Studies On The Role Of Human Wild Type Mstl Gene On The Growth Of Human Tumors

Mammalian STE20-like kinase1(Mst1), also named serine threonine kinase4(STK4) and kinase responsive to stress2(KRS2), is the mammalian homolog ofDrosophila Hippo, a major inhibitor of cell proliferation in Drosophila. Itubiquitously encodes serine threonine kinase that belongs to the family of proteinkinases related to yeast STE20, and is involved in cell proliferation, apoptosis,oncogenesis, and organ growth. Recent studies show that Mst1has tumor-suppressorfunction.Objective:Here we explored the tumor-suppressor function of Mst1and its partialmolecular mechanisms from both molecular, cellular and animal levels, andsimulated gene therapy for cancer to provide it with some reference theory andexperimental basis.Methods:1. Construction of recombinant eukaryotic expression vector pEGFP-N1-Mst1containing human wild type Mst1geneMst1cDNA was directly ligated into enhanced green fluorescent proteinexpression vector pEGFP-N1, the growing positive colons were picked out by usingLuria-Bertani (LB) broth containing kanamycin (Kanr) resistance, and the positiveclones obtaining recombinant plasmids were screened by colony PCR, the insert wasconfirmed by PCR as designed above and sequencing analysis.2. The role of Mst1overexpression on human hepatocellular carcinoma cellsHepG2in vitroThe recombinant pEGFP-N1-Mst1was transfected into human hepatocellularcarcinoma cells HepG2in vitro by PolyJetTMin vitro DNA transfection reagent. Cellgrowth and cell apoptosis were determined by3-(4,5-imethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay, Flow cytometer, and Hoechst33342nuclear staining. To explore the possible molecular mechanisms of thetumor-suppressor function of Mst1, the transcript levels of connective tissue growth factor(CTGF), amphiregulin(AREG), and birc5(Survivin) were detected by Real-timequantitative polymerase chain reaction(RT-qPCR), the phosphorylation status of YAP,the activation of MST1, and caspase-3were detected by Western blot. In addition,we combinated MTT assay, Flow cytometer, and Western blot to explore therelationship between the expression and activation of MST1protein andDDP-induced cell death.3. Effect of Mst1overexpression on the growth of human Non-small cell lungcancerThe recombinant pEGFP-N1-Mst1was transfected into human Non-small celllung cancer(NSCLC) and preliminary investigated the apoptosis of A549and theexpression of MST1, YAP, Phospho-YAP (Ser127) protein and Mst1, CTGF, AREGand Survivin mRNA.We established nude mice models of human NSCLC cells A549xenografts, then used Mst1to simulate gene therapy of xenografts, observed thegrowth of xenografts and detected YAP and Phospho-YAP(Ser127) protein usingimmunohistochemistry assay, to exproe the role of the inhibition of tumor growth byMst1gene in vivo.Results:1. The recombinant plasmid pEGFP-N1-Mst1was successfully constructed andidentified by colony PCR, sequencing and sequence alignment.2. HepG2cells were successfully transfected with pEGFP-N1-Mst1, both theinhibition rate and apoptosis rate were significantly increased, the inhibition rateswere elevated in a time-dependent manner, and significantly downregulated the levelof CTGF, AREG and Survivin mRNA, upregulated the level of MST1aminoterminal truncated form (NT), Caspase-3cleavage (CL), and Phospho-YAP(Ser127),and the founction of Mst1was been promoted by the intervention of DDP. Cellswere treated with DDP after transfected, cell proliferation activity of HepG2-MST1cells was significantly decreased(P<0.05), and the apoptosis rate was significantlyincreased(P<0.05). Levels of MST1(NT) and Phospho-YAP(Ser127) were elevatedin a time-dependent manner after treated with20μg/ml DDP, but the total expressionof MST1and YAP protein had no change.3. After transfected with pEGFP-N1-Mst1, the apoptosis rate of A549cells and the levels of MST1(NT) and Phospho-YAP (Ser127) protein were increased, thelevels of CTGF, AREG and Survivin mRNA were decreased. We used Mst1gene,DDP, combination DDP with Mst1gene to treat nude mice xenografts, the averagetumor size and weights were clearly lower than control and negative control group27days later(P<0.01), their tumor inhibition rate were (32.30±1.87)%,(51.77±3.98)%, and (73.01±1.22)%, respectively, with the decreasing of the nuclearlocalization of YAP and the increasing of the cytoplasmic localization of YAP andPhospho-YAP(Ser127).Conclusion:1. Overexpression of Mst1could inhibit cell proliferation and promote apoptosisof human HepG2cells and A549cells.2. Mst1Overexpression increased the level of MST1(NT), Phospho-YAP(Ser127) and caspase-3(CL), downregulated the expression of CTGF, AREG andSurvivin mRNA, it may be one of the pro-apoptosis molecular mechanisms of Mst1gene.3. There is some relationship between the activation of MST1and DDP-inducedcell death. Mst1overexpression could increase the sensitivity of HepG2cells to DDPand DDP could promote the activation of MST1protein in vitro.4. Mst1expression has some inhibitory effect on the growth of NSCLC cellA549nude mice xenografts in vivo, promoted the cytoplasmic localization andphosphorylation of YAP(Ser127) protein, which may be one of the tumor inhibitorymechanisms of Mst1gene.Overall, our results indicated that Mst1might be developed as a promisinganticancer target.