Regulatory Mechanism Of Baicalein On Proliferation And Cisplatin Sensitivity Of Non-small Cell Lung Cancer Through MiR-424-3p/PTEN/PI3K/AKT Pathway

Background and objectiveLung cancer is a malignant tumor with the highest morbidity and mortality in China,which not only seriously threatens people’s life and health,but also brings great economic and medical burden to the society.The overall 5-year survival rate of lung cancer is still less than 20%although early diagnosis and advanced gene targeted therapy have improved the survival of lung cancer to some extent.According to the pathological type of the disease,lung cancer can be divided into small cell lung cancer(SCLC)and non-small cell lung cancer(NSCLC),of which NSCLC accounts for about 85%of the total lung cancer,which is the main type of lung cancer.Most patients with lung cancer are diagnosed in the late stage of the disease,and platinum-combined chemotherapy is still the most commonly used treatment for advanced lung cancer.However,its application is limited by adverse drug reactions and different individual responses.Therefore,the discovery and application of drugs with anti-tumor effect or chemosensitization effect is of great significance for the treatment of lung cancer.Micro-RNA(miRNA)is a kind of endogenous short RNA with regulatory function,which is about 22 nucleotides in length.miRNA can participate in the translation inhibition or cleavage of its target mRNA through binding,and play an important regulatory role in animals and plants.miRNA plays an important role in the regulation of proliferation,invasion and apoptosis of a variety of solid malignant tumor cells,and may be involved in the regulation of tumor cells’ sensitivity to cisplatin.miRNA has a complex regulatory network in vivo,the application of high-throughput sequencing technology makes it easy to screen differentially expressed miRNA.Multiple miRNA databases such as Targetscan,miRBase,microRNAorg,PITA,miRDB and so on can predict and analyze the target genes of miRNA,and complete the predietion of miRNA pathway.In recent years,traditional Chinese medicine(TCM)has received more and more attention because of its relatively low toxicity.Baicalein is a kind of flavonoids extracted and purified from the dried roots of Scutellaria baicalensis Georgi.Previous studies have shown that baicalein has anti-tumor effects in a variety of cells including NSCLC.In addition,baicalein can affect the cisplatin sensitivity of many kinds of cancer cells in different ways,including MAPK pathway,NF-κB pathway and so on.PTEN(phosphatase and tensin homolog deleted on chromosom eten),located at 10q23.3,is a common tumor suppressor gene.Previous studies have reported that PTEN can regulate the resistance to 5-fluorouracil in gastric cancer cells under baicalein treatment.However,whether and how PTEN is involved in the regulatory mechanism of baicalein on non-small cell lung cancer has not been studied.Previous studies of our group have found a variety of non-coding RNA which may participate in the regulatory mechanism of lung cancer,and a variety of traditional Chinese medicine monomers,which may achieve anti-tumor effect through miRNA regulation pathway.Combined with previous research and literature reports,baicalein may be involved in the regulation of biological behavior of lung cancer cells through the miRNA/PTEN pathway,the research in this area has not been reported yet.This study was to investigate the effects and mechanisms of baicalein on proliferation,apoptosis,invasion and cisplatin sensitivity of non-small cell lung cancer cell lines.The study can be mainly divided into two parts:in the first part,the effects of baicalein on the proliferation,apoptosis,invasion and cisplatin sensitivity of NSCLC cell lines were evaluated;in the second part,the miRNA gene microarray analysis was carried out on A549 cells treated with baicalein to screen out differentially expressed miRNA and further downstream pathway was explored.Part One:Effects of baicalein on proliferation,apoptosis,invasion and cisplatin sensitivity of non-small cell lung cancer cell linesMethods1.Lactate dehydrogenase(LDH)assay and cell counting kit-8(CCK-8)assay were performed to detect the optical density(OD)values of normal bronchial epithelial cells(NHBE),pulmonary epithelial cells BEAS-2B and four kinds of NSCLC cells treated with different concentrations of baicalein for 24 hours,OD value of cells represented the cytotoxicity.2.The CCK-8 experiment was used to detect the OD value of 0,40 μM baicalein-treated NSCLC cells for 0,24,48 and 72 h,which represented the cell proliferation ability.The growth curve was then drew.3.1%agarose-coated 96-well plate was used to simulate 3D cell culture environment,CCK-8 assay was performed to detect the OD value of 0,40 μM baicalein-treated NSCLC cells for 48 hours,and cell relative activity was calculated.4.A 10-day colony formation assay was performed to detect the effect of 0,40 μM baicalein on the number of colonies in NSCLC cells.5.AnnexinV-FITC/PI double-labeled flow cytometry and caspase3/7 activity assay was carried out to detect the effect of 0,40,80 μM baicalein on the apoptosis of NSCLC cells for 48 hours.6.The effects of 40,80 μM baicalein on the migration and invasion of NSCLC cells were detected using 8.0 μm Transwell chamber and Matrigel-coated Transwell chamber,respectively.7.NSCLC cells and A549/DDP cells treated with 0,20,40μM baicalein were treated with different concentrations of cisplatin(0,2,4,8,16,32 μM).After 24 hours,the OD value of cells in each group was detected by CCK-8 assay,and the proliferation inhibition rate and IC50 value of cisplatin in each group were calculated.8.A549 cells with GFP fluorescent label were inoculated with 0.2mL into the armpit of nude mice aL the density of 1×107/mL.After successfully establishing the xenograft nude mice model,the nude mice were treated with intraperitoneal injection of normal saline(0.2ml/day for 3 weeks),baicalein(3mg/kg/day for 3 weeks),cisplatin(3mg/kg,twice a week for 3 weeks),or baicalein combined with cisplatin.The fluorescence signal of transplanted tumor in nude mice was observed by a small animal live imager every week,and the body weight of nude mice was measured every week.Immunohistochemistry was used to detect the positive rate of Ki-67 in transplanted tumor tissues of each group.9.IBM SPSS Statistics 21.0 software was used for statistical analysis of the data,GraphPad Prism5 software was used for drawing figures,t-test was used to compare the measurement data of two groups,one-way ANOVA was used for comparison of data with more than three groups,LSD test or Bonferroni test was used for intra-group comparison,non-parametric test was used for data that did not conform to normal distribution or homogeneity of variance.The test value was defined as 0.05.Results1.Baicalein treatment increased the cytotoxicity in four NSCLC cell lines(NCI-H1299,NCI-H460,NCI-H358 and A549)as a dose dependent manner(P<0.05),while the cytotoxicity of BEAS-2B and NHBE cells was slightly even under high concentration of baicalein.2.Compared with the control group,40μM baicalein significantly inhibited the OD value of A549 and NCI-H460 cells after 24 hours(P<0.05),and the difference between the two groups was gradually significant with time.Results showed that the proliferation ability of NSCLC cells was inhibited by baicalein.3.In the condition of 3D cell culture,the cell activity of A549 and NCI-H460 cells treated with baicalein for 48 h was also significantly lower than that of the control group(P<0.05).4.Compared with the control group,the number of colonies of NSCLC cells in 40μM baicalein group was significantly lower than that in the control group(P<0.05).5.After 48 h treatment with baicalein,the number of apoptotic cells and caspase3/7 activity of A549 and NCI-H460 cells increased in a concentration-dependent manner(P<0.05).6.Baicalein reduced the number of transmembrane cells both in migration and invasion of A549 and NCI-H460 cells(P<0.05).The number of transmembrane cells of 80 μM baicalein was lower than that of 40 μM baicalein(P<0.05).7.the IC50 values of cisplatin in A549,NCI-H460,and A549/DDP cells were 9.15μM,5.23 μM and 31.35 μM,respectively.40μM baicalein decreased the IC50 values of cisplatin in both sensitive cells and resistant A549/DDP cells(P<0.05).8.In the xenograft nude mice model,baicalein inhibited the fluorescence intensity and Ki-67 positive rate of transplanted tumor in vivo when compared with the control group,which represented the ability of proliferation(P<0.05);baicalein with cisplatin combination group had lower fluorescence intensity and Ki-67 positive rate than cisplatin group(P<0.05).That is,the combination of baicalein and cisplatin has a synergistic effect on tumor proliferation.Part Two Regulatory mechanism of baicalein on non-small cell lung cancer cells through miR-424-3p and its downstream pathwayMethods1.A549 cells were treated with 40 μM baicalein and 0.5%DMSO respectively for 24 hours.Total RNA was extracted and analyzed by miRNA microarray to find the differentially expressed miRNA,microarray results were validated by RT-qPCR.2.Lipofectamine 2000 was used to transfect miR-424-3p inhibitor to cells,three groups(miR-inhibitor group,baicalein group,and blank group)were treated with miR-424-3p inhibitor,40 μM baicalein,and vehicle control,respectively.CCK-8 assay,AnnexinV-FITC/PI double labeling flow cytometry,caspase3/7 activity detection and Transwell assay were used to compare the effects of 40 μM baicalein treatment and down-regulation of miR-424-3p on the proliferation,apoptosis,migration and invasion of NSCLC cells.The OD values of three groups of cells treated with different concentrations of cisplatin were detected by CCK-8 assay,and the IC50 values were calculated.3.The target genes of miR-424-3p were predicted using Targetscan,microRNAorg and PIT A3 databases,the wild type and mutant dual-luciferase reporter gene recombinant vectors were constructed and co-transfected with miR-424-3p mimics or unrelated sequence(Scramble),respectively.After 24 hours,the changes of luciferase activity in each group were detected by dual reporter gene kit.4.After 48 h transfection of miR-424-3p mimics and miR-424-3p inhibitor,the protein expression level of the target gene PTEN in NSCLC cells was detected by western blot.5.The relative expression levels of miR-424-3p and PTEN mRNA in chemotherapy-sensitive and resistant human lung cancer tissues were detected by RT-qPCR,and the correlation analysis was also performed.6.Western blot was used to detect the effect of 40,80 μM baicalein on the protein expression of PTEN,survivin,Bcl-xL,PI3K,pAKT,and tAKT.7.Lipofectamine 2000 was used to transfect miR-424-3p mimics and si-PTEN,respectively.CCK-8 assay,AnnexinV-FITC/PI double labeling flow cytometry,caspase3/7 activity assay and Transwell assay were used to compare the restore effects of up-regulation of miR-424-3p and down-regulation of PTEN on baicalein proliferation inhibition,apoptosis promotion,invasion inhibition and cisplatin sensitization in NSCLC cells.8.IBM SPSS Statistics 21.0 software was used for statistical analysis of the data,GraphPad Prism 5 software was used for drawing figures,t-test was used to compare the measurement data of two groups,one-way ANOVA was used for comparison of data with more than three groups,LSD test or Bonferroni test was used for intra-group comparison,Pearson correlation analysis was used for correlation analysis,non-parametric test was used for data that did not conform to normal distribution or homogeneity of variance.The test value was defined as 0.05.Results1.Thirty-three differentially expressed miRNA were found by miRNA microarray of A549 cells treated with baicalein,of which 24 were down-regulated,with a down-regulation ratio of 2.3～5.5,and 9 were up-regulated,with an up-regulation multiple of 2.0～2.6.Among the down-regulated miRNA,the expression of miR-424-3p was the most significant difference between the baicalein group and the control group.RT-qPCR detection showed that the expression level of miR-424-3p in NSCLC cells was down-regulated after treatment with baicalein(P<0.05).2.Compared with the control group,40 μM baicalein treatment and down-regulation of miR-424-3p had similar effects in NSCLC cells,which decreased the OD value of NSCLC cells in CCK-8 assay,increased the number of apoptotic cells and caspase3/7 ratio in apoptosis test,decreased the number of transmembrane cells in Transwell assay,and decreased the IC50 value of cisplatin(P<0.05).3.Target gene prediction analysis showed that there were complementary pairing regions between PTEN mRNA3’ UTR and miR-424-3p.The dual reporter gene recombinant vectors of wild type(WT)and mutant type(MT)of PTEN were successfully constructed.The relative luciferase activity of WT+miR-424-3p group was decreased,which was significantly different from that of the other three groups(P<0.05),suggesting that miR-424-3p could decrease the activity of luciferase gene by acting on the complementary pairing region between miR-424-3p and WT PTEN.4.Compared with blank group,the relative expression level of PTEN protein was decreased in miR-424-3p group,and increased in miR-inhibitor group,indicating that miR-424-3p could negatively regulate the expression level of PTEN protein.5.In tissues of human lung cancer patients,the expression level of miR-424-3p in chemotherapy-sensitive group was lower than that in resistant group(P<0 05).;just the opposite,the relative expression level of PTEN mRNA in chemotherapy-sensitive group was higher than that in resistant group(P<0 05).There was a negative correlation between miR-424-3p and PTEN mRNA(r=-0546,P<0 05).6.40 and 80 μM baicalein both up-regulated the relative expression of PTEN protein and down-regulated the relative expression level of PI3K and p-AKT in A549 and NCI-H460 cells(P<0.05).In addition,baicalein decreased the expression levels of apoptosis inhibitory proteins survivin and Bcl-xL in A549 and NCI-H460 cells(P<0.05).In NCI-H460 cells,the expression levels of survivin and Bcl-xL increased with the increase of baicalein concentration.7.Up-regulation of miR-424-3p or down-regulation of PTEN expression in NSCLC cells restored the proliferation inhibition,apoptosis promotion and cisplatin sensitization of baicalein,which were significantly different from those in baicalein alone group(P<0.05).Conclusions1.Baicalein can inhibit the proliferation,migration and invasion of non-small cell lung cancer cells,promote their apoptosis and increase their sensitivity to cisplatin.2.This study found that baicalein can down-regulate the relative expression of miR-424-3p and the expression of apoptosis inhibitory proteins survivin.and Bcl-xL,regulate the biological behavior of non-small cell lung cancer cells through miR-424-3p/PTEN/PI3K/AKT pathway,which provides a theoretical and experimental basis for the prospect of clinical application of baicalein.