IPO5 Promotes The Proliferation And Migration Of Colorectal Cancer Cells By Mediating RASAL2 Nuclear Transportation

Background and ObjectionKaryopherin nuclear transport receptors play an important role in tumor development and drug resistance,and can be used as potential targets for tumor therapy.Currently reported tumor-related nuclear plasma transporters include KPNA2,XPO5,XPO1 and KPNA7.However,IPO5,a member of the nuclear transporter family of proteins,has not been reported in tumors.In this study,we found that IPO5 was highly expressed in colorectal cancer.We will further explore the in vivo and in vitro functions of IPO5 and its related molecular mechanisms in colorectal cancer.MethodsWestern blot,qRT-PCR and immunohistochemical(IHC)were applied to detect the expression and location of IPO5 in colorectal cancer cell lines and tissues.The expression datas were used to statistically analyze the correlation between IPO5 expression and the pathological parameters of clinical patients.Meanwhile,ROC curve was made to analyze the clinical application value of IPO5.To detect the biological function of IPO5 in colorectal cancer.CCK8 cell proliferation,plate cloning,flow cytometry and transwell assay were used to detect the changes in proliferation and migration of colorectal cancer cell lines after interference and overexpression of IPO5.At the same time,the IPO5 down-regulated cells RKO-SH-IPO5 and RKO-SH-NC were injected into the skin of nude mice respectively,and the effects of IPO5 interfered cell lines and control cell lines on tumor proliferation ability were observed and measured in vivo.Then,in order to preliminarily determine whether IPO5 is resistant to 5-FU,western blot and flow cytometry method were used to analyze the apoptotic response of colorectal cancer cells under the treatment with 5-FU after overexpression of IPO5.In terms of mechanism,the binding protein of IPO5 was pulled down by CO-IP technology,followed by rapid silver staining and mass spectrometry analysis.Then,using bioinformatics databases to further screen the possible transporters of IPO5.And RASAL2 was preliminarily screened by nuclear plasma separation experiment combined with western blot.Then,CO-IP,nuclear plasma separation and immunofluorescence experiment were used to verify whether RASAL2 is the transporter of IPO5 in colorectal cancer cells.Subsequently,RASAL2 nuclear localization signal sequence(NLS)plasmid and point mutation plasmid were constructed and transfected into colorectal cancer cells,and IPO5 was verified to transport RASAL2 by binding the nuclear localization signal sequence(NLS)through nuclear plasma separation experiment,CO-IP technology and immunofluorescence experiment.In order to verify whether IPO5 could regulates RAS pathway activity through RASAL2,IPO5 was interfered and overexpressed in colorectal cancer cells,and changes of major target genes in RAS pathway were detected by western blot.In the meantime,a series of in vivo and in vitro functional recovery experiments were conducted to test whether nuclear localization signal sequence(NLS)mutation of RASAL2 could reverse the promotion effect of IPO5 on colorectal cell proliferation and migration.Finally,cancer tissues of clinical colorectal cancer patients were collected for immunohistochemical(IHC)detection.According to the staining intensity,the correlation test between IPO5 expression and RASAL2 nuclear localization was analyzed to verify whether IPO5 promotes the development of colorectal cancer by translocation of RASAL2 into the nucleus.ResultsIPO5 is highly expressed in colorectal cancer and its expression is related to the size,lymph node metastasis and TNM staging of colorectal cancer.Interfering of IPO5 could inhibit proliferation and migration of colorectal cancer.However,overexpression of IPO5 promoted proliferation and migration.The high expression of IPO5 can reduce the sensitivity of colorectal cancer cells to 5-FU.RASAL2 is the target transporter protein of IPO5,and IPO5 promotes the activation of RAS pathway by increasing the nuclear transport of RASAL2,thus promoting the occurrence and development of colorectal cancer.ConclusionWe found that the high expression of IPO5,by combining the nuclear localization signal sequence(NLS)of RASAL2,leads to the decrease of RASAL2 concentration in the cytoplasm and membrane,resulting in the abnormal activation of the RAS pathway,promoting the proliferation and migration of colorectal cancer cells and inhibiting apoptosis.In addition,the high expression of IPO5 can promote the resistance of colorectal cancer cells to 5-FU.In summary,this study found for the first time that IPO5 plays a role in the pathogenesis and progression of colorectal cancer.This finding will provide new ideas for the mechanism of colorectal cancer development and provide clues and theoretical basis for the treatment of colorectal cancer.