| Hepatocellular carcinoma (HCC) is one of the most common malignant tumors thatseriously threaten human health and life. As the difficulty of early diagnosis, highmalignancy and mortality, rapid progression, and, HCC is called "the king of cancer”.The occurrence of HCC is directly or indirectly associated with multiple genes expressingabnormally, therefore, to explore effective treatment for HCC, the key molecules in tumorprogressing becomes the focus of study. With the development of immunology, molecularbiology, cytobiology and genetics, a large number of tumor antigens have been foundrecently, one of which is MUC1, a member of mucin family. Several researches haveshowed that the transmembrane MUC1as an oncogene can transform normal cells intomalignant ones and inhibit apoptosis, which plays an important role in the developmentof adenocarcinomas, such as breast, lung, ovarian, prostate and pancreatic cancer andseveral hematological malignant tumors. Recent studies have shown that MUC1is alsoexpressed in HCC cells and tissues, but the mechiasm of MUC1in the development ofHCC is not clear.In order to investigate the role of the high expression of MUC1oncoprotein in thedevelopment of HCC, we intended to use RNA interfering to down-regulate theexpression of MUC1in SMMC-7721cells, and explore the effect and mechanism ofMUC1silencing on HCC cells proliferation, cell apoptosis and tumor-forming capacity,which would provide the theoretical basis and therapeutic targets for the pathogenesis andgene therapy of HCC.Our studies include the following contents:Screening and identification of MUC1silencing stable cell clones1. Analysis of MUC1expression on SMMC-7721cells by flow cytometryFirstly, we analyzed MUC1expression of hepatoma cells by flow cytometry, the resultsshowed that the SMMC-7721cells expressed high-level MUC1protein.2. Construction of MUC1siRNA recombinant expression vectorIn order to analyze the function of MUC1, in accordance with GenBank logging inMUC1conserved sequence, we designed and synthetised three pairs of DNA Oligonucleotide which act as siRNA template, three MUC1siRNA recombinantexpression vectors were constructed and identified by DNA sequencing, and named themas pGC-MR1, pGC-MR2, pGC-MR3contrary to the unrelated control sequence pGC-NCrespectively.3. Screening and identification of MUC1siRNA recombinant expression vectorSMMC-7721cells were transiently transfected with pGC-MR1, MR2, MR3andpGC-NC by lipofectamine, and analyzed by flow cytometry, semi-quantitative RT-PCR toidentify the effects of MUC1silencing, the results showed that the efficiency of MUC1silencing in SMMC-7721cells by pGC-MR1, MR2and MR3were64.82±5.17%,32.4±3.19%and39.12±2.07%respectively. Cell proliferation of pGC-MR1,2,3transientlytransfected-SMMC7721cells was measured by MTT assay, and the results showed thatthe proliferation of SMMC-7721cells transfected by pGC-MR1was significantlyinhibited, compared with the control group (P <0.05). Then the vector of pGC-MR1andpGC-NC were transfected into SMMC-7721cells, and the stable clones were selected byG418. The efficiency of MUC1silencing was identified by immunofluorescence, flowcytometry, semi-quantitative RT-PCR and Western blotting. Finally, we established threestable MUC1silencing clones and a control clone, and named as MR1-C6, MR1-D4,MR1-D9and NC. The efficiency of MUC1silencing in MR1-C6，MR1-D4and MR1-D9were74.53%,82.34%and63.22%respectively. MR1-C6and MR1-D4clones were usedfor subsequent experiments together with NC.Effects of MUC1silencing on SMMC-7721cell proliferation and migration1. MUC1silencing inhibits SMMC-7721cell proliferationWe analyzed the effects of MUC1silencing on cell proliferation, colony-formingability and cell cycle distribution of SMMC-7721cells by MTT assay, clone formationassay and flow cytometry. The results showed that the cell proliferation andcolony-forming ability in MR1-C6and MR1-D4groups were significantly decreasedcompared with the control group (P <0.01). We also discovered that the cell cycle wasarrested in S phase. The interaction between MUC1-CT and β-catenin was analyzed byco-immunoprecipitation, and the results showed that MUC1silencing reduced theinteraction between MUC1-CT and β-catenin. The distribution of β-catenin was detectedby Western blotting, and the results showed that MR1-C6and MR1-D4with a higherβ-catenin level in cytoplasmic, while with a lower β-catenin level in nuclear compared with the control group（P <0.05）, suggesting that MUC1silencing blocked nucleartranslocation of β-catenin.We further analyzed the expression of cyclin D1/c-Myc in β-catenin signalingdownstream by Real-Time PCR and Western blotting. We found that the mRNA andprotein levels of cyclin D1/c-Myc in MR1-C6and MR1-D4cells were significantlydecreased compared to the control group (P <0.05).These results suggested that MUC1silencing inhibited cell proliferation by blockingthe nuclear translocation of β-catenin and decreasing the expression of cyclin D1/c-Myc.2. Effects of MUC1silencing on adhesion and migration of SMMC-7721cellsThe changes of cell adhesion and migration capabilities were detected byaggregation, scratch and transwell assays, the results showed that the adhesion ofMR1-C6and MR1-D4cells was increased compared with the control group, but therewas not different in the cell migration capabily. The changes of intercellular adhesionmolecule E-cadherin was analyzed by Western blotting, the results showed that comparedwith the control group, the expression of E-cadherin in MR1-C6and MR1-D4cells wassignificantly increased, suggusting the possible mechanism of the increase of theintercellular adhesion.Effects of MUC1silencing on SMMC-7721cell apoptosis1. MUC1silencing induces cell apoptosisWe observed the cell morphological changes respectively by Giemsa, Hochest33342and Annexin V-PE staining, the results showed that MUC1silencing groups(MR1-C6, MR1-D4) had a quantity of cells with morphological features of apoptosis.DNAs of the experimental cells were analyzed by agarose gel electrophoresis, the resultsshowed that the DNA ladder bands were observed in MUC1silencing groups (MR1-C6,MR1-D4). The expression and activation of caspase-3and its substrate PARP weredetected by Western blotting, the results showed that caspase-3and its substrate PARPwere activated.The results above demonstrate that MUC1silencing induced the apoptosis ofSMMC-7721cells.2. The mechanism of MUC1silencing induced-SMMC-7721cell apoptosisThe expression and activation of signaling molecules in mitochondrial and receptorapoptotic pathways were analyzed by Real-Time PCR and Western blotting, the results showed that, compared with the control group, in MR1-C6and MR1-D4cells, theexpression of p53and Bax were significantly increased, while the expression of Bcl-2was significantly reduced, and the mitochondrial cytochrome C release increased, andcaspase-8and caspase-9cleave band can be seen. The results of co-immunoprecipitationshowed that MUC1silencing can reduced the interaction between MUC1-CT withcaspase-8or Bax. These results indicates that MUC1silencing induced SMMC-7721cellapoptosis by the mitochondrial and receptor apoptotic pathways.Microarray analysisThe proliferation or apoptosis related genes among MR1-C6, MR1-D4or NC cellswere analyzed by microarray analysis. The results showed that, compared with the NCgroup, several genes on proliferation or apoptosis signaling pathway changedsignificantly, including Wnt/β-catenin, NF-κB, Insulin, MAPK, TGF-β, growth factorreceptor signaling pathway and death receptor signaling pathway. These results suggestthat MUC1involved in multiple regulatory mechanisms on cell proliferation.Effects of MUC1silencing on cells tumorigenic capacity of SMMC-7721cells innude miceWe subcutaneously inoculated the back of nude mice with SMMC-7721, NC,MR1-C6and MR1-D4cells (2×106cells) to observe the capability of tumorigenesis.After35days, tumor-forming rate in SMMC-7721and NC groups were100%. Thevolume of tumor in SMMC-7721group was larger and the maximum tumor diameter wasabout1.5cm, while the tumor was not observed in the groups of MR1-C6and MR1-D4,which suggested that MUC1silencing in SMMC-7721cells could significantly inhibittumorigenicity of hepatoma cells in vivo.ConclusionIn conclusion, MUC1silencing can inhibit the proliferation of SMMC-7721cellsand induced apoptosis, suggesting that MUC1plays a important role in the process ofHCC development, which made MUC1as a target and provide a theoretical basis forgene therapy of HCC. |
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