Study On Differential Expression And Mechanism Of TRIM29 In Left-sided And Right-sided Colon Cancer

Worldwide,the incidence of colorectal cancer ranks third among malignant tumors,and the death rate ranks fourth.In China,the incidence and death rate of colorectal cancer have shown an increasing trend in recent years.Lately,the influence of tumor site on the treatment and prognosis of CRC has attracted widespread attention.Some new methods have brought highlights to the treatment of colorectal cancer,especially the effect of tumor site on the treatment effect and prognosis of colorectal cancer,changing the treatment guidelines and providing a new direction for precision treatment.Splenic curvature is currently clinically used as a distinction between LSCC and RSCC because it is the boundary of their different embryonic origins.There are also significant differences in the anatomy,clinical manifestations,intestinal flora,treatment response and prognosis of colon cancer in different tumor sides.It is important but difficult to choose the precise treatment of LSCC and RSCC in the clinic.On the gene level,RSCC and LSCC are two different entities.RSCC is associated with mismatch repair(MMR)gene defects,KRAS and BRAF mutations,and micro RNA-31,while LSCC is closely related to chromosomal instability(CIN),p53,NRAS,micro RNA-146 a,micro RNA-147 b,and micro RNA-1288.However,accurate biomarkers and mechanistic characteristics explaining the difference in LSCC and RSCC are still unknown.To meet the demand for precision treatment,there is an urgent need to find a biomarker and a mechanism for the difference between LSCC and RSCC.Proteins are the main functional components of the body,and they are involved in almost every cellular process.A growing amount of evidence has demonstrated that the practicality protein could be an important biomarker of CRC and a potential target for treating cancer.The purpose of this study is toidentify the protein that expressed differently in RSCC and LSCC and to explore the its function and the molecular regulatory mechanisms.Our study include four parts.Our previous work has already identified different m RNAs that can encode proteins via an m RNA assay(GEO accession number: GSE110204)in CRC and paired normal tissues.In this work,we further selected the four candidate proteins according to the m RNA assay by using a 4.5-fold cutoff and log10 P value >5 cutoff.After that,we focus four m RNAs which can encode KLK6,CDH3,CST1 and TRIM29.Then we tested the expression levels of the 4 candidate proteins in 55 paraffin sections of human colon cancer tissue and matched adjacent normal tissue;the expression levels in RSCC and LSCC were also analyze and combined with bioinformatics analysis.With these analyses,we demonstrated that TRIM29 was expressed at higher levels in CRC tissue than in normal tissue and was much higher in RSCC compared with LSCC.By knocking down and overexpressing TRIM29 in colon cancer cells,XCELLigence RTCA MP system,transwell experiment and clonal growth experiment showed that TRIM29 could increase the proliferation,migration and invasion ability of colon cancer cells.Different embryonic origin is the main cause of the difference in left-sided and right-sided colon cancer.The frequency of amplification and mutation of TRIM29 in the TCGA database is low(http://www.cbioportal.org),so a higher level of TRIM29 is likely regulated by transcription factors.JASPAR database were used to blast the transcription factors and TRIM29 promoter region,and also considered the function of transcription factors.Fortunately,GATA2 was revealed because it has binding sites of TRIM29 promoter and it has embryonic development function.GATA2 is a classic nuclear transcription factor known as the master of promoting hematopoiesis and and monitoring cardiac differentiation by binding directly to the regulating promoters and leading to their downregulation.Some specific kinds of cancers have been reported to be related to the dysfunction of GATA2,such as leukemia and prostate cancer.In addition,GATA2 is reported to beone of the major discriminating transcription factors in CRC.Most importantly,several studies have indicated that GATA2 plays an important role during mesodermal diversification,so it probably plays a more significant role as a biomarker for RSCC than for LSCC.In this study,the expression level of GATA2 was detected in 10 pairs of LSCC and RSCC respectively,indicating that the expression level of RSCC was lower than that of LSCC.Immunohistochemistry was used to detect the expression levels of TRIM29 and GATA2 in the same tissue microarray,and the correlation was analyzed.The results confirmed the negative correlation between GATA2 and TRIM29.Further more,we overexpressed GATA2 in colon cancer cells to detect the level of TRIM29 expression,we confirmed that GATA2 could inhibit TRIM29 expression.Using luciferase-reporter assay,we showed that GATA2 significantly inhibited TRIM29 expression.An immunoprecipitation(IP)assay combined with mass spectrometry(MS)suggested that downregulation of TRIM29 suppresses CRC proliferation,migration and invasion by targeting PKM1/2.TRIM29 mainly degraded PKM1 by ubiquitination in vitro and in vivo,which lead to a decrease in the PKM1/PKM2 ratio.The PKM1 isoform has been associated with reduced tumor cell proliferation,and PKM2 plays a critical role in promoting cancer cells.The PKM1/PKM2 ratio determines the direction of glucose catabolism.The tumor phenotype of CRC has also been verified to be regulated by energy metabolism.The Seahorse Extracellular Flux Analyzer XF96(Seahorse Bioscience)was used to monitor cell metabolic alterations in vitro,and the results confirmed that overexpression of TRIM29 leads to enhanced glycolysis and decreased oxidative phosphorylation via reducing the PKM1/PKM2 ratio.This study include four parts.Part One The Expression of TRIM29 in CRC and its different expre- ssion between RSCC and LSCCObjective: To explore and confirm the molecule that plays the key role in the development of CRC and expresses differently in RSCC and LSCC.Methods: To determine key proteins in CRC,an analysis of our previous m RNA array was performed(GEO accession number: GSE110204);comparison of colon cancer tissues and matched adjacent normal tissues using a4.5-fold cutoff,log10 P value >5 cutoff and high expression in a colon cell line according to CCLE(https://portals.broadinstitute.org/ccle).IHC was used to detect KLK6,CDH3,CST1 and TRIM29 protein levels in 55formalin-fixed,paraffin-embedded CRC tissues and matched adjacent normal tissues,the chi-square test analyzing was used to find out the protein expressed differently in RSCC and LSCC.The q PCR was used to measure the TRIM29 m RNA level in another cohort of CRC tissues and their normal tissue counterparts from 46 patients.The t-test analyzing was used to compare the TRIM29 m RNA expression level between tumor tissues and normal tissues and to compare the different level between LSCC and RSCC.Western Bloting was used to detect the TRIM29 protein express level in a cohort of CRC tissues and their normal tissue counterparts from 8 patients.The chi-square test were used to analyse the relationship between the TRIM29 and TNM stage,lymphatic metastasis.Kaplan-Meier method and Log-rank test were used to analyze the difference of survival rate between different TRIM29 levels.Results:1.Screening of key m RNAs associated with the development of CRC.Our previous work has already identified differentially expressed genes(DEGs)(GEO accession number: GSE110204)between CRC tissues and matched adjacent normal tissues,1605 m RNAs are high expressed and 1606 m RNAs are low expressed in the tumor tissue.CDH3,CST1,KLK6 and TRIM29 were selected by using the cutoff value of 4.5-fold,log10 P value >5and aslo high expressed in a colon cell line according to CCLE.2.Confirming the differentially expressed protein.Immunohistochemistry(IHC)was used to detect KLK6,CDH3,CST1 and TRIM29 protein levels in 55 formalin-fixed,paraffin-embedded CRC tissues and matched adjacent normal tissues.Twenty of these samples wereright-sided tumors,and 35 were left-sided tumors.The IHC results revealed that these four candidate proteins were upregulated in CRC to varying degrees,but only TRIM29 is much more highly expressed in RSCC than in LSCC(P<0.0001).So TRIM29 is highly expressed in CRC tumor tissue than normal tissue,and is also differently expressed between LSCC and RSCC.3.The q PCR results revealed that TRIM29 expression was significantly higher in tumor tissues than in paired normal tissues(P=0.014)and was much more highly expressed in RSCC than in LSCC(P=0.03).4.TRIM29 expression was associated with lymph node metastasis(P=0.018),advanced stage(P<0.001)and poor prognosis(P=0.005).The median overall survival(OS)was 64 months in patients with low TRIM29 expression and 24 months in patients with strong TRIM29 expression;the latter group had shorter.Part Two Effects of TRIM29 on the biological behavior of colon cancer cellsObjective: To explore the effects of TRIM29 on the biological behavior of colon cancer cells.Methods: The plasmid that overexpressed TRIM29 and the Si RNA that knocked down TRIM29 were constructed.The SW480 and DLD-1 cell lines,which exhibited high and moderate expression levels of TRIM29,were transfected with Si RNA targeting TRIM29.The DLD-1 and HCT116 cell lines,with moderate and lower levels of TRIM29 expression,were transfected with a p GV230-TRIM29 overexpression plasmid.After transfection for 48 h,we examined cell proliferation,cloning ability,migration and invasion.T-test analyzing were used to explore the difference between the experimental group and control group.To assess the effect of TRIM29 on CRC in vivo,two pooled sets of SW480 and HT29 cells were stably infected with negative control(lenti-NC)or sh TRIM29 lentiviral vectors(lenti-sh TRIM29).The lenti-NC and lenti-sh TRIM29 cells were subcutaneously injected into their upper backs.The weight of subcutaneously transplantation tumors was analyzed.Two pools ofstably infected HT29 cells were separately injected into the tail veins of 5nude mice.After two months,the presence of lung and liver metastases was analyzed.Results:1.The abilities of cell proliferation was enhanced in DLD-1 and HCT116 cell lines after transfecting with TRIM29 overexpression vector compared to the p GV230-vector.By contrast,the abilities of cell proliferation was weakened in DLD-1 and SW480 cell lines after transfecting with TRIM29-targeted Si RNA compared to that in cells transfected with NC Si RNA.2.After transfected with overexpressed TRIM29 plasmid(TRIM29 GFP)for 48 h,the number of cells migrated to the submembrane surface in DLD-1GFP and DLD-1 TRIM29 GFP groups were 278±21 and 376±32,respectively;and number of cells invaded to the submembrane surface in two groups were173±13 and 282±21,all the above groups had significant statistical significance(P<0.001).After transfected with TRIM29 for 48 h,the number of cells migrated to the submembrane surface in HCT116 GFP and HCT116TRIM29 GFP groups were 149±16 and 211±15,respectively;and number of cells invaded to the submembrane surface in two groups were 158±14 and201±17,all the above groups had significant statistical significance(P<0.001).The ability of cellular migration and invasion was enhanced after transfecting with TRIM29 GFP plasmid.3.After transfected with TRIM29 Si RNAs for 48 h in DLD-1 and SW480 cell lines,the number of cells migrated to the submembrane surface in SW480 TRIM29 NC、SW480 TRIM29 Si1 and SW480 TRIM29 Si2 groups were 150±18,82±8 and 88±9,respectively;and number of cells invaded to the submembrane surface in three groups were 16±3,3±1 and 8±2.The number of cells migrated to the submembrane surface in DLD-1 TRIM29 NC、DLD-1TRIM29 Si1 and DLD-1 TRIM29 Si2 groups were 398±29,163±14 and143±11,respectively;and number of cells invaded to the submembrane surface in three groups were 349±27,133±11 and 125±9.All the above groupshad significant statistical significance(P<0.001).The ability of cellular migration and invasion was weakened after transfecting with TRIM29 Si RNAs.4.DLD-1 and SW480 cell lines were transfected with TRIM29 Si RNA2,HCT116 and DLD-1 cell lines were transfected with TRIM29 GFP for48 h.The number of cell clone formed in SW480 TRIM29 NC and SW480TRIM29 Si RNA2 groups were 103±7 and 25±3.The number of cell clone formed in DLD-1 TRIM29 NC and DLD-1 TRIM29 Si RNA2 groups were13±2 and 3±1.The ability of cell clone was weakened after transfecting with TRIM29 Si RNA.The number of cell clone formed in DLD-1 GFP、DLD-1TRIM29-GFP、HCT116 GFP、HCT116 TRIM29-GFP were 6±2,13±4 and21±3,58±5.All the above groups had significant statistical significance(P<0.001).The ability of cell clone was enhanced after transfecting with TRIM29 plasmid.5.The abilities of proliferation,invasion,migration and clone forming in lenti-sh TRIM29 cell lines were consistented to the TRIM29 Si RNA cell lines.6.Two pooled sets of SW480 and HT29 cells stably infected with negative control(lenti-NC)or sh TRIM29 lentiviral vectors(lenti-sh TRIM29)were injected in mice to observe the cellular function in vivo.Compared with tumors in the lenti-NC group,tumors in the lenti-sh TRIM29 group grew more slowly,which resulted in smaller tumor masses(P<0.01 in SW480 and P<0.001 in HT29).Part Three Study on the upstream regulation mechanism of TRIM29Objective: To investigate and identify the upstream regulatory gene of TRIM29.Methods:We determined the gene amplification and mutation status in cohorts from public databases.The results showed a low frequency of amplification and mutation for TRIM29.We reviewed the literature to understand the possibility of non-coding RNA regulating TRIM29 in CRC.After all,we considered that TRIM29 was regulated by a transcriptionfactor.The JASPAR database was used to blast the transcription factors that could recognize sequences in the TRIM29 promoter region.A total of 220 potential sites were found by using a 90% relative profile score threshold cutoff.The top 15 sites were selected,and their functions as described in the literature were reviewed.GATA2 became our focus because of its regulatory transcription function in mesoderm.GEPIA software was used to analyze the correlation between GATA2 and TRIM29.Constructed the overexpressing GATA2 plasmid,and the m RNA expression levels of GATA2 and TRIM29 were detected by q RT-PCR after overexpressing GATA2.Ch IP assays in DLD-1 cell line and luciferase reporter assays in 293 T cell line revealed that GATA2 effectively bound to the three corresponding binding elements in the TRIM29 promoter and negatively regulated TRIM29 expression.IHC was performed in 20 formalin-fixed,paraffin-embedded tumor tissues and their matched adjacent normal tissues(10 LSCC and 10 RSCC samples,which were previously used to measure TRIM29 expression)to examine the GATA2 expression levels.Results:1.The results in database showed a low frequency of amplification and mutation for TRIM29,less than 1%.2.The results in GEPIA public database showed that the expression of GATA2 significantly negative correlated to TRIM29(R=-0.3,P<0.001).3.Ch IP assays revealed that GATA2 antibody can precipitate three regions of Primer a,Primer b+c and Primer d in the TRIM29 promoter region in the TRIM29 promoter,but Ig G antibody can’t.4.Luciferase reporter assays revealed that luciferase activity in the 293 T cell line,driven by the TRIM29 promoter sequence,was significantly decreased when GATA2 was overexpressed.It confirmed that TRIM29 is a target gene of GATA2,and GATA2 transcription inhibits the expression of TRIM29.Part Four Study on the downstream regulation mechanism of TRIM29Objective: To investigate and identify the downstream regulatory gene of TRIM29.Methods: Immunoprecipitation(IP)combined mass spectrometry(MS)assay was used to obtain the downstream molecular mechanism.Coimmunoprecipitation(Co-IP)experiments were carried out in SW480 and DLD-1 cells and the results confirmed the interaction between endogenous TRIM29 and PKM1/2.After overexpressing and knocking down TRIM29,q PCR and Western blotting were used to measure the m RNA and protein expression levels of PKM1/2 respectively.A cycloheximide(CHX)pulse-chase assay was performed to examine the function of TRIM29 in PKM1/2 stability.After treated with MG132 for 12 h,the ubiquitination levels of PKM1 and PKM2 were examined after transfected with TRIM29-GFP compared with those transfected with the GV230 vector.IHC was used to detect the protein expression levels of GATA2,TRIM29 and PKM1 in three identical microarrays.Kendal’s tau-b test was used to analyze the correlation between the GATA2 and TRIM29,TRIM29 and PKM1.Extracellular acidification rate(ECAR)and oxygen consumption rate(OCR)were measured by using XF96(Seahorse Bioscience),Seahorse XF Glycolysis Stress Test Kit and Seahorse XF Cell Mito Stress Test Kit after knocking down TRIM29 over expressing PKM1 and PKM2,respectively.After overexpressing TRIM29,PKM1 or PKM2 was overexpressed respectively to perform rescue experiments,observed and analyzed whether PKM1 and PKM2 could reverse the changes of TRIM29-induced proliferation,invasion and migration,ECAR and OCR.Results:1.Five proteins(NT5E,Serpin B3,ANXA2,LDHA and PKM1/2)were detected in the corresponding region according to the molecular weight by using immunoprecipitation(IP)combined mass spectrometry(MS)assay.2.The results of coimmunoprecipitation(Co-IP)experiments confirmed the interaction between endogenous TRIM29 and PKM1/2.3.The results showed that the m RNA level of PKM did not change significantly whereas PKM protein levels were negatively correlated with TRIM29 expression.Interestingly,we found that the effect of TRIM29 on PKM1 was very obvious but that on PKM2 was very weak.4.After treated with CHX for 24 h,overexpressing TRIM29 could decrease the protein stability of PKM1 and PKM2.After treated with MG132 for 12h,TRIM29 overexpression allowed more PKM1/PKM2 to enter the degradation process.More PKM1 than PKM2 was degraded by TRIM29.5.TRIM29 protein levels negatively correlated with GATA2/PKM1 protein levels(P=0.023,0.003 respectively;Kendal’s tau-b R=-0.389 and-0.516 respectively).6.Overexpression of PKM1 significantly increased the OCR and decreased the ECAR,whereas overexpression of PKM2 significantly decreased the OCR and increased the ECAR.7.The OCR,ECAR and malignant CRC cell phenotypes were partially rescued by ectopic expression of PKM1 in the TRIM29-GFP plasmid-treated cells,but the effects could not be rescued by ectopic expression of PKM2.Conclusions:1.TRIM29 expression in colon cancer tissues was significantly higher than that in adjacent normal tissues,TRIM29 plays an important role in the development of colon cancer.2.TRIM29 promotes the malignant phenotype of colon cancer,TRIM29 expression was associated with lymph node metastasis,advanced stage and poor prognosis of CRC.3.TRIM29 is higher expressed in RSCC than in LSCC,which suggests that TRIM29 might be a molecular marker for the difference between left and right colon cancer.4.GATA2 transcriptionally represses TRIM29 expression.5.TRIM29 regulates glycolytic metabolism by promoting the ubiquitination degradation of PKM1.