The Biological Function And Mechanism Of LncRNA TEX41 In Lung Adenocarcinoma Bone Metastasis

ObjectivesLung cancer is the largest malignancy that threatens the life and health of the population.The pain and bone destruction caused by bone metastases severely reduce the quality and survival of patients.Finding easy-to-use biomarkers for early detection or assessment of bone metastases is of great clinical value.Our group previously performed transcriptome sequencing of lung adenocarcinoma tissue and lung adenocarcinoma bone metastasis tissue samples and used bioinformatics analysis to explore differentially expressed genes that play an important role in lung adenocarcinoma bone metastasis.lnc RNA TEX41 was shown to play a possible key role in the lung adenocarcinoma bone metastasis signaling pathway.1.In this study,by applying bioinformatics algorithms,we predicted TEX41 TEX41 and Runx2 may play an important role in lung adenocarcinoma bone metastasis,therefore,the relationship between the expression levels of TEX41 and Runx2 and clinicopathological features was examined in clinical samples.3.TEX41 was explored at the cellular and animal levels The effect of TEX41 on the malignant biological behavior of lung adenocarcinoma cells was investigated at the cellular and animal levels.4.The possible molecular mechanisms of TEX41 affecting bone metastasis of lung adenocarcinoma were initially explored.MethodsPart I:Mining raw information from public databases such as Lnc Base,mi RDB and Targetscan,combined with bioinformatics algorithms,predicted the possible target genes of TEX41,and performed GO functional annotation and KEGG pathway enrichment analysis for differentially expressed target genes to explore the possible biological functions and molecular mechanisms of TEX41.Part II:Surgical cases of lung adenocarcinoma without distant metastasis who underwent radical lung cancer surgery at Yunnan Cancer Hospital between January2019 and December 2019 were selected,and specimens of surgically resected lung adenocarcinoma tissues and paracancerous tissues were collected;clinical samples of lung adenocarcinoma diagnosed with bone metastasis and palliative surgery of bone metastases were collected during the same period.After extracting the total RNA from the tissue samples,the expression levels of TEX41 and Runx2 were detected by q RT-PCR,and the gene expression levels were analyzed by the 2^‐ΔΔCt relative quantification method.Part III:In this study,lung normal epithelial cell line Beas-2B,lung adenocarcinoma cell lines A549,H1734,H1299 and H838 were selected for cell experiments.According to the expression of TEX41 in lung adenocarcinoma cell lines,the construction of interfering and overexpressing TEX41 stable transgenic strains were performed in A549 and H838 cell lines,respectively.The effects of TEX41 on lung adenocarcinoma cell growth,proliferation,migration and invasion were investigated using CCK8,clone formation,scratch healing and Transwell assays.Total cell or tissue proteins were extracted and the target proteins were detected semi-quantitatively by protein immunoblotting assay.A bone metastasis model was constructed by intracavitary injection of lung adenocarcinoma cells into the bilateral tibial cavity of nude mice,which were incubated for 5 weeks and then X-rayed to observe bone metastasis,and the material was taken for tumor weighing and pathological examination to further investigate the effect of TEX41 on bone metastasis of lung adenocarcinoma.Part IV:To design a rescue assay,TEX41 was overexpressed in A549 cell line while adding Runx2 specific inhibitor CADD522 to observe the effect on cell migration,invasion and metastatic ability.TEX41 was disrupted in the H838 cell line while adding the autophagy inducer rapamycin（Rapamycin）to observe the effects on cell migration,invasion and metastatic ability.ResultsPart I:294 TEX41 differentially expressed target genes were screened by bioinformatic methods,including 82 positively and 212 negatively expressed genes.t EX41 target genes were mainly enriched in protein regulation,cell surface signaling pathway and tissue morphogenesis,the first 5 GO Term were GO:0007167（enzyme-linked receptor protein signaling pathway）,GO:0009653（anatomical structure morphogenesis）,GO:0071310（cellular response to organisms）,GO:0007166（cell surface receptor signaling pathway）,and GO:0048729（tissue formation）.The results of KEGG pathway analysis suggest that TEX41 target genes are significantly enriched mainly in various cancer-related pathways,PI3K/AKT,and TFBSTools R package predicted the transcription factors and binding sites of TEX41target genes,suggesting that Runx2,a gene that was screened by RNA-seq and shown to be significantly associated with bone metastasis signaling pathway in lung adenocarcinoma,is one of the top 30 genes predicted by TEX41.Runx2,one of the top 30 predicted transcription factors of TEX41.Part II:The relative expression of TEX41 correlated with the TNM stage and the presence or absence of lymph node metastasis of patients.The median OS of patients with lung adenocarcinoma bone metastases was 6.5 months in the TEX41 high expression group and 13.3 months in the TEX41 low expression group,with a statistically significant difference of P<0.001 when comparing OS between the two groups.Runx2 was highly expressed in primary lung adenocarcinoma tissues relative to paracancerous tissues（P<0.001）and had higher expression levels in bone metastases（P<0.001）.The relative expression of Runx2 correlated with the TNM stage,primary tumor size,and the presence of lymph node metastasis of patients.The median OS of patients with bone metastases from lung adenocarcinoma was shorter in the Runx2 high expression group compared with the Runx2 low expression group（P<0.01）.Part III:The expression of TEX41 was significantly higher in lung adenocarcinoma cell lines A549,H1734,H1299 and H838 than in lung normal epithelial cell line Beas-2B,where TEX41 was expressed at a higher level in lung adenocarcinoma metastatic lymph node derived cell line H838 and at a relatively lower level in lung adenocarcinoma primary foci derived cell line A549.The expression of E-cadherin and Vimentin was detected by CCK8,clone formation assay,scratch assay and Transwell assay,and Western blot,suggesting that interference with TEX41 could inhibit the growth,proliferation,migration,invasion and metastatic ability of lung adenocarcinoma cells H838（P<0.05）;overexpression of TEX41 In contrast,overexpression of TEX41 promoted the growth,proliferation,migration,invasion and metastasis of lung adenocarcinoma cells A549（P<0.05）.The experiments of bone metastasis model in nude mice suggested that interference or overexpression of TEX41 could inhibit or promote the formation of subcutaneous tumor and bone metastasis in nude mice,and overexpression of TEX41 promoted the formation of bone metastasis more significantly than subcutaneous tumor.Part IV:Runx2 was highly expressed in lung adenocarcinoma cell lines A549,H1734,H1299 and H838 compared to lung normal epithelial cell line Beas-2B（P<0.001）.western blot assay results showed that Runx2 was expressed at elevated protein levels in lung adenocarcinoma cell lines A549 and H838,with the H838 cell line Runx2 had a higher protein expression level in H838 cell line（P<0.01）.In vitro and in vivo assays showed that interference or overexpression of TEX41 could inhibit or promote Runx2 expression（P<0.05）,and Fish assay suggested that some TEX41co-localized with Runx2 in the nucleus.To design the rescue assay,a Runx2protein-specific inhibitor（CADD522）was added to the A549 stable transgenic strain overexpressing TEX41,and CADD522 was able to effectively counteract the migration（P<0.001）,invasion（P<0.001）and metastasis phenotypes（P<0.01）promoted by overexpression of TEX41.In H838 cells with knockdown of TEX41,p62 expression was elevated（P<0.01）and Beclin1（P<0.01）and LC3II/I expression levels were decreased（P>0.05）;conversely,in A549 cells overexpressing TEX41,p62 expression was decreased（P<0.01）and Beclin1（P<0.01）and LC3II/I expression levels were increased（P<0.01）.The bone metastasis protein assay assay in nude mice in the sh-TEX41 group showed elevated p62 expression（P<0.01）,decreased Beclin1（P<0.05）and LC3II/I expression levels（P<0.05）;while in the bone metastasis autophagy protein assay assay in the OE-TEX41 group,LC3II/I was found to be significantly elevated（P<0.01）,the changes in p62 and Beclin1 protein expression levels were not significant.In a reactive assay,Rapamycin was added to the H838 stable transgenic strain with knockdown of TEX41,and Rapamycin was able to reverse to some extent the cell migration（P<0.01）,invasion（P<0.001）and metastatic ability（P<0.01）inhibited by knockdown of TEX41.In further experiments,CADD522 was added to A549 stable-transformed cells overexpressing TEX41,and it was found that CADD522 effectively counteracted the decrease in P62expression（P<0.01）,as well as the increase in Beclin1 expression level and LC3II/I（P<0.05）caused by overexpression of TEX41.Conclusions1.TEX41 and Runx2 were highly expressed in the primary tissues of lung adenocarcinoma,and the expression levels were higher in the tissues of bone metastases of lung adenocarcinoma.The expression levels of both correlated with clinicopathological signs,and the high expression of TEX41 and Runx2 wasassociated with poor prognosis of patients with bone metastases of lung adenocarcinoma.2.TEX41 is highly expressed in lung adenocarcinoma cell lines and has higher expression in lung adenocarcinoma metastatic lymph node derived cell lines.TEX41can promote the growth,proliferation,migration,invasion and metastasis of lungadenocarcinoma cells and may play an important role in promoting the process of lung adenocarcinoma cell bone metastasis.3.The effect of TEX41 on the ability of lung adenocarcinoma cells to migrate,invade,and metastasize may be partially dependent on cellular autophagy.4.TEX41 may promote autophagy of lung adenocarcinoma cells through Runx2,thus promoting migration,invasion and bone metastasis of lung adenocarcinoma cells.