The Study Of The Antitumous Effect Of Tumor Suppressor Gene RBM5on Lung Adenocarcinoma And Breast Carcinoma

Preface:Researches found that (3p21.3) chromosome abnormality often appeared in the earlystage of lung cancer, breast and kidney cancer patients. There are19genes in this area,display the function of tumor inhibition in different degree (including cell differentiation,proliferation and signal transduction and apoptosis). RBM5(also called g15, LUCA-15andH37) is located in the end of these19genes. Study proved: in patients with breast cancer andlung cancer the chromosome abnormality area only include17genes, except RBM5. Thephenomenon showed that the RBM5has special position and important role in lung cancer.In order to study the function and the mechanism of RBM5on lung adenocarcinoma andbreast cancer, we collected30lung adenocarcinoma tissue and normal lung tissue samples(from the same patients with lung cancer), and detected the expression of RBM5in mRNAand protein levels. In addition, over-expressing pcDNA3.1-RBM5plasmid was transfectedinto A549lung cancer cells and MCF7breast cancer cells on experimental study in vivo andin vitro.Methods:PcDNA3.1-RBM5plasmid was instantaneously transfected into cells by liposomes;Transplant tumor model was obtained by injecting A549, MCF7cells to nude mice for in vivostudy; Cell apoptosis of over-expressing RBM5was detected by Annexin V/PI double dying;The cell cycle of RBM5over-expressing cells was tested by flow cytometry; TUNEL methodwas used to detect the apoptosis situation in tumor tissue; The related gene expression changein the mRNA level of over-expressing RBM5cells was detected by PCR array screening;RT-PCR method/realtime-PCR were used to detect the RBM5and TNF-α mRNA expressionlevel; The protein expression levels of RBM5, pERK, PCNA, TN F-α, cleaved-caspase-3,8,9,and cleaved-PARP were detected by Western blot and immunohistochemical method.Results:Comparing the lung cancer tissues with the normal lung tissues, the correspondingRBM5mRNA and protein levels of lung cancer group were significantly lower (P <0.05); Tansfection pcDNA3.1-RBM5got A549and MCF7cells with high RBM5mRNA andprotein expression (P <0.01); After transfection with pcDNA3.1-RBM5, A549and MCF7cells appeared early apoptosis, and A549cells also appeared the cell cycle arrest in G1phase;After transfection with pcDNA3.1-RBM5, the protein levels of RBM5, TNF-α,cleaved-caspase-3,8,9, and cleaved-PARP were increased significantly in A549cells (P <0.05); After transfection with pcDNA3.1-RBM5, the protein levels of TNF-α,cleaved-caspase-3,8, and cleaved-PARP were increased significantly in MCF7cells (P <0.05); The nude mice model with lung and breast adenocarcinoma cancer was builtsuccessfully, tumor growth was inhibited significantly after intravenous injection withpcDNA3.1-RBM5plasmid(P <0.01). And the RBM5protein expression was high (P <0.01);After intravenous injection with pcDNA3.1-RBM5plasmid appeared apoptosis significantly(P <0.01), with the apparent proliferation inhibition (P <0.01) in lung adenocarcinoma group;The expression levels of RBM5, TNF-α, cleaved-caspase-3,8,9, and cleaved-PARP proteinsincreased significantly after intravenous injection with pcDNA3.1-RBM5plasmid in lungadenocarcinoma tissue (P <0.05); The expression levels of RBM5, TNF-α, cleaved-caspase-3,8and cleaved-PARP protein increased significantly after intravenous injection withpcDNA3.1-RBM5plasmid in breast adenocarcinoma tissue (P <0.05).Conclusion:1. Confirmed that the expression level of tumor suppressor gene RBM5in humanprimary lung adenocarcinoma tissue was lower than normal lung tissue. The results showedthat the deletion of RBM5gene is closely related with lung adenocarcinoma.2. Through instantaneous transfection, A549and MCF7cell models with highexpression of RBM5were obtained, which proved that RBM5could induce cell apoptosis,the cell cycle arrest and proliferation inhibition in A549cells in vitro. Mean while, RBM5induced apoptosis in MCF7cells in vitro.3. By building nude mouse transplant tumor model with lung and breastadenocarcinoma, proved that RBM5inhibited the tumor growth of lung and breast cancer,and confirmed RBM5induced cell apoptosis and proliferation inhibition in lungadenocarcinoma tissue, and inhibited breast cancer apoptosis in vivo.4. The lung and breast adenocarcinoma experiments in vivo and in vitro showed, RBM5could suppress tumor growth by inducing cell apoptosis in lung adenocarcinoma. Themechanism is, first, RBM5may promote TNF-α expression and activate caspase-8to inducethe apoptosis; on the other hand RBM5can inhibit Bcl-2protein, promote Bax protein, cause Bax/Bcl-2ratio increases, thus activate caspase-9to induce apoptosis. And confirmed,RBM5can suppress lung adenocarcinoma growth by inhibiting cancer cell proliferation, itsmechanism may involve in inhibiting Ras/Raf/MEK/Erk signaling pathway.5. Through the breast adenocarcinoma experiments in vivo, proved that RBM5cansuppress tumor growth by inducing cell apoptosis. Its mechanism is that RBM5maypromote TNF-α expression and activate caspase-8to induce the apoptosis. The result isconsistent with lung adenocarcinoma research, which suggested that the effect of RBM5onTNF-α may be the one major mechanism on tumor growth effection.6. In this study, through the lung and breast adenocarcinoma experimental research invitro and in vitro confirmed the inhibition function of RBM5on lung and breastadenocarcinoma. We provided theoretical basis of further study on the function andmechanism of RBM5on lung and breast adenocarcinoma, which had certain directivesignificance on the clinical diagnosis and treatment of lung adenocarcinoma targeted withRBM5.