The Expression And Potential Pathogenesis Of CCR9in Human Non-small Cells Lung Cancer

BackgroundLung cancer is the leading cause of cancer-related mortality worldwide, Approximately1.6million new cases of lung cancer are diagnosed each year throughout the world, the mortality related to lung cancer continues to rise. Non-small cell lung cancer (NSCLC) is the most frequent (approximately85%) type of lung cancer. Due to the lack of effective biomarkers, at least40%of patients with lung cancer are diagnosed in an advanced stage. Therefore, reliable and independent prognostic or predictive markers are needed for further intervention.Chemokines are small (8-14kDa) proteins that play key roles in development and leukocyte trafficking through interactions with cognate cell surface seven-transmembrane G protein-coupled receptors(GPCRs). CC chemokine receptor-9(CCR9) is a GPCRs and plays an important role in T-cell development and tissue-specific homing when binding to its specific ligand CCL25, which is also known as thymus expressed chemokine (TECK). Chemokines are also important for solid tumor apoptosis. CCR9is expressed in some carcinoma cells, and may promote proliferation and suppress apoptosis of cancer cells by activating the PI3K/Akt pathway.We investigated the expression status and functional significance of CCR9in NSCLC tissue and cell lines, and then explored the probable mechanism in vitro and in vivo, so as to provide experimental evidence on searching novel therapeutic targets of NSCLC.Methods1. Telephone following up regarding on life span and postoperative treatment was carried out with119patients who visited at The People’s Hospital of Guangxi Zhuang Autonomous Region; Immunohistochemistry was used on119NSCLC pathological sections to detect the expression of CCR9; the correlation of CCR9expression with clinic pathologic variables and prognosis was analyzed.2. Three precursor microRNAs (Pre-miRNA) sequences targeting to CCR9and a negative control were designed and inserted into pcDNA6.2-GW/EmGFP-miR expression Vector to construct recombinant plasmid. To verify the recombinants, the recombinants were extracted for sequence detection. Using the instruction for lipofectamine2000, we transient transfected the recombinant plasmids in cultured SK-MES-1and A549cells. After48hours, RT-PCR and Western blot testing were used to identify the target site with the highest interfering efficiency. We packaged the recombinant lentiviral vector for CCR9RNAi with the highest interfering efficiency using the BLOCK-iTTPol Ⅱ miR RNAi Expression Vector Kit. The lentiviral vectors were transfceted into SK-MES-1and A549cells. To produce stable transfection cell lines, the cells were cultured in a selection medium.3. MTT assay was used to assess the proliferation ability of NSCLC cell lines SK-MES-1and A549with CCR9-CCL25interaction; FACE (Fast-activated cell-based enzyme-linked immunosorbent) assay was used to assess the influence of activation of PI3K/Akt signal pathway; Cell cycle distributions and related proteins were analyzed by flowcytometry and Western blot; Cell apoptosis and related proteins were analyzed by Annexin V-A and Western blot.4. BALB/c nude mice were injected subcutaneously with stably transfected NSCLC cells groups and control cell groups respectively to construct nude mouse of lung cancer animal models. Times of tumor formation of each group were obtained. The formation and growth of the tumors were observed by measuring. CCR9, PI3K110, pAkt and Akt proteins expression were detected by Western blot test.Results1. The results of immunohistochemistry showed that CCR9was mainly localized in cell membrane and cytoplasm. The positive rate of CCR9in lung cancer was significantly higher than that of CCR9in normal lung tissues (52.1%vs.10.1%,/2=49.028, P=0.000). CCR9expression closely correlated with histopathologic classification, lymph node metastasis and TNM stage in NSCLC by Pearson Chi-square test. The survival time in the CCR9expression group was shorter than those in the none expression group. CCR9was adopted as an independent prognostic factor for survival of NSCLC patients though multivariate Cox proportional hazard model analysis.2. Three recombinant plasmid expression vectors encoding Pre-miRNA against CCR9and a negative control were constructed correctly and veritified by sequencing. GFP was observed under the fluorescence microscope after the plasmids were transfected into the SK-MES-1and A549cells24hours later. Then pcDNA-CCR9-miR-MR-1was identified to have the highest interfering efficiency by RT-PCR and Western blot. We packaged the plenti6/v5recombinant lentiviral expression vector by pDNOR221and plenti6/v5-DEST for CCR9RNA interference with pcDNA-CCR9-miR-MR-1. To construct the stable transfectants, the cells were cultured in a selection medium containing6ug/ml of blasticidin for14days and the stable cell lines after selection were obtained with3ug/ml of blasticidin. Of note, about85-90%knockdown of CCR9expression in the SK-MES-1and A549cells were obtained by miRNA tcehnique.3. Compromised CCR9-CCL25interaction induced cell cycle arrest, reduced proliferation and promoted apoptosis in NSCLC cells in vitro. Importantly, CCR9-CCL25interaction resulted in a significant increase in phosphoinositide3-kinase (PI3K)/Akt activity. In addition, we showed that CCR9-CCL25interaction mediated the activation of the PI3K/Akt pathway in NSCLC cells, resulting in the up-regulation of important cell cycle proteins and antiapoptotic proteins, as well as the down-regulation of apoptotic proteins in a PI3K/Akt-dependent manner. These CCR9-CCL25mediated effects were abrogated in the presence of a PI3K inhibitor (wortmannin) or by inhibiting the CCR9-CCL25interaction through CCR9silencing, which also suggested that the biological function of CCR9-CCL25was mainly regulated by PI3K.4. After lung cancer cells transfected with CCR9RNAi were inoculated subcutaneously in nude mice, tumor growth were inhibited and tumor volume reduced significantly, compared with the control group. Expression of CCR9protein reduced. The expression of activated PI3K/Akt signaling pathway proteins were significantly down regulated.Conclusion1. Our IHC data indicated that CCR9is over-expressed in NSCLC. The expression of CCR9is correlated to histological types, with or without lymphatic metastasis and TNM stages. The survival time in the CCR9expression group was shorter than those in the none expression group. CCR9was adopted as an independent prognostic factor for survival of NSCLC patients.2.Our in vitro and in vivo data indicated that CCR9-CCL25interaction, via activation of the PI3K/Akt signaling pathway, significantly promoted NSCLC cell proliferation and suppressed NSCLC cells apoptosis, which might be a possible cause of NSCLC tumor progression.