KLF6/QKI-5/TGFβR1 Axis Regulates TGF-β/SMAD Signaling To Inhibit EMT,Invasion And Metastasis Of Lung Adenocarcinoma

Backgrounds and Objectives:Lung cancer is the number one killer threatening human life and health worldwide.Non-small cell lung cancer(NSCLC)accounts for～85%of all LC cases,which includes lung adenocarcinoma(LUAD),lung squamous cell carcinoma(LUSC),and large cell carcinoma.LUAD represents the most common subtype of NSCLC and has the most heterogeneity and aggressiveness.At present,metastasis is the leading cause of cancer-associated mortality,and about 90%of cancer patients’ deaths are caused by metastasis.Epithelial-mesenchymal transition(EMT)is a key event in the transformation of early tumors into advanced malignant tumors,which plays important roles in the invasion and metastasis of tumor cells.Transforming growth factor-β(TGF-β)/SMAD signaling triggering EMT plays a predominant role in the invasion and metastasis of NSCLC.Quaking(QKI)proteins belong to the signal transduction and activation of RNA(STAR)family of RNA-binding proteins(RBPs),including three major isoforms QKI-5,QKI-6 and QKI-7,which are the key post transcriptional regulatory factors.It was reported that QKI-5 is the main isoform of QKls,and its abnormal expression is closely related to the occurrence and development of a variety of tumors.Bioinformatics analysis and previous experiments identified a metastatic LUAD-associated RBP,QKI-5,which was dramatically downregulated in LUAD tissues,especially in metastatic LUAD tissues,indicating that QKI-5 may participate in the invasion and metastasis of LUAD.However,whether and how QKI-5 contributes to TGF-β-induced EMT and metastasis of LUAD remains poorly understood.Moreover,the underlying mechanism resulting in QKI-5 downexpression in LUAD remains unclear.This study aims to explore the transcriptional regulation mechanism of QKI-5 lowly expressed in LUAD,and to reveal the crucial role of QKI-5 in the invasion and metastasis of LUAD.We’ll further clarify the molecular mechanism of KLF6/QKI-5/TGFβR1 axis regulating TGF-β/SMAD signaling to inhibit EMT,invasion and metastasis of LUAD,so as to provide new theoretical basis for further understanding the metastasis mechanism of LUAD,as well as provides new scientific basis for early diagnosis and accurate treatment of LUAD.Methods:(1)Bioinformatic analysis of microarray data was used to screen LUAD-associated differentially expressed RNA-binding proteins(RBPs)and transcription factors(TFs).(2)qRT-PCR and Western blot were used to verify the bioinformatic data.We further detected the expression KLF6,QKI-5,TGFβR1 and other molecules,as well as analyzed the correlation between their expression in the collected LUAD tissues.(3)Applying lentiviral transduction systems to establish LUAD cells in which QKI-5 was consistently over-expressed,QKI-5 and TGFβR1 were silenced.The expression plasmids of TGFβR1 was constructed and transiently transected into LUAD cells.siRNAs interference was used to transiently silence interesting genes.(4)The migratory and invasive capabilities of LUAD cells were determined by Wound-healing,Transwell migration and invasion assays.(5)In vivo metastasis assays were conducted to examine the metastatic ability of LUAD cells.(6)We detected the expression levels of KLF6,QKI-5,TGF-βR1,EMT markers(p-smad3,Smad3,E-cadherin,N-cadherin and snail)and other molecules using qRT-PCR and Western blot assays.(7)RNA binding protein immunoprecipitation(RIP),RNA pull-down,dual luciferase report system,qRT-PCR and Western blot assays were performed to explore the targeting effect and underlying mechanism of QKI-5 on TGFβR1.(8)We investigated the molecular mechanism by which KLF6 transcriptional regulate QKI-5 in LUAD cells using chromatin immunoprecipitation(ChIP),dual luciferase report system,qRT-PCR and Western blot analysis.(9)We performed rescue experiments to verify the axial regulation relationship among KLF6,QKI-5 and TGFβR1 in TGF-β-induced EMT,invasion and metastasis of LUAD cells.Results:(1)Through bioinformatics analysis and detection of LUAD tissue samples,we identified a LUAD-associated RBP,QKI-5,which was dramatically downregulated in LUAD tissues,especially in metastatic LUAD tissues.and its low expression was associated with poor prognosis of LUAD.Kaplan-Meier survival analysis showed that low expression of QKI was significantly associated with poor survival in LUAD patients.(2)Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment analysis of QKI-RIP-seq microarray data showed that TGFβR1,an upstream receptor that initiates TGF-β/SMADs signaling pathway,was a putative target of QKI-5.We further confirmed that QKI-5 negatively regulated TGFβR1 at the post transcriptional level by promoting the degradation of TGFβR1 mRNA,and then inhibited the TGF-β/SMAD signaling pathway.(3)QKI-5 overexpression suppressed TGF-β-induced EMT and LUAD cell metastasis in vitro and in vivo;whereas QKI-5 knockdown exhibited the opposite effects on the phenotype of LUAD cells.(4)Rescue experiments confirmed that QKI-5 inhibit TGF-β-induced EMT,invasion and metastasis of LUAD cells via a TGFβR1-dependent manner.(5)Bioinformatics analysis showed that QKI-5 is a potential target of KLF6.We further confirmed that KLF6 regulate QKI-5 at the transcriptional level.In addition,KLF6 is lowly expressed in LUAD tissues,which is a key mechanism resulting in QKI-5 down-expression in LUAD.(6)Rescue experiments revealed that KLF6 negatively regulated TGFβR1,thereby inhibiting TGF-β-induced EMT,invasion and metastasis of LUAD cells via a QKI-5-dependent manner.(7)Clinical analysis showed that KLF6 was lowly expressed and positively correlated with QKI-5 expression,whereas TGFβR1 expression was highly expressed and inversely correlated with QKI-5 expression.Conclusion:In the present study,we identified a metastatic LUAD-specific RBP,QKI-5,which was dramatically downregulated in LUAD tissues,especially in metastatic LUAD tissues.We revealed that QKI-5 can interdict TGF-β/SMADs signaling transduction via post-transcriptionally inhibiting TGFβR1 expression and thereby repressing TGF-β-induced EMT,invasion and metastasis of LUAD cells.Meanwhile,we also discovered that QKI-5 is positively regulated at transcriptional level by KLF6.Our findings clarified a novel mechanistic role of KLF6/QKI-5/TGFβR1 axis regulating TGF-β/SMAD signaling to inhibit EMT,invasion and metastasis of LUAD cells,providing new theoretical basis for further understanding the metastasis mechanism of LUAD.