The Role And Mechanism Of LAMB1 In The Migration And Invasion Of Nasopharyngeal Carcinoma

ObjectiveNasopharyngeal carcinoma(NPC)is a malignancy arising from The epithelium of the nasopharynx,particularly prevaling in south of China.Nasopharyngeal carcinoma is characterized by distinct geographical distribution and familial aggregation.Local invasion and early distant metastasis are common clinical manifestations of nasopharyngeal carcinoma.It is extremely diffcult to detect early because of its deep location and asymptomatic in its early stages,so numerous patients usually are identified in the advanced stage.Although concurrent chemotherapy and radiotherapy,especially IMRT,improves the 5-year survival outcomes,local or regional failure in the form of persistent or recurrent still occurs in some patients.Therefore,novel biomarkers and therapeutic strategies to improve treatment outcomes are urgently required for NPC patients.Laminins constitute the ECM as the main non-collagenous glycoproteins of the basement membrane and affect the migratory behavior of numerous malignant cell types.Each one consists of three subunits,namely α,β,and γchains.LAMB1,also known as lamininβ1(laminins subunits),is composed of laminin subunits and distribute in most tissues.LAMB1 modulates laminin-mediated integrin signaling that promotes cell adhesion,motility and differentiation.Previously,studies have shown that LAMB1 is highly expressed in various cancers,and is closely associated with poor prognosis for patients.By our previous bioinformatics analysis,LAMB1 was revealed highly expressed in the nasopharynx carcinoma tissues.However,the role of LAMB1 in nasopharyngeal carcinoma is not yet illustrated.In the light of this,the study aims to explore the biological function of LAMB1 and its related molecular mechanism in nasopharyngeal carcinoma and to elucidate the biological function of LAMB1 in NPC and to provide new molecular markers and potential therapeutic targets for the diagnosis,treatment and prevention of nasopharyngeal carcinoma.Methods1.We evaluated LAMB1 transcription levels in nasopharyngeal carcinoma from Gene Expression Omnibus(GEO)combined with R language.Enrichment and functional annotation of overexpressed genes,including LAMB1,were analyzed by Gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).2.LAMB1 m RNA and protein expression were detected by q PCR/Western Blot in NP69 and NPC cell lines(HNE1,HNE2,CNE1,5-8F).LAMB1 protein was detected by immunohistochemistry(IHC)in 78-paired patients with nasopharyngeal carcinoma(NPC),and its relationship with clinical features and prognosis was further analyzed.3.The stable LAMB1 cell lines were constructed,and then cell proliferation,migration and invasion were detected by CCK-8,clony formation,wound healing and Transwell assay.4.The metastasis ability of LAMB1 on lung in NPC cells was analyzed by injected into nude mice via the lateral tail vein along with control cells.5.MMP14 is a predictive downstream target gene of LAMB1,and the relationship was analyzed.The expression of LAMB1 and MMP14 in NPC specimens was analyzed by immunohistochemistry,and the relation between them was analyzed by Pearson.The proliferation,migration and invasion ability of the NPC cells were detected by CCK-8,clony formation,wound healing and Transwell assays after given or loss function of MMP14.Finally,rescue experiments were applied to detect the proliferation,migration and invasion ability of cells after mutual interference of LAMB1 and MMP14.6.After treated with PI3K/ Akt agonist or inhibitor in HNE1 and 5-8F cells,PI3 K,p-AKT and MMP14 expression were detected by q RT-PCR and Western blot repectively.7.The expressions of PI3 K,p-Akt and MMP14 were detected by Western blot after overexpressed and knockdown of LAMB1.8.Finally,the rescue experiments further demonstrated that LAMB1 can regulate the MMP14 via PI3K-Akt pathway in nasopharyngeal carcinoma cells.Results1.LAMB1 expression was significantly upregulated in NPC tissues,compared to normal nasopharyngeal tissues,and LAMB1 expression was higher in metastatic nasopharyngeal carcinoma than that in non-metastatic nasopharyngeal carcinoma.2.Analysis of enriched gene ontology(GO)terms indicated that these genes encode proteins localized mainly to basement membrance,collagen-containing extracellular matrix and laminin complex.These proteins are primarily involved in cell substrate adhesion,extracellular matrix and extracellular structure organization,while they also serve as structural constituents of extracellular matrix and intergrin and cell adhesion molecule.Similarly,Kyoto Encyclopedia of Genes and Genomes(KEGG)pathway analysis showed overexpressed genes,including LAMB1,are mainly involved in ECM-receptor interaction,focal adhesion and PI3K-Akt signaling pathways.3.The LAMB1 m RNA and protein were indentified in NP69 and nasopharyngeal carcinoma cells.Compared to NP69,LAMB1 m RNA and protein expression in NPC cells were relatively increased.Among these cells,the expression of HNE1 was lowest(P<0.01),while the expression of 5-8F was the highest(P<0.01).4.As showed by Immunohistochemtry,the expression of LAMB1 in NPC patients was significantly higher than that in adjacent tissues(P<0.01).Similarly,LAMB1 expression was significantly higher in NPC patients with metastasis than that without metastasis(P<0.01).Increased LAMB1 expression is negatively related to OS and PFS.5.CCK-8 and Clony formation assay showed that upregulated LAMB1 could induce cell proliferation(P<0.01),increase the ability of cell clony formation(P<0.01),promote the migration and invasion of cells.On the contrary,downregulated LAMB1 expression could restrain cell proliferation(P<0.01),weaken the ability of cell clony formation,inhibit the migration and invasion of cells(P<0.01).6.We predicted that MMP14 was the putative target of LAMB1 by GEIPA,and MMP14 is positively correlated with LAMB1.Then,we identified that LAMB1 and MMP14 have a positive correlation with each other(r=0.6306)by pearson analysis.CCK-8,Clony formation and Transwell assays showed that overexpressed MMP14 could markedly promote cell proliferation,migration and invasion,while silencing MMP14 could significantly inhibit cell proliferation,migration and invasion.7.Overexpression of LAMB1 resulted in high expression of MMP14,while MMP14 knockdown inhibited the expression of MMP14.LAMB1 knockdown decreased the expression of MMP14,while overexpression of MMP14 could restore the expression of MMP14.8.overexpression of LAMB1 promoted cell invasion and migration(P<0.05),while MMP14 knockdown weakened the these effects of LAMB1(P<0.05)。Silencing LAMB1 inhibited migration and invasion of cells(P<0.05),while overexprssion of MMP14 could restore the effect of LAMB1(P<0.05).9.740Y-P(agonist)can increase the expression of PI3 K,p-Akt and MMP14 in NPC HNE1 and 5-8F cells,while LY294002(inhibitor)can significantly decrease the expression of PI3 K,p-Akt and MMP14 in HNE1 and5-8F cells.Furthermore,Overexpression of LAMB1 can promote the expressions of PI3 K,p-Akt and MMP14 in NPC HNE1 cells,while silence of LAMB1 can significantly inhibit the expressions of PI3 K,p-Akt and MMP14 in5-8F cells,suggesting that LAMB1 can regulate MMP14 via activating the PI3K/ Akt pathway.10.Subsequent rescue experiments showed that overexpression of LAMB1 can promote the expressions of PI3 K,p-Akt and MMP14 in NPC HNE1 cells(P<0.05),while LY294002 can significantly reduce the promoting effects of LAMB1(P<0.05).Silenced LAMB1 significantly inhibited the expression of PI3 K,p-Akt and MMP14 in nasopharyngeal carcinoma 5-8F cells,but 740Y-P significantly reversed the inhibitory effect on the expression of PI3 K,p-Akt and MMP14(P<0.05).Conclusions1.LAMB1 expression was significantly upregulated in NPC compared to normal adjacent tissues,and LAMB1 expression was higher in metastatic nasopharyngeal carcinoma than that in non-metastatic nasopharyngeal carcinoma.Increased expression of LAMB1 was also found to be associated with short OS and PFS in patients with NPC.2.In vitro,overexpression of LAMB1 facilitated cell proliferation,colony formation,invasion and migration.While silencing the expression of LAMB1 decreased these effects in nasopharyngeal carcinoma cells.3.In vivo,the numbers of metastatic nodules was higher than that in the control group by using a lung metastasis model.4.LAMB1 can regulate MMP14 to promote proliferation,invasion and migration in nasopharyngeal carcinoma via PI3K/AKT signaling pathway.