SHISAL1 Suppresses Proliferation,Migration And Invasion Of Hepatocellular Carcinoma Cells Via Wnt5a/FZD5 Noncanonical Wnt Pathway

Objective:Shisa like 1(SHISAL1,KIAA1644)is a novel protein found in vertebrates with domain sequence similarity in computational biology in 2012 by Pei et al.However,little is known about the role of SHISAL1,particularly in hepatocellular carcinoma(HCC).The aim of the research team was to prepare polyclonal antibodies against SHISAL1 protein and study the localization of SHISAL1 in HCC cell line SMMC-7721 and the effects of SHISAL1 on the proliferation,migration and invasion of HCC cell lines.Methods:(1)Expression and purification of SHISAL1 recombinant protein: Human SHISAL1 gene was cloned from HCC cell line Hep G2 cells by RT-PCR,and then inserted into prokaryotic expression vector p ET-28 a to generate the p ET-SHISAL1 expression vector.The recombinant plasmid was then introduced into E.coli BL21 (DE3).The expression of SHISAL1 was induced by IPTG,followed by purification with Ni-NTA affinity column.(2)Preparation of polyclonal antibodies against SHISAL1 protein: The SHISAL1 protein was used to immunize 8-week-old female Kunming mice.The specificity of polyclonal antibody was verified by Western blot.(3)Subcellular localization of SHISAL1 protein: The expression of SHISAL1 in SMMC-7721 cells was detected by immunofluorescent cytochemistry.Golgi complexes were signed by Golgi-Tracker Red to analyze the subcellular localization of SHISAL1 protein in SMMC-7721 cells.(4)Construction of recombinant vectors and recombinant lentivirus packaging:human SHISAL1 gene was cloned from Hep G2 cells by RT-PCR,and then inserted into p LVX-Ac GFP-N1 to construct eukaryotic expression vector of pLVX-Ac GFP-SHISAL1.Short hairpin DNA(sh DNA)targeted SHISAL1 was cloned into lentiviral interference vector p GLV3/H1/GFP+Puro(LV3).After validation,Human Embryonic Kidney cells 293 T were cotransfected with the mixture of lentivirus packaging plasmids to package into lentivirus solution.(5)Construction of exogenous SHISAL1 overexpressed(OE-SHISAL1)HCC cells and knockdown of SHISAL1(sh-SHISAL1)in HCC cells: SMMC-7721 and Hep G2 cells were infected with lentivirus solution.The stable infected cells were screened with puromycin.After amplification,colonies were identified by qRT-PCR and Western blot to evaluate the expression of target genes.(6)Detection of cell function: Cell proliferation activity was detected by real time cellular analysis(RTCA).Cell clonogenesis and tumor stemness were detected by Colony formation assay.Cell cycle was detected by flow cytometry.Cell migration and cell invasion were tested by wound healing experiment and transwell assay.(7)The expression and interaction of Wnt receptor Frizzled5(FZD5)and related proteins were detected by Western blot,immunoprecipitation and immunofluorescent cytochemistry.Results:(1)The SHISAL1 gene was cloned and recombinant vector,named p ET28a-SHISAL1 was successfully constructed.Pure SHISAL1 protein expression in E.coli BL21 was confirmed.(2)Polyclonal antibodies against protein SHISAL1 was obtained from Kunming mice.(3)Immunofluorescent cytochemistry showed that SHISAL1 was expressed in the cytoplasm and co-localized with Golgi-Tracker Red in SMMC-7721 cells.(4)Successful packaging and collection of lentivirus solution.OE-SHISAL1 HCC cells and sh-SHISAL1 HCC cells were obtained successfully.(5)We uncovered the facts that a forced over-expression of SHISAL1 inhibited cell proliferation,migration and invasion of human HCC cells while sh-SHISAL1 significantly promoted the ability in HCC cells.(6)SHISAL1,locating in Golgi apparatus,inhibited the maturation and cell surface localization of Wnt receptor FZD5,resulting in the inactivation of noncanonical Wnt pathway through Fyn-Stat3 axis.(7)Over-expression of FZD5 partly abolisheed the inhibitory effects on cell proliferation,migration and invasion elicited by SHISAL1 deficiency in HCC cells.Conclusion:(1)Polyclonal antibodies against protein SHISAL1 was obtained successfully from Kunming mice.(2)SHISAL1 was expressed in the cytoplasm and co-localized with Golgi-Tracker Red in SMMC-7721 cells.(3)SHISAL1 inhibited cell proliferation,migration and invasion of HCC cells,which acts as a tumor suppressor mainly through regulating the establishment of FZD5 in HCC.