Investigation Of Protamine 1 In Colorectal Cancer Diagnosis And Treatment

Background:Colorectal cancer(CRC)is one of the most common malignancies of digestive tract worldwide.In our country,due to the low efficacy of early diagnosis,83% of CRC patients were diagnosed in middle or late stage for the first time,and the clinical outcome was frustrating because radical section was nearly impossible and the disease developed rapidly associated with increased recurrence and distant metastasis.In that case,screening of CRC in population is of great clinical significance,which will increase the detection rate of early tumor and reduce the incidence and development of late-stage CRC.Among the diagnostic strategies of CRC,serological detection of tumor markers is thought to be suitable for population screening due to the less invasive method,low cost,and easy to repeat character.However,there are only approximately 20 markers routinely used in clinical practice among the hundreds of biomarkers,and those are even more rare which are suitable for screening in large-scale population.Therefore,developing novel biomarkers of early diagnosis and recurrence monitoring is believed to be essential to successfully prevent and cure CRC,as well as prolong the overall survival.It is well known that CTA is a protein family expressed only in male testis and malignancies,and is the promising target library for tumor diagnosis and biotherapy due to the tissue-specific expression pattern and immunogenicity to some extent.So far,several CTAs have been identified in CRC,including MAGE family,SPAG9,AKAP4,but the expression frequency of which were low.Therefore,to develop novel CRC diagnostic marker and to enrich the profile of CTAs in CRC,we carried out a high through-put RNA-sequencing of 5 paired CRC tissues,and identified 9 CTAs abnormally expressed in CRC tissues.Among them,Protamine 1(PRM1)is upregulated in CRC tissues.The literature concerning PRM1 as an CTA in CRC is very limited,and the exploration of diagnostic value and biofunction are yet to be investigated.Based on the above research background and RNA sequencing result,this study intended to clarify the expression level of PRM1 in CRC by detection of large clinical samples and analyze the diagnostic value through carrying out a double-centered large sample retrospective clinical research;meanwhile,five CRC cell lines(DLD-1,HCT116,RKO,SW480 and SW620)were cultured,and expression and secretion of PRM1 in CRC cells were detected by various experimental methods to clarify the effect of PRM1 on CRC growth;Flow cytometry,western blot and other molecular biology related technologies were used to explore the biological function and relative molecular mechanism of PRM1;Finally,CRC xenograft were established in Balb/c nude mice to investigate the effect of PRM1 on tumor growth and metastasis in vivo,and to explore the antagonistic effect of antibody against PRM1 on growth of CRC.Methods:1.Collect 5 paired CRC tissues for transcriptome analysis to investigate the expression of CTAs in CRC tissues.2.Collect clinical samples,including 90 paired fresh CRC tissues(270 tissues in all),128 paraffin sections of CRC tissues,10 paired fresh CRC tissues(20 tissues in all),and 2 colorectal mucosa tissues from nonmalignant tumor patients.The expression of PRM1 in tumor tissues were detected,and the correlation was analyzed between the expression level of PRM1 and the clinicopathological features of CRC by means of Chi-square analysis.3.Recruit 520 subjects from two centers,including 304 patients with CRC and216 healthy controls,who were divided into testing cohort and validation cohort.The concentration of serum PRM1 was analyzed,and receiver operating characteristic curves(ROC)were established to calculate the diagnostic accuracy by comparing area under curves(AUC).The correlation was analyzed between serum PRM1 and the clinicopathological features of CRC by means of Chi-square analysis.4.Follow-up patients in the test cohort and record the survival information.The correlation between serum PRM1 concentration and the survival status of CRC patients was analyzed.Taking the median concentration of serum PRM1 as threshold,the patients were divided into PRM-high group and PRM1-low group.Kaplan-Meier survival analysis was performed to clarify the correlation between serum PRM1 and the prognosis of CRC.5.Culture five CRC cell lines,including DLD-1,HCT116,RKO,SW480 and SW620,and adult nonmalignant cell line MCF-10 A.The expression,secretion,and subcellular localization of PRM1 in CRC cells were detected by q PCR,immunofluorescence(IF)and ELISA.6.Observe the regulatory mechanism of PRM1 expression and secretion from CRC cells under different nutritional conditions.Five CRC cell lines were cultured in media containing different concentrations of fetal bovine serum(FBS).The expression and secretion of PRM1 were detected by q PCR,IF,ELISA and western blot.7.Investigate the cellular function of PRM1 in CRC:(1)RKO and SW480 were incubated with PRM1 protein or specific antibody.CCK8 and Ed U experiments were used to detect cell viability and DNA replication;wound healing and transwell assays were used to detect the change of cell metastasis ability;flow cytometry and western blot were used to analyze CRC cell cycle,apoptosis,and the activation level of related signal transduction pathways;(2)Observe the cellular effect of PRM1 overexpression or knockdown after transfection of Pc DNA-PRM1 and si-PRM1 s into RKO and SW480 cells.The increased or decreased expression level of PRM1 was verified by q PCR,ELISA,and western blot.The proliferation rate,cell cycle,and activation level of related signal transduction pathways were detected by CCK8,Ed U,flow cytometry and western blot.8.Observe the effects of PRM1 protein and specific antibody on the CRC growth in vivo.Establish CRC xenograft by subcutaneous injection of SW480 cells into the back of Balb/c nude mice.After formation of tumors,we injected PRM1 protein,antibody against PRM1,or PBS solution subcutaneously into the tumor every day.By the end of experiment on the 21 st day,the mice were anesthetized and sacrificed,tumor tissues and organs including brain,lung,heart,liver,spleen,and kidney were obtained and fixed in formalin.The effects of PRM1 protein stimulation and antibody antagonism on CRC growth were analyzed by drawing tumor growth curve and Ki67 staining.The metastasis and biosafety of antagonism treatment were also analyzed.Results:1.We found 9 CTAs abnormally expressed in CRC.Through RNA sequencing analysis,a total of 9 CTAs exhibited increased or decreased expression in tumor tissues,among which the expression of were up-regulated,and we decided to carry out a systematic study of PRM1.Go analysis of PRM1 predicted that PRM1 might be involved in important bioprocesses such as cell metabolism,DNA conformation changes,cell component organization or biogenesis in CRC.2.We detected the elevated expression level of PRM1 in CRC.Both mRNA and protein expression of PRM1 were significantly upregulated in CRC tissues,and PRM1 protein was negatively detected in nonmalignant tissues by IHC.Statistical analysis showed that the expression level of PRM1 protein was significantly correlated with tumor differentiation level and T stage,but not with other clinicopathological features.3.The detection of serum PRM1 demonstrated high accuracy for CRC diagnosis.(1)The level of serum PRM1 was much higher in CRC patients than healthy controls.The ROC curve of test cohort revealed the best cutoff was at 271.94 pg/m L(AUC=0.962,95%CI 0.939-0.985),the sensitivity and specificity of CRC diagnosis were 84.2% and 96.7%,moreover,diagnostic sensitivity of early-CRC was higher at85.5%(AUC=0.971);taking 271.94 pg/m L as the threshold,we observed the same results in validation cohort by establishment of ROC curve,the sensitivity and specificity of CRC diagnosis were 67% and 84.7%(AUC=0.837),and higher sensitivity was also detected in early-CRC diagnosis at 71%(AUC=0.851);(2)Concentration of serum PRM1 was significantly correlated with the differentiation level of CRC in test cohort,but no relevance was observed with other clinicopathological features;(3)The diagnostic accuracy of PRM1 was better than either of commonly used clinical markers for CRC(including CEA,CA19-9,CA24-2,CA50,CA72-4 and AFP);the performance of combination use of these six markers(AUC=0.942)was much lower than that of PRM1(AUC=0.962).4.There was no relevance between serum PRM1 and 3-year survival rate of CRC.We followed-up 101 patients in test cohort for 3-76 months,with a median follow-up at 36 months.A total of 75 cases were recorded.After statistical analysis,we found no significant difference in the 3-year survival rate of CRC between PRM1-high and PRM1-low concentration groups.5.We observed the higher expression and secretion level of PRM1 in 5 CRC cell lines.The results showed that expression and secretion of PRM1 in DLD-1,HCT116,RKO,SW480,and SW620 cells were significantly higher than that in immortalized glandular epithelial cells MCF-10 A,and PRM1 protein was distributed in the cytoplasm of 5 CRC cell lines.6.The expression and secretion of PRM1 were up-regulated in CRC cells under nutrient stress conditions.When cultured in serum-reduced or serum-free medium,the expression and secretion level of PRM1 protein in 5 CRC cell lines were increased significantly as compared with those cultured in 10% FBS group.7.PRM1 promotes CRC proliferation.(1)Incubation with PRM1 protein promoted the progression of G1/S phase transition,upregulated the activation level of PI3K/AKT/mTOR pathway,and enhanced RKO and SW480 cells proliferation.Antibody against PRM1 reversed the enhanced proliferation rate of CRC.At the same time,PRM1 had no effect on metastatic ability and apoptosis of CRC cells;(2)After PRM1 overexpression,the transition of G1/S phase of RKO and SW480 cells were promoted,phosphorylation level of PI3K/AKT/mTOR signal pathway was elevated,and cell proliferation rate was enhanced which could be antagonized by antibody against PRM1;After PRM1 knockdown,RKO and SW480 cell cycles exhibited arrest at G1/S phase,declined activation level of PI3K/ AKT/mTOR signal pathway,and impaired proliferation rate which could be rescued by addition of PRM1 protein to the culture medium.8.PRM1 protein stimulation promoted tumor growth and antibody against PRM1 inhibited tumor growth in vivo:(1)Compared with the control groups,the mice harbored larger tumors and faster rate of tumor growth,as well as the higher rate of Ki67 staining;while the mice treated with antibody against PRM1 had smaller tumor in volume and a rather slower rate of tumor growth,as well as the lower positive rate of Ki67 staining;(2)PRM1 protein stimulation or antibody antagonistic treatment had no effect on CRC metastasis;(3)The structure and morphology of mice organs from the antibody antagonistic treatment group suffered no alternation,and there was no tissue damage or inflammatory cell infiltration.Conclusions:1.The abnormal expression of 9 CTAs(including PRM1)in CRC tissues is identified which will serve as theoretical basis for the development of new diagnostic and therapeutic strategies in the future.2.Upregulated expression level of PRM1 in CRC tissues and circulation were confirmed.At the same time,the performance of serum PRM1 detection was impressive for CRC diagnosis,especially early CRC.We propose PRM1 as a promising biomarker for clinical application.3.PRM1 plays an important role in the growth of CRC cells.The expression and secretion level of PRM1 were upregulated when CRC cells were cultured in nutritional deficient environment,indicating that PRM1 facilitates CRC cells in keeping viability in ischemia or nutrition-deficient environment.4.PRM1 protein incubation or endogenous overexpression can activate PI3K/AKT/mTOR signal pathway,promote the transition of G1/S phase,and enhance the proliferation rate of CRC cells.It provided valuable indications for further studies concerning the autocrine and paracrine effects of PRM1 on CRC growth.5.Through neutralization of PRM1 protein and specific antibody,the impaired CRC growth is achieved after antibody antagonistic treatment which will become a novel individual therapeutic strategy for CRC patients with high expression level of PRM1.