Korean J Physiol Pharmacol.  2024 May;28(3):265-273. 10.4196/kjpp.2024.28.3.265.

RNA polymerase I subunit D activated by Yin Yang 1 transcription promote cell proliferation and angiogenesis of colorectal cancer cells

• Shan J
• Liang Y
• Yang Z
• Chen W
• Chen Y
• Sun K

• KMID: 2555668
• DOI: http://doi.org/10.4196/kjpp.2024.28.3.265

Abstract

This study aims to explore possible effect of RNA polymerase I subunit D (POLR1D) on proliferation and angiogenesis ability of colorectal cancer (CRC) cells and mechanism herein. The correlation of POLR1D and Yin Yang 1 (YY1) expressions with prognosis of CRC patients in TCGA database was analyzed. Quantitative realtime polymerase chain reaction (qRT-PCR) and Western blot were applied to detect expression levels of POLR1D and YY1 in CRC cell lines and CRC tissues. SW480 and HT-29 cells were transfected with si-POLR1D or pcDNA3.1-POLR1D to achieve POLR1D suppression or overexpression before cell migration, angiogenesis of human umbilical vein endothelial cells were assessed. Western blot was used to detect expressions of p38 MAPK signal pathway related proteins and interaction of YY1 with POLR1D was confirmed by dual luciferase reporter gene assay and chromatin immunoprecipitation (ChIP). TCGA data showed that both POLR1D and YY1 expressions were up-regulated in CRC patients. High expression of POLR1D was associated with poor prognosis of CRC patients. The results showed that POLR1D and YY1 were highly expressed in CRC cell lines. Inhibition or overexpression of POLR1D can respectively suppress or enhance proliferation and angiogenesis of CRC cells. YY1 inhibition can suppress CRC progression and deactivate p38 MAPK signal pathway, which can be counteracted by POLR1D overexpression. JASPAR predicted YY1 can bind with POLR1D promoter, which was confirmed by dual luciferase reporter gene assay and ChIP. YY1 transcription can up-regulate POLR1D expression to activate p38 MAPK signal pathway, thus promoting proliferation and angiogenesis ability of CRC cells.

Keyword

Colorectal neoplasms; POLR1D; p38 mitogen-activated protein kinases; YY1 transcription factor

Figure

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  Fig. 1 Increased expression levels of POLR1D and YY1 in CRC tissues and cell lines. (A, B) The expression levels of POLR1D and YY1 in CRC patients were analyzed by GEPIA in TCGA database. (C) Kaplan–Meier method was used to analyze the correlation between POLR1D expression level and overall survival of CRC patients in TCGA database. (D, E) qRT-PCR and Western blot were applied to detect the mRNA and protein expression levels of POLR1D and YY1 in CRC cell lines. (F, G) CRC tissues and their adjacent normal tissues were collected from 22 CRC patients. qRT-PCR was used to detect the mRNA expressions of POLR1D and YY1. (H) Pearson method was used to analyze the correlation between POLR1D and YY1. Data were presented as mean ± SD. CRC, colorectal cancer. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 2 POLR1D silencing inhibits the cell proliferation of CRC cells and angiogenesis ability of HUVECs. After SW480 and HT-29 cells were transfected with si-NC or si-POLR1D, qRT-PCR (A) and Western blot (B) were applied to detect the mRNA and protein expressions of POLR1D. The cell proliferation ability was assessed by CCK-8 (C) and clone formation assay (D). Tube formation assay was applied to detect the angiogenesis ability of HUVECs. Scale bar, 20 μm (E). Data were presented as mean ± SD. CRC, colorectal cancer; HUVECs, human umbilical vein endothelial cells. *p < 0.05, **p < 0.01, ***p < 0.001.

• 

  Fig. 3 YY1 can bind with the promoter of POLR1D to increase its transcription and elevate its expression level. SW480 and HT-29 cells were transfected with pcDNA3.1, YY1, si-NC or si-YY1. JASPAR was used to predict the binding sites and the mutant sequences between YY1 and POLR1D promoter (A). The interaction between YY1 and POLR1D was verified by dual luciferase reporter gene assay (B) and ChIP (C). qRT-PCR (D) and Western blot (E) were used to detect POLR1D mRNA and protein expression levels. Data were presented as mean ± SD. CRC, colorectal cancer. *p < 0.05, **p < 0.01.

• 

  Fig. 4 POLR1D activated by YY1 can regulate CRC cell proliferation and angiogenesis ability through p38 MAPK signal pathway. After SW480 and HT-29 cells were transfected with si-NC, si-YY1, pcDNA3.1 and POLR1D or 10 μM SB 203580, the cell proliferation was detected by using CCK-8 assay (A) and clone formation assay (B). The angiogenesis of HUVECs was assessed by tube formation assay. Scale bar, 20 μm (C). Western blot was used to detect the protein expression levels of p38 MAPK signal pathway related proteins (D). Data were presented as mean ± SD. CRC, colorectal cancer; HUVECs, human umbilical vein endothelial cells. *p < 0.05, **p < 0.01, ***p < 0.001.

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