The Role And Molecular Mechanism Of TUSC3 Gene In Regulating Stemness And Drug-resistance Of Human Colorectal Cancer

BACKGROUND&OBJECTIVEColorectal cancer(CRC)is one of the most common malignant tumors of human digestive system,ranking at the third place of malignant tumor-associated mortality in China.At present,the major treatment of CRC is surgical resection,supplemented by chemical drug treatment.Nonetheless,there are still a considerable number of patients have postoperative recurrence and metastasis.The formation of sternness and drug-resistance are the two main reasons responsible for the failure of chemotherapy.Leading to the recurrence and metastasis of CRC.Therefore,it is important to clarify the molecular mechanism of the formation of stemness and drug-resistance of CRC cells,which may provide new ideas and solutions for clinical treatment of CRC.TUSC3(tumor suppressor candidate 3)gene,also known as D8S1992,M33,MRT22,MRT7,N33,and OST3A,expresses widely in human tissues such as brain,heart,lung,and liver.TUSC3 mainly locates in the endoplasmic reticulum,and catalyzes the N-glycosylation of the protein.Aberrant N-glycosylation of protein may promote tumor development.What's more,TUSC3 is also an integral member of Mg2+transportion on the cell membrane.TUSC3 plays an important role in the balance between the total magnesium concentration and the free magnesium concentration.Lack of TUSC3 may affect the absorption of magnesium and lead to developmental disorders.It has been reported that loss of TUSC3 results in the intellectual disability,Alzheimer's disease and Parkinson's disease.Besides,TUSC3 is considered as a tumor suppressor gene in a variety of cancers,such as prostate cancer,ovarian cancer,pancreatic cancer,and breast cancer etc.However,earlier studies in our group suggested that TUSC3 has a significant effect in promoting CRC invasion and metastasis.Since the role of TUSC3 in CRC is not fully understood,we further explore the relationship between TUSC3 and CRC stemness and drug-resistance based on our previous bioinformatic analysis.In this study,we explored the expression and clinical significance of TUSC3 in CRC.The relationship between TUSC3 and CRC cell stemness and drug-resistance was also detected.To clarify the underlying molecular mechanism,we further examined the correlation between TUSC3 and Hedgehog signaling pathway.METHODS1.Expression level and clinical significance of TUSC3 in CRCThe qRT-PCR assay was used to detect mRNA expression levels of TUSC3 in 38 cases of CRC tissue and paired normal mucosa.Immunohistochemistry was used to detect TUSC3 protein expression levels in 90 cases of CRC tissue and paired normal mucosa.The relationship between TUSC3 expression and clinical parameters of CRC patients was also analyzed.The protein and mRNA expressions of TUSC3 in 7 different CRC cell lines and a normal colon epithelial cell line were detected by western blot and qRT-PCR.The GEPIA Web site(http://gepia.cancer-pku.cn/)and Genomics of Drug Sensitivity in Cancer(GDSC,https://www.cancerrxgene.org/)databases were used to research the expression level of TUSC3 and its clinical significance in CRC.2 TUSC3 regulates stemness and drug-resistance of CRC cellsTwo cell lines with low expression levels of TUSC3 were over-expressed,two cell lines with higher expression levels of TUSC3 were silenced.Tumorsphere Formation Assays in vitro was used to induce CRC stem cells,and the expression level of TUSC3 in CRC stem cells was detected by western blot.In order to confirm whether TUSC3 plays a role in the regulation of CRC cell sternness,the expression of sternness-related protein was detected in TUSC3 over-expressed or silenced CRC cell lines by western blot.MTT assay was used to detect the changes of drug-resistance in CRC cells after the over-expression and silence of TUSC3,flow cytometry assay was used to detect the changes of apoptosis in CRC cells after the over-expression and silence of TUSC3,and the expression of drug-resistance related protein was detected by western blot.The correlation of TUSC3 and the sternness marker CD 133 or drug-resistance marker ABCC1 was detected by Immunofluorescence.To clarify whether TUSC3 regulates sternness and drug-resistance in CRC cell lines.3.The molecular mechanism of TUSC3 in regulating the stemness and drug-resistance of CRC cellsWe analyzed TCGA database and gene chip,and screened out that the HH signaling pathway was associated with aberrant TUSC3 expression.Then the expression levels of SMO and Glil,two activator of Hedgehog pathwy,were detected by immunohistochemistry assay in 90 cases of CRC tissue and paired normal mucosa.Western blot assay was used to detect the expression of proteins of HH signaling pathway in TUSC3-overexpressed and TUSC3-silenced CRC cell lines.The TUSC3 overexpressed and silenced cell lines were treated with HH signaling pathway inhibitors and HH signaling pathway agonists respectively,changes of function and protein expression were detected,with an expection to further clarify the molecular mechanism of TUSC3 on the regulation of stemness and drug-resistance in CRC cell lines.Furthermore,Co-Immunecoprecipitation,western blot and immunofluorescence assays were also used to detect the correlation between SMO and TUSC3.Tunicamycin and GNPase F were used to detect N-glycosylation level of SMO protein.RESULTS1 The expression of TUSC3 in CRC cell lines and tissues and its clinical significance(1)The results of qRT-PCR and immunohistochemistry showed that TUSC3 overexpressed in CRC tissues,positively associated with T,N,and M stages of CRC patients.(2)The results of qRT-PCR and Western Blot showed the expression of TUSC3 mRNA and protein are higher in most CRC cell lines than in normal colon epithelium FHC.(3)GEPIA(http://gepia.cancer-pku.cn/)analysis also revealed that TUSC3 is positively correlated with the TNM stage of CRC patients while negatively correlated with postoperative disease-free survival time and postoperative overall survival time.2 TUSC3 regulates the sternness of CRC cells(1)The whole-genome expression patterns for LS174T-NC and LS174T-TUSC3 were detected by gene chip,the expression level of TUSC3 is associated with that of CD133 and Lgr5.(2)Immunofluorescence assay showed the expression level of TUSC3 is associated with that of CD 133.(3)Results of In Vitro Tumorsphere Formation Assays showed the expression of TUSC3 is up-regulated in the CSC cells which was validated by western blot.(4)The expression levels of CRC stem markers Lgr5 and CD 133 were up-regulated in TUSC3-overexpressed cell lines,while both Lgr5 and CD 133 were down-regulated in TUSC3-silenced cell lines.The opposite effect could be observed in the TUSC3-silence CRC cells.(5)The results of qRT-PCR and tissue microarray immunohistochemistry showed that TUSC3 was significantly correlated with CD133 in protein levels.3 TUSC3 regulates drug-resistance of CRC cells(1)TUSC3 gene was related with drug-resistance of 5-fluorouracil and cisplatin in CRC cell lines.(2)Immunofluorescence showed that TUSC3 was co-expressed with CRC drug-resistance marker ABCC1.(3)MTT assay and Flow Cytometry assay showed that CRC cell lines with overexpression of TUSC3 were resistant to 5-fluorouracil and cisplatin treatment.The expression levels of ABCC1 and ABCC6 proteins were increased in TUSC3-overexpressed CRC cell lines,while decreased in TUSC3-silenced CRC cell lines.(4)Knocking out TUSC3 had no statistically significant effect on the drug-resistance of CRC cells in vivo.4 The molecular mechanism of TUSC3 in regulating the stemness and drug-resistance of CRC cellsWe screened the HH signaling pathway from the TCGA database and gene chip.Immunohistochemistry confirmed that the HH signaling pathway is widely activated in CRC.Western blot experiments verified that the HH signaling pathway is indeed the downstream signaling pathway of TUSC3.Afterwards,the TUSC3 overexpressed cell lines was treated with HH signaling pathway inhibitors,the TUSC3 silenced cell lines was treated with HH signaling pathway agonists,changes of related functions and protein expressions was detected.The results showed that the significant changes in sternness,drug-resistance,and expression differences disappeared,thus further confirm that TUSC3 is responsible for the sternness of colorectal cancer cell lines via the HH signaling pathway.The results of co-immunoprecipitation indicated that TUSC3 interacts directly with SMO protein in HH signaling pathway,but not direct interaction with CD 133 and ABCC1.There is a spatial overlap between TUSC3 protein and SMO protein detected by immunofluorescence.The glycosylation results showed that the SMO protein is associated with N-glycosylation modification.We speculated that TUSC3 may involved in the modification of N-glycosylation of SMO protein,activatied the HH signaling pathway,so as to promote CRC cell stemness and drug-resistance.CONCLUSIONTUSC3 is up-regulated in CRC cells in vivo and in vitro,and is positively correlated with the stage of CRC patients and negatively correlated with the survival of CRC patients.TUSC3 promotes the stemness and drug-resistance of CRC cells via Hedgehog signaling pathway,which may involved in the aberrent N-glycosylation modification of SMO protein.Therefore,knocking out of TUSC3 may provide new ideas and protocols for the clinical treatment of CRC.INNOVATION1.This study explores overexpression of TUSC3 in CRC is associated CRC stage and poor prognosis,elucidates the function of TUSC3 in regulating the sternness and drug-resistance of CRC cells from clinical specimen and bioinformatics analysis.2.This study reveals the molecular mechanisms of TUSC3 in regulating the stemness and drug-resistance,so as to provide the theoretical basis and experimental evidence for the treatment of CRC.