Histone H3 modifications of Cancer/Testis Antigens in the colorectal cancer cell lines

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 𝛽- actin and 𝛽-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 (𝑝 < 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 (𝑝 < 0.05).

 

 

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