| PTEN(Phosphatase and tensin homology deleted on chromosome ten), (MMAC1or TEP1)is a novel anti-oncogene cloned by three research groups alternatively in 1997, and was named in different view. PTEN locates in chromosome 10q23,200kb in length with 9 exons and 8 introns. The mRNA is 5.5 kb in length. The protein which encodes contains 403amino acid,weigh 56000D, and lives in cytosol. PTEN is conservative because it is homogeneous in fruit fly, rat, and human being. It is important in cell life.PTEN regulates cell activities by dephosphorylation. PTEN encodes for a phosphatase with double specific substrates, which has ability of lipophosphatase and protein phosphatase. PTEN is the first tumor-repressor with double specific phosphatase which has been found. PTEN could dephosphorylated poly acidic protein by get rid of the third phosphate residue in PIP3, or dephosphorylated the key kinase in tumor signal transduction pathway, then decrease the phosphorylation level and block signal transduction pathway. So it inhibits cell growth, transduction, adhesion, dilation and transplantation, promotes apoptosis, and then inhibit tumor growth, invasion and transfer. PTEN deletion or mutation may cause loss-of-function. PTEN mutation may happen in each of its nine exons, and the mutation rate is correlated with malignant degree. PTEN inactivation is corresponding to different stage of tumor. In early stage of tumor in endometrium, PTEN deletion or mutation could be found. Expression lost of PTEN could be detected in 83% of tumor which show obvious histological malignance. PTEN deletion or mutation happen before cytological atypical changes, and the latter is the sign which differents benign endometrium tumor from malignant in panthohistology. In malignant melanoma, prostate carcinoma, glioma, PTEN deletion is a sign of their high malignancy and metastasis. Though metastasis was considered as the late accident in tumor progress, PTEN inactivity may happened early. For prostate carcinoma that metastasis has happened, PTEN deletion was show much early in main locus. So, PTEN mutation is important in late phase of tumor with metastasis and invision though it happens in early phase.Angiogenesis is the base of tumor growth and metastasis. VEGF is considered as the most important factor in tumor angiogenesis, whose main accepter is KDR. PTEN could lower down the expression of VEGF. Hwang et al transduct PTEN into PTEN-/- murine melanoma cell line B16F10,and confirmed that PTEN could decrease the secretion of VEGF,and exogenous PTEN could inhibit B16F10 growth. And PI3/Akt signal pathway could induce angiogenesis in CAM and up-regulated the expression of VEGF.Lung cancer happens most frequently in China. Eight hundred thousand people die of it every year. In them, patients with non-small cell lung cancer is about 80%. Until now, there is little research about PTEN expression and function in set off, progress and metastasis in lung cancer. And no report about the effect of PTEN on VEGF in set off, progress and metastasis in lung cancer, especially the relationship between PTEN and VEGF as well as its receptor KDR. So, research on mechanism of PTEN in inhibiting tumor angiogenesis may be significant in tumor diagnosis, treatment. And it may play a role in looking for new drug in tumor treatment, and with great foreground in market.Materials and methods1. Cell culturePulmonary carcinoma cell line A549 was stored in our lab. After cell thawing, A549 cell line was cultured in RPMI 1640 as routine.2. pcDNA-PTEN recombinant construction and transfectionThe target gene was obtained by RT-PCR, and digested by restriction enzyme together with pcDNA3.1(+)vector. After ligation, the recombinant was transformed into E. coli DH5α. A positive clone was chose to be cultured by shaking. Then the plasmid were extracted, identified by enzyme digestion and analyzed by sequencing.3.Plasmid transfectionPulmonary carcinoma cell line A549 was transfected by pcDNA-PTEN recombinant. And at the mean time, the other two group as blank vector and non-transfection were set as control. After regular culture, the positive clone could be screened by G418.4.Inhibition function assayCell number was counted by cell counter for 7 days continuously, and cell growth curve was draw according to data. KDRmRNA lever in cell line were detected by RT-PCR.Result1. Cell culturePulmonary carcinoma cell line A549 were thawed successfully, and cultured appropriately. Adherent cells grow well.2. pcDNA-PTEN recombinant construction and transfection pcDNA-PTEN recombinant was transfected into pulmonary carcinoma cell line A549 by Lipofectamine 2000 as vector. After screened by G418, the positive clone could be obtained. 3. Inhibition effect on different groupsA549 cell lines in each group which include pcDNA-PTEN recombinant transfection group, blank vector group and non-transfection group were implanted into 96 well plates for 7 well each. Cell number in one well each was counted by cell counter in 7 continuous days. Numbers were used to draw a growth curve. It could be found that compared with blank vector group and non-transfection group, cell grow much slower in pcDNA-PTEN recombinant group. The cell number is also smaller and the grow proliferation is decreased. (table 1)4. KDR mRNA level detected by RT-PCRBright bands could be found at 399bp site in blank vector group and non-transfection group under UV light, while there is only a faint bands in pcDNA-PTEN recombinant group at the same site. There were bright bands at 326bp in all of three groups (figure 1). The result shows that KDR mRNA is highly expressed in blank vector group and non-transfection group, while just expressed in low level in pcDNA-PTEN recombinant group.DiscussionPTEN is a novel anti-oncogene that was found in 1997, which encodes a cytosol protein with double phosphorase, and was the first tumor suppressor gene with phosphorase that was found until now. PTEN play an important role in tumor progress, invasion, angiogenesis and metastasis. The mechanism about tumor suppression of PTEN is not clear yet. PTEN was considered that it could get rid of the third phosphate residue in PIP3 by its phosphorase, lower down the function of PIP3, block PIP3-AKT signal transduction pathway. Then it inhibits cell growth, division, and promotes apoptosis. PTEN deletion or mutation is related with malignant potential of tumor. The higher malignant, the more prominent of deletion.Lung cancer is the one kind of tumors with most frequently happened rate and highest mortality rate now. Though there is many way for treatment, the survival rate is relative low with bad prognosis. Gene therapy is more and more regarded as a novel model in tumor treatment. There are only few reports about PTEN expression in lung cancer. Angiogenesis is the base of tumor growth and metastasis. VEGF is considered as the most important promoter in tumor angiogenesis, whose main accepter is KDR. PTEN expression is negative related with VEGF expression. PTEN may lower down the level of VEGF mRNA, inhibit angiogenesis in tumor. Until now, no report about the effect of PTEN on tumor set off, progress and metastasis, especially the relationship between PTEN and VEGF as well as its receptor KDR. In our research, we try to constructe PTEN-pcDNA recombinant DNA, and transfect lung cancer cell line A549. Meantime, we set up blank vector and non-transfction group as control, and observe their anti-tumor function in vitro. Results show that in PTEN-pcDNA transfected group, cell growth is lower, cell number is smaller, cell proliferation rate is decrease; and KDR mRNA band is weaker in electrophoresis, which means KDR expression is lower. The above results support the conclusion that PTEN-pcDNA recombinant DNA could inhibit cell growth by several way in vitro, and then has anti-tumor function is cell line A549. So, our research may provide an evidence for gene therapy via PTEN, and explore the mechanism of PTEN in inhibiting tumor progress, and may offer a new idea in looking for new drug target and gene therapy.ConclusionConstructed PTEN-pcDNA recombinant DNA was transfected into lung cancer cell line by Lipofectamine. After screened by G418, the positive clone could be obtained. Then lung cancer cell line A549 with stable expression of PTEN was successfully constructed. Cell inhibition experiment demonstrated that, comparing with non-transfction group and blank vector group, cell number in PTEN-pcDNA transfected group is decreased, cell proliferation is slow. While, RT-PCR demonstrated that, comparing with non-transfction group and blank vector group, expression of KDR mRNA in PTEN-pcDNA transfected group was decreased, which went against angiogenesis, growth and metastasis of tumor. All above came to the conclusion that PTEN-pcDNA cloud inhibit cell growth in several way, and processes anti-tumor function.
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