Wnt Signaling and Cadherin Expressions in Different Staging of Colorectal Cancer as Biomarkers for Metastasis: Study of SW480, COLO 320DM, and HCT116 Cellines

BACKGROUND: Early metastases is still unresolved problem in cancer management, eventually in colorectal cancer (CRC). In addition, many markers are useful just only in the late stage of CRC. AIM: This study evaluates the differences in the expression intensity of nuclear β-catenin, cytoplasmic β-catenin, E-cadherin, and N-cadherin between CRC SW480 cell line as control group and COLO320DM and HCT116 cell lines as case groups. MATERIALS AND METHODS: This study applied experimental research design with the different test methods. Culture growing and subcultures manufacturing for the CRC cell line models were done initially and followed by the immunofluorescence method by administering antibodies on β-catenin, E-cadherin, and N-cadherin, and continued with staining process using fluorescein-5-isothiocyanate and 4’, 6-diamidino-2-phenylindole. Observations were done using an immunofluorescence microscope. Calculation of area density in each cell to perceive the expressions of cytoplasmic and nuclear β-catenin, E-cadherin, and N-cadherin was conducted using ImageJ software, resulted in mean fluorescence intensity. RESULTS: There are significant differences in the expressions of cytoplasmic β-catenin, nuclear β-catenin, E-cadherin, and N-cadherin among SW480, COLO320DM, and HCT116 cell lines (p < 0.05). Despite no significant differences in cytoplasmic and nuclear β-catenin expressions between SW480 and HCT116 cell lines, and in E-cadherin and N-cadherin expressions between COLO320DM and HCT116 cell lines (p > 0.05). SW480 cell line has a higher expression of nuclear β-catenin than the cytoplasm (p < 0.05). CONCLUSION: This study reveals differences in the expression of nucleic and cytoplasmic β-catenin, E-cadherin, and N-cadherin in three stages of CRC (Duke B, C, and D) refer to different activation invasion, migration, and metastatic processes. Furthermore, the high expression of nuclear β-catenin and N-cadherin in the early stage of CRC indicate there is a metastatic process in that stage, so nuclear β-catenin and cadherin can be considered as potential biomarkers in the early stage of this cancer.

Mir-186-5p Regulates WNT Signaling Pathway by Targeting TCF4 Transcription Factor

The evolutionarily conserved Wnt signaling pathway plays essential roles during embryonic development, tissue homeostasis and differentiation. This pathway is deregulated in many cancers especially colorectal cancer. MiRNAs are a class of small noncoding RNAs that may play a major role in post transcriptional regulation of many genes and signaling pathway such as WNT signaling pathway. Here, we intended to investigate if miR-186-5p is capable of regulating WNT signaling pathway wia suppression TCF4 gene expression. miR-186-5p was bioinformatically predicted as a candidate regulator of TCF4 gene expression and then, in this experimental study, miR-186-5p was overexpressed in SW480 cell line and its increased expression was detected through quantitative reverse transcription polymerase chain reaction (RT-qPCR). The effect of miR-186-5p on WNT pathway was analysied with TOP/FOP flash assay in SW480 cell line. Finally, flow cytometery was used to inves tigate the effect of miR-186-5p overexpression on cell cycle progression in SW480 cell line. miR-186-5p was overexpressed in the SW480 cell line and its overexpression resulted in significant reduction of the TCF4 mRNA level. TOP/FOP flash assay, confirmed the negative effect of miR-186-5p on the Wnt pathway in SW480 cells. Finally, Overexpression of miR186-5p in SW480 cells resulted in cell cycle arrest in subG1 phase, detected by flow cytometry. Overall, accumulative results indi-cated that miR-186-5p by targeting TCF4 is potentially one of the regulators of the WNT signaling pathway.

Mir-186-5p Regulates WNT Signaling Pathway by Targeting TCF4 Transcription Factor

The evolutionarily conserved Wnt signaling pathway plays essential roles during embryonic development, tissue homeostasis and differentiation. This pathway is deregulated in many cancers especially colorectal cancer. MiRNAs are a class of small noncoding RNAs that may play a major role in post transcriptional regulation of many genes and signaling pathway such as WNT signaling pathway. Here, we intended to investigate if miR-186-5p is capable of regulating WNT signaling pathway wia suppression TCF4 gene expression. miR-186-5p was bioinformatically predicted as a candidate regulator of TCF4 gene expression and then, in this experimental study, miR-186-5p was overexpressed in SW480 cell line and its increased expression was detected through quantitative reverse transcription polymerase chain reaction (RT-qPCR). The effect of miR-186-5p on WNT pathway was analysied with TOP/FOP flash assay in SW480 cell line. Finally, flow cytometery was used to inves tigate the effect of miR-186-5p overexpression on cell cycle progression in SW480 cell line. miR-186-5p was overexpressed in the SW480 cell line and its overexpression resulted in significant reduction of the TCF4 mRNA level. TOP/FOP flash assay, confirmed the negative effect of miR-186-5p on the Wnt pathway in SW480 cells. Finally, Overexpression of miR186-5p in SW480 cells resulted in cell cycle arrest in subG1 phase, detected by flow cytometry. Overall, accumulative results indi-cated that miR-186-5p by targeting TCF4 is potentially one of the regulators of the WNT signaling pathway.