WNT5B Functions As A Potential Oncogene And Is Negatively Regulated By Mir5587-3p In Lung Adenocarcinoma Progression

Background:According to the latest cancer epidemiology,lung cancer has become the leading cause of cancer-related death and the highest incidence among malignant tumors worldwide.Non-small cell lung cancer(NSCLC)is the most popular pathological type of lung cancer,including lung adenocarcinoma(LUAD),lung squamous cell carcinoma(LUSC)and large cell carcinoma.Among them,LUAD approximately accounts for 40%of all lung cancer.The average age of patients with LUAD is usually lower than those with other types of lung cancer.Besides,LUAD grows fast and is rich of blood vessels,leading to early local infiltration and hematogenous metastasis.The five-year survival rate of LUAD is less than 15%.The rapid progression of LUAD results from dysfunctions of various signaling pathways,therefore,understanding the mechanisms of tumor proliferation and metastasis will help new therapeutic directions towards LUAD.WNT signaling pathway is one of the major players for maintaining homeostasis of the lung.Dysregulation of WNT signaling axis is a key factor of lung cancer development.WNT signaling is not only related with lung carcinogenesis,but also regulates tumor growth,angiogenesis and drug response.WNT5B is a well-known ligand of the WNT pathway.However,its expression in LUAD,the correlation between WNT5B and clinicopathological features,as well as its effect on the tumor growth remain unknown.Materials and Methods:1.The public databases TCGA and GEO were used to analyze the mRNA expression of WNT5B.Real-time PCR and Western blot were performed to evaluate the mRNA and protein expression levels of WNT5B in LUAD and paired adjacent normal tissues.A tissue microarray of LUAD was applied to test the protein expression profile of WNT5B in LUAD and matched adjacent normal specimens with immunohistochemistry(IHC).The correlation between WNT5B expression and clinicopathological features,and prognosis of enrolled LUAD patients were further analyzed.2.The LUAD cell lines A549,H1299 and PC9 with high expressions of WNT5B were used for shRNA knockdown experiments.MTT,colony formation and transwell assays were performed to detect the proliferation and migration abilities of LUAD cells.3.In WNT5B knockdown cells,flow cytometry of Annexin V-FITC/PI staining labeled cells were performed to detect cell apoptosis,and flow cytometry of PI staining labeled cells were used to analyze cell cycle.The expression levels of proteins related to cell cycle were detected by Western blot.4.Western blot was used to assess whether the non-canonical WNT pathway contributed to the oncogenic role of WNT5B in LUAD.5.In the WNT5B knockdown A549 cells,the downregulated genes were mainly enriched in metabolic-related signaling pathways by Next Generation Sequencing(NGS)and bioinformatics analysis.In addition,non-targeted metabolomics mass spectrometry was applied to explore the corresponding metabolic pathway.A combination analysis of genomics and metabolomics was performed to figure out the specific mechanism of WNT5B on modulating metabolism of LUAD cells.Finally,western blot was used to further verify the metabolic mechanism.6.The PC9 cells stably transfected with WNT5B or negative control were used to construct a subcutaneous xenograft model in nude mice.The volume and weight of subcutaneous tumors were measured.Immunohistochemical staining and western blot analyses targeting specific biomarkers were performed to evaluate cell proliferation,cell cycle and metabolism.7.Twenty-three LUAD tissues were used to screen for WNT5B-related miRNAs by real-time PCR.Western blot,real-time PCR and dual luciferase reporter gene assays were conducted to verify the regulation of WNT5B by miR-5587-3p in LUAD cells.The effect of miR-5587-3p on proliferation and cell cycle of LUAD cells were assessed by colony formation and flow cytometry.Besides,real-time PCR and Western blot assays were used to explore the metabolic molecules and cell cycle related proteins.Results:1.The TCGA and two GEO public databases showed that WNT5B was highly expressed in NSCLC compared to the normal lung tissues.The mRNA and protein expression levels of WNT5B were significantly higher in LUAD than that in the matched para-cancerous lung tissues.Moreover,the IHC results of LUAD tissue microarray demonstrated that WNT5B protein level was significantly upregulated in LUAD compared with paired adjacent specimens.In addition,higher WNT5B protein expression is correlated with lymph metastasis and advanced TNM stage.2.The expression of WNT5B was tested in seven NSCLC cell lines(A549、H1299、SPC-A1、H1703、H1650、PC9、H1975),showing the high expression of WNT5B in three LUAD cell lines A549,H1299 and PC9.Knockdown of WNT5B in A549,H1299 and PC9 cell lines suppressed their proliferation and migration abilities.3.The cells of G1 phase were significantly increased after WNT5B knockdown in A549,H1299 and PC9 cells,indicating the cell cycle was mainly arrested in G1 phase.The protein levels of G1 phase positive regulatory molecules(Cyclin D3、cdc25A、CDK2、CDK4、CDK6)were decreased,as well as the target protein E2F1 in G1 phase pathway.On the contrary,G1 phase negative control factor P27 was significantly up-regulated after knockdown of WNT5B.4.WNT5B activates non-canonical WNT/Ca2+signaling pathway in LUAD cells.5.After knocking down WNT5B in A549 cells,the down-regulated genes were mainly enriched in metabolic and cell cycle signaling pathways by second-generation sequencing(NGS).KEGG analysis of non-targeted metabolomics mass spectrometry revealed that the different metabolites in ABC transporters,central carbon metabolism and protein absorption and metabolism showed highest significance.A combination analysis of genomics and metabolomics showed that WNT5B might be closely related to protein absorption and metabolism pathway.Further study showed that the tumor-associated amino acid transporter LAT1 was down-regulated in LUAD cells after WNT5B knockdown.The intracellular amino acid sensing pathway GCN2/eIF2α/ATF4 was activated and the mTORCl was inhibited.6.The xenograft experiment showed that WNT5B knockdown inhibited tumor growth in vivo.The volume and weight of subcutaneous tumor were significantly lower in WNT5B depletion group.In addition,a lower expression of Ki-67 was presented in the tumor tissue from WNT5B-knockdown group.The expression of G1 phase related molecules including Cyclin D3、cdc25A、CDK2、CDK4 and E2F1 were decreased,and the tumor-associated amino acid transporter LAT1 was down-regulated as well.7.The mRNA level of WNT5B was negatively correlated with the level of miR-5587-3p in 23 LUAD tissues.Further study verified that miR-5587-3p could target WNT5B and suppress the expression of WNT5B in LUAD.Moreover,the number of clones was decreased and the G1 phase arrest was observed in miR-5587-3p highly expressed cells of LUAD.Additionally,the expression of Cyclin D3,cdc25A,CDK2 and E2F-1 proteins of G1 phase was down-regulated,and the tumor-associated amino acid transporter LAT1 was decreased after overexpressing miR-5587-3p in LUAD cells.Conclusion:Together,WNT5B is highly expressed in LUAD tissues and WNT5B is an independent prognosis biomarker for LUAD.Knockdown of WNT5B can inhibit cell proliferation,attenuate cell invasion and metastasis,and induce cell cycle G1 phase arrest.Moreover,the tumor-associated amino acid transporter LAT1 is down-regulated in LUAD cells with WNT5B-knockdown,suggesting that WNT5B is closely related to the amino acid metabolism of LUAD.In addition,the oncogenic role of WNT5B in LUAD might be explained by non-canonical WNT/Ca2+pathway.At the same time,miR-5587-3p can target and inhibit the expression of WNT5B in LUAD cells and regulate the biological functions of LUAD.