The Molecular Mechanism Of RPL34 Deubiquitination Mediated By CAND1 Promoting The Invasion And Metastasis Of Colorectal Cancer Cells

Objective:Colorectal cancer(CRC)is the third most common cancer and the second most common cause of cancer death in the world.The International Agency for Research on Cancer(IARC)reported more than 193,000 new cases of CRC worldwide in 2020,accounting for 10.0%of the total incidence of cancer and more than 953,000 deaths.Among them,the overall incidence of CRC decreased in middle-aged and elderly people,but showed an increasing trend in young people.CRC is a heterogeneous disease with many risk factors that increase the likelihood of developing it,including a history of inflammatory bowel disease,changes in diet and lifestyle.Currently,treatment for CRC patients includes surgery,radiotherapy,chemotherapy,immunotherapy and biotargeted therapy.Although extensive CRC screening and surgical excision have improved overall survival for CRC patients,about 25%of CRC patients are diagnosed with distant organ metastases at a later stage and are difficult to manage surgically.Treatment of advanced CRC remains a challenge due to the lack of highly specific biomarkers and the complex biology of CRC,the lack of drugs targeting colorectal cancer stem cells,and resistance or intolerance to current therapies.Therefore,the discovery of new CRC targets and elucidation of relevant mechanisms are essential to improve treatment outcomes for CRC patients.Ribosomal Proteins(RP)are an important component of ribosomes,and the expression patterns in various human tumors are highly specific to tumor type and tissue.The Ribosomal Proteins L34(RPL34),a member of the RPs L34E family,are located in the cytoplasm and are mainly involved in the formation of the 60S subunit.In addition to its role in ribosome biogenesis,RPL34 has been found to be involved in other cellular functions such as cell proliferation,cycle,and biosynthesis.And RPL34 has carcinogenic or tumor suppressive properties in different malignancies.For example,Zhu et al.previously reported that overexpression of RPL34 inhibited the proliferation and ability of cervical cancer cells to migrate and invade.Yang et al.showed that RPL34 gene knockout could reduce the malignant proliferation of non-small cell lung cancer cells.However,whether RPL34 is involved in CRC tumorigenesis and metastasis and its upstream regulation mechanism has not been clarified.Cullin RING Ligases(CRLs)are the largest class of E3 ubiquitin ligases involved in protein ubiquitination mediated by the ubiquitin-proteasome system.Dysregulation of CRL is associated with many diseases and may be a potential anti cancer target.Cullin-associated NEDD8-dissociated protein 1(CAND1),a substrate receptor exchange factor for CRL,is a protein containing a 120kDa HEAT repeat sequence that inhibits the assembly of the multisubunit E3 ubiquitin ligase complex.It binds unacylated Cullin proteins in a manner that is reciprocal with the substrate receptor.CAND1 acts as a mediator to regulate protein degradation and stabilization.Accumulating evidence highlights the importance of CAND1 in several human cancers.However,the significance of CAND1 in CRC and mechanisms remains to be determined.We found that the expression of RPL34 was significantly up-regulated in colon cancer tissues through the website prediction results,so we speculated that RPL34 might be involved in the biological process of CRC.In addition,it is reported recently that ribosomal proteins,such as RPL23,were shown to be degraded by the ubiquitin-proteasome system.And we found that CAND 1 interacts with RPL34 through the analysis of bioinformatics.In summary,we speculate that CAND1 plays an important role in CRC progression by possibly modulating RPL34.This study provides a new research target for CRC treatment and is of great significance for improving the clinical efficacy of cancer treatment and the survival rate of patients.Methods:(1)RPL34 expression and clinical correlation analysis in colorectal cancer tissues:Bioinformatics analysis of the mRNA expression of RPL34 in all stages,lymph node metastasis stages,and pathological subtypes of CRC.30 pairs of fresh colorectal cancer and paracancer tissue samples without chemoradiotherapy were collected,and the expression of RPL34 in cancer and paracancer tissues was detected by RT-qPCR.The expressions of RPL34 in cancer and adjacent tissues were detected by Western blot.In situ colorectal cancer samples from 36 CRC patients without chemoradiotherapy were collected for paraffin section preparation.The expressions of RPL34 at the central location of cancer tissue were detected by immunohistochemistry,and the correlation between the expression of RPL34 and the pathological indexes of patients was analyzed.(2)In vitro and in vivo experiments were combined to explore the regulatory effect of RPL34 on the biological behavior of colorectal cancer cells:RT-qPCR was used to detect the expression of RPL34 in colorectal cancer cells(HCT116,SW620,LoVo,HT29,SW480).RPL34 high expression cell line H and low expression cell line L were screened.Three siRNA and one negative control for RPL34 were designed and synthesized.The siRNA group was treated with SirNA-pool method,and RPL34 was knocked down using cell lines(H)with high RPL34 expression.Empty Vector or RPL34 overexpression vector(RPL34)was used to transfect L cells with RPL34 overexpression.The experiment was divided into 6 groups:(H)Control,NC siRNA,siRPL34,(L)Control,Vector and RPL34.48 h after transfection,the expression of RPL34 was verified by RT-qPCR and Western blot.Cell proliferation was verified by CCK-8 assay at 0,12,24,48,and 72 h.48 h after transfection,cells in S phase were detected by EdU staining for 2 h.The cell cycle was detected by flow cytometry 48 h after transfection.Cell migration ability was detected by scratch assay(0,12,24 h).Transwell assay was used to detect cell invasion ability(24 h).48 h after transfection,the expression levels of E-cadherin,N-cadherin and ZEB1 in the cells were detected by Western blot.The expression levels of p-JAK2,JAK2,p-STAT3(Tyr705)and STAT3 in tumor tissues were determined by Western blot.JAK2 inhibitor AG490(20μM)was added 48 h after transfection of RPL34 into L cells.The experiment was divided into three groups:(L)Vector,RPL34 and RPL34+AG490.Cell viability was detected 24 h after transfection by CCK-8 assay.Cell migration ability was detected by scratch assay(0,12,24 h).Purchase male BALB/c nude mice aged 5-6 weeks.Tumor cells with stable knockout or overexpression of RPL34 were screened,and a certain amount of tumor cells in logarithmic growth stage were injected subcutaneously into the right axilla of nude mice.The experiment was divided into four groups:(H)shNC,shRPL34,(L)Vector and RPL34.Tumor diameter was measured every 3 days and tumor volume was calculated.30 days later,mice were sacrificed and tumor tissues were collected.Take pictures and plot the growth of the tumor.RPL34 was detected by Western blot.The expressions of Ki67,p-STAT3 and p-JAK2 were detected by immunohistochemical staining.Western blot was used to detect p-JAK2,JAK2,p-STAT3 and STAT3 in tumor tissues.A certain number of RPL34 stable knockdown or overexpressed tumor cells were injected into the distal spleen to suture the skin.After 5 weeks,the mice were killed and spleens and livers were collected.Detection of liver metastases by colorectal cancer bioluminescence system.The overall appearance of the spleen and liver was photographed and the number of metastatic nodules was quantified.Liver metastasis was observed by HE staining.(3)RPL34 influences the biological behavior of rectal cancer cells by regulating ubiquitination level through direct interaction with CAND1 protein:Bioinformatics methods were used to analyze the RPL34 PPI network and its functions.Thirty pairs of fresh colorectal cancer and adjacent tissue samples without radiotherapy and chemotherapy were taken,andthe expression of CAND1 in the cancer and adjacent tissue were detected using RT-qPCR;Western blot was used to detect the expression of CAND1 in cancer and adjacent tissues;The expression of CAND1 in the center of cancer tissue was detected by immunohistochemistry.The co-localization of RPL34 and CAND1 in H cells was detected by immunofluorescence.The presence of RPL34 and CAND1 binding was verified by co-immunoprecipitation in H and L cells.Three siRNA and one negative control for CAND1 were designed and synthesized.The siRNA group was treated with siRNA-POOL method and transfected into H cells.CAND1 overexpressed plasmid and its control were transfected into L cells.The experiment was divided into 6 groups:(H)Control,siNC,siCAND1,(L)Control,Vector and CAND1.48 h after transfection,the CAND1 expression was detected by RT-qPCR and Western blot.The expression of RPL34 was verified by Western blot.The ubiquitization level of RPL34 was determined by immunoprecipitation.The expression of RPL34 was detected by western blot after 4 h with or without the addition of 15 μM MG132.The experiment was divided into two groups:(H)siNC and siCAND1.After transfection 48 h and 72 h,CCK-8 assay was used to verify cell viability.Cell migration ability was detected by scratch assay(0,12,24 h).Transwell assay was used to detect cell invasion ability(24 h).CAND1 OE and RPL34 siRNA-pool were co-transfected into L cells to explore whether RPL34 mediates CAND1 function in CRC.The experiment was divided into four groups:Vector,CAND1,CAND1+siNC and CAND1+siRPL34.After transfection 48 h and 72 h,CCK-8 assay was used to detect cell viability.Cell migration ability was detected by scratch assay(0,12,24 h).Transwell assay was performed to observe the cell invasion ability.RPL34,E-cadherin,N-cadherin and ZEB1 were detected by Western blot.Results:(1)Compared with normal tissues,RPL34 was highly expressed at both transcription and translation levels in CRC tumor tissues,and correlated with cancer stage,lymph node metastasis and histological type.In five clinical samples,compared with the normal tissue,amount of CRC RPL34 expression in cancerous tissue were significantly higher(P<0.05).With the increase of CRC TNM staging,IHC score increased,and the expressions of RPL34 gradually increased.This indicates that the later the CRC stage,the higher the expression levels of RPL34.(2)In CRC cell lines(HCT116,SW620,LoVo,HT29,SW480),the expression level of RPL34 mRNA was the highest in SW620 cell line,which was named H cell,and RPL34 knockdown treatment was performed.In SW480 cell line,the lowest was L cell,which was overexpressed with RPL34.Compared with the control group,there were no significant changes in RPL34 mRNA and protein levels in the siNC group and the Vector group,indicating that plasmid had no effect on cells and would not interfere with the experimental results.And siRPL34 group RPL34 mRNA and protein levels were significantly decreased(P<0.01),and RPL34 group RPL34 mRNA and protein levels significantly elevated(P<0.01),to ensure that the transfection efficiency.SiRPL34 group compared with that of siNC group cell viability decreased significantly(P<0.01),EdU marked positive cells decreased significantly(P<0.01),cell number of G1 phase increased significantly(P<0.01),a significant reduction in the number of S phase and G2 cells(P<0.01).Compared with the Vector to be RPL34 group of cell viability increased significantly(P<0.01),EdU marked positive cells increased significantly(P<0.01),cell number of G1 phase decreased significantly(P<0.01),significantly increased the cell number of S phase and G2(P<0.01).siRPL34 group when compared with the siNC group,24 h cell migration distance and hit number drop significantly decreased(P<0.05),E-cadherin protein expression level increased,N-cadherin and ZEB1 levels drop(P<0.01).Compared with the Vector to be RPL34 group cell migration distance and hit number drop significantly increased(P<0.05),E-cadherin protein expression level decreased,N-cadherin and ZEB1 levels(P<0.01).Compared with siNC group,the expression levels of p-JAK2 and p-STAT3 proteins in siRPL34 group were significantly decreased.Compared with Vector group,the expression levels of p-JAK2 and p-STAT3 proteins in RPL34 group were significantly increased.Compared with the Vector group,RPL34 group of cells increased CCK 8 OD value and migration distance(P<0.01).Compared with group RPL34,RPL34+AG490 group cell vitality and migration distance decreased by(P<0.01).In vivo:Compared with the shNC group,the tumor volume of siRPL34 nude mice was significantly increased,the expression level of RPL34 was significantly decreased,and the expression of Ki-67 was significantly decreased.Western Blot and HE results showed that compared with shNC group,the expression levels of p-JAK2 and p-STAT3 in tumor tissue of nude mice in shRPL34 group were significantly decreased.RPL34 overexpression was the opposite.Compared with the shNCgroup,the shRPL34 group had significantly reduced abdominal signals,fewer metastatic nodules on the liver surface,and fewer metastases formed in the liver.RPL34 overexpression was the opposite.(3)The results of bioinformatics analysis indicated that RPL34 related genes may be involved in ubiquitin modification and cancer,such as CAND1.RT qPCR and Western blot results showed that compared with normal tissue,the expression level of CAND1 in CRC tissue was significantly increased(P<0.01).The results of immunofluorescence double staining showed that RPL34 staining and CAND 1 staining overlapped in SW620 cells,indicating that they were co-expressed in CRC cells.Immunocoprecipitation showed that the ubiquitination level of RPL34 was increased in siCAND1 group.Overexpression of siCANDl inhibited the ubiquitination level of RPL34.Addition of MG132 could inhibit the ubiquitination level of siCAND1 group and increase RPL34.Compared with siCAND1,the ubiquitination process was inhibited and the expression of RPL34 was increased after adding MG132,cell viability decreased significantly(P<0.01);Compared with group siCAND1 siCANDl+MG132 group of ubiquitin level enhanced obviously(P<0.01).SiCAND1 group compared with that of siNC group within 48 h and 72 h cell viability decreased significantly(P<0.01),cell migration rate decreased significantly(P 0.01),invasion of cells decreased significantly(P<0.05).Subsequently,CAND1 was overexpressed in SW480 cells and RPL34 was knocked out.Compared with the Vector,CAND1 overexpression of group had a significantly enhance the cell vitality(P<0.01),cell mobility and cells was significantly increased(P<0.01);Compared with CAND1+siNC group,the cell vitality of CAND1+siRPL3 4 group decreased significantly(P<0.01),cell mobility and cells was significantly reduced(P<0.01).Compared with the Vector,CAND1 across groups and CAND1+siNC RPL34 protein expression and EMT related protein expression significantly raised(P<0.01);Compared with CAND1+siNC group,group CAND1+siRPL34 RPL34 protein and EMT related protein expression significantly lowered(P<0.01).Conclusions:1.The results of bioinformatics analysis and clinical sample detection showed that the level of RPL34 mRNA and protein were high in CRC tissue.The results of IHC showed that RPL34 expression was positively correlated with TNM stage of CRC.2.The expression of RPL34 is up-regulated in CRC cell lines,and up-regulated RPL34 can promote the proliferation,migration and invasion of CRC cells,and affect the biological process of CRC by activating the JAK2/STAT3 signaling pathway.3.Overexpression of RPL34 can promote tumor growth,activate JAK2/STAT3 signaling pathway,and promote liver metastasis of CRC in nude mice.4.CAND1 promoted CRC cell migration and EMT invasion by binding with RPL34 to deubiquitinate RPL34 and stabilize RPL34.