The Molecular Mechanism Of HMGA1 Modulating Autophagy,migration,and Invasion Of Bladder Cancer Via MiRNA-221/TP53INPL/p-ERK1/2 Axis

Background and Aim:Bladder cancer(BC)is the fourth most common cancer in men in the United States with an estimated 62,380 new cases and 12,520 deaths in 2018.Approximately75% of cases are non-muscle-invasive BC(Ta,T1),which has a good prognosis with94% of patients surviving ≥5 years and is usually treated by transurethral resection and intravesical chemotherapy.However,approximately half of these patients will experience cancer recurrence and 20% will progress to muscle-invasive bladder cancer(MIBC).Although few patients are initially diagnosed with MIBCs,the 5-year survival of patients with MIBC is low: nearly 50.1% for regional stage,and 10.2% for distant stage.Despite the crucial advances in BC research this year,the mortality rate of BC has remained unchanged due to the lack of specific targets.Autophagy,which is a cellular self-degradative process mobilizing intracellular nutrient resources,plays an important role in cell survival under stress conditions.However,hyperactivated autophagy can lead to nonapoptotic programmed cell death.The deregulation of this process is associated with multiple human diseases,including cancer.In cancer,autophagy has a complex role,and shows either an oncogenic or tumor suppressor activity.A feasible strategy to treat cancer is to reprogram cancerous cells to undergo toxic autophagy.Micro RNAs(miRNAs)have been implicated in regulating the development and metastasis of human cancers.Mi RNAs play an important regulatory role in the biological functions of tumor cell proliferation,apoptosis,pyrolysis,ferroptosis,invasion,metastasis,drug resistance,immune escape,and stemness of tumor stem cells.MiR-221 is reported to be an oncogene in multiple cancers,including bladder cancer(BC).Deregulation of autophagy is associated with multiple human malignant cancers.Whether and how miR-221 regulates autophagy and how miR-221 been regulated in BC are poorly understood.Here,this study explored the potential functions and mechanisms of miR-221 in the autophagy and tumorigenesis of BC.HMGA1 has been reported to play an important role in many types of cancers.HMGA1 contributes to tumor formation and progression through several mechanisms:inactivation of the apoptotic function of p53,enhancement of the expression of genes involved in stem cell and inflammatory pathway,modulation of the expression of miRNAs and genes involved in cell cycle and epithelial-mesenchymal transition.However,the role of HMGA1 in BC remains unknown.Previously,miR-221 is targeted and regulated by transcriptional factor HMGA1 during cervical cancer.In addition,as a key regulator of the autophagic pathway in cancer cells,HMGA1 might contribute to cancer progression.We want to investigate whether the miR-221-mediated autophagy,migration and invasion of bladder cancer cells was regulated by HMGA1.Methods:1.The expression of miR-221 in bladder cancer cells and immortalized normal bladder epithelial cells was detected by q RT-PCR,and the relationship between the expression of miR-221 and the prognosis of bladder cancer was analyzed by bioinformatics.Western blot,transmission electron microscopy,Cyto-ID autophagy detection kit and LC3 staining were used to detect the changes of autophagy in bladder cancer cells after knocking down miR-221.The effect of knocking down miR-221 on the invasion and migration of bladder cancer cells was verified by transwell assay and wound healing assay.The effect of TP53INP1 on autophagy and invasion and migration of bladder cancer cells induced by knockdown of miR-221 was verified by rescue assay.In addition,the effect of miR-221 knock down on the proliferation of bladder cancer in vivo was verified by xenografts in nude mice.2.The effects of ERK1/2 pathway and autophagy on the invasion and migration of bladder cancer cells mediated by miR-221 were verified by rescue assay using ERK1/2 inhibitor U0126 and ATG5 siRNA.3.The relationship between the expression of miR-221 and HMGA1,the expression of HMGA1 and prognosis of bladder cancer were studied by bioinformatics.The expression of miR-221 was detected by q RT-PCR after HMGA1 silencing by HMGA1 si RNA.In addition,the expression of HMGA1 in bladder cancer was detected by immunohistochemistry.4.CCK8 assay was used to detect the effect of HMGA1 knock down on the proliferation of bladder cancer cells.Western blot,autophagy detection kit and LC3 staining were used to detect the changes of autophagy in bladder cancer cells after HMGA1 knock down.The effect of HMGA1 knockdown on the invasion and migration of bladder cancer cells was verified by transwell assay and wound healing assay.5.The role of miR-221 in autophagy and invasion and migration of bladder cancer cells induced by knockdown of HMGA1 was verified by rescue assay.Results:1.Downregulation of miR-221 induces autophagy and inhibits invasion and migration of bladder cancer cells in vitro via TP53INP1/p-ERK axis.We showed that miR-221 was significantly upregulated or unchanged in invasive BC lines(T24,J82,EJ,UM-UC-3)but downregulated in the non-invasive bladder cancer cell line(5637).Moreover,we performed Kaplan-Meier analysis in the database(http://kmplot.com/analysis/)and found that patients with BC with high miR-221 expression had worse prognosis and shorter survival time.Our results also indicated that the suppression of miR-221 expression promoted autophagy in BC cells.By using Western blot and luciferase reporter assay,we found that TP53INP1 is a target of miR-221.In addition,further rescue assay showed that the autophagy and subsequent decrease of invasion and migration of bladder cancer cells induced by miR-221 knock down depended on TP53INP1/p-ERK1/2 axis.In addition,we also found that suppression of miR-221 inhibits the proliferation of bladder cancer in vivo.2.The oncogenic effect of miR-221 is ERK pathway-dependent and miR-221 suppression induced inhibition of invasion and migration is autophagy-dependent.Western blot analysis showed that the ectopic overexpression of miR-221 decreased TP53INP1 expression and enhanced the phosphorylation of ERK1/2 in5637 cells.By contrast,the suppression of endogenous miR-221 expression increased the expression of TP53INP1 and repressed the phosphorylation of ERK1/2 in T24 cells.Transwell and wound-healing assays showed that the ectopic overexpression of miR-221 could remarkably promote the invasive and migratory capacity of 5637 BC cells,whereas the suppression of endogenous miR-221 expression could impede the invasive and migratory capacity of T24 BC cells.The introduction of U0126 attenuated in vitro cell migration and invasion abilities,suggesting that ERK signaling is needed to drive invasion and migration in BC cells with ectopic miR-221 overexpression.We found that ATG5 knock down partly blocked miR-221 suppression induced-inhibition of invasion and migration.Overall,these results suggested that the oncogenic effect of miR-221 is ERK pathway-dependent and the effects of miR-221 suppression on cell invasion and migration are autophagydependent.3.HMGA1 was upregulated in bladder cancer and correlated with miR-221.We evaluated the correlation between HMGA1 and miR-221 in the Cancer Genome Atlas(TCGA)database by bioinformatics,in which we observed a strong and positive correlation between miR-221 and HMGA1,while a negative correlation between miR-221 and TP53INP1,HMGA1 and TP53INP1 in 402 bladder cancer tissues.We further evaluated the effect of HMGA1 silencing on the expression of miR-221 and TP53INP1 by q RT-PCR(Quantitative Real time PCR),observing a strong reduction of miR-221 and increase of TP53INP1.Altogether,these data suggest that HMGA1 is correlated with miR-221 in bladder cancer.HMGA1 expression was significantly increased in BC tissues and cell lines.The high expression of HMGA1 was correlated with poor prognosis of patients with BC as revealed by provisional TCGA database analysis in the database.HMGA1 was highly expressed in 45/64(70.31%)BC and 12/48(25%)para-cancer tissues(p < 0.001).4.Down-regulation of HMGA1 inhibits the proliferation,colony formation,invasion and migration of BC cells by promoting autophagy.HMGA1 silencing decreased colony formation and inhibited cell migration and invasion.HMGA1 knockdown increased TP53INP1 expression and repressed the phosphorylation of ERK1/2 in T24 cells.The effects of HMGA1 knock down on cellular proliferation,invasion and migration were due to autophagy induction.5.Down-regulation of HMGA1 modulates autophagy and inhibits the proliferation,colony formation,invasion and migration of BC cells partly by miR-221/TP53INP1 axis.HMGA1 knockdown markedly inhibited cell invasion and migration and miR-221 could rescue their invasive and migratory potential.Mi R-221 mimic significantly antagonized autophagy and the increase in TP53INP1 expression and reduction in ERK1/2 phosphorylation mediated by HMGA1-silencing.Conclusions:Mi R-221 was significantly upregulated or unchanged in invasive BC lines(T24,J82,EJ,UM-UC-3)but downregulated in the non-invasive bladder cancer cell line(5637).We found that patients with BC with high miR-221 expression had worse prognosis and shorter survival time.And downregulation of miR-221 induces autophagy and inhibits invasion and migration of bladder cancer cells in vitro via TP53INP1/p-ERK axis.We also observed a strong and positive correlation between miR-221 and HMGA1,while a negative correlation between miR-221 and TP53INP1,HMGA1 and TP53INP1 in 402 bladder cancer tissues by bioinformatics.Through immunohistochemistry experiments we found that HMGA1 was upregulated in bladder cancer tissues compared with adjacent bladder tissues.We also found that HMGA1 was significantly upregulated in most of BC lines(T24,5637,EJ,UM-UC-3).We found that the high expression of HMGA1 was correlated with poor prognosis of patients with BC as revealed by provisional TCGA database analysis.Down-regulation of HMGA1 inhibits the proliferation,colony formation,invasion and migration of BC cells by promoting autophagy.And down-regulation of HMGA1 modulates autophagy and inhibits migration,and invasion of bladder cancer cells partly by miR-221/TP53INP1/p-ERK axis.Above all,in this study,we investigated the role of miR-221 and HMGA1 and demonstrated that the down-regulation of HMGA1 inhibits migration,and invasion partly via miRNA-221/TP53INP1/p-ERK axis-mediated toxic autophagy in BC.Both of miR-221 and HMGA1 are valuable therapeutic targets for bladder cancer.