Korean J Gastroenterol.  2024 Jan;83(1):6-16. 10.4166/kjg.2023.132.

Transcription Silencing and CpGs Hypermethylation as Therapeutic Gene Editing in Clinical Colorectal Adenocarcinoma Repression

• Obaid AL-Jumaili MM

• KMID: 2552237
• DOI: http://doi.org/10.4166/kjg.2023.132

Abstract

Background/Aims
Colorectal cancer is the most common cancer in oncopathology, with an increasing incidence among the elderly during the last decade. Various genetic and environmental factors play important roles in the emergence of colorectal adenocarcinoma. Non-coding RNAs, approximately 20–22 nucleotides, are transcribed irregularly in many cancer cells and play a critical role in many metabolic pathways in clinical cancer cases. DNA methylation is a critical epigenetic alteration that controls gene expression. In the current study, transcriptional silencing and CpG hypermethylation were developed as a therapeutic gene editing strategy for the clinical repression of colorectal adenocarcinoma.
Methods
A human colorectal adenocarcinoma cell line (Caco2) and a normal lung fibroblast cell line (Wi38) were utilized as the paradigms in this research to examine the effect of mir155 molecule transfection and CpGs-island (CGI) methylation. Cell counting was achieved using six-well and 24-well plates before transfection using a hemocytometer. The two cell lines were transfected with the mir155 agomir and antagomir molecules. The transfection efficiency, cell viability, cell IC 50 , and target gene expression were measured, and CGIs-methylation was achieved by bisulfate conversion.
Results
The outcomes revealed the downregulation of oncogenes (AKT1 and VCAM1 genes as cancer-associated genes) and the upregulation of tumor suppressor genes (TSGs, Tp53 and KEAP1). In addition, CpG-islands methylation showed significant blocking of the oncogene promoter regions, and the switch on of TSG promoter regions was continuous.
Conclusions
miRNA-CGI-methylation led to the regression of Caco2 cell proliferation, suggesting the potential use of RNA silencing and DNA methylation in targeted gene therapy for colorectal cancer. (

Keyword

Colorectal cancer; Transcriptional silencing; CpGs-islands methylation; Mir155 agomir and antagomir

Figure

• 

  Fig. 1 Inverted fluoresce microscope images of Caco2 and WI38 cells before and after transfection with the mir155 agomir and antagomir artificial sequences under ×40 magnification. (A) Caco2, antagomir. (B) Caco2, agomir. (C) Caco2, before. (D) Wi38, antagomir, (E) Wi38, agomir and (F) Wi38, before.

• 

  Fig. 2 Amplification products of conventional and MS-PCR of AKT1gene in the Caco2 (left) and Wi38 (right) cell lines: line M, molecular marker (100 bp); upper line, bands of DNA with/without agomir sequence of mir155 transfection; lower line, bands of DNA with/without antagomir sequence of mir155 transfection amplicon size 180–190 bp. The blue arrows represent significant results. Electrophoresis conditions, 5v/cm2 and agarose concentration 1.5%.

• 

  Fig. 3 Amplification products of conventional and MS-PCR of the KEAP1 gene in the Caco2 (left) and Wi38 (right) cell lines: line M, molecular marker (100 bp); upper line, bands of DNA with or without the agomir sequence of mir155 transfection; lower line: bands of DNA with or without antagomir sequence of mir155 transfection amplicon size 180–190 bp. The blue arrows represent significant results. Electrophoresis conditions, 5v/cm2 and agarose concentration 1.5%.

• 

  Fig. 4 Amplification products of conventional and MS-PCR of the TP53 gene in the Caco2 (left) and Wi38 (right) cell lines: line M, molecular marker (100 bp); upper line, bands of DNA with or without the agomir sequence of mir155 transfection; lower line: bands of DNA with or without the antagomir sequence of mir155 transfection, amplicon size180-190 bp. The blue arrows represent significant results. Electrophoresis conditions: 5 v/cm2 and agarose concentration of 1.5%.

• 

  Fig. 5 Amplification products of conventional and MS-PCR of the TP53 gene in the Caco2 (left) and Wi38 (right) cell lines: line M: Molecular marker (100 bp); upper line, bands of DNA with or without the agomir sequence of mir155 transfection; lower line, bands of DNA with or without antagomir sequence of mir155 transfection, Amplicon size180-190 bp. The blue arrows represent significant results. Electrophoresis conditions: 5 v/cm2 and agarose concentration of 1.5%.

• 

  Fig. 6 Amplicon density of the target genes with/without bisulfate modification. The yellow color represents the AKT1 oncogene, with a significant promoter blocking value surrounded by a black line. The blue color represents the KEAP1 antioxidant-responsible gene, with an insignificant blocking value surrounded by a black line. The red color represents the TP53 tumor suppressor gene, with a significant unblocking value surrounded by a black line. The grey color represents the VCAM1 metastasis-responsible gene, with a significant blocking value surrounded by a black line.

• 

  Fig. 7 Amplicon density of the target genes with/without bisulfate modification. The yellow color represents the AKT1 oncogene, with a significant promoter blocking value surrounded by a black line. The blue color represents the KEAP1 antioxidant-responsible gene, with a significant blocking value surrounded by a black line. The red color represents the TP53 tumor suppressor gene, with a significant unblocking value surrounded by a black line. The grey color represents the VCAM1 metastasis-responsible gene, with a significant blocking value surrounded by a black line.

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