Testis Antigens.
Table 1: Knockdown Tex19 by siRNA7 using RQ- PCR
PCR Products
Knockdown TEX19
by siRNA7
effects
PCR Products Gel Images
TEX19
Knockdown
OCT4
No effects
RAD51
Knockdown
KLF4
Reduction
PLK1
No effects
KRAS
No effects
TGFB1
No effects
DKK1
No effects
WT1
No effects
SOX2
No effects
SEPT12
Slight intensity
ABL1
No effects
MYC
No effects
LRIG1
No effects
PLOE-3
No effects
Expression of CTAs in Clinical Samples.
The samples collected from normal colonic mucosa that acted as the control and tissues with tumour. The samples of RNA from cancerous and normal tissues were reverse transcribed. Assessment of Cancer/Testis Antigens expression were made possible by real-time PCR and ?- actin and ?-microglobulin expression played a significant role in expression as endogenous controls. The Wilcoxon signed-rank test therefore gave 33% of the tested Cancer/Testis Antigens which were overexpressed significantly (? < 0.05) in the tumour tissue in comparison to the normal cells. Statistical analysis allowed cancer progression and CTA overexpression according to a system known as Dukes staging. Additionally, correlation test of the Spearman rank shoed that disease progression and Absolute expression of the CTAs that were overexpressed were significantly positive (? < 0.05).
