| Background:Colorectal cancer is one of the five leading cancers in prevalence and mortality rate,and poses a major threat to the public health in china and worldwide.The initiation and progression of colorectal cancer is a multi-step pathological process regulated by a number of dysfunctional genes and signaling pathways.While pathological alterations of genes such as TP53,APC?KRAS,BRAF and CTNNB has been well recognized in the initiation of colorectal cancer,advancement in gene sequencing technology has identified splice variants of certain genes that may also take part in the pathology of colorectal cancer.Splice variants are product of RNA splicing,a process in which pre-mRNA is cut and reconnected at specific splicing sites to generate different mRNA transcripts.This leads to the generation of a variety of structuraly similar but functionally different protein products from a single gene.The expression profile of splice variants from certain genes may be altered in tumor tissues in comparison to normal tissues,resulting in changes in tumor biology.For example,CD44 gene splice variant CD44v6 is overexpressed in a variety of tumors and participates in tumor metasatasis and invasion.In addition,CD44v6 is implicated in the prognosis of colorectal cancer.LGR5(Leucine-rich repeat-containing G-protein coupled receptor 5)belongs to the family of G-protein-coupled receptor(GPCR).It is predominantly expressed in highly proliferative tissues such as the digestive tracts,hair follicles,ovary,and endometrium.LGR5 is overexpressed in colorectal cancer.It generates 4 splice variants(SPV).While SPV1,SPV2 and SPV3 can be translated into mature proteins,SPV4 is a non-coding mRNA,and cannot be appropriately translated.Early studies have shown that LGR5 splice variants SPV 1-3 can be detected in normal intestinal mucosa,with different abundancy in different compartments of the intestinal crypt,suggesting that these splice variants may play different roles in the differentiation and development of intestinal cells.However,the distribution of LGR5 splice variants in colorectal cancer tissue,its clinical significance as well as biological functions in tumor biology has not been investigated.Objective:In this study,we measured the abundancy of LGR5 splice variants SPV1,SPV2 and SPV3 in colorectal cancer tissues and analysed its clinical significance in terms of pathological characteristics.We further investigated the function of the respective splice variants in cancer cell motility and proliferation,and its role in associated signaling pathways.Methods:?.The expression and clinical significance of LGR5 splice variants in colorectal cancer1.We designed specific primers for LGR5 splice variants SPV1-3,and constructed eukaryote expression plasmids containing the respective splice variants for primer validation.2.We selected 93 pairs of colorectal cancer and normal mucosa for measurement of the three LGR5 splice variants by RT-qPCR,which was further analyzed in terms of pathological characteristics.?.Functional study of LGR5 splice variants1.RKO and HT29 colorectal cell-lines were used for stable overexpressiong of SPV1,SPV2 and SPV3 respectfully.2.CCK8,Transwell assay and wound healing assay was used evaluate the effect of overexpressing LGR5 splice variants on cell proliferation and migration.3.Alterations in EMT pathway associated proteins such as E-cadherin and Vimentin as well as Wnt pathway associated proteins such as ?-catenin and phosphorylated LRP6 was measured by western blotting to evaluate the effect of LGR5 splice variant overexpression on the respective pathways.4.Wnt pathway specific target gene AXIN2 mRNA was measured to evaluate the regulation of Wnt pathway by LGR5 splice variants.Results:?.The expression and clinical significance of LGR5 splice variants in colorectal cancer1.The level of all three LGR5 splice variants SPV1-3 was significantly higher in normal colorectal mucosa in comparison to cancer tissue(n=93)(SPV1:42.73±5.26 vs 24.60±3.34,p<0.0001;SPV2:43.43±5.71 vs 36.78±2.66,p=0.0041;SPV3:53.77±7.63 vs 27.76±4.15,p=0.0009).2.SPV1 levels were not correlated with clinical staging,differentiation state,lymph node metastasis and distant metastasis(p>0.05).3.SPV2 levels were significantly higher in poor-differentiated versus medium-to-high-differentiated colorectal cancer(SPV2:31.69±7.15 vs 56.49±8.75,p=0.0367);In stage ?/? versus stage ?/? colorectal cancer(SPV2:71.72±14.26 vs 37.10±6.03,p=0.0184);And showed no correlation with lymph node or distant metastasis(p>0.05).4.SPV3 levels were significantly higher in poor-differentiated versus medium-to-high-differentiated colorectal cancer(SPV3:97.84±24.69 vs 43.91±7.16,p=0.0057);In stage ?/? versus stage ?/? colorectal cancer(SPV3:35.14±7.64 vs 74.51 ± 13.12,p=0.0074);And lymph node(SPV3:39.53±7.35 vs 75.28±14.26,p=0.0138)and distant metastasis(SPV3:47.37±5.98 vs 71.641 ± 12.46,p=0.0363)versus non-metastatic colorectal cancer.?.Functional study of LGR5 splice variants1.Overexpression of LGR5 splice variants did not affect the proliferation of RKO and HT29 in vitro.2.Overexpression of SPV1 and SPV2 facilitated migration of RKO and HT29,while SPV3 suppressed this process.3.Overexpression of SPV1 and SPV2 in RKO and HT29 cells facilitated the EMT pathway and strenghted Wnt pathway activation,while overexpression of SPV3 inhibited the EMT pathway and alleviated Wnt pathway activation.Conclusions:1.LGR5 splice variants SPV1,SPV2 and SPV3 levels are significantly higher in colorectal cancer tissues compared to normal colorectal mucosa,suggesting a role in the initiology of colorectal cancer.2.SPV2 and SPV3 showed a similar pattern in terms of pathological characteristics:High abundancy in low-differentiated and high clinical grade colorectal cancer in comparison to medium-to-high-differentiated and low clinical grade colorectal cancer.In particular,SPV3 level was significantly correlated with colorectal cancer metastasis.SPV1 did not show significant correlation to pathological characteristics.Therefore,SPV3 could be a potential marker for the differentiation and metastatic characteristics of colorectal cancer.3.SPV1 and SPV2 show similar biological functions,opposite to that of SPV3.The former facilitates migration of colorectal cancer cells and enhance EMT and Wnt signaling which are inhibited by the latter.Therefore,SPV1 and SPV2 may take part in the initiation and metastasis of colorectal cancer.The biological function of SPV3 was not consistent with its clinical features,and additional investigation is required to explain this finding. |
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