The Role And Molecular Mechanism Of MiR-29b-3p In EGFR-TKI Acquired Resistance By Targeting "PTEN/PI3K/Akt" Cascade In Lung Cancer

Background and objective:With the advancement of molecular diagnostic technology and precise targeted therapy,the individualized diagnosis and treatment of lung cancer are more targeted.For patients with EGFR gene exon 19 deletion or exon 21 L858R mutation,the clinical application of first-generation EGFR-TKIs(Gefitinib,Erlotinib)or second-generation EGFR-TKIs(Afatinib)as standard first-line treatments have achieved clear curative effect.However,most patients will develop acquired resistance after 6-8 months of targeted drug treatment,which limits the long-term efficacy of EGFR inhibitors.The most common mechanism of drug resistance is T790M mutation of EGFR gene.For NSCLC patients with T790M mutation,third-generation EGFR-TKIs,such as Osimertinib,have been used in clinical patients with T790M mutation and have achieved definite curative effect.However,the third-generation of targeted drugs is the same as the firstgeneration EGFR-TKIs,and patients inevitably develop acquired resistance after a period of continuous medication.The long-term efficacy of EGFR TKIs is still unsatisfactory.MicroRNA(miRNA)is a kind of highly conserved single stranded noncoding RNA molecule with length of about 22 nucleotides,which is widely present in various organisms.It can block the translation of messenger RNA or induce mRNA degradation by completely or incomplete binding with target gene 3 ’UTR region,thus regulating the gene after transcription.miRNA can be used as a biomarker for early diagnosis,and a predictor of prognosis and a potential therapeutic target.Our previous study found that there was a significant difference in the expression level of miR-29b-3p between the EGFR-TKI acquired resistance lung cancer cell line PC9GR and EGFR-TKI sensitive lung cancer cell line PC9.MiR-29b-3p may be involved in the EGFR-TKI acquired resistance of lung cancer.However,what is the signaling pathway involved in miR-29b-3p’s regulation of acquired resistance to EGFR-TKI in lung cancer?What are the downstream target genes of miR-29b-3p regulating EGFRTKI acquired resistance in lung cancer?Does Inhibition or Blocking of miR-29b-3p Reverse Acquired Resistance to EGFR-TKI in Lung Cancer,and what is the molecular mechanism of miR-29b-3p regulating EGFR-TKI acquired resistance to EGFR-TKI in lung cancer,there are no reports at home and abroad.In order to study and explore the signal pathway and molecular mechanism of miR-29b-3p regulating acquired resistance to EGFR-TKI in lung cancer,based on previous research work,Gefitinib acquired resistance cell lines PC9GR and Osimertinib acquired resistance cell lines H1975OR were constructed by in vitro drug resistance induction technology.The Expression microarray,miRNA microarray and RT-PCR were used to screen and identify differentially expressed gene profiles and differentially expressed miRNA profiles related to acquired drug resistance to Gefitinib and Osimertinib.The role and molecular mechanism of miR-29b-3p in regulating Gefitinib and Osimertinib acquired resistance in lung cancer in vivo and in vitro,find the molecular target of miR-29b-3p in regulating acquired resistance of Gefitinib and Osimertinib in lung cancer,and provide theoretical basis and experimental evidence for the development of small molecular drugs to reverse the acquired resistance of EGFR-TKI in lung cancer.The role and molecular mechanism of miR-29b-3p in the regulation of acquired drug resistance to Gefitinib and Osimertinib was studied in lung cancer in nude mouse xenograft model for finding the molecular target of miR-29b-3p in the regulation of acquired drug resistance to Gefitinib and Osimertinib in lung cancer,and for provide theoretical basis and experimental evidence for the development of small molecule drugs to reverse acquired resistance of EGFR-TKI in lung cancer.Materials and Methods:(1)The Gefitinib acquired drug resistant cell lines PC9GR and Osimertinib drug resistant cell lines H1975OR were screened and constructed by in vitro drug resistance induction technology.(2)CCK-8 assay,flow cytometry,plate cloning assay and scratch healing assay were used to detect in vitro proliferation,apoptosis,in vitro cloning formation and in vitro migration of Gefitinib and Osimertinib resistant lung cancer cell lines and their differences.(3)Mutations and differences in genes associated with sensitive and resistance to EGFR-TKI were detected in both the Gefitinib and Osimertinib sensitive lung cancer cell lines,and the Gefitinib and Osimertinib resistant lung cancer cell lines by DNA sequencing.(4)Gene expression microarray,miRNA microarray and second-generation gene sequencing technology were used to screen the differentially expressed gene profiles,differentially expressed miRNA profiles and EGFR gene exon mutations related to the acquired drug resistance to Gefitinib and Osimertinib in lung cancer.(5)RT-PCR was used to verify the differentially expressed gene and miRNA profiles associated with acquired drug resistance of Gefitinib and osimertinib.(6)Targetscan and miRTarBase database were used to predict the downstream target gene of related to miR-29b-3p related to acquired drug resistance to EGFR-TKI.(7)Double luciferase reporter genes were used to verify the downstream regulatory target gene PTEN of miR-29b-3p and the binding sites and binding conditions of miR-29b-3p and PTEN gene 3’UTR.(8)Human Gefitinib acquired drug resistant lung cancer cell line PC9GR and Osimertinib acquired drug resistant lung cancer cell line H1975OR were used to construct xenograft model of lung cancer in nude mice.(9)Antagomir,a miR-29b-3p inhibitor,and Antagomi-NC,a miR-29b-3p control,were used for intratumoral injection of xenograft tumors in nude mice with lung cancer.The effects of the two drugs on the inhibition of tumor formation in xenograft tumors were observed.(10)Western Blot was used to detect the protein expression levels of PTEN,p-Akt and Akt pathway in the tumor tissues of the four groups of nude mice transplanted with lung cancer.(11)Western Blot was used to detect the expression levels of PTEN,pAkt and Akt protein between Gefitinib and Osimertinib sensitive lung cancer cell lines,and Gefitinib and Osimertinib resistant lung cancer cell lines.(12)RT-PCR was used to detect the effects of transfection of miR-29b-3p-micmic and miR-29b-3p-inhibitor on PTEN mRNA and miR-29b-3p expression levels in the Gefitinib and Osimertinib resistant lung cancer cell lines.(13)CCK-8 assay was used to detect the Changes and differences in in vitro proliferative potential in the Gefitinib and Osimertinib Sensitive lung cancer cell line and Gefitinib and Osimertinib resistant lung cancer cell lines after transfection with miR-29b-3p-mimi and miR-29b-3p-inhibitor.(14)The apoptosis induced by Gefitinib and Osimertinib was detected by flow cytometry in lung cancer cell lines with overexpression and low expression of miR-29b-3p.(15)The effect of Gefitinib and Osimertinib on the colony forming ability of lung cancer cell lines with overexpression and low expression of miR-29b-3p was detected by plate cloning assay.(16)Scratch healing assay was used to detect the migration ability of lung cancer cell lines with overexpression and low expression of miR-29b-3p after treated with Gefitinib and osimertinib.(17)Western blot was used to detect the effects of overexpression and low expression of miR-29b-3p on the expression of miR-29b-3p and PTEN/PI3K/Akt cascade related proteins in lung cancer.Results:1.Compared with the Gefitinib sensitive lung cancer cell line PC9,the IC50 of Gefitinib resistant lung cancer cell line PC9GR was significantly increased(P<0.001).2.Compared with the Osimertinib sensitive lung cancer cell line H1 975,the IC50 of Osimertinib resistant lung cancer cell line H1975OR was significantly increased(P<0.001).3.Compared with the Gefitinib sensitive lung cancer cell line PC9,the proliferation of Gefitinib resistant lung cancer cell line PC9GR was significantly increased in vitro(P<0.001);Compared with the Osimertinib sensitive lung cancer cell line H1 975,the proliferation ability of Osimertinib resistant lung cancer cell line H1975OR was significantly increased in vitro(P<0.001).4.Compared with the Gefitinib sensitive lung cancer cell line PC9,the apoptosis level of Gefitinib resistant lung cancer cell line PC9GR was significantly reduced(P<0.001);Compared with the Osimertinib sensitive lung cancer cell line H1975,the apoptosis level of Osimertinib resistant lung cancer cell line H1975OR was significantly reduced(P<0.001).5.The results of clone formation experiment showed that Gefitinib resistant lung cancer cell line PC9GR was significantly higher than Gefitinib sensitive lung cancer cell line PC9 in vitro(P<0.001),and the in vitro clone formation ability of Osimertinib resistant lung cancer cell line H1975OR was significantly higher than that of Osimertinib sensitive lung cancer cell line H1 975(P<0.001).6.The scratch healing test results showed that In vitro migration ability of Gefitinib resistant lung cancer cell line PC9GR was significantly higher than that of Gefitinib sensitive lung cancer cell line PC9(P<0.001),and Osimertinib resistant lung cancer cell line H1975OR had significantly higher in vitro migration ability than that of Osimertinib sensitive lung cancer cell line H1975(P<0.001)。7.The exon sequencing results of EGFR gene showed that Geiftinib resistant lung cancer cell line PC9GR had sensitive mutation with deletion of 19 exons,and there is no secondary T790M resistant mutation,and the sensitive mutations of L858R and T790M were found in Osimertinib resistant lung cancer cell line H1975OR,while acquired resistance mutations such as C797S were not found.8.A total of 806 differentially expressed genes were found between Gefitinib sensitive lung cancer cell line PC9 and Gefitinib resistant lung cancer cell line PC9GR,among which 327 genes were highly expressed and 479 genes were low expressed in Gefitinib resistant lung cancer cell line PC9GR(the up-regulation and downregulation ratio was more than 2 times)(P<0.05).9.The qRT-PCR results showed that the expression level of miR-29b-3p in Gefitinib resistant lung cancer cell line PC9GR was significantly higher than that in Gefitinib sensitive lung cancer cell line PC9(P<0.001),and the expression level of miR-29b-3p in Osimertinib resistant lung cancer cell line H1975OR was significantly higher than that in Osimertinib sensitive lung cancer cell line H1975(P<0.001)。10.The results of theTargetscan search showed that Eight bases at the 3’UTR end of PTEN gene were complementary paired with miR-29b-3p sequence,suggesting that PTEN gene is a downstream regulatory target gene of miR-29b-3p.11.The dual luciferase reporter gene results showed that Compared with empty vector mimic-NC transfection,transfection of miR-29b-3p mimic could significantly inhibit the luciferase activity of 3’UTR-WT of transgenic lung cancer cell line wildtype PTEN,and There were significant differences between the two groups(P<0.001);However,there was no significant difference in luciferase activity between the 3’UTR-MUT transfected with mutant PTEN and the transgenic lung cancer cell line transfected with no-load plasmid,which further proved that miR-29b-3p binds to the 3’ UTR terminal of PTEN gene.12.The tumorigenicity of Gefitinib acquired resistance lung cancer cell line PC9GR in miR-29b-3p-antagomir inhibitor group was significantly lower than that in human miR-29b-3p-antagomir NC control group Tumor into tumor(P<0.001).13.Tumorigenicity of xenograft in nude mice of Osimertinib acquired drugresistant lung cancer cell line H1975OR in nude mice treated with miR-29b-3pantagomir inhibitor group was significantly lower than that of Osimertinib acquired drug resistant lung cancer cell line H1975OR in the control group treated with miR29b-3p-antagomir-NC(P<0.001).14.Expression of PTEN protein in xenograft tumor tissue of nude mice in the MiR-29b-3p-antagomir inhibitor treatment group was significantly higher than that in the miR-29b-3p-antagomir NC control group in the Gefitinib acquired drug-resistant lung cancer cell line PC9GR(P<0.001)。15.The expression level of p-Akt protein in the MiR-29b-3p-antagomir-inhibitor treatment group was significantly lower than that of miR-29b-3p-antagomir-NC control group in Gefitinib acquired drug-resistant lung cancer cell line PC9GR in nude mice(P<0.001).16.The Expression level of PTEN protein of MiR-29b-3p-antagomir inhibitor treatment group was significantly higher than that of miR-29b-3p-antagomir-NC control group in xenograft tumor tissues of Osimertinib acquired resistant lung cancer cell line H1975OR in nude mice(P<0.001).17.The expression level of p-Akt protein in xenograft tissue of acquired drugresistant lung adenocarcinoma cell line H1975OR in miR-29b-3p-antagomir inhibitor treatment group was significantly lower than that in miR-29b-3p-antagomir-NC control group in nude mice.(P<0.001).18.In vitro proliferative ability and IC50 of Gefitinib sensitive lung cancer cell line PC9-miR-29b-3p-mimic with supplement of miR-29b-3p-mimic were significantly higher than those of Gefitinib sensitive lung cancer cell line PC9-miR29b-3p-mimic-NC with supplement of miR-29b-3p-mimic-NC(P<0.001),and the In vitro proliferative ability and IC50 of Osimertinib sensitive lung cancer cell line H1975-miR-29b-3p-mimic with supplement of miR-29b-3p-mimic were significantly higher than those of Osimertinib sensitive lung cancer cell line H1975-miR-29b-3pmimic-NC with supplement of miR-29b-3p-mimic-NC(P<0.001).19.The flow cytometry results showed that the apoptosis level of Gefitinib sensitive lung cancer cell line PC9-miR-29b-3p-mimic with supplement of miR-29b3p-mimic were significantly lower than that of Gefitinib sensitive lung cancer cell line PC9-miR-29b-3p-mimic-NC with supplement of miR-29b-3p-mimic-NC(P<0.001),and the apoptosis level of Osimertinib sensitive lung cancer cell line H1975-miR-29b3p-mimic with supplement of miR-29b-3p-mimic were significantly lower than that of Osimertinib sensitive lung cancer cell line H1975-miR-29b-3p-mimic-NC with supplement of miR-29b-3p-mimic-NC(P<0.001).20.The apoptosis level of Gefitinib resistant lung cancer cell line PC9GR-miR29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly higher than that of Gefitinib resistant lung cancer cell line PC9GR-miR-29b-3p-inhibitor-NC with supplement of miR-29b-3p-inhibitor-NC(P=0.0424),The apoptosis level of Osimertinib resistant lung cancer cell line H1975OR-miR-29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly high than that of Osimertinib resistant lung cancer cell line H1975OR-miR-29b-3p-inhibitor-NC with supplement of miR-29b-3p-inhibitor-NC(P=0.0161).21.The results of plate clone formation experiment showed that the In vitro clone formation ability of Gefitinib sensitive lung cancer cell line PC9GR-miR-29b-3pmicmic with supplement of miR-29b-3p-micmic was significantly higher than that of Gefitinib sensitive lung cancer cell line PC9-miR-29b-3p-mimic-NC with supplement of miR-29b-3p-mimic-NC(P<0.001),and the In vitro clone formation ability of Osimertinib sensitive lung cancer cell line H1975-miR-29b-3p-mimic with supplement of miR-29b-3p-mimic were significantly lower than that of Osimertinib sensitive lung cancer cell line H1975-miR-29b-3p-mimic-NC with supplement of miR-29b-3p-mimic-NC(P<0.001).22.The results of plate clone formation experiment showed that the in vitro clone formation ability of Gefitinib sensitive lung cancer cell line PC9-siPTEN with supplement of siPTEN was significantly higher than that of Gefitinib sensitive lung cancer cell line PC9-siPTEN-NC with supplement of siPTEN-NC(P<0.000),and the In vitro clone formation ability of Osimertinib sensitive lung cancer cell line H1975-siPTEN with supplement of miR-siPTEN was significantly lower than that of Osimertinib sensitive lung cancer cell line H1975-siPTEN-NC with supplement of siPTEN-NC(P<0.000).23.The PTEN protein expression level of Gefitinib resistant lung cancer cell line PC9GR-miR-29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly higher than that of Gefitinib resistant lung cancer cell line PC9GR-miR29b-3p-inhibitor-NC with supplement of miR-29b-3p-inhibitor-NC(P=0.0053).24.The p-AKT protein expression level of Gefitinib resistant lung cancer cell line PC9GR-miR-29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly lower than that of Gefitinib resistant lung cancer cell line PC9GR-miR29b-3p-inhibitor-NC with supplement of miR-29b-3p-inhibitor-NC(P=0.0011).25.The PTEN protein expression level Osimertinib resistant lung cancer cell line H1975OR-miR-29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly higher than that of Osimertinib resistant lung cancer cell line H1975ORmiR-29b-3p-inhibitor-NC with supplement of miR-29b-3p-inhibitor-NC(P=0.0098).26.The p-AKT protein expression level of Osimertinib resistant lung cancer cell line H1975OR-miR-29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly lower than that of Osimertinib resistant lung cancer cell line H1975ORmiR-29b-3p-inhibitor-NC with supplement of miR-29b-3p-inhibitor-NC(P=0.0015).27.The PTEN protein expression level Gefitinib sensitive lung cancer cell line PC9-miR-29b-3p-micmic with supplement of miR-29b-3p-inhibitor was significantly lower than that of Gefitinib sensitive lung cancer cell line PC9-miR-29b-3p-micmicNC with supplement of miR-29b-3p-micmic-NC(P<0.001).28.The p-AKT protein expression level of Gefitinib sensitive lung cancer cell line PC9-miR-29b-3p mimic with supplement of miR-29b-3p-micmic was significantly higher than that of Gefitinib sensitive lung cancer cell line PC9-miR29b-3p mimic-NC with supplement of miR-29b-3p mimic-NC(P<0.001).29.The PTEN protein expression level of Osimertinib sensitive lung cancer cell line H1975-miR-29b-3p-micmic with supplement of miR-29b-3p-micmic was significantly lower than that of Osimertinib sensitive lung cancer cell line H1975miR-29b-3p-micmic-NC with supplement of miR-29b-3p mimic-NC(P=0.0036).30.The p-AKT protein expression level of Osimertinib sensitive lung cancer cell line H1975-miR-29b-3p-micmic with supplement of miR-29b-3p-micmic was significantly higher than that of Osimertinib sensitive lung cancer cell line H1975miR-29b-3p-micmic-NC with supplement of miR-29b-3p mimic-NC(P<0.001).31.The PTEN protein expression level of Gefitinib sensitive lung cancer cell line PC9-siPTEN with supplement of siPTEN was significantly lower than that of Gefitinib sensitive lung cancer cell line PC9-siPTEN-NC with supplement of siPTENNC(P<0.001).32.The p-AKT protein expression level of Gefitinib sensitive lung cancer cell line PC9-siPTEN with supplement of siPTEN was significantly lower than that of Gefitinib sensitive lung cancer cell line PC9-siPTEN-NC with supplement of siPTENNC(P<0.001).33.The PTEN protein expression level of Osimertinib sensitive lung cancer cell line H1975-siPTEN with supplement of siPTEN was significantly lower than that of Osimertinib sensitive lung cancer cell line H1975-siPTEN-NC with supplement of siPTEN-NC(P=0.0087).34.The p-AKT protein expression level of Osimertinib sensitive lung cancer cell line H1975-siPTEN with supplement of siPTEN was significantly higher than that of Osimertinib sensitive lung cancer cell line H1975-siPTEN-NC with supplement of siPTEN-NC(P<0.001).35.The vitro migration ability of Gefitinib sensitive lung cancer cell line PC9miR-29b-3p-micmic with supplement of miR-29b-3p-micmic was significantly higher than that of Gefitinib sensitive lung cancer cell line PC9-miR-29b-3p-micmicNC with supplement of miR-29b-3p-micmic-NC(P=0.000),and The vitro migration ability of Gefitinib resistant lung cancer cell line PC9GR-miR-29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly lower than that of Gefitinib resistant lung cancer cell line PC9GR-miR-29b-3p-inhibitor-NC with supplement of miR-29b-3p-inhibitor-NC(P=0.000).36.The vitro migration ability of Osimertinib sensitive lung cancer cell line H1975-miR-29b-3p-micmic with supplement of miR-29b-3p-micmic was significantly higher than that of Osimertinib sensitive lung cancer cell line H1975miR-29b-3p-micmic-NC with supplement of miR-29b-3p-micmic-NC(P=0.000),and The vitro migration ability of Osimertinib resistant lung cancer cell line H1975OR-miR-29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly lower than that of Osimertinib resistant lung cancer cell line H1975ORmiR-29b-3p-inhibitor-NC with supplement of miR-29b-3p-inhibitor-NC(P=0.000)..37.The results of EDU staining and proliferation experiments showed that the in vitro proliferation ability of Gefitinib resistant lung cancer cell line PC9GR-miR29b-3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly lower than that of Gefitinib resistant lung cancer cell line PC9GR-miR-29b-3p-inhibitorNC with supplement of miR-29b-3p-inhibitor-NC(P<0.001),and the in vitro proliferation ability of Osimertinib resistant lung cancer cell line H1975OR-miR-29b3p-inhibitor with supplement of miR-29b-3p-inhibitor was significantly lower than that of Osimertinib resistant lung cancer cell line H1975OR-miR-29b-3p-inhibitorNC with supplement of miR-29b-3p-inhibitor-NC(P<0.001)。Conclusions:(1)Overexpression of miR-29b-3p was existed in the Gefitinib resistant lung cancer cell line PC9GR and Osimertinib resistant lung cancer cell line H1975OR,and the in vitro proliferation,migration and anti-apoptostic abilities were increased both in the Gefitinib resistant lung cancer cell line PC9GR and Osimertinib resistant lung cancer cell line H1975OR;(2)Geiftinib acquired resistant lung cancer cell line PC9GR had sensitive mutation with deletion of 19 exons,and there is no secondary T790M resistant mutation,and the sensitive mutations of L858R and T790M were found in Osimertinib acquired resistant lung cancer cell line H1975OR,while acquired resistance mutations such as C797S were not found;(3)Overexpression of miR-29b-3p significantly reduced the sensitivity of EGFR-TKI sensitive lung cancer cell lines PC9 and H1975 to EGFR-TKI,and in vitro proliferation,migration and anti-apoptotic ability were significantly enhanced;(4)After in vivo inhibition of miR-29b-3p expression in transplanted lung cancer tumors,the expression level of PTEN gene is significantly up-regulated and the expression level of p-Akt pathway is significantly down-regulated;miR-29b-3p-Antagomir inhibitor can remarkably inhibit the tumorigenicity of Gefitinib acquired resistant lung cancer cell line PC9GR and Osimertinib acquired resistant lung cancer cell line H1975OR in nude mice;(5)PTEN is a downstream regulatory target gene of miR29b-3p,By binding with the 3’UTR end of PTEN gene,miR-29b-3p negatively regulates the expression of PTEN gene and positively regulates the expression of pAkt,which gives acquired drug resistance to EGFR-TKI in lung cancer and MiR-29b3p might be a potential molecular target to inhibit and/or reverse acquired resistance to EGFR-TKI in lung cancer.