Dysregulation of transcripts SMAD4-209 and SMAD4-213 and their respective promoters in colon cancer cell lines

Abstract

Background: The pervasive role of alternative promoters in context-specific isoform expression and the importance of promoter choice over its level of transcriptional activity have been recently implied based on pan-cancer in silico studies. We aimed to explore this phenomenon at the cellular level on the example of a major tumor suppressor SMAD4 in search of molecular mechanisms in colorectal cancer that could be exploited for novel biomarkers or therapeutic approaches. Methods: Multi-omics technologies, in silico tools and in vitro functional assays were applied to analyze the transcripts expression and the alternative promoters' function of the SMAD4 gene in colon cell lines HCEC-1CT, HCT116, DLD-1, SW480 and SW620. Results: High expression of the transcript SMAD4-213 emerged as a hallmark of colon cancer cells, while in silico tools point to its possible additional role and potential for sponging miRNAs. Based on the observed dysregulation of SMAD4-209 and SMAD4-213 in malignant vs. non-malignant colon cells, we propose that their expression ratio might be a solid biomarker candidate for colorectal cancer detection. Conclusions: A differential pattern of the respective promoters' activity was observed that corresponds to the expression of transcripts, confirming the role of alternative promoters in context-specific isoform expression. The investigated SMAD4 promoters and transcripts harbor translational potential that should be further investigated.

Keywords: SMAD4; alternative promoter; colorectal cancer; transcript; transcriptional regulation.

Figure 1

Expression of the SMAD4 transcripts in colon cell lines based on FPKM values obtained through RNA sequencing. Presented are SMAD4 transcripts with moderate/high abundance in non-malignant HCEC-1CT and malignant DLD-1 and HCT116 cell lines. LogFC and p values are presented only for transcripts that have |logFC| values>2.5 being in the same direction (both positive or both negative) in both malignant cell lines in comparison to non-malignant and which p values are <0.05. FPKM - Fragments Per Kilobase of transcript sequence per Millions base pairs. logFC - log 2-fold change, * p<0.05, *** p<0.001.

Figure 2

Schematic representation of the predicted microRNA binding sites positions in the secondary structure of the SMAD4-213 transcript obtained by AnnoLnc2 and miRDB tool.

Figure 3

Fluorescence measurement of proteins originating from reporter vector constructs for promoter C and D in malignant SW620 and non-malignant HCEC-1CT cell lines. The fluorescence values of the reporter vectors with cloned promoters from which the values for the empty vectors were subtracted are presented.

Figure 4

Promoters C and D activity estimated as the total transcription initiated at each promoter in FPKM in non-malignant HCEC-1CT and malignant HCT116 and DLD-1 cell lines.

Figure 5

Schematic representation of the SMAD4 gene promoters C and D with binding sites of transcriptional regulators predicted to interact with these sequences. Promoter C is presented as a green box, while promoter D as a red box. Transcriptional regulators predicted to bind to promoter C - NF-kB, EGR3, PPAR-γ, E2F1, MAZ, ELF1, ELK1, ETF1, SP1 and NRF. Transcriptional regulators predicted to bind to promoter D - GFI1, NF-Y and SOX9. TSS - transcription start site.