MicroRNA-218Regulates Invasion And Lymphatic Metastasis Of Pancreatic Cancer By Targeting The ROBO1Receptor

Objective To explore miRNAs related to lymphatic metastasis of pancreatic cancer, investigate how miRNA regulats invasion and migration of pancreatic cancer and reveal the effect of inhibiting lymphatic metastasis via miRNA in vivo.Methods (1) miRNAs expression profiling was conducted in primary PDAC tissues with matched adjacent benign tissues as control, and pancreatic cancer cell colony BxPC-3-LN with its parental cell line BxPC-3as control, to find novel miRNAs between groups. Real time quantitative PCR was performed to validate the expression of interest miRNA in pancreatic cancer tissues and cell lines. The relationships between the expression of interest miRNA and clinicopathological features were studied. Trial of in situ hybridization (ISH) was carried out to reveal the in site features of interest miRNA in PDAC, IPMN and matched adjacent benign tissues. Real time quantitative PCR and Western blotting were used to identify the expression of target gene of interest miRNA in pancreatic cancer.(2) Transient transfection of Mimics-218, a analogue of miRNA-218, in cell colony BxPC-3-LN was conducted to up-regulate the expression of miRNA-218, aiming to study the inhibitory effect of miRNA-218on its target gene ROBO1. A luciferase-based transcription reporter assay was performed to verify the interaction site between miRNA-218and ROBO1. Stable transfection of Lentivirus vector with miRNA-218(Lenti-218) to increase the expression of miRNA-218and transient transfection of plasmid pcDNA3.1with muROBO1(loss of3’-UTR) to restore the expression of ROBO1in cell colony BxPC-3-LN were designed, to elucidate that miRNA-218regulate the invasion and migration of pancreatic cancer cell via ROBO1, using transwell assay. CCK-8assay and colony forming assay were conducted to examine the influence of miRNA-218on proliferation of pancreatic cancer cell.(3) miRNA-218stably expressing cell by Lenti-218transfection was used to establish lymphatic metastasis model of human pancreatic cancer in BALB/C nude mice, to investigate whether miRNA-218inhibit the metastasis and growth of pancreatic cancer in vivo.Results (1) Microarray results revealed that the expression of70miRNAs significantly differed between PDAC and matched adjacent benign tissues. Compared with parental cell line BxPC-3,63miRNAs were differentially expressed in cell colony BxPC-3-LN. miRNA-663, miRNA-145, miRNA-218and Let-7were disregulated miRNAs in both pancreatic cancer and cell colony BxPC-3-LN, of these, miRNA-218was the most novel gene (down-regulate more than10fold change). Real time quantitative PCR validated the down-regulation of miRNA-218in both pancreatic cancer tissues and cell colony BxPC-3-LN. Clinically, the group with lymph nodes metastasis showed lower expression of miRNA-218(P=0.003), compared to patients without lymph nodes metastasis. The in site expression of miRNA-218was decreased in PDAC compared to normal acinous epithelium, normal ductal epithelium and IPMN, while the tumor cells in metastatic lymph nodes of pancreatic cancer displayed even lower expression of miRNA-218than primary tumor. The mRNA and protein expression of target gene ROBO1were over-expressed in pancreatic cancer.(2) Transient transfection of Mimics-218in cell colony BxPC-3-LN significantly up-regulated the expression of miRNA-218at the timepoint of24hr,48hr and72hr (P<0.001、P<0.001and P=0.001respectively), and inhibited the mRNA expression (P=0.002、0.002and0.006respectively) and protein expression of ROBO1. The interaction site between miRNA-218and ROBO1located at the3’-UTR of ROBO1with a length of8bases (971-978). Stable transfection of Lenti-218in cell colony BxPC-3-LN increased miRNA-218levels and eliminated repression of both mRNA (P=0.002) and protein of ROBO1, which inhibited the invasion and migration of tumor cells (P<0.001), while enforced expression of miRNA-218resistant ROBO1by transient transfection of pcDNA-muROBO1restored the invasion and migration of tumor cells (P<0.001). The over-expressed miRNA-218in cell colony BxPC-3-LN decreased the density of cytoskeleton and exerted a negative effect on cell junction. Up-regulation of miRNA-218also inhibited proliferation of tumor cells and leaded to lower colony formation ratio (P=0.004)(3) The cell colony BxPC-3-LN transfected with Lenti-218and Lenti-NC were inoculated in BALB/C nude mice and cells grew into tumor2weeks after inoculation. No significant difference of animal weight between group Lenti-218and group Lenti-NC was found. When the trial terminated, the volume of primary tumor was smaller in group Lenti-218, compared to group Lenti-NC (P=0.001). The metastasis ratio of popliteal lymph nodes was lower in group Lenti-218, compared to group Lenti-NC (P=0.007). No significant difference of the metastasis ratio of inguinal lymph nodes was indicated (P=0.108). No metastasis lymph nodes was found among para-iliac lymph nodes. Total metastasis ratio of lymph nodes showed no significant difference between group Lenti-218and group Lenti-NC either (P=0.083)Conclusions (1) miRNA-218was down-regulated in both pancreatic cancer tissues and cell colony BxPC-3-LN with high metastasis ability, while the target gene ROBO1was over-expressed in pancreatic cancer tissues, in a negative feedback loop.(2) miRNA-218regulated ROBO1by interacting with a definite site on the3’-UTR of ROBO1.(3) miRNA-218inhibited invasion and migration of pancreatic cancer cells via ROBO1pathway.(4) miRNA-218exerted a negative effect on lymphatic metastasis and growth of pancreatic cancer in vivo.