Long noncoding RNA VPS9D1-AS1 promotes angiogenesis in colorectal cancer by regulating the VEGFA signalling pathway

Abstract

To clarify the mechanism of long non-coding RNA VPS9D1-AS1 affecting angiogenesis in colorectal cancer (CRC). Western blot and qRT-PCR assays were performed to detect the expression of VPS9D1-AS1 in colorectal cancer. The effects of VPS9D1-AS1 regulating VEGFA and affecting the proliferation, migration and invasion of human umbilical vein endothelial cells (HUVECs) were examined using cell biology, in vitro tubeformation and Chorioallantoic membrane vascular assay. Chromatin Immunoprecipitation (ChIP) and dual luciferase assays were performed to verify the specific sites of transcription factor binding to the promoter region of VPS9D1-AS1. VPS9D1-AS1 is highly expressed in colorectal cancer. Interfering with VPS9D1-AS1 inhibited the proliferation, invasion and migration of HUVECs. Mechanistically, VPS9D1-AS1 can promote angiogenesis by upregulating VEGFA expression and activating the downstream PI3K/AKT pathway. In addition, CEBPB is a transcription factor of VPS9D1-AS1 predicted by database, and the results of ChIP experiments showed that CEBPB could directly bind to the VPS9D1-AS1 promoter region at the -698 bp to -794 bp site. The results of dual luciferase assay showed that CEBPB could enhance VPS9D1-AS1 promoter activity and promote its transcription. VPS9D1-AS1 can be activated by CEBPB transcription factor and target VEGFA to activate its downstream pathway to promote colorectal cancer angiogenesis, which may suggest that VPS9D1-AS1 is critical for regulating colorectal cancer angiogenesis.

Keywords: CEBPB; Colorectal cancer; VEGFA; VPS9D1-AS1; angiogenesis.

Figure 1

VPS9D1-AS1 is highly expressed in colorectal cancer. A. Data were downloaded from the TCGA database and collated and analyzed for differentially expressed LncRNAs in colorectal cancer. B. Volcano plot analysis of the screened LncRNA expression trends. The horizontal axis represents ploidy changes. The vertical axis represents the p-value of the difference. Green, down-regulated genes; red, up-regulated genes. C. Expression of Lnc VPS9D1-AS1 was predicted in 275 colorectal tissues and 349 normal tissues adjacent to cancer using the GEPIA database. *P<0.05. D. qRT-PCR assay to detect the expression of VPS9D1-AS1 in five cell lines of colorectal cancer. E. qRT-PCR assay to detect VPS9D1-AS1 expression in 8 matched pairs of colorectal cancer clinical tissues.

Figure 2

VPS9D1-AS1 promotes proliferation, migration, and angiogenesis in Colorectal Cancer. A. GEPIA database predicted the correlation of VPS9D1-AS1 with ATG2B, ATG3, ULK1, SLC7A11, TP53, ACSL4. B. GEPIA database predicted the correlation of VPS9D1-AS1 with VEGFA, MVD, VEGFR, *P<0.05. C. VPS9D1-AS1 siRNA was transfected into HCT-116 and SW480, and the expression of VPS9D1-AS1 was detected (n=3); *P<0.05. D. Two siRNAs of VPS9D1-AS1 with different interference sites were transfected into HCT116 and SW480 cells, and supernatants and HUVEC were co-cultured after 48 h. cck8 detection of the effect of VPS9D1-AS1 on the proliferation of HUVEC cells (n=3); *P<0.05. E. Two siRNAs of VPS9D1-AS1 with different interference sites were transfected into HCT116 and SW480 cells, and supernatants and HUVEC were co-cultured after 48 h. Scratch experiment detection of the effect of VPS9D1-AS1 on the proliferation of HUVEC cells (n=3); *P<0.05. F. Two different interfering sites of siRNA of VPS9D1-AS1 were transfected into HCT-116 and SW480 cells, and the supernatants were co-cultured with HUVEC after 48 h. Transwell assays were performed to evaluate the effect of VPS9D1-AS1 on HUVEC cell invasion, and compared with NC group (n=3); *P<0.05. G, H. In vitro tubule, chick embryo chorioallantoic membrane assay to detect angiogenesis after three co-culture supernatant treatments of NC, si-VPS9D1-AS1-1, and si-VPS9D1-AS1-2, and the branching nodes of blood vessels were reduced compared with the NC group (n=3); *P<0.05.

Figure 3

VPS9D1-AS1 promotes angiogenesis through VEGFA in colorectal cancer. A. Correlation of VPS9D1-AS1 with VEGFB, VEGFC and VEGFD was predicted by GEPIA database. B. siRNA of VPS9D1-AS1 was transfected in HUVEC cells and VEGFA expression was detected using western blot. C. VEGFA was overexpressed in HCT-116 and SW480 transfected with si-VPS9D1-AS1, and western blot was used to detect the expression of VEGFA. D, E. The siRNA of VPS9D1-AS1 was cotransfected with VEGFA into HCT-116 and SW480 cells, and the supernatant and HUVEC were co-cultured after 48 h. cck8, scratch assay to detect the effect of VPS9D1-AS1 on the proliferation and migration of HUVEC cells. si-VPS9D1-AS1-1 and si-VPS9D1-AS1-2 were compared with NC group (n=3); *P<0.05. si-VPS9D1-AS1-1+VEGFA and si-VPS9D1-AS1-2+VEGFA were compared with si-VPS9D1-AS1-1 and si-VPS9D1-AS1-2 groups, respectively (n=3); *P<0.05.

Figure 4

VPS9D1-AS1 promotes angiogenesis through VEGFA in colorectal cancer. A. The siRNA of VPS9D1-AS1 was cotransfected with VEGFA into HCT116 and SW480 cells, and the supernatant and HUVEC were co-cultured after 48 h. The effect of VPS9D1-AS1 on HUVEC cell invasion was detected by Transwell. si-VPS9D1-AS1-1 and si-VPS9D1-AS1-2 were compared with NC group (n=3); *P<0.05. B, C. In vitro tubule and chick embryo chorioallantoic membrane assays were performed to detect the effects of NC, si VPS9D1-AS1-1+VEGFA, si-VPS9D1-AS1-2+VEGFA after three co-culture supernatant treatments, and the branching nodes of blood vessels were reduced compared with si-VPS9D1-AS1-1 and si-VPS9D1-AS1-2 groups, respectively, (n=3); *P<0.05. D. qRT-PCR assay to detect VPS9D1 expression in HCT-116 and SW480 cells with knockdown of VPS9D1-AS1. E. Correlation analysis of GEPIA database predicted VPS9D1-AS1 with AKT1 and AKT2. F. Western blot detection of p-PI3K, p-AKT and VEGFA expression in HUVEC cells after incubation with supernatants of the indicated cells for 48 h.

Figure 5

CEBPB binds to the promoter of VPS9D1-AS1 and activates its transcription. A. Correlation of molecular VPS9D1-AS1 with transcription factors in the GEPIA database. B. Predicted protein expression of CEBPB in 100 colorectal cancer tissues and 97 paracancerous normal colorectal cancer tissues from the CPTAC database. C. Predicted promoter sites of CEBPB binding to VPS9D1-AS1 according to the database. D. Chip-PCR detected no binding at the -1419 bp to -1539 bp site and binding at -698 bp to -794 bp. E. Construction of dual luciferase reporter gene plasmids with wild-type and mutant sequences of the promoter of VPS9D1-AS1 inserted before Luciferase. F. Co-transfection of CEBPB, PRL-TK, PGL3-698-WT/MT plasmids to detect dual luciferase activity (Firely/Renilla) MT group < WT group (n=3); *P<0.05. G. Detection of mRNA expression level of VPS9D1-AS1 after overexpression of CEBPB by qRT-PCR. H. qRT-PCR to detect the expression of CEBPB and VPS9D1-AS1 in 40 colorectal cancer tissues (n=3); *P<0.05.