RNF8 Induces ?-catenin-mediated C-myc Expression And Promotes Colon Cancer Proliferation

Objective: Colorectal cancer(CRC)is the fourth most commonly diagnosed and the second leading cause of cancer mortality worldwide.In recent years,new treatments for CRC have been developed,which have improved the overall survival of CRC patients significantly.Nevertheless,50% of CRC patients undergo disease recurrence.Therefore,it is urgent to unravel the underlying molecular mechanisms of CRC oncogenesis and progression to identify novel therapeutic targets.In the Wnt/?-catenin pathway,?-catenin cannot be phosphorylated by GSK3?.Non-phosphorylated ?-catenin cannot be degraded by the proteasome,causing ?-catenin to accumulate in the cytoplasm and move into the nucleus.In turn,it activates the expression of its downstream target gene c-Myc,which is closely related to tumor cell proliferation.Ring finger protein 8(RNF8),which belongs to the RING finger E3 ligase family,is an essential factor for transducing DNA double-strand breaks(DSB)signaling.It has been reported to be highly expressed in breast cancer and positively correlated with the progression of breast cancer by activating the Wnt/?-catenin pathway or enhancing the activity of Twist and estrogen receptor ?(ER?).These findings suggest an essential role of RNF8 in the progression of cancer.However,the exact function and mechanism of RNF8 in regulating the progression of CRC remain largely unknown.In this study,we found a positive correlation between RNF8 and c-Myc in colon cancer tissues.Subsequently,our results showed that RNF8 induced c-Myc expression via binding to ?-catenin and facilitating its nuclear translocation by conjugating K63 polyubiquitination onto it.Furthermore,knockdown of RNF8 significantly inhibited the growth of colon cancer cells in vitro and in vivo.These results indicate that RNF8 promotes colon cancer proliferation by upregulating ?-catenin-mediated c-Myc expression,and provide a new insight into the mechanism of colon cancer progression.Methods: Part I: Analysis of the expression of RNF8 and c-Myc in colon cancer and the correlation with the prognosis of colon cancer patients:(1)Using colon cancer RNA-seq data in the TCGA database,478 patients with colorectal cancer were detected And the m RNA levels of RNF8 and MYC in tissue samples of 41 normal control patients.(2)The difference between RNF8 and c-Myc protein expression in colon cancer tissues and benign adjacent tissues was detected by immunohistochemical staining.The second part: RNF8 regulates the function and mechanism of colon cancer cells:(1)Design siRNA of RNF8,detect the effect of RNF8 on MYC transcription level by Realtime PCR;design the overexpression plasmid and sh RNA of RNF8,The expression efficiency was detected in SW480;the protein levels of RNF8,c-Myc,and ?-catenin in colon cancer cell lines HCT116 and SW480 were subsequently detected by Western blot to reflect the exogenous overexpression of RNF8 and the endogenous knockout of both influences.(2)Use immunoprecipitation experiments to detect whether there is an interaction between RNF8 and ?-catenin.Overexpression and silencing of RNF8 in HCT116 and SW480 cells to explore whether it can affect the distribution of ?-catenin in the nucleus.(3)Overexpress RNF8 and ubiquitin in HCT116 and SW480 cells,and then detect the ubiquitination of ?-catenin by the ubiquitination test.(4)Obtain the gene fragment of RNF8 interference sequence,construct the recombinant plasmid of the lentiviral vector,and then conduct the lentiviral packaging and titer detection,and use the lentivirus to transfect the colon cancer cell lines HCT116 and SW480 to construct the HCT116 which stably silences RNF8 And SW480 cell line,and these cells were used for clone formation experiments and CCK8 proliferation assay experiments.Results:(1)RNF8 induces c-Myc expression through ?-catenin: compared with the negative control NC group,the m RNA level of si RNF8 is significantly reduced;and when RNF8 is knocked down,the m RNA level of MYC is significantly reduced.This suggests that RNF8 can up-regulate the transcription of MYC.When the expression of RNF8 protein increased,the expression of c-Myc protein also increased;secondly,we found that after stable silencing of RNF8,the expression of c-Myc protein also decreased correspondingly compared with the normal control group.The above results further suggest that RNF8 may up-regulate the expression of c-Myc.Next,we overexpressed RNF8 and silenced ?-catenin in HCT116 and SW480 cells,and detected the expression level of c-Myc.The results showed that when ?-catenin in HCT116 and SW480 cells was silenced,the induction of c-Myc expression by RNF8 overexpression was greatly reduced.These results indicate that RNF8 induces the expression of c-Myc dependent on ?-catenin.(2)RNF8 facilitates ?-catenin nuclear translocation.We performed co-immunoprecipitation between RNF8 and ?-catenin in HCT116 and SW480 cells,respectively.Found that there is an interaction between the two.Next,we performed nuclear extraction in HCT116 and SW480 cells,and the results showed that overexpression or silencing of RNF8 increased or decreased the protein level of ?-catenin in the nuclear component.(3)RNF8 promotes K63 polyubiquitination of ?-catenin.We overexpressed RNF8 and ubiquitin in HCT116 and SW480 cells,and then detected the ubiquitination of ?-catenin by the ubiquitination assay.To further analyze its ubiquitin type,RNF8 assembled a polyubiquitin chain on ?-catenin on the K63 ubiquitin bond.(4)We constructed HCT116 and SW480 cell lines containing sh RNA lentiviral infection and stably silenced RNF8 for clone formation experiments and CCK8 proliferation assay experiments.It was found that the loss of RNF8 would lead to a weakening of colon cancer proliferation ability.Conclusion:1.RNF8 expression increases in colon cancer and is positively correlated with c-Myc;2.RNF8 induces c-Myc expression via ?-catenin;3.RNF8 facilitates ?-catenin nuclear translocation;4.RNF8 promotes K63 polyubiquitination of ?-catenin;5.RNF8 is required for the proliferation of colon cancer cellsIn the review,we conclude that RNF8 promotes nuclear translocation of ?-catenin in colon cancer cells through K63-linked polyubiquitination,thereby enhancing the expression of its downstream target gene MYC,thereby promoting colon cancer cells proliferation.These findings reveal new mechanisms for colon cancer development,which could be potential therapeutic targets for colon cancer treatment.