The Function And Mechanism Of LRP5 Gene In Promoting The Colorectal Carcinogenesis

BackgroundColorectal cancer(CRC)is one of the common malignancies in digestive system,which is the third most common in worldwide.The abnormal activation of Wnt/β-catenin signaling is one of the foundations of colorectal carcinogenesis.However,the exact role of LRP5,an auxiliary receptor of Wnt ligand,in the colorectal carcinogenesis is still unclear.Therefore,it is necessary to explore the effect of LRP5 on the cancer stem cells(CSC)-like traits of CRC cells and the mechanisms in the occurrence and development of CRCObjectiveTo explore the effect of LRP5 on the tumorigenesis of CRC and the molecular mechanisms from a new perspective.Our finding could provide new biomarkers and drug target for the diagnostic and therapeutic management of CRCMethod1.To analyze the relationship between the expression level of LRP5 gene and colorectal carcinogenesis(1)The expression levels of LRP5 gene in CRC tissues with different pathological stages and the commonly used CRC cell lines,such as Caco2,HCT-116,LoVo and SW480 were analyzed by quantitative real-time PCR(RT-PCR)and immunohistochemistry(IHC)methods(2)The data on the expression levels of LRP5 gene and other genes in CRC tissues and corresponding normal ones were analyzed from the tumor databases,such as Oncomine and TCGA,and the correlation between mRNA level of LRP5 gene and clinical indicators such as survival time,tumor stage and grade,and tumor metastasis were also determined.At the same time,we used the data from the Human Protein Atlas database to analyze the LRP5 protein expression in CRC tissues and corresponding normal ones.2.CRC cell lines with stable activation or inhibition of LRP5 gene were constructed(1)CRC cell lines with stable activation or inhibition of LRP5 genes were constructed according to the following methods:CRISPR/Cas9 KO plasmid specifically targeting LRP5 gene(LRP5-KO),CRISPR plasmid activating LRP5 gene(LRP5-ACT)or their corresponding negative control plasmid was transfected into HCT-116 cells separately.Purinomycin was used as the drug to select the positive tranfected cells.The change of LRP5 mRNA levels in different cells was identified by RT-PCR,and cells with the expected changing trend of the LRP5 mRNA levels were regarded as the positive ones(2)Screening out the stable monoclonal cell lines:Single cell was selectively into 96-well plate by flow cytometer and expanded.Clones with the most significant activation or downregulation of LRP5 gene compared with their controls were regarded as stable monoclonal cell lines and used for further analysis3.To identify whether activating LRP5 gene could promote the development of CRC and to reveal the underlying mechanism(1)The selected monoclonal CRC cell lines were used to investigate the effect of activation of LRP5 gene on the malignant behavior of CRC and xenograft tumor assay in nude mice.Meanwhile,cells were treated with cisplatin to analyze the change of apoptosis rate of HCT-116 cells after activating LRP5 gene(2)The expression levels of crucial genes in canonical Wnt/β-catenin signaling pathway were detected in LRP5-ACT CRC cells and control ones.(3)The mRNA and protein levels of crucial genes in IL-6/STAT3 signaling pathway were detected in LRP5-ACT CRC cells and control ones.(4)The expression levels of crucial genes related to the CSC-like traits were detected in LRP5-ACT CRC cells and control ones.(5)HCT-116 cells were divided into CD133+and CD133’ groups by flow cytometry,and the expression levels of crucial genes in canonical Wnt/β-catenin and IL-6/STAT3 pathways were detected Meanwhile,the expression levels of LRP5 gene in other CD133+and CD133-CRC cell lines were determined by analyzing the online datasets about CRC.4.To identify whether silencing LRP5 gene could inhibit the development of CRC and to reveal the underlying mechanisms(1)The selected monoclonal CRC cell lines were used to investigate the effects of downregulation of LRP5 gene on the malignant behavior of CRC and xenograft tumor assay in nude mice.(2)The expression levels of crucial genes in canonical Wnt/β-catenin and IL-6/STAT3 signaling pathways,and the apoptosis rate were detected in LRP5-KO CRC cells and control ones.(3)The expression levels of crucial genes related to the CSC-like traits were also detected in LRP5-KO CRC cells and control ones.Result1.The relationship between the mRNA levels of LRP5 gene and colorectal carcinogenesisThe results of RT-PCR and 1HC showed that mRNA levels of LRP5 gene in CRC tissues was increased compared with normal ones.Moreover,the mRNA levels of LRP5 gene was positively correlated with the degree of malignant progression of CRC.2.CRC cell lines with stable activation or inhibition of LRP5 gene were successfully constructed3.Activation or inhibition of LRP5 gene could promote or inhibit the progression of CRC(1)The results of RTCA showed that activation of LRP5 gene could increase the proliferation capacity of HCT-116 cells.Conversely,knockdown of LRP5 gene decreased the proliferation capacity of HCT-116 cells.(2)The results of wound healing migration assay showed that the scratch healing rate of CRC cells increased after the activation of LRP5 gene,whereas knockdown of LRP5 gene in HCT-116 cells decreased the proliferation capacity.(3)The results of colony formation assay showed that activation of LRP5 gene could enhance the tumorigenic ability of HCT-116 cells.On the contrary,knockdown of LRP5 gene reduced the tumorigenic ability of CRC cells.(4)The results of tumorsphere formation results showed that activation of LRP5 gene could increase the volume of tumorspheres of HCT-116 cells.Conversely,silencing of the LRP5 gene decreased the volume of tumorspheres of HCT-116 cells.(5)The results of cisplatin sensitivity test showed activation of LRP5 gene could decrease the sensitivity of HCT-116 cells to cisplatin.On the contrary,knockdown of LRP5 gene increased the sensitivity of HCT-116 cells to cisplatin.(6)The results of xenograft tumor assay showed activation of LRP5 gene could accelerate the tumor formation in nude mice.On the contrary,knockdown of LRP5 gene inhibited the tumor formation in vivo4.The mechanisms of activation or inhibition of LRP5 gene in promoting or inhibiting CRC development(1)The mRNA levels of crucial genes in canonical Wnt/β-catenin pathway,such as Wnt3,CTNNB1,c-Myc,CCND1 and COX2 were all increased in LRP5-ACT CRC cells.On the contrary,the mRNA levels of these genes were decreased in LRP5-KO CRC cells.(2)The activation of LRP5 gene in HCT-116 cells upregulated the mRNA levels of several stemness-related genes,such as CD133,ALDH1A1,Bmil,OCT3/4 and Nanog.On the contrary,the mRNA levels of these genes were decreased in LRP5-KO CRC cells.(3)Bioinformatics data showed that mRNA levels of LRP5 gene was positively correlated with that of CD 133 gene.(4)We separated CD133 positive(CD133+)cells and CD133 negative(CD133-)ones from HCT-116 cells by flow cytometry.The mRNA levels of Wnt3,LRP5,CTNNB1,c-Myc,CCND1 and COX2 gene in CD133+HCT-116 cells were all upregulated compared with those in CD133-ones.(5)The mRNA levels of lL-6 and STAT3 genes were upregulated in CD133+HCT-116 cells compared with that in CD 133-ones.(6)The results of RT-PCR and Western bolt showed that the expression levels of IL-6 and STAT3 genes in HCT-116 cells were upregulated when LRP5 gene was activated.On the contrary,those in HCT-116 cells were all reduced after the silence of LRP5 gene.(7)Bioinformatics data showed that mRNA level of LRP5 gene was positively correlated with that of STAT3 gene,and mRNA level of CD133 gene was also positively correlated with that of STAT3 geneConclusion1.The expression level of LRP5 gene was upregulated in CRC tissues and corresponding cell lines and exerted a carcinogenic effect on the progression of CRC.2.Activation of LRP5 gene could promote colorectal carcinogenesis by enhancing the CSC-like traits,activating the canonical Wnt/β-catenin and IL-6/STAT3 signaling pathways,thus to reduce the sensitivity of CRC cells to chemotherapy drugs.3.Inhibiting endogenous LRP5 gene could inhibit colorectal carcinogenesis by weakening the CSC-like traits,inhibiting the canonical Wnt/β-catenin and IL-6/STAT3 signaling pathways,thus to enhance the sensitivity of CRC cells to chemotherapy drugs.4.LRP5 gene could be taken as the novel biomarker for the diagnosis of CRC and potential drug target for the prevention and treatment of CRC.