The Role And Mechanism Of MYH9 Promoting Renal Cell Carcinoma Development And Sunitinib Resistance Via AKT Signaling Pathway

ObjectiveRenal cell carcinoma(RCC)is one of most common cancers,and accounts for 2%to 3%of adult malignant tumors.The prognosis of advanced renal cancer is very poor due to lack of effective treatment.RCC is a highly heterogeneous tumor.There are significant differences in the growth rate,invasive ability,and drug sensitivity of tumor cells in the same renal cell carcinoma patients.Single-cell RNA-sequencing(scRNA-seq)is a new technique for high-throughput sequencing and analysis of RNA at a single cell level and has significantly advanced our knowledge of cell biology.In the recent study,we identified 5 cell subgroups from renal cancer tissues and adjacent normal tissues using scRNA-seq clustering analysis.In order to identify the cancer promoter gene,we compared the differences between renal tube cells and renal cancer cells and found 5 differentially expressed genes(DEGs)that were significantly higher in renal cancer cells than that in renal tube cells.Among them,Myosin heavy chain 9(MYH9)was the most remarkable DEG,closely related to RCC prognosis.Myosin heavy chain 9(MYH9),as a skeleton protein,plays an important role in many cellular movements such as cell adhesion,migration and division.MYH9 has been shown to be involved in cancer progression and metastasis,while it exerts paradoxical roles in different cancers.The role of MYH9 in renal cell carcinoma(RCC)and its underlying mechanism has not been investigated.This study aimed to explore the function of MYH9 in the RCC development and its potential mechanism.Materials and Methods1.In this study,single-cell data from GEO database were selected for data analysis.In order to identify the cancer promoter gene,we selected renal tuble cell subgroup and cancer cell subgroup for different expression genes(DEGs)analysis.The DEGs were verified by bioinformatics.2.The study adopted Western blot,immunohistochemistry,RT-PCR for detecting the expression of MYH9 protein and mRNA in RCC tissues and adjacent normal tissues.We compared the PFS and OS of RCC patients in MYH9 high expression group and low expression group to analyze the correlation between MYH9 expression and prognosis of RCC patients.3.Two shRNA-MYH9 and MYH9 overexpression plasmid were constructed,transfected and screened for cell lines stably downexpressing or overexpressing MYH9.RT-PCR and western blot were used to detect transfection efficiency.4.The effect of MYH9 on cell proliferation were detected by CCK8 and colony formation assay.5.The effect of MYH9 on cell migration were detected by wound-healing assay and transwell migration assay.6.We constructed the subcutaneous tumorigenesis model of nude mice by LV-MYH9 786-O cells,recording the tumor tissue weight and volume for monitoring RCC cell proliferation in vivo.7.Bioinformatics analysis was used to screen the downstream pathway of MYH9 and found MYH9 activated AKT signaling pathway.Western blot was used to detect the protein expression of p-AKT in LV-MYH9 293-T cells.The activity of AKT signaling pathway in LV-MYH9 293-T cells was further evaluated by dual luciferase reporter assay.8,The p-AKT protein expression in the transplanted tumor tissue was detected by western blot to further verify the effects of MYH9 on the proliferation of RCC in vivo.9.Through the "rescue assay" method,CCK8 and wound-healing assay further verified that MYH9 affected the proliferation and migration of RCC through AKT signaling pathway.10.The shMYH9 RCC and LV-MYH9 RCC cells were treated by SC79 or A6730.CCK8,colony formation assay,and apoptosis cells assay were used to investigate the effects of MYH9/AKT signaling pathway on sunitinib sensitivity of RCC cells.11.We compared the PFS and OS of RCC patients with or without sunitinib treatment in MYH9 high expression group and low expression group to analyze the correlation between MYH9 expression and prognosis of RCC patients treated with sunitinib.Results1.A total of 3 RCC patients’ tissues,including 2 tumor tissues and 1 adjacent tissues,were used for scRNA-seq analysis.We use the t-SNE algorithm to precisely cluster the populations of cells,successfully classifying the samples into five independent cell subgroup.In order to identify the cancer promoter gene,we selected clusters renal tuble cell and cancer cell for DEGs analysis.Finally,we found that the mRNA expression of MYH9 in RCC cells was significantly higher than that in normal kidney cells.So we chose MYH9 for subsequent experiments to explore its impact on RCC progression.2.In RCC tissues and adjacent tissues of 42 RCC patients,IHC staining and western blotting were performed and found that MYH9 was significantly upregulated in RCC tissues,compared to normal kidney tissues.The RT-PCR also showed that RCC tumor was of higher level of MYH9 than adjacent tissue in this cohort.Moreover,we also found patients’ OS and PFS were significantly poorer in RCC with high MYH9 levels in cohort 2.3.Based on the transfection efficiency assay,following shMYH9-1 and shMYH9-2 transfection,MYH9 mRNA and protein expression were decreased significantly in ACHN cells and 786-O cells,compared to negative control group.By contrast,MYH9 mRNA and protein expression presented an obvious increase due to LV-MYH9 transfection,compared to negative control group.4.Cell growth was assessed by CCK8 assay and plate colony formation assay and we found MYH9 knockdown significantly inhibited the proliferation,while MYH9 overexpression promoted the proliferation of RCC cells.5.Cell migration was assessed by wound-healing assay and transwell migration assay and we found MYH9 knockdown significantly inhibited the migration,while MYH9 overexpression promoted the migration of RCC cells.6.The results of subcutaneous tumorigenesis model of nude mice showed that the transplanted tumors of MYH9 overexpression group mice grew faster and larger than those of the control group.Western blot showed that transplanted tumors in MYH9 overexpression group had higher Ki-67 protein expression than negative control group mice tumors.7.With the purpose to detect the underlying mechanism of MYH9 in RCC,KEGG and WIKI pathway analysis were performed.The results showed that there was a widespread impact of MYH9 on the AKT signaling pathway.Western blot showed that phosphorylation of AKT was significantly upregulated in LV-MYH9293-T cells.AKT reporter assay further confirmed that MYH9 up-regulation incurred higher level of AKT activation.Moreover,AKT was also remarkably activated in LV-MYH9 xenografts by western blotting.8.The capacity of proliferation and migration were suppressed in shMYH9-2 RCC cells,while these abilities could be reversed by co-incubation with SC79,an AKT activator.While the capacity of proliferation and migration were enhanced in LV-MYH9 RCC cells,while these abilities could be reversed by co-incubation with A6730,an AKT inhibitor.Taken together,these results indicated that MYH9 promoted RCC proliferation and migration through activating AKT signaling pathway.9.CCK8,colony formation assay and apoptosis cells assay revealed that MYH9 knockdown RCC cells presented an increased sensitivity to sunitinib treatment compared with control group,while activation of AKT remarkably restored sunitinib resistance.CCK8,colony formation assay and apoptosis cells assay revealed that MYH9 overexpression RCC cells presented an reduced sensitivity to sunitinib treatment compared with control group,while inhibition of AKT remarkably restored sunitinib resistance.These data indicated that the MYH9-mediated AKT activation might be required for sunitinib resistance in RCC cells.10.In 50 sunitinib-treated RCC patients,the MYH9 levels were higher in the RCC tissues from poor response group than that from well response group.11.Patients with low MYH9 expression group displayed more notable improvement in PFS after receiving sunitinib than control group.While,compared with control group,high MYH9 expression patients did not present obvious clinical effect.Furthermore,patients with low MYH9 expression levels in their RCC had a more significant improvement in PFS after receiving sunitinib when compared with those in patients with high MYH9 expression.Conclusion1.There is a significant increase in MYH9 expression in RCC tissues.MYH9 expression is related to the prognosis of RCC patients.2.MYH9 promotes the proliferation and migration of RCC cells.3.MYH9 promotes RCC sunitinib resistance.4.MYH9 promotes the proliferation,migration,and sunitinib resistance of RCC cells via AKT signaling pathway.Taken together,our findings uncovered MYH9 as a novel carcinogenic protein and revealed the significant role of the MYH9/AKT axis in RCC development and sunitinib response,rendering MYH9 as an optimal marker for the prevention and intervention of RCC.