Role On Proliferation And Invasion Potentiality Of Wnt5a In Non-Small Cell Lung Cancer

Background: Lung cancers, which are the highest occurrence ratio in the whole world, are the leading cause of death among all kinds of caners. It has been reported that among the newly occurrence lung caners, 75-80% of them are non-small cell lung cancer (NSCLC). The prognosis of NSCLC is still very poor even with the accumulating improvements therapies, mainly because of the underlying mechanisms, especially that of metastasis, of the occurrence of lung cancers are still unclear. Therefore, it is very important to answer these questions in order to make better prognosis and improve the treatment of NSCLC. Wnt5a is one of the most important members of Wnt family, it activates the classic Wnt/β-catenin pathway as well as the non-classic Wnt/Ca2+ pathway, and these two pathways interact. Studies have shown that Wnt5a is closely related many malignant cancers, plays very important roles in the proliferation, differentiation and metastasis of tumor cells. Studies have also shown that Wnt5a acts differentially among different metastasis tumors. Some of them show this gene processes the characteristics of oncogene, high expression of Wnt5a was detected in most malignant tumors such as melanoma and stomach cancer, and it can promote tumor occurrence and metastasis, thus its high expression is related to the metastasis potential and poor prognosis; however, some other researches argue Wnt5a share the characteristics of anti-oncogenes, it can decrease or delay the occurrence and metastasis of leucocythemia, malignant lymphanode tumors as well as colon tumors. By now most of the studies about the relationship between Wnt5a and lung cancers were focused on the expression of Wnt5a in clinic samples, but the underlying mechanisms are poorly understood.Aims: To explore the expression of Wnt5a and role on the proliferation and invasion potentiality in NSCLC, aim at help effective treatment and prognosis of lung cancer.Methods:1. Tumor tissues of 43 cases of NSCLC were examined by Northern Blot, and the protein expression of these tissues were examined by Western Blot and immunohis- tochemistry. Survival analysis was carried out between the expression and clinical pathological characteristics by statistics.2. By gene recombination technique, human Wnt5a gene sense plasmid and its RNAi eukaryotic expression plasmid were constructed, human cell lines of H157 and A549 were transfected, and semi-quantitative RT-PCR and Western Blot were employed to detect the change of Wnt5a mRNA and protein before- and after transfection, respectively.3. MTT was used to test the change of cell proliferation; flow cytometry was used to test the changes of cell cycle; nick test was used to examine cell migration, and modified Transwell chamber test was used to examine cell invasion.Results:1. The positive ratio of Wnt5a mRNA in the tumor tissue of NSCLC and tissue from normal lung was 79.1% and 45.4%, respectively. Difference of expression level also existed: in the former it was 0.96±0.65 and the later was 0.44±0.23. This suggested higher expression of Wnt5a was detected in the NSCLC, statistically significant when compared with normal (P<0.05).2. There is some degree of relationship between Wnt5a expression and pathological grade, cell differential stage, with- or without lymphonode metastasis and its tissue type, and smoking or not. Higher expression was detectable inⅢ-Ⅳphase(1.24±0.47) compared withⅠorⅡphase(0.58±0.71); in lower differentiation(1.27±0.46) compared with higher differentiation(0.65±0.47); in with lymphanode metastasis(1.10±0.63) compared with non-lymphanode metastasis(0.67±0.66); in squamous cell carcinoma(1.31±0.65) compared with adenocarcinoma(0.58±0.46), and in smokers(1.26±0.56) compared with non-smokers(0.57±0.62)(P<0.01 or 0.05).3. The protein expression of Wnt5a in all the tissue was in coincidence with the mRNA expression, showing a significant positive correlation (correlation coefficient: 0.74, P<0.01), which highly suggested a consistency.4. In NSCLC patients, two-year surviving ratio in Wnt5a positive patients(31.1%, 9/29) was lower than Wnt5a negative controls (71.4%, 10/14), showing statistically significant(P<0.01).5. Restrictive enzyme analysis was carried to identify the recombined plasmid pcDNA3.1+—XE58 and pAVU6-si Wnt5a, the results showed the size of the cutting segment was identical to the inserting target segment; Sequencing analysis showed it was identical to the reported sequence in GeneBank, no missing or mutation was detected. 6. The proliferation index in untreated H157 cell line and A549 cell line was 37.26% and 29.01%, respectively; it increased in sense-transfected H157-XE58 and A549-XE58 (62.94% and 54.49%, respectively); decreased RNAi treated H157 and A549 cells(20.81% and 17.66%, respectively), suggesting a positive role of Wnt5a in the promoting potential of cell proliferation.7. Untreated H157 and A549 cells had some degree of migration and invasion, respectively. Sense-transfection increased the migration after 24h,the number of cells in the nick area increased significantly, and the distance of migration was longer, too; the number of cross Matrigel cells increased (71.3±3.2 and 61.7±3.5 in H157-XE58 and A549-XE58 cells, respectively). However, all the above parameters were obviously inhibited by siRNA treatment.Conclusion:1. In vivo studies showed an increased expression of both Wnt5a mRNA and protein in NSCLC, furthermore the expression level of Wnt5a was closely related to the cell differentiation, histology and TNM stages of NSCLC. These suggested Wnt5a may play an important role in the occurrenence and progression of NSCLC.2. In vitro studies showed the proliferation increased with the overexpression of Wnt5a and the ability can be inhibited by RNAi targeting Wnt5a efficiently in vitro and in vivo. confirmed this gene may play an important role in the proliferateion of NSCLC.3. In vitro studies showed the invasion increased with the overexpression of Wnt5a and the ability can be inhibited by RNAi targeting Wnt5a efficiently in vitro and in vivo. confirmed this gene may play an important role in the invasion of NSCLC.4. Wnt5a gene may be the target of gene therapy of lung cancer, this open a new field for the clinical application of RNA interference or designer drug to aim directly at the gene.5. Higher expression of Wnt5a was related to the poor prognosis and shorter survival, suggesting Wnt5a may be used as one of the prognosis index.6. The successful construction of sense plasmids and RNAi eukaryotic expression plasmid provided an experimental platform for further study of the role of this gene .