MiR-27a/miR-335 Enhances Gefitinib Sensitivity In Human Non-small Cell Lung Cancer Through Targeting HOXC6

miR-27a/miR-335 enhances gefitinib sensitivity in human non-small cell lung cancer through targeting HOXC6BackgroundLung cancer is the most general type of cancer worldwide with the highest incidence and mortality.According to statistics,the estimated new incidence and the number of deaths of lung cancer in China in 2015 were respectively 733,300 and 610,200 cases,ranking first in the national cancer.The major type of lung cancer is non-small cell lung cancer(NSCLC),which is a most devastating cancer with rapid progression,easy to relapse and poor prognosis.At present,the first-line chemotherapy regimens for NSCLC include platinum-based combination therapy and small molecule targeting therapy such as EGFR tyrosine kinase inhibitors(EGFR-TKIs).EGFR-TKIs,represented by gefitinib(Gef),have been widely used clinically and exhibited favorable effects especially on non-smokers and Asian patients,bringing a ray of hope for patients with advanced NSCLC.However,the emergence of acquired resistance after EGFR-TKIs therapy leads to treatment failure,greatly hinders its application.Acquired resistance is a key factor correlated with poor prognosis in NSCLC,to explore its mechanisms in order to solve this problem and improve the survival of patients is extremely urgent.Homeobox genes(HOX)are a class of highly conserved genes in evolution,which encode transcription factors that control cell growth and differentiation in embryonic development.In recent years,it was found that HOX genes can be "oncogene" or "tumor suppressor gene" through the regulation of tumor stem cell self-renewal and participation in the biological processes of tumor cell proliferation,apoptosis,cell cycle and invasion and migration.In addition,some HOX genes are confirmed to get involved in tumor resistance.HOXC6,as an important member of the HOX family,has been reported to be expressed abnormally in a variety of cancers and can serve as a favorable biomarker for the diagnosis and prognosis of malignant tumors.However,the role of HOXC6 in the development and progression of NSCLC,whether HOXC6 is involved in EGFR-TKIs resistance in NSCLC,have rarely been reported so far.MicroRNAs are a class of endogenous noncoding small RNAs that can control multiple biological processes(such as cell growth,proliferation,differentiation,apoptosis by their ability to regulate their target mRNAs,therefore affecting tumor development and regulating tumor resistance.Recently,many miRNAs have been reported to play crucial roles in the occurrence,recurrence and metastasis of lung cancer as well as modulation of the response to anti-tumor therapy(especially EGFR-TKIs).MiR-27a and miR-335 are expressed abnormally in a variety of cancers,such as gastric cancer,liver cancer and breast cancer.However,there have been no reports about the expression and significance of miR-27a and miR-335 in NSCLC.In addition,it was found that the expression of HOX genes are subjected to target regulation of a variety of miRNAs,and then affect tumor development and drug resistance.This study aim to investigate the role of HOXC6 in gefitinib sensitivity in NSCLC and elucidate its mechanism,further to explore whether HOXC6 is target of miR-27a/miR-335 and whether miR-27a/miR-335 can affect Gef sensitivity in NSCLC.We hope this study can provide new molecular targets for NSCLC treatment and new reference for clinically reversal of gefitinib resistance.The study is divided into two parts:Part 1 The effect of HOXC6 on Gef sensitivity in NSCLC and its mechanismObjective:To inquiry the expression of HOXC6 in NSCLC and explore the role of HOXC6 gene in gefitinib resistance and elucidate its mechanism.Methods:1)The expression of HOXC6 in tissue samples of 93 NSCLC patients was detected by immunohistochemistry(IHC),and the relationship of HOXC6 expression with clinical characteristics as well as the survival of NSCLC patients was analyzed.2)The mRNA and protein expression of HOXC6 in NSCLC Gef-sensitive cells(PC9,HCC827)and Gef-resistant cells(PC9/G,H1299,A549,H1975)were detected by qRT-PCR and Western blot,respectively.3)PC9/G cells were transfected with HOXC6 siRNA,then the expression of HOXC6 protein is detected by Western blot.The effect of Gef on the viability of PC9/G cells was detected by CCK assay,the effect of Gef on PC9/G cell proliferation was detected by clone formation assay,the effect of Gef on cell apoptosis and cell cycle were detected by flow cytometry(FCM),the effect of Gef on cell migration and invasion were detected by Transwell assay.4)The expression of apoptosis,cell cycle,migration and invasion-related proteins and ABC transporters after HOXC6 silencing were detected by Western blot in PC9/G cells.Results:1)The positive expression rate of HOXC6 in NSCLC and their adjacent tissues were respectively 98.92%and 77.01%,with statistically significant difference(?2=20.945.P<0.001).The IHC score of HOXC6 staining in NSCLC tissues was also higher than that of their adjacent tissues(6.74 ± 3.11 vs 2.92 ± 1.63,P<0.001).High expression of HOXC6 was positively correlated with age(P=0.015),lymph node invasion(P=0.005)and TNM clinical stage(P=0.007).Kaplan-Meier analysis demonstrated that high expression of HOXC6 predicted poor survival(P=0.043).2)The mRNA and protein expression of HOXC6 were significantly increased in Gef-resistant NSCLC cells(PC9/G,H1299,A549,H1975)compared with Gef-sensitive NSCLC cells(PC9,HCC827)(P<0.05;P<0.05).3)After PC9/G cells transfected with HOXC6 siRNA,the protein expression of HOXC6 decreased in a dose-dependent manner;the cell viability and cell colony formation of PC9/G cells inhibited by Gef was significantly decreased(P<0.01,P<0.001);Gef-induced cell apoptosis was significantly higher(P<0.001);Gef-induced G2/M phase cell cycle arrest was increased(P<0.001);the inhibition of Gef on PC9/G cells migration and invasion were enhanced(P<0.001,P<0.001).4)After HOXC6 silencing,the protein expression of survivin and Bcl-2 was decreased,while the expression of Bim was increased(P<0.05);the expression of Cyclin B1 was increased,and the expression of Cyclin D1 was decreased(P<0.05);the expression of Akt was decreased,the expression of GSK3? was increased,the expression of ?-catenin was decreased(P<0.05),the expression level of Vimentin was unchanged;the protein expression levels of ABC family ABCG2,MDR1 and MRP1 were all significantly decreased(P<0.05),but the expression of ABCB5 protein was significantly increased(P<0.001).Conclusions:1)HOXC6 was significantly upregulated in NSCLC tissues,HOXC6 may be involved in NSCLC development and influence survival of patients with NSCLC.2)The expression of HOXC6 was significantly upregulated in Gef-resistant NSCLC cells,the mechanisms of HOXC6 silencing reversing Gef resistance in NSCLC cells are probably as follows:Increasing Gef-induced cell apoptosis by upregulation of Bim and down-regulation of Survivin and Bcl-2;inducing G2/M phase cell cycle arrest by regulation of cyclin B1 and cyclin D1;inhibiting cell migration and invasion through the regulation of Akt/GSK3?/?-catenin signaling pathway;affecting the accumulation and release of Gef by regulating ABC transporter family proteins.This study clarified the role and significance of HOXC6 in NSCLC development as well as in Gef resistance,which will help to enhance our understanding of the progression of NSCLC and the mechanism of Gef resistance in NSCLC.Part 2 miR-27a/miR-335 enhances gefitinib sensitivity in NSCLC by targeting HOXC6Objective:To investigate the targeting relationship between miR-27a/miR-335 and HOXC6 and inquiry the role of miR-27a/miR-335 in gefitinib sensitivity in NSCLC.Methods:1)TargetScan and other bioinformatics tools were used to predict the miRNAs that can target HOXC6;PC9/G cells were transfected with different miRNA mimics and then HOXC6 protein expression was detected by Western blot to screen target miRNAs;whether HOXC6 is a direct target of miR-27a was detected by dual luciferase reporter assay.2)PC9/G cells were transfected with miR-27a/miR-335 mimics or cotransfected with plasmid pcDNA3.1-HOXC6,the effect of Gef on the viability and cell proliferation of PC9/G cells was detected by CCK assay and colony formation assay,respectively,the effect of Gef on cell apoptosis and cell cycle were detected by flow cytometry(FCM),the effect of Gef on cell migration and invasion were detected by Transwell assay.3)5×106 PC9/G cells were transplanted into BALB/c nude mice for xenograft model,then the tumor-bearing mice were randomly divided into six groups(n=5):the miR-NC group,miR-27a group,miR-335 group,miR-NC+Gef group,miR-27a+Gef group,miR=335+Gef group.The effect of combined miR-27a/miR-335 Agomir with Gef treatment on tumor growth was measured after treatment of multi-point intratumoral injection of miR-NC/miR-27a/miR-335 Agomir(10 nmol,two times a week)and/or intragastric administration of Gef(15 mg/kg,five times a week)for 3 weeks.Then the mice were sacrificed and the tumor tissue were fixed and paraffin embeded.H&E staining,Ki-67 staining and HOXC6 staining of tumor tissues were assessed by IHC.4)The expression of miR-27a/miR-335 in tissue samples of 93/91 NSCLC patients was detected by in situ hybridization(ISH),then the relationship of miR-27a/miR-335 expression and clinical characteristics as well as the survival of NSCLC patients was analyzed.Results:1)MiR-27a and miR-335 can down-regulate the expression of HOXC6 in a dose-dependent manner;miR-27a mimics transfection significantly inhibited the luciferase activity of HOXC6 3'-UTR wt rather than HOXC6 3'-UTR mut.MiR-27a can bind directly to the 3'-UTR region of HOXC6,and miR-335 cannot bind directly to the 3'-UTR region of HOXC6.2)After PC9/G cells transfected with miR-27a/miR-335 mimics,the cell viability and cell colony formation of PC9/G cells inhibited by Gef was significantly increased(P<0.05,P<0.01;P<0.01,P<0.001),through cotransfection of miR-27a/miR-335 mimics with pcDNA3.1-HOXC6 in PC9/G cells,the cell viability of PC9/G cells inhibited by Gef was significantly decreased(P<0.05,P<0.01).Besides,after PC9/G cells transfected with miR-27a/miR-335 mimics,Gef-induced cell apoptosis was significantly higher(P<0.05,P<0.01);Gef-induced G2/M phase cell cycle arrest was increased(P<0.01,P<0.001);the inhibition of Gef on PC9/G cells migration and invasion were enhanced(P<0.001,P<0.001;P<0.01,P<0.001).3)MiR-27a or miR-335 Agomir could significantly enhanced the inhibition of Gef on tumor growth(P<0.001,P<0.001).HOXC6 expression in the miR-27a/miR-335 agomir-treated group or the combination groups was markedly decreased.H&E and Ki67 staining revealed that miR-27a/miR-335 agomir enhanced Gef-induced inhibition of cell proliferation.4)The positive expression rate of miR-27a in NSCLC tissues and their adjacent tissues were respectively 6.45%and 52.87%,with statistically siginificant difference(?2=47.153,P<0.001);the positive expression rate of miR-335 in NSCLC tissues and their adjacent tissues were respectively 53.85%and 89.53%,with statistically siginificant difference(x2=27.456,P<0.001).Besides,the IHC score of miR-27a staining in NSCLC tissues was also lower than that of their adjacent tissues(0.68± 0.56 vs 2.02 ± 1.61,P<0.001);the IHC score of miR-335 staining in NSCLC tissues was also lower than that of their adjacent tissues(1.68 ± 1.05 vs 2.86 ± 1.47,P<0.001).The low expression of miR-27a was positively correlated with gender(P=0.047).lymph node invasion(P=0.042),lymph node-positive(P=0.007),and TNM clinical stage(P=0.043);the low expression of miR-335 was positively correlated with TNM clinical stage(P=0.028).Kaplan-Meier analysis demonstrated that low miR-27a/miR-335 expression predicted poor survival(P=0.009;P=0.000).Conclusions:1)miR-27a binds directly to the binding site on the 3'-UTR of the HOXC6 promoter region while miR-335 indirectly regulate the expression of HOXC6,miR-27a and miR-335 could down-regulate the protein expression of HOXC6;2)MiR-27a or miR-335 overexpression could increased the sensitivity of PC/G cells to Gef through enhancing Gef-induced cell apoptosis,cell cycle arrest,inhibition of cell migration and invasion.3)MiR-27a or miR-335 agomir could significantly enhance Gef-induced inhibition of tumor growth by targeting HOXC6 in PC9/G xenograft model.4)MiR-27a/miR-335 was significantly downregulated in NSCLC tissues,miR-27a and miR-335 may be involved in NSCLC development and progression as well as the survival of patients.This study elucidated the target regulatory effects of miR-27a/miR-335 on HOXC6 and its influence on Gef resistance,which will provide new prospects for adjuvant targeted therapy in patients with NSCLC.