The Effects Of ?B-crystallin On Proliferation,Invasion And Metastasis In Colorectal Cancer

Colorectal cancer(CRC)is the third most prevalent cancer worldwide,of which the incidence and mortality is 1360,000 and 694,000,respectively.Moreover,CRC is the second most common cancer in Europe,with an estimated overall incidence of 447 per 100,000.In China,both the incidence and mortality of CRC rank the top 5 of all cancers.It is reported that there are estimated 746,000 newly diagnosed cases of CRC and approximately 694,000 cancer deaths due to CRC in China annually.Of patients with primary CRC,nearly 20%encounter distant metastasis at the time of diagnosis and only 10-30%of patients with distant metastasis can have potentially curative resection of the primary tumor and the distant focus.For CRC prognosis,survival is closely related to stage at diagnosis,with five-year survival rates of 90%for localized cases,while 67%for regional cases and only 10%for distant metastatic cases.Although the great developments for diagnosis and treatment for CRC have been made,the overall survival rate of CRC patients rarely shows the encouraging result,post-operative recurrence and metastasis remain the two most challenging obstacles for CRC patients.Hence it is valuable and necessary to identify molecular predictive markers,which would facilitate the selection of targeted therapeutic strategies and further ameliorate patients' survival for CRC.aB-crystallin(Alpha B-crystallin,CRYAB,HSPB5)is primarily found as the major structural protein of the ocular lens and is a principal member of the small heat shock protein(sHSP)family.It is widely accepted that CRYAB acts as a molecular chaperone by preventing the aggregation of denatured proteins after the exposure to stresses including heat shock,radiation,and oxidative stress.In addition to being a molecular chaperone,CRYAB is suggested to play an important role in apoptosis inhibition and cellular protection.Recently,the importance of CRYAB in cancer development,invasion and metastasis has drawn great attention.As a result,a growing number of studies indicate the intricate relationship between high CRYAB expression and the prognosis of various human cancers,including breast carcinoma,head and neck carcinoma,renal cell carcinoma,thyroid carcinoma,glioblastoma,and osteosarcoma.In this study,we focus on the effects and mechanism of aB-crystallin on proliferation,invasion and metastasis in CRC.Part One:CRYAB Correlates with Poor Survival in CRCObjective:To study the expression of CRYAB and evaluate the association between the expression and clinicopathological characteristics of CRC.Methods:18 fresh CRC cancer tissue samples and 18 normal tumor-adjacent tissue samples were collected for quantitative real-time polymerase chain reaction(qPCR)analysis,formalin-fixed,paraffin-embedded tumor samples from 100 CRC cases and 94 matched peritumoral tissue specimens were collected in CRC patients for tissue microarrays(TMA).QPCR analysis of 18 samples of CRC and normal tumor-adjacent tissue and immunohistochemistry(IHC)analysis with 100 cases of CRC sample and 94 matched peritumoral tissue specimens in TMA were employed to evaluate the expression of CRYAB in CRC.Results:The results suggested that CRYAB mRNA and protein expressions were significantly higher in CRC tissues than those in corresponding non-cancerous tissues.The expression of CRYAB protein in CRC was significantly associated with distant metastasis and overall survival.Kaplan-Meier method and multivariate survival analysis indicated that high expression of CRYAB and distant metastasis showed significant correlations with poor prognosis of CRC patients.Conclusions:The data imply that CRYAB expression is correlated with substantial clinical characteristics of CRC,and it may be identified as an unfavorable prognostic factor for CRC.Part Two:The tumor biological influence of the Expression of CRYAB on CRC SW480 CellsObjective:To study the influence of the expression of CRYAB on the proliferation,apoptosis,cell cycle,migration,invasion and tumorigenicity in nude mice.Methods:The expression of CRYAB mRNA and protein on HT29,SW480 and HCT116 cells was detected by qPCR and Westem blot,respectively.The Si-CRYAB cells were constructed by lentiviral vector on SW480 cells,and the expression of CRYAB mRNA and protein was detected by qPCR and Western blot.Growth curve was used to estimate proliferation,and cell migration was detected by transwell.Apoptosis was detected by FCM and Tunel.Cell cycle was detected by FCM.The cancer cells were injected to the backside of the nude mice to observe the tumorigenicity.Results:The expression CRYAB mRNA and protein on SW480 was higher than that on HT29 and HCT116,but the mRNA and protein level on Si-CRYAB was lower than that on SW480 and the control(empty vector).The ability of proliferation,migration,and tumorigenicity in nude mice on Si-CRYAB was lower than that on SW480 and the control,but apoptosis on Si-CRYAB was higher.The inhibition of CRYAB induced the arrest of G1 phase on Si-CRYAB.Conclusions:High expression of CRYAB can promote the proliferation,cell cycle transformation,migration,invasion and tumorigenicity in nude mice and inhibit apoptosis on CRC SW480 cells.Part Three:The Mechanism of CRYAB in Invasion and Metastasis of CRCObjective:To study the mechanism of CRYAB in invasion and metastasis of CRC.Methods:Formalin-fixed,paraffin-embedded tumor samples from nude mice and 54 CRC patients were collected for IHC.The patients were divided into 3 groups,namely cancer in situ(control)group,invasion group,and hepatic metastases group.The expression of CRYAB,p53,CD31,cyclin D1,and epithelial mesenchymal transition(EMT)markers,such as E-cadherin,fibronectin,vimentin,slug were examined by IHC on Control and Si-CRYAB.And the expression of CRYAB,E-cadherin,fibronectin,vimentin and slug were examined by IHC on the tumor samples of CRC patients.Results:The expression of CRYAB,cyclin D1,fibronectin,vimentin and slug on Si-CRYAB were lower than those on Control,while the expression of E-cadherin was higher on Si-CRYAB than that on Control.The expression of p53 and CD31 had no significant differences among the two groups.The expression of CRYAB,fibronectin,vimentin and slug were higher on the invasion group and hepatic metastases group than those on control group while the expression of E-cadherin was lower than that on control group.Conclusions:CRYAB promotes CRC invasion and metastasis by enhancing the expression of cyclin D1 and EMT.