| Objective:In this study,we focus on the effect of Yifei Sanjie Pill(YFSJ)on cisplatin(DDP)resistance in non-small cell lung cancer(NSCLC)and explore the potential molecular mechanism of its inhibition of DDP resistance in NSCLC.Methods:The first part:(1)NSCLC DDP resistant cell line s were constructed by gradually increasing DDP concentration and intermittent induction in vitro to provide high-quality biological cell resources for follow-up experimental research.(2)After continuous intragastric administration of YFSJ pill for 7 days in adult SD rats,blood samples from abdominal aorta of SD rats were collected after anesthesia,and the drug-containing serum of SD rats was further extracted.(3)Drug sensitivity test was used to analyze the half inhibitory concentration(IC 50)of YFSJ pill containing serum combined with DDP in NSCLC cells and DDP resistant cells.(4)Flow cytometry was used to detect the effect of YFSJ pill containing serum combined with DDP on apoptosis of NSCLC cells and its DDP resistant cell lines.(5)The apoptosis-related proteins were detected by western blot.(6)The subcutaneous xenograft model of nude mice was used to further detect the effect of YFSJ pill combined with DDP on the subcutaneous tumorigenesis of NSCLC cells and its DDP resistant cell lines in nude mice.The second part:(1)The differentially expressed genes of A549 and its DDP resistant cell line A549DDP were detected by whole genome sequencing.(2)6 genes with the greatest difference(3 up-regulated and 3 down-regulated)were identified by RT-PCR,and the possible targets of YFSJ pills were screened.(3)The expression of FMNL1 in A549 and its DDP resistant cell line A549DDP was verified by RT-PCR and westernblot.(4)The sublocalization of FMNL1 protein in cells was studied by Genecard analysis and immunofluorescence experiment.(5)NSCLC cell lines stably overexpressing or knocking down FMNL1 were established by lentivirus transfection technique.(6)Drug sensitivity test was used to detect the effect of FMNL1 on DDP sensitivity of NSCLC cells.(7)Cell clone formation experiment was applied to detect the effect of FMNL1 on cell clone formation ability of NSCLC cells and DDP resistant cell lines.(8)Apopto sis assay was used to detect the effect of FMNL1 on apoptosis of NSCLC cells and DDP resistant cell lines with or without DDP.(9)Co-IP silver staining and protein spectrum were used to screen the proteins associated with FMNL1,and IP and immunofluorescence experiments were used to further study the interaction between FMNL1 and its downstream target proteins in the cytoplasm.(10)The effect of FMNL1 protein on the protein level of its interacting protein TRAP1was detected by western blot assay.(11)RT-PCR and Western blot were used to expore the effect of YFSJ pill on the m RNA and protein levels of FMNL1/TRAP1in NSCLC DDP resistant cells.The third part:(1)A total of 107 patients with stage III NSCLC who received DDP adjuvant chemotherapy were colle cted and tissue microarray was constructed.(2)Immunohistochemical staining was used to detect the expression of FMNL1protein in 107 cases of NSCLC.(3)Chi-square test,Pearson Chi-square test or Fisher exact probability method were used to analyze the relationship between FMNL1 expression level and clinical characteristics,including age,sex,tumor grade,T and N stage and survival status.Cox regression model was used for univariate and multivariate analysis.(4)The relationship between the expressio n of FMNL1 protein and the overall survival rate was analyzed by Kaplan-Meier method,and different survival curves were detected by Log-ranktest.Results:The first part:(1)NSCLC DDP resistant cell lines A549DDP and PC9DDP,were established by gradually increasing DDP concentration and intermittent action in vitro.It was found that the cell morphology of A549 and PC9 cells changed from oval to slender fusiform after DDP resistance.The results of drug sensitivity test showed that the drug resistance index was about 10 times,indicating that we have successfully constructed NSCLC DDP resistant cell lines A549DDP and PC9DDP.The successful establishment of NSCLC DDP resista nt cell lines A549DDP and PC9DDP provides a high-quality biological cell resource for us to further study the inhibitory effect and mechanism of YFSJ pill on DDP resistance in NSCLC.(2)Through the drug sensitivity test,we found that the drug-containing serum of YFSJ pill could reduce the DDP IC50 value of A549,PC9,A549DDP and PC9DDP cells with a concentration-dependent manner.It is suggested that the drug-containing serum of YFSJ pill can enhance the sensitivity of NSCLC cell lines A549 and PC9 to DDP and inhibit the DDP resistance of A549DDP and PC9DDP cells.(3)The apoptosis analysis experiment showed that the drug-containing serum of YFSJ pill could induce the apoptosis of NSCLC cells and DDP resistant cells,but its effect was not as obvious as th at of DDP,but the combination of YFSJ pill and DDP could significantly enhance the effect of DDP on the apoptosis of NSCLC cells and DDP resistant cells.(4)Apoptosis-related proteins were detected by westernblot.In NSCLC cell line A549 and DDP resistan t cell line A549DDP,both YFSJ pill and DDP could inhibit the expression of Caspase-3 and Bcl-2,while up-regulate the expression of Bax,and the effect of YFSJ pill combined with DDP were more obvious.(5)In the xenograft tumor model of nude mice,it was found that YFSJ pill could inhibit the subcutaneous tumorigenesis of NSCLC cells and their cisplatin-resistant cells,and inhibit the tumor value and weight of NSCLC cells,and the inhibitory effect was weaker than that of DDP.However,the combination of YFSJ pill and DDP could significantly inhibit the subcutaneous tumorigenesis of NSCLC cells and their DDP resistant cells.In addition,we also found that YFSJ pill had no significant effect on the body weight of nude mice,and could alleviate the weight loss effect of DDP on nude mice.These results suggest that YFSJ pill combined with DDP c ould play a synergistic effect.The second part:(1)In order to further study the molecular mechanism of DDP resistance in NSCLC and the mechanism of YFSJ pills in inhibiting DDP resistance in NSCLC,we further analyzed the differences in gene transcriptome between NSCLC cell line A549 and DDP resistant cell line A549DDP by hi gh-throughput genome sequencing and bioinformatics.The results showed that FMNL1 and other genes were abnormally over-expressed in NSCLC DDP resistant cell line A549DDP.(2)Through the RT-PCR experiment,we further verified the 6 genes with the greatest difference(3 up-regulated and 3 down-regulated).It was found that the drug-containing serum of YFSJ pill had a certain inhibitory effect on the m RNA level of FMNL1,but had no significant or no effect on other differential genes.Therefore,we focus on the role of FMNL1 in DDP resistance of NSCLC and the effect of YFSJ pill on FMNL1 and its downstream signaling molecules.(3)The m RNA level of FMNL1 in A549 cells and DDP resistant cells A549DDP detected by RT-PCR showed that the m RNA level of FMNL1 in A549DDP was significantly higher than that in A549 cells.Western blot found that the protein level of FMNL1 in A549DDP was also significantly higher than that in A549 cells.(4)In order to further study the sublocalization of FMNL1 in NSCLC cells and DDP resistant cells,we first predicted that FMNL1 may be mainly located in the cytoplasm of NSCLC cells and DDP resistant cells on the website of Gene Cards online analysis,and we further confirmed that FMNL1 was mainly located in the cytoplasm of NSCLC cells and DDP-resistant cells by immunofluorescence experiment.(5)The cell lines stably overexpressing or knocking down FMNL1 were established by lentivirus transfection technique,and the m RNA and protein levels of FMNL1 were verified by RT-PCR and western blot.It was found that the cell lines A549-FMNL1 and PC9-FMNL1 that stably overexpressed FMNL1and the cell lines A549DDP-sh6,A549DDP-sh7,PC9DDP-sh6,PC9DDP-sh7 and the control cell lines A549DDP-sh C and PC9DDP-sh C that stably knocked down FMNL1 were successfully constructed.(6)Drug sensitivity test showed that overexpression of FMNL1 could reduce the sensitivity of NSCLC cell line to DDP and promote DDP resistance,while knocking down FMNL1 could enhance the sensitivity of NSCLC cell line to DDP and reverse DDP resistance.(7)Cell clone formation assay showed that overexpression of FMNL1 could enhance the clone formation ability of NSCLC cell line s,while knocking down FMNL1 could weaken the clone formation ability of NSCLC DDP resistant cell lines.(8)Apoptosis experiment showed that knocking down FMNL1 could enhance the sensitivity of NSCLC DDP resistant cell line to DDP,promote apoptosis and reverse DDP resistance.Overexpression of FMNL1 can reduce the sensitivity of NSCLC cell line to DDP,inhibit apoptosis and promote DDP resistance.(9)In order to furt her study the specific molecular mechanism of FMNL1inhibiting apoptosis,Co-IP silver staining and mass spectrometry were used to screen the interacting protein TRAP1.The interaction between FMNL1 and TRAP1 protein in NSCLC cells was further confirmed by co-immunoprecipitation technique.In addition,cellular immunofluorescence assay also showed that FMNL1 and TRAP1 co-located in the cytoplasm of NSCLC DDP resistant cell line A549DDP cells.(10)It was found that in NSCLC DDP resistant cell line A549DDP,the expression level of TRAP1 was down-regulated after knocking down FMNL1,while in NSCLC cell line A549,the expression level of TRAP1 was also up-regulated after overexpression of FMNL1,indicating that TRAP1 is a downstream effector molecule of FMNL1.In addition,we also found that the half-life of TRAP1 protein was shortened after knocking down FMNL1.Th ese suggests that FMNL1 can stabilize TRAP1 protein in cells and maintain the protein level of TRAP1.(11)RT-PCR experiment showed that compared with the control group,the drug-containing serum of YFSJ pill could significantly inhibit the m RNA transcription level of FMNL1 in NSCLC DDP resistant cell line A549DDP,and showed a concentration-dependent manner,and similar results were found in another NSCLC DDP resistant cell line PC9DDP.Compared with the control group,the drug-containing serum of YFSJ pill could significantly inhibit the m RNA transcription level of TRAP1 in NSCLC DDP resistant cell line A549DDP in a concentration-dependent manner,and similar results were found in another NSCLC DDP resistant cell line PC9DDP.Western blot also found that,compared with the control group,the serum containing YFSJ pill could significantly inhibit the protein expression levels of FMNL1 and TRAP1,and similar results were found in another DDP resistant cell line PC9DDP.Th ese suggests that YFSJ pill may enhance the sensitivity of NSCLC cells to DDP by inhibiting the level of m RNA transcription and protein expression of FMNL1 and TRAP1,and then inhibit DDP resistance of NSCLC cells.The third part:(1)In order to further clarify the effect of FMNL1 protein o n DDP sensitivity in NSCLC,we collected 107 patients with stage III non-small cell lung cancer treated with DDP adjuvant chemotherapy in the Cancer Center of Sun Yat-sen University,and constructed the tissue microarray of NSCLC.Immunohistochemical metho d was used to investigate the expression of FMNL1protein in NSCLC and its clinicopathological significance.We used immunohistochemical method to detect the expression of FMNL1 in 91.6%(98/107)NSCLC samples,of which 9 samples were detached or had too f ew tumor cells(less than 300 in each sample),so they were not included in our data analysis.(2)In 98 cases of III stage non-small cell lung cancer,61 samples showed high expression of FMNL1,accounting for 62.2%of stage III NSCLC(61/98).We found that the expression level of FMNL1 was not significantly correlated with age,sex,T stage of primary tumor and N stage of lymph node metastasis.When comparing the correlation between FMNL1 and pathological types of lung cancer,we found that the expressi on of FMNL1 in lung adenocarcinoma was higher than that in squamous cell carcinoma(P<0.001),and the expression level of FMNL1 was related to the survival status of NSCLC patients.(3)According to Kaplan-Meier survival analysis and log-rank test,the survival time of patients with stage III NSCLC treated with DDP adjuvant chemotherapy was compared.It was found that the overall survival rate of patients with abnormally high expression of FMNL1 was significantly lower than that of patients with low expre ssion of FMNL1.Conclusion:YFSJ pill combined with DDP can synergistically inhibit the viability and proliferation of NSCLC cells and DDP resistant cells,promote apoptosis,and inhibit the tumor value and tumor weight of NSCLC cells and DDP resistant cells in nude mice.YFSJ pill inhibits the m RNA and protein levels of FMNL1 and TRAP1,which may be the main mechanism of its inhibition of DDP resistance in NSCLC.Abnormal high expression of FMNL1 promot es the progress of NSCLC DDP resistance and indicates poor prognosis.FMNL1 may be used as a predictor of the effect of adjuvant chemotherapy with DDP in NSCLC patients,while targeted FMNL1 may improve the treatment effect of NSCLC. |
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