Lentivirus-mediated RNA Interference Targeting WWTR1in Human Colon Cancer Cells Inhibits Cell Proliferation In Vitro Andtumor Growth In Vivo

Colon cancer is one of the most prevalent malignancies worldwide. Despite theimprovement in its prognosis and therapy in the last few decades, there is presently noefficient cure for disseminated colon cancer and nearly half of colon cancers haveaggressive re-growth of the tumor mass replete with metastatic disease after curativesurgery or chemotherapy. It is essential to develop new targets and therapeuticapproaches, and therapeutic target development requires identification of novelfunctional molecules, their mechanisms of action and strategies for intervention.WW-domain containing transcription regulator1(WWTR1), also called TAZ(transcriptional co-activator with PDZ binding motif), activates many transcriptionalfactors and has important roles in the development of various tissue in mammals..WWTR1has also been shown to regulate stem cell differentiation and self-renewalthrough binding with the transcription factors PPAR-γ, Runx2and Smad. Mostrecently, enhanced expression of WWTR1has been found in both breast and lungcancer cell lines. Moreover, overexpression of WWTR1has been shown to causeenhanced cell proliferation and migration, transformation and the epithelial–mesenchymal transition (EMT) in human immortalized mammary epithelial cells,whereas knockdown of WWTR1in breast cancer cells inhibits tumor formation.suggesting that WWTR1is a novel oncogene and may have important roles in thedevelopment of breast cancer. However, whether WWTR1is also involved in thetumorigenesis of colon cancer has not been explored.In this study, we demonstrate that WWTR1is important to colon l cancer,knockdown of WWTR1by lentivirus-mediated RNA interference in colon cancercells strongly repressed the cell proliferation in vitro and tumor growth in vivo, suggesting WWTR1is a novel oncogene in colon cancer cells.The First SectionStudy on designing and synthesis of WWTR1-siRNA,andhuman colon cancer cells of the line RKO were cultured andtransfected with pGCSIL-GFP-si-WWTR1vectorObjective To design and synthesize siRNA targeted on human WWTR1andclone the recombinant eukaryotic expression plasmid of specific small interferingRNA (siRNA)against WWTR1gene and transfecte the plasmid into human coloncancer RKO cell. Methods To follow the principle of selecting RNA interferencetarget sequence, to utilize the web resource,design complementary small interferenceRNA (siRNA) of WWTR1. The annealed siRNA template was inserted into pGCL-GFP-Neo plasmid. The recombinant plasmid (pshWWTR1) was transformed into293T cell and identified by restrictive enzyme digestion and sequence analysis. The effectof the recombinant plasmid on the WWTR1expression of human colon cancer RKOcells was detected by RT-PCR and Western blot after optimizing transfectionconditions. Results The siRNA targeted on WWTR1were successfully constructed.It was confirmed by restrictive enzyme digestion and sequence analysis that therecombinant plasmid was cloned and the aim sequence was obtained. The WWTR1expression of RKO cells was inhibited at mRNA and protein levels after transfectedwith the recombinant plasmid. The difference is significant between the grouptransfected with the recombinant plasmid and the control groups (P<0.001).Conclusions The siRNA targeted on WWTR1were successfully designed andconstructed. WWTR1shRNA expression plasmid pshWWTR1successfullyconstructed can lastingly inhibit the expression of WWTR1gene in human coloncancer RKO cells. It may be a tool to study the relations between WWTR1and coloncarcinoma in vitro and in vivo. The second sectionKnockdown of WWTR1by lentivirus-mediated RNAinterference in human colon cancer cells significantly decreasedcell proliferation and the colony formation of RKO cells in vitroand tumor growth in vivo.Objective To observe the effect on Proliferation, Cell cycle and apoptosis ofRKO cells with short hair-pin RNA targeted on WWTR1gene in vitro,and investigatethe effect of knockdown of WWTR1on tumorigenicity in vivo. Methods AfterRKO cells were transient transfected with pshWWTR1plasmid, the proliferation andcolony formation of KD cells and NC cells were examined based on the expression ofGFP. The cell cycle was examined by FACS and the cell dividing rate was measuredby BrdU incorporation assay. The rate of apoptosis was evaluated using Annexin V/PIdouble staining coupled flow cytometry analysis. The effect of knockdown ofWWTR1on tumorigenicity was assessed by subcutaneous injection of NC cells andKD cells into severe combined immune deficiency (SCID) BALB/c mice. ResultsThe effects of knockdown WWTR1to the proliferation of RKO cells were examineddaily using the Cellomics ArrayScan high content screening (HCS) platform.Strikingly, RKO KD cells showed decreased proliferative capacity at day4comparedto the NC cells, and this effect continually increased on Day5accompanied by astatistically significant between the two groups (P <0.01). RKO KD cells showedsignificantly fewer colonies than NC cells (P<0.05). Flow cytometric analysisrevealed that the G0/G1-phase DNA content is higher in KD cells (53.61%) than inNC cells (43.3%)(P<0.05), while the S-phase DNA content is lower in KD cells(31.7%) than in NC cells (42,9%)(P<0.05).The rate of DNA synthesis in RKO KDcells was not affected at day1of infection, but showed significantly decreased at day4compared to NC group (P<0.05).The apoptosis rate in RKO KD cells (4.34%) was significantly higher than RKO NC cells (2.05%)(P<0.05).RKO NC cells developedvisible subcutaneous tumors about2weeks post-inoculation and grew rapidly in thenext3weeks. In contrast, KD cells developed tumors slowly and the tumors werevisible only at4weeks post-inoculation, and the average volume of tumors in KDgroup was significantly smaller than NC group after6weeks of inoculation ((P<0.01)Conclusion The knockdown of WWTR1markedly inhibited cells proliferation,colony formation in vitro and tumor growth in vivo, and induced cell cycle arrest,andenhanced cell apoptosis. WWTR1may be a potential therapeutic target in coloncancer. The third sectionExamination the downstream genes of WWTR1in knockdowncellsObjective To understand the underlying mechanism of the anti-proliferation incells with knockdown of WWTR1. Methods we selected several potentialdownstream genes (ASNS, SMAD3, LTBR,et al) from the published DNA microarraydata of WWTR1-overexpressed cells. The quantification of mRNA levels of thetargeting genes was carried out using the Thermal Cycler Dice Real-time PCRSystem (Takara, Japan). We investigated the related pathway by DAVID and KEGGdatabases. Results Quantitative real-time PCR showed ASNS was significantlyrepressed and SMAD3,BTC and ID1were significantly upregulated in WWTR1KDcells, respectively.(P <0.01). Three genes (LTBR, BAX, BAK1) was showed middleupregulation in KD cells (P<0.05), while no change was found in BTC gene. Exceptthe Hippo pathway, the TGF-beta signaling pathway and colon cancer seems to berelative with the anti-proliferation in cells with knockdown of WWTR1. ConclusionWWTR1-KD induced the downregulation of proliferation promoting gene andupregulation of apoptosis genes in KD cells,which may be the possible mechanism for si-WWTR1induced anti-proliferation in the colon cancer cell.