The Prognostic Significance And Mechanism Study Of ALDH3B2 In The Progression Of Cholangiocarcinoma

BackgroundCholangiocarcinoma(CCA)is a cancer type arising from the biliary system.It is of high malignancy.The morbidity of CCA is increasing worldwide and the mortality remains high.According to the anatomical location,CCA is further classified as intrahepatic cholangiocarcinoma(iCCA),perihilar cholangiocarcinoma(pCCA),and distant cholangiocarcinoma(dCCA)by AJCC and UICC.Different subtypes of CCA have different clinical features,treatment strategies,prognosis,and molecular genetic characteristics.Currently,complete tumor resection is the only curative option on CCA treatment.However,CCA is asymptomatic in its early stage.Most patients are diagnosed when symptoms such as jaundice and abdominal pain occur.The patient is often in an advanced stage of the CCA and lost the opportunity to receive radical resection,resulting in a relatively low resection rate of CCA.Cholangiocarcinoma is not sensitive to chemotherapy or radiotherapy,and no targeted drugs have been approved as first-line treatment for CCA.Effective adjuvant therapy is lacking for advanced cholangiocarcinoma.All these reason,the 5-year survival rate for patients with cholangiocarcinoma is less than 10%.At present,chemotherapy is still the first-line treatment for advanced cholangiocarcinoma.With the development of individual treatment,the role of targeted therapy and immunotherapy for specific patients cannot be ignored.The studies of biomarkers and drug targets of CCA are rare,resulting in the slow progression of CCA targeted therapy.Hence,both effective pre-and post-operational biomarkers are urgently needed for early diagnosis and individualized treatment of CCA.Further research on the molecular mechanism of tumor markers will be helpful to reveal whether they are possible therapeutic targets for cholangiocarcinoma,and provide reference for further research and application of targeted drugs.Aldehyde dehydrogenase(ALDH)gene superfamily is closely related to the occurrence and development of various diseases,such as Sjogren-Larsen syndrome,cancer,Alzheimer’s disease and other serious diseases.The main physiological function of ALDH is to encode an enzyme that catalyzes the oxidation of aldehydes to the corresponding carboxylic acids.The enzymatic activity disappeared or decreased after Gene mutations or single nucleotide polymorphisms of ALDH.At present,19 members of this family have been identified,and some genes are associated with cancer.For example,ALDH1,a ALDH family member,has been identified as an important cancer stem cell marker in a range of malignancies.Gene polymorphism of ALDH2 has also been proved to be significantly correlated with the occurrence of oropharyngeal cancer,esophageal cancer,hepatocarcinoma,colorectal cancer and gastric cancer.ALDHs are found to play an important role in the progression of several malignant tumors and cardiovascular diseases,but little is known about the function of the ALDH family members in CCA.The expression patterns and function of ALDH family members need to be systematically studied in cholangiocarcinoma.Objective1.To clarify the differential expression of ALDH family members between the tumor and paired tumor-adjacent tissues,and to screen new tumor markers or therapeutic targets in CCA.2.To define the mRNA expression of ALDH3B2 in iCCA,pCCA and dCCA and explore the relationship between ALDH3B2 expression and the prognosis of CCA patients.3.To explore the relationship between ALDH3B2 expression and the prognosis and clinicopathologic characteristics in different subtypes of CCA.4.To identify the effects of ALDH3B2 on the migration,invasion of CCA cells by using in vivo and in vitro experiments and exploring the mechanism.Methods1.High-throughput sequencing and fresh tissues was used to obtain differential gene expression profiles on tumor tissues and corresponding adjacent tissues.The mRNA expression of 19 ALDH family members were screened to make variance analysis.2.A total of 18 pairs of CCA tissues and their adjacent normal tissues were collected,including 6 pairs each for iCCA,pCCA,and dCCA.Extracting RNA and using qRT-PCR to identify the mRNA expression of ALDH3B2.3.The TMA(including 27 cases of iCCA,87 cases of pCCA,80 cases of dCCA)was used to make IHC staining.The expression of ALDH3B2 protein was evaluated in Semiquantitative scoring.4.The IHC score of six was used to divided the patients into the group of low ALDH3B2 expression and the group of high ALDH3B2 expression.Univariate analysis was utilized to analyze the prognostic significance of ALDH3B2 and other clinicopathological factors.Cox regression model was next used to clarify the independent prognostic elements.5.Chi-square test or Fisher’s precision probability test was applied to evaluate the correlation of ALDH3B2 expression with clinicopathological characteristics of cholangiocarcinoma.6.Wound-healing assay,transwell migration or invasion assay and mouse models of metastasis were applied to analysed the abilities of migration and invasion in CCA cells.7.Western blot was utilized to detected the protein expression of EMT-related indicators in order to determine the effect of ALDH3B2 on EMT of CCA.Results1.The results of high-throughput sequencing showed that the mRNA expression of ALDH3B2 in pCCA cancer tissues was obviously higher than normal tissues near CCA(p<0.01).2.The results of qRT-PCR verified that the mRNA expression of ALDH3B2 in iCCA(p<0.001),pCCA(p<0.01)and dCCA(p<0.01)tissues was markedly increased compared with adjacent para-tumor tissues.3.By IHC staining,ALDH3B2 protein was showed to be expressed in every sub-types of CCA4.Using univariate analysis,high expression of ALDH3B2 protein was proved to be distinctly associated with poor prognosis of iCCA(p=0.038),pCCA(p=0.001)and dCCA(p=0.026).Multivariate analysis showed that ALDH3B2 was an independent prognostic factor of iCCA(p=0.047)and pCCA(p=0.042).5.Correlation analysis showed that patients with high ALDH3B2 expression were more likely to have advanced T stage(p=0.030)and M stage(p=0.048),positive neural invasion(p=0.012)in pCCA.While there was no obvious correlation between the expression level of ALDH3B2 and clinicopathological features in iCCA or dCCA.6.When ALDH3B2 was knocked down in CCA cell lines,the migration and invasion abilities of CCA cells in vitro were decreased,and the liver and lung metastasis of CCA cells in vivo was also inhibited.7.Overexpression of ALDH3B2 in CCA cells enhanced their abilities of migration and invasion and induced EMT progression.Conclusion1.Differential expression of ALDH3B2 exists in cancer tissues and adjacent tissues of iCCA,pCCA and dCCA,and compared with para-tumor tissues,the expression of ALDH3B2 is higher significantly in tumor tissues.2.In iCCA,pCCA and dCCA,poor prognosis occurs in patients with high expression of ALDH3B2.Futhermore,high expression of ALDH3B2 is an independent risk factor for poor prognosis in iCCA and pCCA patients.3.The high expression of ALDH3B2 can promote the metastasis ability of CCA cells in vivo and in vitro,and induce epithelial mesenchymal transformation of CCA cells.