GOLPH3 Promotes The Mechanism Of Stem Cell-like Phenotype Enhancement In Non-small Cell Lung Cancer (NSCLC)

Background/Aims: Golgi phosphoprotein 3(GOLPH3)is a new oncogene and mainly located at trans-Golgi network(TGN).Recent research had shown that GOLPH3 highly implicated in the development of several human tumors.Abundant evidences have revealed cancer stem cells are strongly associated with self-renewal,proliferation,differentiation,tumorigenesis,invasion,metastasis,drug-resistance and recurrence.It also has been reported that the development of NSCLC is closely related to cancer stem cells.However,the clinical significance and biological function of GOLPH3 in stem cell-like phenotypeof NSCLC remains little to known.Our aim is to study the relationship between GOLPH3 and stem cell-like phenotype of NSCLC,and explore its molecular mechanism.Method: Western Blot was used to check the expression of GOLPH3 in the previously constructed NSCLC cell lines(A549,NCI-H460).And then,investigating the effect of GOLPH3 on NSCLC stem cell-like phenotype related protein markers via Gene Set Enrichment Analysis(GSEA)and Real-time quantitative PCR.Furthermore,Sphere formation assay,Flow cytometric analysis and Tumor xenografts assay were used to explore whether GOLPH3 can enhance the stem cell-like phenotype in NSCLC.GSEA and the Luciferase reporter assay examined Wnt/β-catenin signalingpathway correlated with GOLPH3 and stem cell-like phenotype.Furthermore,we used Immunofluorescence assay and Subcellular fractionation assay to explore whether GOLPH3 can promoteβ-catenin accumulation in the nucleus of NSCLC cell lines.Then we used si RNA(LEFI-si RNA,TCF4-si RNA)transfect the GOLPH3-overexpressing cell lines,added with Luciferase reporter assay,Flow Cytometry Analysis,and Real-time PCR to study how GOLPH3 affect on the stem cell-like phenotype of NSCLC cells after silencing TCF4/LEF1.Result: GOLPH3 was stable express in the previously constructed NSCLC cell lines(A549,NCI-H460).From the SPSS soft analysis we found GOLPH3 expression has a positive relation with the expression of cancer stem cell markers,and has statistically significant.The result of Quantitative real-time PCR showed that GOLPH3 over expression can result in the high expression of these stem cell makers.Flow cytometric analysis found the proportion of side-population-positive(SP+)is higher in GOLPH3 stable expression NSCLC cell lines than control.Sphere formation assay consequence displayed that the GOLPH3-transduced cell lines get more ability of sphere formation,both number and volume,than control cell lines.On the contrary,silenced the express of GOLPH3 can lead to the low expression of these stem cell markers.The proportion of side-population-positive(SP+)was less in GOLPH3 silenced cell lines than the control group.And the GOLPH3-silenced cell lines almost loss the ability of sphere formation,neither the number nor the volume.The outcome of Animal experiments also exhibited that volume tumors formed by A549-GOLPH3-Over cells are bigger than those formed by the paired A549-GOLPH3-Vector cells,and the speed of tumourigenesis is faster.By contrast,volume tumors formed by A549-GOLPH3 RNAi cells were obviously less than those formed by the paired A549-GOLPH3 Vector cells,and the speed of tumourigenesis was lower.GSEA outcome indicated that it was a positive correlation between GOLPH3 and Wnt/β-catenin signaling pathway.TOP/FOP Flash consequence showed that the over expression of GOLPH3 could active Wnt/β-catenin signaling pathway.Ncleocytoplasmic separation assay and Immunofluorescence assay demonstrated GOLPH3 over-expression could promote theβ-catenin accumulated in cell nucleus.The result of Quantitative real-time PCR displayed that GOLPH3 can enhance Wnt signaling downstream genes m RNA levels by activating Wnt/β-catenin signaling pathway.On the contrary,silenced GOLPH3 suppress the activation of Wnt/β-catenin signaling pathway,inhibit β-catenin entrancing into cell nucleus.And it could decrease them RNAlevel of Wnt signaling downstream genes.Conclusions: The experimental results showed that the oncogene GOLPH3 play an important role in regulating the biological function of cancer stem cell-like phenotype in NSCLC.Over-expression of GOLPH3 could activate the Wnt/β-catenin signaling pathway,promoteβ-catenin entrancing into the nucleus and bind to the transcription factor TCF4/LEF1,which could initiate the expression of downstream target genes and enhance the stem cell-like phenotype of NSCLC.Silencing TCF4/LEF1 could block the Wnt/β-catenin signaling pathway,and decrease the stem cell-like phenotype of NSCLC.