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Transgelin/PARP1 regulates colorectal cancer metastasis through Rho GTPase pathway
Background
Our previous studies showed that transgelin may promoted the migration of HT29 and HCT116 colon cancer cells through nuclear translocation. Proteomics analysis demonstrated that transgelin interacted with 297 proteins, while the gene expression profile showed that TAGLN affected the expression of 256 downstream genes. Bioinformatics analysis identified 7 key genes, the Rho GTPase signaling pathway as the key pathway, and suggested that PARP1 may participate in the regulation of the key genes with transgelin. This study aims to verify the interaction between PARP1 and transgelin, and explore the mechanism of transgelin/PARP1 in regulating the metastasis of colorectal cancer.
Methods
Co-immunoprecipitation and immunoblotting were used to verify the interaction between PARP1 and transgelin. The expression of PARP1 in colorectal cancer and non-cancerous tissues were analyzed with the mRNA data from GEO microarrays and TCGA database, and were also examined with a tissue microarray by immunohistochemistry. Kaplan-Meier Analysis was performed to analyze the influence of PARP1 expression on overall survival. We then explored the nuclear localization of transgelin in siRNA-mediated knockdown of PARP1 in colon cancer cells by immunoblotting and immunofluorescence. The proliferation, migration and invasion of PARP1-knockdown SW480 and RKO cells were detected by CCK8, wound healing and transwell invasion assays. An expression plasmid containing RAC1 which was one of the key genes identified from the TAGLN-overexpression cDNA microarray analysis, and also a key member of the Rho GTPase family was constructed. This plasmid was co-transfected with PARP1-siRNA into SW480 and RKO cells to replenish the expression of RAC1. The proliferation, migration and invasion assays of the co-transfected colon cancer cells were also performed.
Results
Immunoprecipitation and immunoblotting showed that PARP1 interacted with transgelin in HCT116 cells. The expression of PARP1 in colon cancer was increased as compared to the non-tumor samples p < 0.05). The expression of PARP1 was correlated with disease-free survival (p < 0.05) and overall survival p < 0.05. Knockdown of PARP1 reduced the nuclear transgelin in RKO and SW480 cells, and simultaneously decreased the proliferation migration and invasion of colon cancer cells, respectively. Overexpression of TAGLN led to increased expression of RAC1, while knockdown of PARP1 resulted in reduced expression of RAC1 in colon cancer cells. While the PARP1-knockdown cells were re-introduced with RAC1 expression, their proliferation migration and invasion ability were re-gained as compared to the control groups.
Conclusions
Transgelin interacted with PARP1 in HCT116 cells. PARP1 was an adverse prognostic marker in colorectal cancer and participated in the nuclear translocation of transgelin. Transgelin / PARP1 may regulate the metastasis of colorectal cancer through RAC1 of the Rho GTPase signaling pathway.
Legal entity responsible for the study
The authors.
Funding
The Natural Science Foundation of Guangdong Province (grant no. 2017A030313603, YL) supported the present study.
Disclosures
All authors have declared no conflicts of interest.
