The Study Of Tumor Suppressor Gene RBM5Expression And Its Correlation With EGFR And KRAS In NSCLC

BackgroundLung cancer is a significant worldwide health problem.The5-year survival rateof lung cancer still remains at13to15%for the past3decades, despite recentadvances in lung cancer early diagnosis, surgical techniques, and the development ofnovel chemotherapeutic agents. Molecularly, NSCLC development is believed to beinitiated by the activation of oncogenes or inactivation of tumor suppressor genes. Inrecent years, there is great progress in the NSCLC treatment as the development ofdrugs targeting tumor regulatory genes like human EGFR inhibitors. Therefore try tofind more tumor related genes and study their interrelationship will facilitate the studyof molecular mechanism for tumor development and exploit new gene targetingdrugs.Our preliminary study showed the tumor suppressor gene RBM5(RNA BindingMotif Protein5, also called Lucal5、H37) is closely related to NSCLC. RBM5is oneof the approximately19genes locued on chromosome3p21.3, loss of which is themost frequent and earliest event in NSCLC. Tumor suppressor RBM5plays animportant role in the induction of cell cycle arrest and apoptosis through pre-mRNAsplicing of multiple target genes, and inhibits tumor transformation and theprogression of several malignancies, including NSCLC. However, there are only afew studies to date on RBM5expression in NSCLC.Epithelial growth factor receptor (EGFR, HER-1/ErB1) is the tyrosine kinasereceptor family member. KRAS belongs to RAS gene family. It was shown that EGFRand its downstream signaling gene KRAS are over-expressed and gene mutated in thetumor samples, and they play important roles in tumor formation and progression.HER2is also a tyrosine kinase receptor family member, and previous studydemonstrated that HER2over-expression could changes RBM5expression. Moreover, RBM5was down-regulated by the constitutively activated RAS mutant protein RAS(G12V) in rat embryonic fibroblast cells. Down-regulation of EGFR expression didnot up-regulate RBM5expression in the NCL-H975lung adenocarcinoma cancercells. According to the above, we hypothesize that the roles of RBM5in regulatingcell apoptosis and inhibiting cell proliferation are related to EGFR signaling pathway.In our study we will examine1) whether RBM5is the upstream gene in EGFRsignaling pathway;2) whether regulating the RBM5-EGFR signaling gene expressionwill inhibit tumor development;3) whether RBM5can be a new potential target toenhance the treatment effects of EGFR targeting drugs. And also we will examine themolecular mechanisms and treatment effects of RBM5and EGFR signaling inNSCLC.PurposeWe aim to explore the anti-cancer roles of RBM5gene in NSCLC and thechanges of EGFR and KRAS gene expression after RBM5gene over-expression; toclarify the molecular mechanisms of RBM5-EGFR signaling in NSCLC, and exploitnew target gene for lung cancer treatment.Method1. Semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR)，western blotting and immunohistochemistry methods are performed to detectRBM5, EGFR and KRAS mRNA and protein expression in120paired non-tumorand tumor samples of NSCLC. Statistics are performed to analyze RBM5andEGFR, KRAS expression correlation and their clinical relevance, includingsmoking history, lymph node metastasis, lung cancer stages, histology, gender andage etc).2. PcDNA3.1-RBM5plasmid is transfected into A549cells by liposomes to overexpress RBM5in vitro;human Cancer Drug Targets mRNA expression in RBM5over-expressing A549cell group was detected by PCR array; RT-PCR and westernblotting were used to examine EGFR and KRAS mRNA and protein expression andconfirm the PCR array results. 3. Tumor model was set up by transplanting A549cells into nude mice. Thetumor-beating mice treated with attenuated Salmonella carrying pcDNA3.1-RBM5or PcDNA3.1plasmid through tail vein injection to examine the effects of RBM5on tumor growth. RT-PCR, western blotting and immunohistochemistry wereperformed to detect RBM5, EGFR and KRAS mRNA and protein expression in thetransplanted tumors before and after the treatment. Statistics is performed toanalyze these3genes relevance in NSCLC.Results1. RBM5, EGFR and KRAS gene expression in human NSCLC tissues(1) The expression of RBM5mRNA and protein was significantly decreased inNSCLC compared to normal tissues (P<0.05); decreased in the smoking groupcompared to non-smoking group (P<0.001); and decreased in lymph nodemetastasis group compared to non-metastasis group (P<0.007); decreased inIIIA and IIIB stages compared to I and IIA stages in tumor staging (P=0.029);and was not correlated with tissue classification, gender and age. There was noRBM5gene expression in NSCLC and its loss was related to smoking, lymphnode metastasis and tumor staging.(2) The expression of EGFR mRNA and protein was significantly increased inNSCLC compared to normal tissues (P<0.05); and increased in lymph nodemetastasis group compared to non-metastasis group (P=0.009); increased inIIIA and IIIB stages compared to I and IIA stages in tumor staging (P=0.031);and was not correlated with smoking, tissue classification, gender and age.There was EGFR gene over-expression in NSCLC and its over-expression wascorrelated with lymph node metastasis and tumor staging.(3)The expression of KRAS mRNA and protein was significantly increased inNSCLC compared to normal tissues (P<0.05); and increased in lymph nodemetastasis group compared to non-metastasis group (P=0.01); increased inIIIA and IIIB stages compared to I and IIA stages in tumor staging (P=0.022);increased in the smoking group compared to non-smoking group (P=0.002); and was not correlated with tissue classification, gender and age. There wasEGFR gene over-expression in NSCLC and its over-expression was correlatedwith lymph node metastasis and tumor staging.(4) In NSCLC tissues, the mRNA and protein expression of RBM5and EGFR,KRAS was negatively correlated (P<0.01) indicating up-regulation of RBM5gene expression can down-regulate EGFR, KRAS gene expression.2. The EGFR and KRAS mRNA and protein expression after over-expression ofRBM5in A549cells(1) A549cells showed obvious growth inhibition after transfection withpcDNA3.1-RBM5. RBM5over-expression can inhibit A549cell growth.(2) PCR array result showed that there were20out of91human Cancer DrugTargets genes showed3fold increase of mRNA expression, includingPIK3CA, PIK3C3, PLK4, KRAS, EGFR etc. Over-expression of RBM5candown-regulate the above gene expression.(3) RT-PCR and western-blotting results showed that EGFR and KRAS mRNAand protein expression was significantly decreased after transfection withpcDNA3.1-RBM5(P<0.05). RBM over-expression can down-regulate EGFRand KRAS expression in A549cells.3. The EGFR and KRAS expression after over-expression of RBM5in A549celltransplanted nude mice tumor model.(1) Tumor growth was significantly inhibited after intravenous injection withpcDNA3.1-RBM5plasmid (P<0.01) indicating that RBM5over-expressioncan inhibit tumor growth.(2) Expression of EGFR and KRAS proteins on A549xenograft nude mice treatedwith attenuated Salmonella carrying RBM5was decreased compared to thecontrol group (P <0.05).Conclusion1. In human NSCLC tissues, RBM5expression was down-regulated, while EGFR,KRAS expression was up-regulated. The results indicated that RBM5gene loss and EGFR, KRAS gene over-expression are all involved in NSCLC progression.2. RBM5gene low expression and EGFR, KRAS high expression are related to thetumor staging and lymph node metastasis, and RBM5and KRAS expression isrelated to smoking. The results indicate that RBM5gene loss and EGFR, KRASover-expression participate in lung cancer progression and metastasis. Smokingmay increase NSCLC risks through increasing RBM5and KRAS expression.3. RBM5expression is negatively related to EGFR and KRAS in human NSCLC,suggesting that tumor suppressor gene RBM5normal or over-expression mayinfluence EGFR and KRAS expression. Furthermore RBM5may inhibit lungcancer initiation, or even become a new potential biomarker for lung cancerscreening.4. RBM5may functions upstream of EGFR signaling pathway. In both in vitro andin vivo environment, up-regulation of RBM5expression to decrease EGFR andKRAS expression and further inhibit lung tumor and tumor cell growth maybecome a new potential target for NSCLC treatment.