| Colorectal cancer is one of the four most common worldwide malignancies,the incidence of colorectal cancer all over the world each year is rising at a rate of 2%in recent years,and the incidence of colorectal cancer in China is also rising,2 times of the world level.The vast majority cause of patients with colorectal cancer is the death induced by the liver metastases of colorectal cancer.Because the invasion and metastasis of colorectal cancer is a very complex, multi-step process,although some invasion and metastasis mechanism on colorectal cancer has been clarified,there are still a lot of uncertainties.Researchers have reported that many genes involved in regulating the process of colorectal cancer has been identified,which provide an important clue to clarify the molecular mechanism, but they are still far from explaining the complexity and diversity progression of colorectal cancer.At present,the treatment of colorectal cancer surgery,supplemented by local or systemic chemotherapy and radiotherapy failed to significantly improve colorectal cancer 5-year survival rate,with the molecular biology and the development of genetic engineering,gene therapy has been becoming a focus. Even though there is a lot of in-depth studies on gene therapy for colorectal cancer,even there have been made in certain clinical efficacy,there are still the following questions:①the safety on gene therapy has not been resolved,such as genes transfected into the host chromosome bring a series of questions;②It is still difficult to find an ideal safety,efficiency,targeting,high-capacity vector applied to the clinical requirements;③more useful target genes are still urgently needed for effective treatment.The first part of this article focuses on roles of tumor control protein translation (TCTP) in the metastasis ability of high-metastatic colon cancer cell lines LoVo.The second part focuses on improvement and optimization of a new type lentiviral vector production system.The translationally controlled tumor protein(TCTP),a highly conserved protein, has been suggested as a tumor associated antigen.Its mRNA and protein expression levels tend to be higher in the colorectal cancers(CRC),compared to the corresponding normal tissue.Biological models of tumor reversion were established from human leukemia and breast cancer cell lines by using the H-1 parvovirus as a selective agent.Differential gene expression analysis was performed between the parental malignant cells and their revertants.TCTP was found to be the most strikingly down-regulated in tumor reversion.Furthermore,The levels of TCTP in the revertants from other three major solid cancers:colon,lung and melanoma cell lines have the same results,in addition,it was verified that inhibition of TCTP expression could induce changes in the malignant phenotype,when the v-src-transformed NIH3T3 cells transfected with antisense TCTP.The expression levels of TCTP were also found to be associated with Caco-2 differentiation by proteomics approach. Based on recently prognosis studies,TCTP has the potential to be identified as a new predictive marker for better understanding CRC progression involved in metastasis, however,the TCTP biological characteristics in colorectal cancer progression were still remained to be elucidated.Double-stranded RNA-mediated interference(RNAi) has recently emerged as a powerful genetic tool to silence gene expression in multiple organisms and widely used for gene function analysis.Small interfering RNA (siRNA) expression mediated by specific plasmids enables efficient and specific suppression of target gene expression in mammalian cells with maintenance of stable loss-of-function phenotypes.To investigate the biological role of TCTP in colorectal cancer progression,we used an RNAi-based proteomics approach with which we compared proteomes of LoVo cells before and after TCTP knockdown.LoVo initiated from a fragment of a metastatic tumor nodule in the left supraclavicular region of a male patient with a histologically proven diagnosis of colon adenocarcinoma.We investigated the phenotypic changes including the tumor proliferative,invasive and metastatic activities.Our results showed that knockdown of TCTP by shRNA inhibited proliferation,invasion and metastasis of LoVo cells both in vitro and in vivo.TCTP knockdown related proteins were identified by the comparative proteomes strategy.Several different spots were identified as components of the Ubiquitin-Proteasome System,proteins involved in the cytoskeleton biosynthesis and tumor metastasis,furthermore,gel electromobility shift assay(EMSA) and luciferase reporter assay revealed that down-regulation of TCTP might inhibit NF-κB binding activity in LoVo cells.These results imply that the expression of TCTP in CRC cell line LoVo is likely to partially associate with the ability of proliferation,migration and invasion,In addition,the NF-κB signaling pathway might be involved in these TCTP-related biological processes.The inhibition efficiency of TCTP specific shRNA in LoVo cells were first examined by reverse transcription-PCR usingβ-actin as the internal reference. Western blotting also showed the reduced expression of TCTP protein in TCTP-shRNA transfected cells.TCTP and OAS1 mRNA levels were also measured by QRT-PCR.The TCTP mRNA levels were obviously reduced in the shRNA-TCTP LoVo cells;however,The IFN-response gene OAS1 mRNA levels have no significant changed in the three groups.To investigate the effect of TCTP knockdown on functional alteration,growth of cells was firstly assessed by CCK-8 assay.As shown in Figure 1-6,compared with those TCTP-shncRNA transfected cells,the growth of TCTP-shRNA transfected cells was inhibited to 78.0%(P＜0.05),67.2%(P＜0.05),81.8%(P＜0.05) and 84.9% (P＜0.05) at 4th,5th,6th and 7th day,calculated by absorbance,respectively.The effect of TCTP RNAi on motility of LoVo cells was examined using transwell(Costar,NY;pore size,8-mm) in 24-well dishes.Fewer TCTP-shRNA-transfected cells than TCTP-shncRNA-transfected and parental cells were observed when the polycarbonate filters were stained with crystal violet.The migration assay showed the number of cells invaded to the bottom chamber was much greater both in TCTP-shncRNA-transfected cells and in parental LoVo cells than in TCTP-shRNA-transfected cells,TCTP knockdown significantly inhibited the invasiveness of LoVo cells,by 87.5%.To determine whether adhesion was affected by inhibiting expression of TCTP in LoVo cells,we tested attachment of LoVo cells to the extracellular matrix (fibronectin,vitronectin,laminin,and collagen I).Under identical experimental conditions,shRNA-TCTP plasmids transfected cells showed reduced adhesion to fibronectin,vitronectin,laminin and collagen I as compared to shncRNA-TCTP vector transfected cells.The result of adhesion assay demonstrated that TCTP silencing significantly inhibited the adhesive power of LoVo cells to laminin up to 59.6%.Since knockdown of TCTP attenuated proliferation,migration,invasion activity of LoVo cells in vitro,we further investigated whether TCTP suppression would alter tumor growth and metastatic potential in nude mice.Cells(5×106/mouse) suspended in 0.2 ml DMEM were injected subcutaneously into the 6-week-old BALB/c nude mice at the right flanks.The animals were sacrificed on the 21st day after injection and the tumors were dissected and weighed.The tumorigenicity inhibition rate was up to 71.9%.shRNA-TCTP transfected,shncRNA-TCTP transfected and parental LoVo cells were injected into the spleen of nude mice.When the mice were sacrificed 5 weeks after injection,the number of the hepatic surface metastases of shRNA-TCTP group was significantly less than that of the tumors formed by shncRNA-TCTP transfected LoVo cells.These data indicated that suppressed TCTP expression in LoVo cells could inhibit tumor growth and metastasis in nude mice. Histopathologic examinations revealed relatively normal liver tissue of the mice injected with TCTP-shRNA transfected cells and malignant liver tissue of the mice injected with TCTP-shncRNA transfected cells,there are more carcinoma cell colonies presented in liver samples from mice injected with shncRNA-TCTP transfected LoVo cells.To explore the functional consequences of TCTP knockdown,we compared whole-cell proteomes on 2D-SDS-PAGE before and after knockdown of TCTP. Comparison of whole-cell proteomes yielded an alteration in 2D gel spots that were changed upon the removal of TCTP.These spots were excised from the gel,digested with trypsin,and analyzed by mass spectrometry.Information was gathered from 27 individual spots in total,from which 23 were down-regulated and 4 were up-regulated accompanied by the reduced levels of TCTP.The altered spots include Ubiquitin -Proteasome subunits,cancer proliferation,metastasis,cytoskeleton metabolism and ion binding related protein.To validate our mass spectrometry results,we obtained antibodies against a subset of the identified proteins and analyzed their expression levels in the shRNA-TCTP,shncRNA-TCTP and parental LoVo cells groups by Western blotting.We confirmed reduced expression of proteasome subunits of ubiquitin-proteasome system:PSMA1,PSMA3,PSME3,PSMA6,PSMD8 upon knockdown of TCTP;translational related protein HMGB1,the cell cytoskeleton metabolism related protein STMN1,LASP1 could also be confirmed.The mechanism of TCTP-induced attenuated LoVo liver metastasis ability is not clearly understood.From the bioinformatics analysis on the proteomics results,some spots including proteasome subunits PSMA1,PSMA6,PSMD8, ubiquitin-proteasome system activator PSME3 and translationlly and related protein HMGB1 are closely involved in the NF-κB signaling pathways.The transcription factor nuclear factor-κB(NF-κB) family regulates the expression of genes involved in immune response,inflammation,cell proliferation,angiogenesis,metastasis,survival and suppression of apoptosis.To investigate whether NF-κB involved in the TCTP knockdown induced process,the shRNA-TCTP,shncRNA-TCTP cells and the parental LoVo were measured by the NF-κB luciferase reporter and EMSA assays.As shown in Figure.1-14,shRNA-TCTP plasmids transfected cells were associated with a six fold reduction of luciferase activity compared to negative control vector transfected cells,showing that knockdown TCTP inhibits the NF-κB binding activity. NF-κB activation was also examined by using electro mobility shift assays to measure binding of nucleus-located NF-κB to aγ-32P labeled probe containing an NF-κB consensus sequence.As expected,nuclear extracts from shRNA-TCTP transfected cell populations showed the decreased binding activity to the labeled probe.There was no mobility-shifted band detected in no nucleus-extracted protein samples plus labeled probe as a control.To determine whether the probe bind specifically to NF-κB, we determined binding specificity by competition assays using the cold probe and the cold mutant probe.In the superfamily of Rel,p65 plays an important role in transcription after translocation to nuclei after phosphorylation and subsequent degradation of its inhibitor,IκBα.To study the nuclear transactivation of p65 in LoVo cells,we prepared nuclear extracts from three cells cultured for 24 h.A proper aliquot of protein from each cell lysate was loaded per lane onto a SDS-PAGE,transferred to polyvinyldifluoride membrane,and immunoblotted with anti-NF-κB,p65 specific antibodies.Results revealed that the enriching level of p65 was higher in nuclei of shncRNA-TCTP and parental LoVo cells than that in nuclei of shRNA-TCTP LoVo. The usual mechanism of NF-κB induction involves phosphorylation of IκBαby the IKK complex as a key step.The reverse results were detected for IκBαin cytoplasmic extracts compared to p65 in nuclear extracts.Lentiviral vectors are attractive tools for human gene therapy.The goal of the present study was to develop an efficient transient-hybrid system for large-scale production of high titer lentiviral vector stocks.In this system,BHK21 was co-transfected by three main plasmids consisting of transuding plasmid pVECRNA,packaging plasmid pgagpol,envelop plasmid pVSVG, and thereafter infected with the vaccinia vTF-3 containing bacteriophage T7 RNA polymerase gene using Lipofectamin2000TM.After 4 days,the culture supernatant of lentiviral vectors was collected and then followed by filtration through 0.22-um microporous membrane. The culture supernatant was confirmed by the fragment of 96bp generated by RT-PCR and sequencing.After 48h,BHK21 was co-transfected by the three main plasmids and the helper virus.The expression of p24 was observed in BHK21 through indirect immunofluorescence by confocal microscopy,the p24 was mainly distributed in cytoplasm,rarely in cell nucleus.GFP was also expressed in BHK21 and 293T 48h later after they were infected with the culture supernatant.3*3*3 factorial design was used to explore the yield optimization of this system. The type of cell lines,plasmids dosage and the MOI(the proportion between cell numbers and virus copies) were considered critical to the output of this system.The SPSS analysis results demonstrated that the three factors have significant effects on the output of the system respectively,and these three factors have significant interaction.3*3 factorial design was used to explore the yield optimization of this system when BHK21 was utilized as the production cell line for that it was the best cell line for the LV production among the three mentioned cell lines containing BHK21,Vero, and HepG2.The SPSS analysis results illustrated that the plasmids dosage and the MOI have significant effects on the system output respectively,and these two factors have significant interaction.In short,the system yield archived to the favorable condition that BHK21 was co-transfected with 10μg pVECRNA,10μg pGAGPOL and 2μg pVSVG with the MOI 1 under the condition of 37℃and 5%CO2.The LV copy number achieved to 1×1011c/ml and the titers of LV stocks achieved to 1.3×108tu/ml.In summary,the present study suggests that knockdown of TCTP in colon carcinoma LoVo cells might play at least a partial role in effectively inhibiting their migration,invasion in vitro and liver metastasis in vivo.the NF-κB signaling pathway might be involved in these TCTP-induced biological processes.Further work will be required to explain the concrete biological mechanism of TCTP on tumor growth and metastasis of colon adenocarcinoma LoVo cells.The LV has been successfully prepared with the new manufacturing system,which established a solid foundation for the optimization of further manufacture and clinical application. |
