| Background and objective:Lung cancer is one of the most common malignant tumors,and various risk factors such as smoking and air pollution are closely related to the occurrence of lung cancer.Despite significant advances in surgery,radiation therapy,and chemotherapy,the survival rate of lung cancer remains low.Lung cancer includes two types: SCLC(small cell lung cancer)and NSCLC(non-small cell lung cancer).NSCLC causes more than 80% of lung cancers.At present,the research on early biomarkers of NSCLC is still limited,and early diagnosis is difficult.Most NSCLC patients are diagnosed at an advanced stage and die after missing the optimal period of surgery.Therefore,the study of the mechanism of NSCLC tumorigenesis is crucial for both the early prognosis of NSCLC and the development of new treatments.Micro-RNAs(miRNAs)are non-coding RNAs of approximately 19 – 25 bp that repress gene expression by inhibiting mRNA stability or translation.Recent studies have shown that many miRNAs exhibit aberrant expression in tumor tissues,suggesting that miRNAs may be involved in tumorigenesis.Although the human genome encodes more than 400 small RNAs,the function of many small RNAs remains unknown.It will be fruitful to investigate the role of miRNAs in NSCLC progression.In the present study,we found that the expression of miR-520 b was significantly up-regulated in NSCLC samples,and miR-520 b levels were positively correlated with Hedgehog(Hh)pathway activity,so we speculated that miR-520 b regulated NSCLC development through the Hh pathway.In this paper,we will investigate the role of miR-520 b in NSCLC development and its regulatory mechanism,identify the target genes of miR-520 b,verify whether miR-520 b regulates NSCLC development through the Hh pathway,and determine the molecular mechanism by which miR-520 b regulates the Hh pathway in NSCLC cells.This paper will provide a new theoretical basis and new direction for the prevention and treatment of NSCLC.Materials and Methods:1.The expression of miRNA-520 b in NSCLC and its influence on the progression of NSCLC(1)q-PCR analysis of miR-520 bexpression in 12 pairs of NSCLC samples and paratumour normal tissues;(2)MTT assays of A549 and H1299 cells treated with miR-520 b mimic or miR-520 b inhibitor;(3)Wound healing assays of A549 cells treated with miR-520 b mimic or miR-520 b inhibitor.Quantificationof wound closure at indicated time-points;(4)Transwell assay of A549 cells treated with miR-520 b mimic or miR-520 binhibitor.2.Identification of the target gene of miR-520b(1)Predictionof target gene of miR-520 b using bioscience information analysis(www.targesan.org);(2)Westernblottinganalyses ofSPOP protein levels in 12 pairs of NSCLC samples and paratumour normal samples;(3)TCGA cohor analysis of therelationship between miR-520 b expression and survival rate of NSCLC patients;(4)Theendogenous and exogenousSPOP expression of A549 cells treated by miR-520 b mimic or miR-520 b inhibitor using western blotting assay;(5)MTT assays of A549 cells transfected with indicated siRNA or plasmids.(6)Construction of SPOP knockout A549 cells by CRISPR/CAS9 Technology,Luciferase reporter assaysof the interation of miR-520 b with of3'-UTR of WT or mutant SPOP in WT or SPOP KO A549 cells treated with miR-520 b mimic or miR-520 b inhibitor.3.miR-520 b regulates the progression of NSCLC through Hh pathway(1)Relative mRNA levels of GLI1 and BCL2 from NSCLC samples and matched normal samples were examined by q-PCR;(2)The mark protein expression related with Hh,Wnt and Hippo pathway in A549 cells with miR-520 b mimic or miR-520 b inhibitor treatment using Westernb-lotting;(3)MTT and transwell assays of A549 cells under Cyclopamine treatment;(4)MTT and wound healing assays of A549 cells transfected with GLI2-siRNA and GLI3-siRNA.4.Molecular Mechanisms of miR-520 b regulating Hh pathway in NSCLC Cells(1)Validation the interation of SPOPand GLI2/3 using Co-IP experiment;(2)Validation the interation of N-terminal MATH domain of SPOPand GLI2/3using Co-IP experiment;(3)Ubiquitination assay of GLI2/3 in A549 cells transfected with WT SPOP or different SPOP mutants(Y87N,F102 C,S119N,F125 L,K129E,W131 G,F133V,K134N)using western blotting and co-IP experiment;(4)MTT assays of A549 cells and H1299 cellstransfected with WT SPOP or different SPOP mutants(Y87N,F102 C,S119N,F125 L,K129E,W131 G,F133V,K134N).Results:1.The expression of miRNA-520 b in NSCLC and its influence on the progression of NSCLC(1)miR-520 bis up-regulated in NSCLC samples;(2)miR-520 bpromotes proliferation of NSCLC cells;(3)miR-520 bpromotes migration of NSCLC cells;(4)miR-520 bpromotes invasion of NSCLC cells.2.Identification of the target gene of miR-520b(1)Bioscience information analysis identified SPOP is target gene of miR-520b;(2)SPOP expression is down-regulated in NSCLC samples;(3)TCGA cohor showed survival rate of NSCLC cancer patients is positively correlated with SPOP expression;(4)miR-520 b regulates the expression of SPOP;(5)SPOP modulates cell proliferation;(6)miR-520 b targets SPOP to regulate NSCLC cell proliferation and invasion.3.miR-520 b regulates the progression of NSCLC through Hh pathway(1)GLI1 and BCL2 genes in Hh pathway are up-regulated in NSCLC samples;(2)miR-520 b activates Hh pathway in NSCLC cell;(3)miR-520 b promotes NSCLC cell proliferation and invasion through Hh pathway;(4)miR-520 b promotes NSCLC cell proliferation and migration through GLI2/3.4.Molecular Mechanisms of miR-520 b regulating Hh pathway in NSCLC Cells(1)SPOP interacts withGLI2/3;(2)N-terminal MATH domain of SPOP interacts withGLI2/3;(3)SPOP promotes ubiquitination degradation of GLI2/3;(4)miR-520 b regulates the proliferation of NSCLC cells by regulating SPOP-GLI2/3 axis.Conclusion:1.miR-520 b is up-regulated and plays a oncogenic role in NSCLC2.SPOP is a target gene of miR-520b3.miR-520 b activates Hh pathway4.miR-520 b exerts oncogenic effect on NSCLC cells through GLI2/3.5.SPOP suppresses Hh pathway through ubiquitinating GLI2 and GLI3... |
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