Dopamine and cyclic adenosine monophosphate-regulated phosphoprotein with an apparent Mr of 32000 promotes colorectal cancer growth

Figure 1 Expression and clinical relevance of dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 in colorectal cancer. A: Dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 (DARPP-32) mRNA level in primary human colorectal cancer (CRC) tumors (n = 376) and normal tissues (n = 667) were analyzed from the Gene Expression Profiling Interactive Analysis Database; B: DARPP-32 expression was validated in 70 pairs of CRC patient samples by polymerase chain reaction; C-E: Representative images and quantification of DARPP-32 staining in 94 CRC tissues and 86 normal tissues (red scale bars, 200 μm; black scale bars, 50 μm); F: Overall survival curves for CRC patients expressing DARPP-32 according to Kaplan-Meier curves; G-I: DARPP-32’s mRNA and protein levels in cell lines (NCM460, HCT116, SW480, HT-29, LOVO). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001.

Figure 2 Dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 promotes colorectal cancer proliferation in vitro. A and B: Detecting the expression level of dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 (DARPP-32) by western blot analysis in HCT116/SW480 stable cell lines overexpressing DARPP-32 and in HT-29/LOVO cell lines with DARPP-32 knockdown; C-G: DARPP-32 overexpression promoted the growth of HCT116 and SW480 cells by ethynyl-2′-deoxyuridine (EdU) and Cell Counting Kit-8 (CCK-8) (black scale bars, 100 μm); H-K: Representative images of the proliferative capabilities of cells in HT-29 and LOVO cells with small interfering RNA (siRNA) knockdown of DARPP-32 detected by EdU and CCK-8 (black scale bars, 100 μm). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001; ^dP < 0.0001. GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; NC: Negative control.

Figure 3 Dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 promotes colorectal cancer proliferation in vivo. A: Dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 (DARPP-32)-overexpressing HCT116 cells were subcutaneously implanted into the shoulders of naked mice (n = 5) to assess proliferative potential; B: After injection, carcinoma volumes were measured in the negative control (NC) group and DARPP-32 group; C: DARPP-32 and Ki67 immune staining were performed on tumor tissues from the NC and DARPP-32 groups by immunohistochemistry (blue scale bars, 100 μm; black scale bars, 50 μm). ^aP < 0.05; ^cP < 0.001.

Figure 4 Dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 reduces apoptosis in colorectal cancer cells. A and B: The apoptotic capabilities of cells in HCT116 and SW480 cells with dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 (DARPP-32) overexpression detected by analysis of cellular apoptosis; C and D: The apoptotic capabilities of cells in HT-29 and LOVO cells with DARPP-32 knockdown small interfering RNA (siRNA) detected by analysis of cell apoptosis. ^aP < 0.05; ^bP < 0.01. NC: Negative control.

Figure 5 Dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 promotes colorectal cancer cell invasion and migration. A and B: By using the wound healing assay, dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 (DARPP-32) overexpression improved the ability of HCT116 and SW480 cells to migrate; C and D: By using a wound healing experiment, DARPP-32 knockdown reduced the ability of HT-29 and LOVO cells to migrate; E and F: Images illustrating the capacity for cell migration and invasion in HCT116 and SW480 cells where DARPP-32 overexpression was found in the transwell assay; G and H: Images illustrating the capacity for cell migration and invasion in HT-29 and LOVO cells where DARPP-32 knockdown was found in the transwell assay (black scale bars, 100 μm). ^aP < 0.05; ^bP < 0.01; ^cP < 0.001. NC: Negative control.

Figure 6 Effect of dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 on progression is partially dependent on the phosphoinositide 3-kinase/AKT signaling pathway. A: Our group examined the differences in mRNA expression between the normal control and dopamine and cyclic adenosine monophosphate regulated phosphoprotein with an apparent Mr of 32000 (DARPP-32) groups using RNA sequencing (RNA-seq) analysis; B: Heat map of a DARPP-32 downstream target assessed by RNA-seq analysis. Red indicates upregulation of the corresponding gene and green indicates downregulation; C: Bubbly graph of Gene Ontology enrichment terms; D: Analysis of the DARPP-32 level Kyoto Encyclopedia of Genes and Genomes data; E: Western blot analysis of DARPP-32, AKT, p-AKT, phosphoinositide 3-kinase (PI3K) and phosphorylated PI3K proteins in DARPP-32-overexpressing cells or DARPP-32 small interfering RNA (siRNA)-infected cells. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) served as a loading control. NC: Negative control.