Experimental Research Of The Correlation Between PC4and VEGF-C/D-VEGFR-3Signaling Molecules Of Lymphangiogenesis On The Lung Adenocarcinoma Cells In Vitro

Background and objectiveLymphatic metastasis is an important early event occurred in human lung carcinoma.However, the molecular mechanism of lymphatic metastasis has not been clear. It is urgentto find a new target molecule for the diagnosis and treatment of human cancers. The humanpositive cofactor4(PC4) is a novel marker for cancer cell transformation and the diagnosisand treatment of lung carcinoma, which is a highly abundant nuclear protein whichfacilitates activator-dependent transcription. Vascular endothelial growth factor-C (VEGF-C)and VEGF-D are the major molecules involved in development and pathologicallymphangiogenesis of lung carcinoma. Therefore, we doubt whetherPC4,VEGF-C,VEGF-D and VEGFR-3effect each other in this progress. This study we willtry to detect the mRNA and protein levels of PC4, VEGF-C, VEGF-D and VEGFR-3andanalys the correlation between each other after inhibiting PC4expression by gene silencing.MethodsFour synthesized siRNAs targeting PC4were amplified and cloned into pSES-HUSrecombinant plasmid respectively. These plasmids were transiently transfected into293Tcell line by Lipofectamine2000. Then we detected the PC4mRNA and protein levels by realtime quantitative PCR(qRT-PCR)and Western blot. We further designed and synthesisedpGCL-siRNA PC4and the NC-GFP-Lentiviral Vector recombinant plasmids. And then,these plasmids were infected into A549cell line. We detected the mRNA and protein levelsof PC4, VEGF-C,VEGF-D and VEGFR-3by qRT-PCR and Western blot. Meanwhile, weanalysed the correlation between each other by using SPSS17.0statistical analysissoftware.ResultsThe four recombinant plasmids were successfully constructed as confirmed by enzyme digestion and DNA sequencing, named pSES-HUS-PCi1, pSES-HUS-PCi2,pSES-HUS-PCi3and pSES-HUS-PCi4recombinant plasmids. The pSES-HUS-PCi1recombinant plasmids(sense,5′-AACAGAGCAGCAGCAGCAGATTTT-3′; antisense,5′-ATCTGCTGCTGCTGCTCTGTTTT-3′)had the most significant knockdown effect onPC4mRNA and protein (P＜0.01)，with inhibition rates of85.30%at mRNA level and80.57%at protein level. We successfully constructed the pGCL-siRNA PC4LentiviralVector recombinant plasmids, which were had the most significant knockdown effect onPC4mRNA and protein (P＜0.05)，with inhibition rates of85.40%and77.50%at mRNAand protein level. Along with the expression of PC4were knockdown, the expression ofVEGF-C,VEGF-D and VEGFR-3mRNA and protein levels were declined. Fouther more,the PC4mRNA expression and VEGF-C, VEGF-D and VEGFR-3mRNA expression werepositively correlated (r=0.792, P=0.005; r=0.592, P=0.046; r=0.661, P=0.026). Similarly,PC4protein expression and VEGF-C, VEGF-D and VEGFR-3protein expression werepositively correlated (r=0.966, P=0.000; r=0.812, P=0.004; r=0.856, P=0.003).ConclusionsThe small interfering RNA (siRNA) used for PC4knockdown is PC4siRNA#1(sense,5′-AACAGAGCAGCAGCAGCAGATTTT-3′; antisense,5′-ATCTGCTGCTGCTGCTCTGTTTT-3′). The expression of PC4is positively related with VEGF-C, VEGF-D andVEGFR-3. PC4may be a upstream molecules of VEGF-C, VEGF-D and participate in theregulation of VEGF-C/D-VEGFR-3signaling pathways of lymphangiogenesis in lungadenocarcinoma.