The Mechanism Of Berberine And Its Combination With Gefitinib In Inhibiting Proliferation And EMT Of NSCLC Cells

Objective:The main purpose of this study was to explore the sensitizing effect of berberine on EGFR-TKI in the treatment of NSCLC and related molecular mechanisms.In order to provide a more theoretical basis for the potential anti-cancer effect of berberine,and expected to further improve the effect of targeted therapy,and provide new methods and experimental evidence for the clinical use of EGFR-TKI.Methods:The first part of the in vitro experiments:1.MTT assay and BrdU incorporation assay were used to detect the effects of di fferent doses of berberine on the proliferation of A549 and H1975 cells at different times.Flow cytometry was used to detect the changes of cell cycle after different doses of BBR treatment to confirm the inhibition effect of berberine on cell proliferation.2.The effect of berberine combined with gefitinib on the proliferation of A549 and H1975 cells was detected by MTT assay,and the combination index of the two drugs was calculated using CompuSyn software.The effect of the combination was analyzed and the optimal combination dose was confirmed.At the same time,the effect of berberine combined with gefitinib on the proliferation of the two cells was detected by EdU staining.3.Western blot was used to detect the effects of berberine and its combined with gefitinib on the expression of PDPK1,Spl and DNMT1 in A549 and H1975 cells.Real-time PCR was used to detect the effects of berberine on the expression of DNMT1 mRNA.A dual luciferase reporter system was used to examine the effect of berberine on the activity of DNMT1 promoter.4.In order to detect the effect of berberine on the relationship between PDPK1,Spl and DNMT1,the overexpression plasmids of three of them were transiently transfected in vitro:pCMV6-PDPK1,pcDNA3.1-Spl,pCMV6-DNMT1,and then BBR treatment.The effect of overexpression of the corresponding protein on the expression of the other two proteins in each treatment group was detected by Western blot,and the interaction between the three proteins was determined.5.After overexpressing DNMT1 in A549 and H1975 cells,the effect of BBR on cell proliferation was examined by MTT assay to determine the role of DNMT1 in BBR-mediated inhibition of NSCLC cell proliferation.The seconed part of the in vitro experiments:1.Transwell assay and Wound-healing assay were used to detect the effects of berberine and its combination with gefitinib on the invasion and metastasis of A549 and H1975 cells.2.Western blot was used to detect the effects of berberine and its combined with gefitinib on the expression of EMT-related markers:E-cadherin,Vimentin and Snail in A549 and H1975 cells..3.The effect of berberine and its combination with gefitinib on the expression of LncRNA HOTAIR and miR-34a-5p in A549 and H1975 cells was detected by Real-Time PCR.4.HOTAIR siRNA was used to silence the expression of LncRNA HOTAIR in A549 and H1975 cells;In addition,A549 and H1975 cells were transiently transfected with pcDNA3.1-HOTAIR overexpression plasmid,BBR treatment;Then,The expression of E-cadherin and Snail protein were detected by Western blot,and Real-Time PCR was used to detect the expression of miR-34a-5p in cells.To clarify the effect of LncRNA HOTAIR on EMT-related genes and miR-34a-5p expression.5.Transfection of mimic,overexpression of miR-34a-5p in A549 and H1975 cells;Real-Time PCR detect the expression of LncRNA HOTAIR,Western blot analysis of Snail protein expressionin cells.In addition,after transfected HOTAIR-Wt and Mut or Snail 3' UTR-Wt and Mut plasmids,which containing the dual luciferase reporter gene,detected the effect of overexpression of miR-34a-5p on the luciferase activity of each cell.To confirm whether there is a targeted regulation relationship between miR-34a-5p and LncRNA HOTAIR or Snail.6.A549 and H1975 cells were transfected with pEZ-M02-Snail overexpression plasmid.After BBR treatment,the expression of E-cadherin protein in lung cancer cells was detected by Western blot,The activity of E-cadherin promoter was detected by dual luciferase reporter gene system.To clarify the regulation of E-cadherin expression by the Snail.In vivo experiments:1.A nude mouse model of a surface tumor-bearing was established,and treated with berberine in combination with gefitinib.The general condition of the mice was observed.The changes of fluorescence intensity of the tumors in mice before and after administration were measured by bioluminescence imaging.The tumor volume and weight of the mice were measured.To examine the effect of each drug treatment on the tumor growth of mice.2.A lung metastasis model was established by injecting tumor cells into the tail vein of nude mice,and treated with berberine in combination with gefitinib.The general condition of the mice was observed.The growth of tumors in lung was detected by bioluminescence imaging.After the mice were sacrificed,the lungs were perfused,and observe the metastases of tumors in lungs,then the lungs were removed and fixed with paraformaldehyde.After paraffin embedding,HE staining was used to observe the pathological changes of the lung in each group.3.Western blot and Real-Time PCR were used to detect the expression of cell proliferation-related genes and EMT-related genes in tumor tissues of each group.Results:The first part of the in vitro experiments:1.Berberine inhibited the proliferation of A549 and H1975 cells in a time-and dose-dependent manner;and it combination with gefitinib has synergy effect on the inhibition of the growth of lung cancer cells.The combination group on the inhibited of proliferation of A549 and H1975 cells was significantly better than that of berberine and gefitinib treated alone.2.Berberine inhibited the expression of PDPK1,Spl,and DNMT1 proteins in A549 and H1975 cells;and the combined effect of gefitinib on the expression of these proteins was significantly better than that of the two drugs alone.In addition,berberine also inhibited the activity of DNMT1 promoter and mRNA expression in A549 and H1975 cells.3.In A549 and H1975 cells,overexpression of Spl or PDPK1 reversed the inhibition effect of berberine on DNMT1 and each other protein expression;In addition,overexpression of PDPK1 also blocked the inhibition of DNMT1 promoter activity by berberine.4.Overexpression of DNMT1 in A549 and H1975 cells reversed the inhibition effect of berberine on proliferation of A549 and H1975 cells,but had no significant effect on the expression of Spl and PDPK1 proteins.The seconed part of the in vitro experiments:1.Berberine and its combination with gefitinib inhibited the invasion and metastasis of A549 and H1975 cells;and the combination group had the best inhibition effect.2.Berberine up-regulated E-cadherin protein expression,down-regulated Vimentin and Snail protein expression in A549 and H1975 cells;Combined with gefitinib,the up-regulation of E-cadherin and the inhibition of Snail in the combination group significantly better than the two drugs treated alone group.3.In A549 and H1975 cells,berberine combined with gefitinib inhibited the expression of LncRNA HOTAIR and induced the expression of miR-34a-5p;What' s more,the inhibition effect of LncRNA HOTAIR and the induction of miR-34a-5p in the combination group were significantly better than those in the two drugs treated alone group.4.Silence HOTAIR can up-regulate the expression of E-cadherin,down-regulate the expression of Snail and miR-34a-5p in A549 and H1975 cells.And overexpression of HOTAIR in A549 and H1975 cells can reverse the induction of E-cadherin and inhibition of Snail,miR-34a-5p by berberine.5.Overexpression of miR-34a-5p inhibited the luciferase activity of cells transfected with HOTAIR-Wt plasmids in A549 and H1975 cells.What' s more,overexpression of miR-34a-5p down-regulated the luciferase activity of the transfected Snail 3' UTR-Wt plasmid and inhibited the expression of Snail in A549 and H1975 cells.6.Overexpression of Snail reversed the inhibition effect of berberine on E-cadherin promoter activity and protein expression in A549 and H1975 cells.In vivo experiments:1.Berberine combined with gefitinib significantly inhibited the growth of tumors in tumor-bearing mice;At the end point of drug administration,the weight of tumors and the tumor fluorescence intensity of the combination group were the minimum values of each group,and there was a statistically significant difference between the group administered with gefitinib alone.2.Berberine combined with gefitinib significantly inhibited the metastasis tumor growth in the lung.The number of metastasis tumors in lung of drug treated group was significantly lower than that in the control group,and the combined group was the least.3.Compared with the control group,the expression of PDPK1,Spl and DNMT1 protein in the tumor tissue of each drug treatment group was significantly less,and the combination group was the most obvious.In addition,compared with the control group,the expression of E-cadherin and miR-34a-5p was increased,the expression of LncRNA HOTAIR and Snail was decreased in the tumor tissue of each drug treatment group,and the change was most pronounced in the combination group.Conclusions:1.Berberine combined with gefitinib can synergistically inhibit the proliferation of NSCLC cells via the PDPK1/Spl/DNMT1 pathway.2.Berberine combined with gefitinib can synergistically inhibit the EMT process of NSCLC cells through the LncRNA HOTAIR/miR-34a-5p/Snail pathway.3.In vivo experiments,berberine combined with gefitinib significantly inhibited the growth of tumor-bearing tumors and the metastasis in lung in nude mice;And the expression of genes in vitro was also obtained in mouse tumor tissues.Corresponding results are verified.