Study On The Regulation Of Growth Function And Molecular Mechanism Of Karyopherin ? 2 In Melanoma Cell

Objective: Melanoma is a common and highly malignant cutaneous tumor originating from melanocytes,which is related to many factors such as race,skin color,ultraviolet radiation,age,immune status,and genetic mutation.The morbidity of melanoma is increasing year by year,about 96,480 people are diagnosed with melanoma in America in2019.Despite great progress has been made in early diagnosis of melanoma,there is no effective treatment currently,the five-year survival rate for advanced patients is only 17 percent.As melanoma progresses to advanced stage once metastasis occurs,early diagnosis and therapeutic interventions have a great impact on the prognosis of melanoma patients.It is of great significance to explore the potential molecular mechanism of melanoma pathogenesis and to search for new potential therapeutic targets.The nucleocytoplasmic transport mechanism is commonly observed in cancer.Karyopherins act as carrier proteins in the selective bi-directional shuttling of proteins between the cytoplasm and the nucleus,called nucleocytoplasmic transport.Karyopherin? 2(KPNA2),a protein composed of 529 amino acids,plays an important role in nucleocytoplasmic transport.KPNA2 protein comprises an N-terminal hydrophilic importin ?1-binding domain,a central hydrophobic region consisting of 10 armadillo repeats,which binds the cargo's NLS,and a short acidic C-terminus with no reported function.The mechanism is that importin ?1 brings a complex of KPNA2 and nucleoprotein into the nucleus via nuclear pore complexes and binds to Ran GTP to release KPNA2 and cargo proteins into the nucleus.Then importin ?1 returns directly to the cytoplasm with the help of another transporter,protein CAS,for the next cycle.KPNA2 has been involved in promoting tumor cell proliferation,colony formation,migration and invasion through this mechanism.Previous researches had demonstrated that the expression level of KPNA2 was upregulated in breast cancer,gastric cancer tea.al and correlated with the prognosis of patients.Yuntao Lu teams found that KPNA2 was involved in the nuclear transport of TP53 transcription factor,and silence of KPNA2 inhibited autophagy via disruption of the nuclear import of TP53.KPNA2 silence could inhibit nuclear importation of NF-?B-p65 and notably attenuates m RNA expression of MMP(Matrix Metalloproteinase)-1,MMP-3,and MMP-9 induced by IL-1b in oral squamous cell carcinoma.Winnepenninckx et al.demonstrated that melanoma patients with high expression level of KPNA2 had significantly reduced survival rate.However,to the best of our knowledge,the roles of KPNA2 in the function of melanoma cells and its in-depth molecular mechanism have not been reported yet.In this study,we analyze the expression and prognosis of KPNA2 in melanoma.Furthermore,we find that KPNA2 promotes cell proliferation migration and invasion via NF-?B/p65 signaling pathways in melanoma cells and NF-?B inhibitor JSH-23 can reverse the pro-tumor effects of KPNA2 on melanoma cells.In addition,upregulation of KPNA2 increases the tumorigenicity of melanoma cells in the hypodermis of nude mice.Methods: In this study,we investigated the expression and prognosis of KPNA2 in melanoma using the GEPIA database(http://gepia.cancer-pku.cn/).Then,human melanoma cell lines A375 and A875 were used as experimental group,and primary Human Epidermal Melanocytes adult line(HEMa)was used as control.Real-time PCR was used to detect the m RNA expression of KPNA2 in A375,A875 and HEMA,and Western blot and cellular immunofluorescence were used to detect the expression of KPNA2 in each cell line.Subsequently,liposomal transfection was placed on A375 and A875.Cell counting kit-8(CCK-8)assay and clone formation assay were used to detect the proliferation ability of the cells.Western blot was used to detect the expression of proteins related to cell proliferation such as PCNA,Ki67 and c-Myc.Cell migration ability was detected by wound healing assay,cell invasion ability was detected by Transwell assay,and invasiveness related proteins MMP2 and MMP9 were detected by Western blot.The expression levels of the proteins on NF-?B/p65 pathway,COX-2,ICAM-1,i NOS,MCP1 and nuclear p65 were detected by Western blot.Meanwhile,the intracellular localization of p65 was detected by immunofluorescence assay,and the binding activity of NF-?B/p65 was detected by EMSA.Then,in this study,NF-?B inhibitor JSH-23 was used to intervene the overexpression group.CCK-8 test,clone formation test,wound healing assay and Western blot were repeated to detect cell proliferation,migration,invasion ability and related proteins' expression again.Finally,A375 cell with stable overexpression or silence of KPNA2 were selected for tumorigenesis experiment in nude mice model in vivo.The expression levels of KPNA2,PCNA,MMP2 and nuclear p65 in tumor tissues were detected by Western blot and immunohistochemistry to evaluate the effect of on KPNA2‘s tumorigenicity of melanoma cells.Results: 1.Through database information mining,it was obvious that melanoma patients with higher expression of KPNA2 had lower overall survival rate and disease free survival rate.2.After western blot,real-time PCR and immunofluorescence used to detect the expression of KPNA2 in HEMA,A375 and A875 cells,and the results showed that KPNA2 expression in A375 and A875 cells was significantly increased compared with that in HEMa cells.3.The transfection of overexpressed and silence KPNA2 cell lines was successfully established.CCK-8 assay and clone formation assay showed that KPNA2 promoted the proliferation ability of melanoma cells.Western blot assay showed that the overexpression of KPNA2 increased the expression of PCNA,Ki67 and c-Myc,while silence of KPNA2 had the opposite result.4.The wound healing assay and transwell assay showed that KPNA2 could promote the migration and invasion ability of melanoma cells.Western blot showed that the overexpression of KPNA2 increased the expression of MMP2 and MMP9,while silence of KPNA2 showed the opposite result.5.Western blot assay and EMSA assay showed that overexpression of KPNA2 activated the NF-?B/p65 signaling pathway by increasing the expression of COX-2,ICAM-1,i NOS,and MCP1 in the nucleus of melanoma and led to nucleocytoplasmic transport of p65,while silence of KPNA2 had the opposite result.6.CCK-8 assay,clone formation assay,wound healing and Western blot were repeated to detect the proliferation,migration,invasion ability and related protein expression of cells after applied the NF-?B inhibitor JHS-23.NF-?B inhibitor JSH-23 could reverse the pro-tumor effects of KPNA2 on melanoma cells.7.Tumorigenesis model was successfully established in nude mice.Tumor was larger in nude mice after overexpression of KPNA2,western blot and immunohistochemical assay showed the related protein expression was increased.While when silent KPNA2,the tumors were smaller,and the expression of related proteins was significantly down-regulated.Moreover,upregulation of KPNA2 facilitated the tumorigenicity of melanoma cells.Conclusion: 1.KPNA2 promotes proliferation,migration and invasion through enhancing NF-?B/p65 signaling pathways in melanoma cells.2.The overexpression of KPNA2 facilitated the tumorigenicity of melanoma cells in nude mice,which was consistent with the results in vitro study 3.KPNA2 may be regards as a potential therapeutic target for the therapy to melanoma.