SMYD3 Confers NSCLC Cells Chemoresistcance To Cisplatin In An ANKHD1-dependent Manner

Background:Lung cancer is one of the most important causes of cancer-related deaths in the world.It is mainly divided into two subtypes:non-small-cell lung carcinoma（NSCLC）,which accounts for 85%of cases,and small-cell carcinoma（SCLC）,which accounts for 15%of cases.NSCLC accounts for a large proportion of cancer deaths and is characterized by low response rate and poor overall prognosis.Although chemotherapy was effective for NSCLC patients at the beginning,but the 5-year survival rate is still very low.In the absence of specific treatable mutations,cisplatin（DDP）-based chemotherapy plays an important role in the treatment of the disease.However,acquired drug resistance in cancer cells limits the clinical application of DDP,and the molecular mechanism of this resistance remains to be fully explored.SET and MYND domain-containing protein 3（SMYD3）is a member of the SET and MYND domain family of methyltransferases,and its high expression has been associated with poor prognosis various cancers.In liver and colon tumors,SMYD3 is localized in the nucleus,where it interacts with RNA Pol II and H3K4me3 as a selective transcriptional amplifier of oncogenes and genes that control cell proliferation and metastasis.The expression of SMYD3 has a high ability to distinguish liver tumors and is positively correlated with poor prognosis.In lung and pancreatic cancer,SMYD3 acts on the cytoplasm,amplifying the oncogenic RAS/ERK signal by methylation of the MAP3K2kinase and subsequent release of its inhibitors.Studies have found that SMYD3 may influence the sensitivity of cells to DDP,but the mechanism of its role in the regulation of DDP resistance in NSCLC has not been clarified.Recently,it has been reported that Ankyrin repeat and KH domain-containing 1（ANKHD1）are potential members of the Hippo signaling pathway.Significant up-regulation of ANKHD1 protein expression was observed in NSCLC cells and tissues,which was associated with pathological grade,lymph node metastasis and poor prognosis of NSCLC patients.However,can ANKHD1 mediate the progression in NSCLC by binding to its specific histone markers?And whether it is involved in the chemotherapy resistance of NSCLC regulated by SMYD3?It remains to be further discussed and verified.Objective:The purpose of this study was to investigate the role and mechanism of SMYD3 in the resistance of NSCLC to DDP and whether ANKHD1 could participate in the resistance of NSCLC to DDP mediated by SMYD3.Provides potential ways and good laboratory basis for clinical improvement of NSCLC treatment.Methods:1.q PCR detection to examine the expression of SMYD3 in cancer and adjacent tissue samples,and tissue samples of different pathological grades;Kaplan-Meier survival analysis for adverse prognosis in non-small cell lung cancer;q PCR was used to detects the expression of SMYD3 in DDP sensitive tissues,DDP resistant tissues,normal human epithelial cells,NSCLC cells and NSCLC DDP-resistant cells;MTT detection of cell IC₅₀;flow cytometry detection of cell apoptosis.2.q PCR detection of transfection efficiency of si-SMYD3 and over-expressing SMYD3 plasmid;MTT detects the cell IC₅₀ of si-SMYD3 and over-expressing SMYD3cells;colony formation assay was used to detect the generation of si-SMYD3 cells and over-expressing SMYD3 cells;the apoptosis level and cell cycle of si-SMYD3 cells and over-expressing SMYD3 cells were detected by flow cytometry.3.Co-IP observed the interaction between ANKHD1 and H3K4me3 after overexpression of SMYD3;the co-localization of ANKHD1 and H3K4me3 in cells after overexpression of SMYD3 was observe by nuclear-cytoplasmic separation WB and confocal microscope;the expression level of ANKHD1 was detected by q PCR in cancer and adjacent tissue;Pearson correlation analysis was used to observe the correlation between the expression level of ANKHD1 and SMYD3;MTT detects the cell IC₅₀ after overexpression of SMYD3 and si-ANKHD1 cells;cell cloning experiment detects the generation of monoclonal cells after overexpression of SMYD3 and si-ANKHD1;flow cytometry to detect the level of apoptosis and cell cycle changes after overexpression SMYD3 and si-ANKHD1.4.The m RNA expression level of CDK2,CDK4 and CDK6 were detected by q PCR after overexpression of SMYD3 and si-ANKHD1;the expression levels of SMYD3,ANKHD1 and CDK2 were detected by western blot after overexpression of SMYD3 and si-ANKHD1;CHIP detection and analysis of the enrichment of SMYD3 and H3K4me3 on the CDK2 promoter.5.The growth of xenograft tumor was observed,the expression of si-SMYD3 and Ki67 after overexpression of ANKHD1 were analyzed by immunohistochemistry.Results:1.SMYD3 expression in NSCLC was significantly higher than that in control group;the expression level of SMYD3 in advanced pathological graded tissues was significantly increased;higher level of SMYD3 also indicates a poor prognosis;compared with DDP-sensitive tissues,the expression level of SMYD3 in DDP-resistant tissues was significantly increased;compared with 16HBE cells,the expression level of SMYD3 in NSCLC cells was significantly increased,and the expression level of SMYD3 in NSCLC DDP-resistant cells was up-regulated more significantly;compared with the control group,the IC₅₀ of NSCLC DDP-resistant cells was significantly increased,while the proportion of apoptosis induced by DDP was significantly reduced.2.Compared with the si-NC group,when SMYD3 expression was silenced,the IC₅₀ of NSCLC DDP-resistant cells was significantly decreased,the number of single cell clones was significantly decreased,the proportion of apoptosis was significantly increased,and the cell cycle was arrested;compared with the empty plasmid group,after overexpression of SMYD3,the IC₅₀ of NSCLC DDP-resistant cells was significantly increased,the number of single-cell clones was significantly increased,the proportion of apoptosis was significantly reduced,and the ratio of S phase and G2/M phase was significantly increased.3.Compared with the control group,overexpression of SMYD3 can significantly promote the interaction between ANKHD1 and H3K4me3 in NSCLC cells,which is located in the nucleus;the expression of ANKHD1 in NSCLC was significantly higher than that in normal tissues,and there was a positive correlation between the expression of ANKHD1 and SMYD3;compared with the empty plasmid group,after overexpression of SMYD3,the IC₅₀ of NSCLC DDP-resistant cells increased significantly,the number of single-cell clones was significantly increased,the proportion of apoptosis was significantly reduced,and the ratio of S phase and G2/M phase was significantly increased.After silencing the expression of ANKHD1 at the same time,the effect of SMYD3 on cells can be reversed.4.Compared with the empty plasmid group,after overexpression of SMYD3,the expression levels of CDK2,CDK4,CDK6 and ANKHD1 in NSCLC cells were significantly up-regulated,and after the simultaneous silence of ANKHD1 expression treatment,the effect of SMYD3 on cells could be reversed;SMYD3 can bind to the CDK2promoter near the transcription start site;Overexpression of SMYD3 significantly increased the H3K4me3 level of CDK2’s proximal promoter;si-SMYD3 can reduce the level of H3K4me3 in the proximal promoter of CDK2.5.Compared with the si-NC group,the tumor size in the si-SMYD3 group was significantly reduced,the tumor weight was significantly decreased,and the tumor growth was significantly inhibited.However,the inhibition of si-SMYD3-induced tumor growth was reversed by overexpression of ANKHD1.Compared with the si-NC group,the positive expression of Ki67 in the si-SMYD3 group was significantly suppressed,and this phenomenon can be reversed after the simultaneous overexpression of ANKHD1 treatment.Conclusion:1.SMYD3 promotes DDP resistance in NSCLC cells.2.As a co-regulator,ANKHD 1 is involved in SMYD3-mediated NSCLC chemotherapy resistance.3.CDK2 is a downstream target gene involved in NSCLC DDP resistance regulated by the SMYD3-ANKHD1 axis.4.SMYD3 regulates NSCLC chemotherapy resistance in an ANKHD1-dependent manner in vivo.