The Significance Of MTOR Expression In Cervical Cancer And The Influence Of SiRNA And Rapamycin On Gene Expression Of MTOR In Human Hela Cell

Objective:Cervical cancer is one of the common maglignant tumors from female reproductive system. On a global scale, about20millins women die from cervical cancer each year, which seriously harm to women’s health. Cervical cancer incidence of occult, aggressive, early lymph node metastasis, high recurrence rate and poor prognosis.Target of rapamycin (target of rapamyein, TOR) gene is a kind of evolutionarily and highly conservative protein kinase family, and is widely present in a variety of organisms. TOR gene was first found in the yeast and subsequently found in mammals:the structure and function of conserved TOR proteins, known as mammalian target of rapamycin(mammalian target of rapamyein, mTOR, also known as FRAP, RAFT, RAPT). mTOR is a Ser/Thr kinase and its gene is located in1p36.2.The protein encoded by it has2,549amino acids, the molecular weight is289kDa. Because the C-terminal segment of mTOR has the same Catalytic domain homology as a PEG-PI3-kinase (P13K),it belongs to the family members of phosphatidylinositol kinase-related kinase (phosphatidylinositol kinase-related kinase, PIKK). mTOR is involved in the basic functions of the cell activities, which plays a pivotal role in the feeling the nutritional signals,regulating the cell growth and proliferation. mTOR is also an important signal transduction molecule, and is one important downstream effector of the phosphatidylinositol3-kinase/protein kinase B/mammalian target of rapamycin (P13K/Akt/mTOR) signaling pathway. Research shows that the abnormal signal transduction pathway mediated by mTOR is related to a variety of malignant tumors, such as breast cancer, renal cell carcinoma, non-small cell lung cancer. Other studies have shown that the mTOR signaling pathway in esophageal cancer cells is also active, and has a higher expression in esophageal cancer tissues.Translational control has a key role in regulating cell growth. The mRNA binding translation factors (eIF4s) selectively modulate the translation of different mRNAs based on their differing properties, especially the extent of inhibitory secondary structure in their5’untranslated regions. Overexpression of eIF4E leads to cell transformation or disordered growth. It also enhances the translation of certain mRNAs, including that for cyclin Dl. Decreased expression of eIF4E leads to reduced rates of cell growth. Transformed cells express elevated levels of eIF4E. However, the role of eIF4E phosphorylation and the signalling pathways through which it is controlled remain to be elucidated. Oncogenic ras leads to increased phosphorylation of eIF4E, and the ability of ras to transform cells is diminished when eIF4E expression is decreased, implying that eIF4E is a key mediator of ras mediated transformation. The recent discovery of inhibitors of eIF4E (eIF4E binding proteins,4EBP1/2) adds another regulatory component to the system. Although a role for4EBP1in cell transformation (or rather in impeding it) has yet to be established, this is an exciting and likely possibility. It should also be noted that4EBP1seems to be under the control of the p70S6K pathway, which has a role in cell cycle progression. mTOR and4EBP1has become a new target for cancer therapy.RNA interference (RNAi) at the mRNA level, with double chain RNA (double strand RNA, dsRNA) as a tool, the induction of specific gene silencing process, namely sequence-specific post-transcriptional gene silencing (post-transcriptional gene silencing, PTGSRNA). DsRNA in the cell specificity of nuclease (Dicer) role, cracked into small interfering RNA (small interfering RNA, siRNA), about21bp-23bp of siRNA and multimeric nucleic acid enzyme chelate, formation of RNA induced silencing complex (RNA-induced silence complex, RISC), the latter is combined with siRNA, like RNA enzyme function mRNA degradation. RNAi technology has a precise, efficient, simple operation, and is widely applied in gene function research, antitumor, antiviral, signal transduction and gene therapy, RNAi is expected to become the life science and genetic engineering of important tool.Rapamycin (Rapamycin), firstly discovered as one of mTOR inhibitors, is a macrolide antibiotic isolated from the bacterium Streptomyces hygroscopicus in1970s. Rapamycin can combine FKBP (FK506binding protein), inhibit the mTOR expression, indirectly inhibit other related protein transcription and translation, have a strong immune inhibition and have the anti-tumor effectIn this study, immunohistochemical method was used to detect the expression of mTOR protein in45cases of cervical squamous cell carcinoma,20cases of CIN tissues and30cases of normal cervical squamous epithelium; the rapamycin and the mTOR RNAi were used to inhibit specifically the mTOR expression in human Hela cells. The immunocytochemistry and the in situ hybridization were used to detect the expressions of mRNA and mTOR,4EBP1protein in each group of human Hela cells; By Boyden chamber in vitro invasion assays, understand mTOR siRNA transfected Hela cell invasiveness change, for the target of rapamycin to the treatment of cervical carcinoma and the clinical application of RNAi technique provide a theoretical basis.Materials and methods1.Detected mTOR protein expression in45cases of cervical cancer,20cases of CIN tissues and30cases of normal cervical squamous epithelium, then observed the morphology.2.Cervical cancer Hela cells in culture:Hela cells were recovered and cultured adherently in10%fetal bovine serum (RPMI-1640100u/ml culture liquid containing penicillin, streptomycin100u/ml) at37℃,5%CO2hatch box adherent culture condition.3. MTOR siRNA synthesis, identification:Design and synthesis of siRNA nucleotide template,5’end of T7promoter sequence, in vitro and T7RNA polymerase binding specificity, transcribed from the destination siRNA. Synthesis of two segments respectively specific siRNA and a nonsense control siRNA,4%agarose gel electrophoresis experiments, the template DNA for reference, the detection of synthetic siRNA base pairs in length and concentration; through the experiment selected a jamming effect better specificity siRNA.4. The transfected cells:application of LipofusinX liposome siRNA transfected Hela cells.5. Application of immunocytochemistry and in situ hybridization technique to detect the transfection of mTOR siRNA before and after treatment of rapamycin in human cervical carcinoma Hela cell mTOR,4EBP1protein and mRNA expression.6.By Boyden chamber invasive experiments in vitro transfection were observed before and after siRNA cell invasiveness and change.7. Statistics:the measurement data with the mean±Standard deviation (X±S), using SPSS15.0software processing, each group rate comparison by χ2test, analysis of variance to take test groups were for the statistical significance of differences between groups, using t test, when variances of the transformation of variables, significant standard for α=0.05.Results1. Successful in vitro synthesis of two siRNA, length of21bp.2. The changes of the cell morphology:The experimental group has an uniform cell morphology, and have a expressly outline; Cells are anomalous polygon, and a close contact between cells.24h after rapamycin treatment, as compared with the control cells, cell morphology changes, as the drug concentration increase, cells become smaller variable length, and some become spindle-shaped, and the floating cells emerge, the cell growth slowed;and with the time increases, these changes become more pronounced. After transfection of mTOR-specific siRNA, compared with the experimental groups, it was observed that the Hela cells shrinkage, gradually became round, refractive enhancement, a small amount of cells are dangling in the culture medium, and with the increase in the concentration of the transfection and the extension of time, these changes are more evident.3. The expression of mTOR protein is mainly located in the cytoplasm of tumor cells. The positive rates of mTOR in30cases of normal cervical squamous epithelium were36.7%(11/30), in20cases of CIN tissues were55.0%(11/20), and in45cases of cervical cancer were71.1%(32/45).There was a significant difference between the group of cervical cancer and the other two groups of normal cervical squamous epitheliums or CIN tissues (P<0.05). Each experimental group compared with the control group, the difference was statistically significant (P<0.05). The positive rates of expression of mTOR protein on the stage I to stage II were50.0%(8/16) and82.8%(24/29), There was a significant difference between them (P＜0.05). The positive rate of expression of mTOR protein in the group without lymph node metastasis was61.3%(19/31), While in the group with lymph node metastasis, it was92.9%(13/14), there was a significant difference between the group (P＜0.05).4. The expressions of mTOR,4EBP1protein and mRNA in each group.4.1Cervical cancer Hela cells were respectively treated by three different concentrations of rapamycin(100nmol/L,150nmol/L,200nmol/L) for24h,48h,72h, the volumes of expressions of mTOR,4EBP1protein and mRNA decreased compared with the DM/EM control group and the normal control group. Each experimental group compared with the control group, the difference was statistically significant (P<0.05). The expressions of mTOR,4EBP1protein and mRNA decresed with the increased concentration of rapamycin during the same time period, each experimental group compared with the control group, the difference was statistically significant (P<0.05); The expressions of mTOR,4EBP1protein and mRNA decresed with the extension of time at the same concentration of rapamycin, each experimental group compared with the control group, the difference was statistically significant (P<0.05).4.2Cervical cancer Hela cells were respectively transfected by three different concentrations of mTOR siRNAs (50nmol/L,100nmol/L,150nmol/L) for24h,48h,72h, the volumes of expressions of mTOR,4EBP1protein and mRNA decreased compared the DM/EM control group and the normal control group. Each experimental group compared with the control group, the difference was statistically significant (P<0.05).The expressions of mTOR,4EBP1protein and mRNA decresed with the increased concentration of rapamycin during the same time period, there was a significant difference between experimental groups and each control groups (P<0.05); The expressions of mTOR,4EBP1protein and mRNA decresed with the extension of time at the same concentration of rapamycin, each experimental group compared with the control group, the difference was statistically significant (P<0.05).5. Boyden chamber mTOR siRNA:in vitro invasive experiments in vitro transfection of cervical cancer cell Hela, through Matrigel cell number was significantly less than that of the blank group and unrelated control group, the difference was statistically significant(P<0.05).Conclusions1. The overexpression of mTOR protein may be play a promote role in the development and metastasis of cervical cacinomar.2. Rapamycin, mTOR siRNA can specifically control the expressions of mRNA and mTOR protein in the cervical cancer Hela cells of.3. MTOR siRNA in lower cervical cancer Hela cell mTOR gene expression at the same time,4EBP1protein and mRNA expression also then cut, prompting mTOR as4EBPlupstream regulatory factors, Specifically regulates4EBP1expression.4. mTOR siRNA inhibited the invasion ability of cervical cancer Hela cells.