| Background and objectiveBreast cancer is one of the most common malignancies in women,it can be divided into non-invasive cancer,microinvasive cancer and invasive cancer in histologically.According to the St Gallen classification criteria,breast cancer is divided into four subtypes according to the expression of molecular markers:Luminal A,Luminal B,Her-2,and basal-like.Clinical treatment is based on molecular subtypes.molecular typing has important guiding significance for clinical treatment and prognosis..Luminal A/Luminal B/Her-2 subtypes breast cancer can obtain obvious treatment effect and improve long-term survival in patients,while basal-like breast cancer lacks a good treatment method and susceptible to drug resistance,most of them have a poor prognosis.Finding new genes related to breast cancer and revealing its functional mechanism is of great significance for clarifying the mechanism of breast cancer and digging new molecular targets.WTX(Wilms Tumor gene on the X chromosome),also known as FAM123B,is a tumor suppressor gene located on the X chromosome.It has been reported to be associated with Wilm’s tumor and OSCS.In the previous study of our research team,it was found that WTX showed a preliminary anti-tumor effect in gastrointestinal tumors,and WTX expression in breast cancer tissue was lower than normal mammary tissue,but its role in the progression of breast cancer is unclear.This project intends to study the effect of WTX on the biological behavior of breast cancer,understand its relationship with breast cancer proliferation,invasion,metastasis and to explore the molecular mechanism,further reveal its funtion and the molecular mechanism in tumordevelopment,and provide new molecular targets for clinical tumor treatment.Methods1、Clinical significance of WTX in invasive breast cancer(1)Detection of WTX expression in breast cancer by immunohistochemistry;(2)Statistical analysis of the relationship between WTX expression and clinical parameters;(3)Statistical analysis of the relationship between the expression of WTX and ER,PR,Her-2,and Ki-67.2.、WTX function in breast cancer cells(1)Establish the stable forced WTX over-expression and stable forced WTX knocked down breast cancer cell linesEstablish the stable forced WTX over-expression breast cancer cell lines MDAMB-231-W,Hs578T-W and matched negative control cell lines,establish stable WTX knocked down breast cancer cell lines MDA-MB-231-shW,MDA-MB-435shW,SKBR3-shW and matched negative control cell lines.Detecting WTX mRNA expression and WTX protein expression with real-time fluorescence quantitative PCR and western blot.(2)functional analysis of breast cancer cellsBreast canCCK8,Transwell cell migration,Wound healing assay and Cell colonies formation assays to detect cell proliferation and invasion ability after WTX was forced over-expressed or knocked down.(3)Investigating the effect of WTX on the ability of breast cancer cell line SKBR3 to form tumors in nude mice.3.Preliminary study on the mechanism of WTX in breast cancerThe KEGG pathway enrichment analysis was performed by GSEA to find molecular pathways that are closely related to WTX low expression enrichment.Results1、Immunohistochemical detection of WTX expression on paraffin sections of human breast cancer tissueWTX is expressed in mammary tissue and cancer tissue,and primary focus in the cytoplasm.WTX expression in cancer tissue was lower than normal mammary tissue in four types of breast cancer:Luminal A,Luminal B,Her-2,and Basal-like,the difference was statistically significant(P<0.05,x2).Breast cancer lymph node metastasis,tumor tissue size and histological grade were negatively correlated with WTX in invasive breast cancer tissue(P<0.05,x2),and the WTX may have little relationship with the age,molecular type,and immunohistochemical indexes such as ER,PR,Her-2,and Ki-67 of breast cancer patients(P>0.05,x2).2、Proliferation of breast cancer cells in vitro after WTX was forced overexpressed or knocked downCCK8(cell counting kit8)results showed that when compared with MDA-MB231-NC or Hs578T-NC groups,the in vitro proliferation speed of MDA-MB-231-W or Hs578T-W groups were decreased((P<0.001,P<0.001).when compared with MDAMB-231-NC,MDA-MB-231-NC and SKBR3-NC groups,the in vitro proliferation speed of MDA-MB-231-shW,MDA-MB-435-shW and SKBR3-shW groups were increased((P<0.001,P<0.001,P<0.001).Using Cell colonies formation assays to detect breast cancer cell proliferation ability changes between groups after WTX was forced over-expressed,the number of form clones in MDA-MB-231 cell was decreased(P<0.001),Cell colonies formation assays carried out in breast cancer cells after WTX was forced knocked down showed,the number of form clones in MDA-MB-231,MDA-MB-435 and SKBR3 cell was increased(P<0.01,P<0.01,P<0.01).Indicating that WTX forced over-expression reduces cell proliferation ability in breast cancer cells suggest that the WTX gene can inhibit the proliferation ability of breast cancer MDA-MB-231 and Hs578T cells;the knocked down of the WTX gene can promote the proliferation ability of breast cancer MDA-MB-231,MDA-MB-435 and SKBR3 cells.3、The ability of breast cancer cells to migrate in vitro after WTX was forced over-expressed or knocked downUsing Transwell migration experiment to detect breast cancer cell migration ability between groups after WTX was forced over-expressed,MDA-MB-231-W and Hs578T-W cells showed much slower migration ability when compared to the negative control MDA-MB-231-NC and Hs578T-NC cell lines,and the reduce was statistically significant(P<0.001,P<0.001).After WTX was knocked down,the migration ability of MDA-MB-231-shW,MDA-MB-435-shW and SKBR3-shW group was significantly enhanced compared with MDA-MB-231-NC,MDA-MB-435-NC and SKBR3-NC cell group,and the difference was significant(P<0.01,P<0.05,P<0.001).Wound healing assay result showed that,MDA-MB-231-W and Hs578T-W cells showed the weakest migration ability,the negative control MDA-MB-231-NC and Hs578T-NC cells showed the strongest,MDA-MB-231-shW,MDA-MB-435-shW and SKBR3-shW cells showed the strongest migration ability,the negative control MDA-MB-231-NC,MDA-MB-435-NC and SKBR3-NC cells showed the weakest.To sum up,the results suggest that the forced over-expression of WTX in MDAMB-231 or Hs578T cell lines inhibit the migration ability in vitro;the knocked down of WTX in SKBR3 cell lines promote the migration ability in vitro.4、The effect of lentivirus-mediated WTX gene knocked down on the growth of breast cancer xenografts in nude miceAfter WTX gene knocked down,SKBR3-shW group of nude mice orthotopic transplantation tumor grew faster and than SKBR3-NC cell group and the tumor volume is larger than the SKBR3-NC cell group,proved that knocked down WTX gene can promote breast cancer cell SKBR3 growth and proliferation in vivo(P<0.05).5、Through the enrichment analysis of KEGG pathway through GSEA,it can be found that WTX is related to various pathways such as calcium signal pathway and autophagy.Conclusions1.WTX is low-expressed in breast cancer tissues and has a negative correlation with lymph node metastasis,tumor tissue size and histological grade.2.WTX inhibits the proliferation,migration and invasion of breast cancer cells.3.WTX is related to calcium ion signaling pathway,autophagy and other pathways. |
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