The Functions And Potential Mechanisms Of Ribosomal Protein L34 In Perihilar Cholangiocarcinoma

According to the physical anatomical location,bile duct carcinoma can be divided into three types: intrahepatic cholangiocarcinoma(iCCA),perihilarl cholangiocarcinoma(pCCA) and distal cholangiocarcinoma(dCCA),of which the most common type is pCCA,accounting for more than 60%.Although different in location,the risk factors associated with the development of various types of cholangiocarcinoma are similar: primary sclerosing cholangitis,calculi in the biliary tract,and parasitic liver disease.Whether lymph node infiltration and marginal tumor cells remain is the most important determinant of postoperative prognosis.The 5-year survival rate of perihilarl cholangiocarcinoma after radical surgical resection is between 10% and 40%,however,the recurrence rate is as high as 50% to 70% even after R0 resection.High invasiveness and high recurrence are two major pathological characteristics of pCCA.The exploration of the molecular mechanism of the occurrence and development of perihilarl cholangiocarcinoma as well as the prediction of prognosis and the selection of therapeutic targets have been the focus of clinical research.Ribosomes are vital organelles in cell activity,responsible for the biosynthesis process of translating RNA into proteins.In addition,ribosomal protein(RPs) can still regulate cell cycle and cell growth and promote tumorigenesis and development.RPL34 is a member of the ribosomal protein RPL34 E family.RPL34 has previously focused on non-human specimens such as insects and plants.In recent years,a large number of data have shown that RPL34 is abnormally expressed in different types of malignant tumors,such as nonsmall cell lung cancer,gastric cancer,esophageal cancer,prostate cancer,etc.Its main function is to regulate cell cycle and promote cell proliferation.These findings suggest that RPL34 may be a candidate target for the treatment of patients with perihilar cholangiocarcinoma.However,the study of RPL34 and its function in perihilar cholangiocarcinoma is rarely reported.This study intends to investigate the expression of RPL34 in pCCA and analyze its correlation with clinicopathological parameters and prognosis of recurrence.Furthermore,its oncogenic function was further demonstrated by cytology and animal experiments.Finally,flux screening was used to find the regulation or related target genes by RPL34.Through a series of studies,we will explore whether RPL34 and its target gene can become the markers with predictive effect on the recurrence and prognosis of pCCA,and whether it can become a potential clinical therapeutic target for pCCA.Part Ⅰ Pathological features and expression of ribosomal protein in perihilar cholangiocarcinoma and its clinical significanceObjective: To detect the expression pedigree of ribosomal protein in pCCA,and analyze its correlation with clinicopathological parameters and recurrence and prognosis of pCCA patients,so as to preliminarily study the potential significance of ribosomal protein in the occurrence and development of pCCA.Methods: Collect the samples of perihilarl cholangiocarcinoma and some of peri-tumor samples,first the morphology studies observed the pathological characteristics of perihilarl cholangiocarcinoma.Secondly,fresh perihilar cholangiocarcinoma samples were collected,and RT-PCR was used to detect the expression of RPs family members in tumor samples.Then using tissue microarray(TMA) technology combined with immunohistochemical(IHC) technology,from the level of protein detection RPL34 proteins in pCCA cells and expression of the tumoral tissue and the peri-tumoral tissue,and then to analyze statistical dyeing results,with clinical and pathological information associated with recurrence and overall survival time,to explore the expression pedigree and clinical value of RPL34 in human perihilar cholangiocarcinoma.Results:(1)Clinicopathological analysis of perihilarr cholangiocarcinoma: irregular glandular tubular arrangement of tumor cells,ethmoidal structure and invasive growth were observed.Nuclear atypia is evident,nucleoli are visible,mitotic or pathological mitotic figures are more common.The interstitium is marked by a connective tissue reaction.According to the optimal cut-off value of TSR=50%,the patients were divided into the "stroma-rich,stromapoor," group.In this study,85 patients(70.2%) were stroma-poor,and 36 patients(29.8%) were stroma-rich.Patients with perihilar cholangiocarcinoma with low TSR are more prone to lymph node metastasis and rapid disease progression.The median recurrence-free survival time of tumor patients with high TSR was 1.5 years,and that of tumor patients with low TSR was 0.5 years,P=0.073.The median overall survival time of tumor patients with high TSR was 1.7 years,and the median recurrence-free survival time of tumor patients with low TSR was 1.0 years,P=0.028.Perineural invasion(PNI)is another significant pathological characteristic of perihilar cholangiocarcinoma.In this study,there were 94 cases(77.7%) with nerve invasion in tissue samples,and 27 cases(22.3%) with no obvious nerve invasion in tissue samples.PNI was more common in patients with lymph node metastasis and advanced cholangiocarcinoma.(2)The expression of RPs family members was up-regulated to varying degrees in perihilar cholangiocarcinoma,suggesting that the main members of this family play an important role in the occurrence of cholangiocarcinoma.The expressions of RPSA(P=0.0266),RPS2(P=0.0309),RPL34(P=0.0034),RPL37(P=0.0584)and RPP0(P=0.0345) in tumor tissues were significantly higher than those in adjacent non-tumor tissues.(3)RPL34 is not expressed or weakly expressed in non-tumor epithelial cells adjacent to pCCA cancer.RPL34 staining in pCCA tissue cells is located in the cytoplasm and nucleus,and is brownish yellow or dark brown.RPL34 expression rate in pCCA organizations was 77.7%(94/121).(4)High expression of RPL34 in pCCA is highly correlated with poor differentiation of tumor cells and lymph node metastasis.The main manifestations were as follows: the lower the degree of differentiation of tumor cells,the higher the expression of RPL34;The positive rate of the low-differentiated group(82%) was significantly higher than that of the middle-highly differentiated group(30%)(P=0.001).RPL34 expression was positively correlated with lymph node metastasis rate(84.6% in the positive group vs 69.6% in the negative group,P=0.049).There was no statistical correlation between positive RPL34 and age,sex,tumor volume or clinical stage(P>0.05).(5)Univariate survival analysis showed that positive margin(P=0.024),regional lymph node(P=0.015),and late stage(P=0.023)were associated with pCCA tumor recurrence.Patients with positive RPL34 had a shorter recurrence-free survival time than those with negative expression of RPL34(1.46 years vs 3.73 years,P<0.001).In Cox model,RPL34 expression was the independent predictor of tumor recurrence.Survival analysis showed that the poor prognosis of patients with perihilar cholangiocarcinoma was positively correlated with positive surgical margin(P=0.013),T stage(P=0.019),lymph node metastasis(P=0.001),TSR(P=0.028),clinical stage(P=0.003)and degree of tissue differentiation(P=0.025).Patients with positive RPL34 had a shorter overall survival time(1.70 years vs 3.63 years,P<0.001)compared with those with negative RPL34 expression.RPL34 expression and TSR were independent prognostic predictors of perihilar cholangiocarcinoma in a multifactorial model.Conclusions: pCCA has higher TSR and PNI,both of which are associated with recurrence and poor prognosis of pCCA.RPs is generally expressed in pCCA,suggesting that ribosomes play an important role in the development of perihilar cholangiocarcinoma.RPL34 is highly expressed in pCCA,and its high expression is associated with disease progression and lymph node metastasis.Patients with overexpression of RPL34 are more prone to having the relapse and adverse outcome.This study suggested that RPL34 has the function of oncogenic gene in perihilar cholangiocarcinoma and is expected to be a candidate molecular marker to predict disease progression and adverse outcome.Part ⅡThe effect of ribosomal protein RPL34 on the invasion and growth of perihilar cholangiocarcinoma cellsObjective: To explore the mechanism of RPL34 promoting pCCA,observe its effect on the proliferation,invasion and tumor formation and metastasis of cancer cells in vivo.Methods: Synthesizing RPL34-shr NA,packaging lentivirus particles,targeting the endogenous RPL34 of perihilar cholangiocarcinoma cells,western blotting to detect the expression of RPL34,CCK8 method to observe its effect on pCCA cell proliferation,Transwell detection of cell invasion ability change in pCCA cell,tumor formation in nude mice experiment to observe the effect of RPL34-shrNA on tumor formation and metastasis ability in vivo.Results: The RPL34 gene was successfully knocked down by lentiviral vector carrying shRNA.The decreased expression of endogenous RPL34 inhibited the growth and migration of perihilar cholangiocarcinoma cells,as well as the tumorigenesis in vivo and liver metastasis of tumor cells.Conclusion: RPL34 plays the role of oncogene in the development of perihilar cholangiocarcinoma.Part Ⅲ The mechanism of ribosomal protein RPL34 in promoting the invasion and growth of perihilar cholangiocarcinoma cellsObjective: To detect the difference of gene expression between perihilar cholangiocarcinoma cells infected with RPL34-shrNA for knockdown of RPL34 and control perihilar cholangiocarcinoma cells with gene chip,and to preliminarily explore the molecular mechanism of RPL34 promoting tumor invasion and growth.Methods:(1)Expressions of EMT-related and metabolism-related indexes were detected by western blotting after RPL34-shrna infection.(2)Perihilar cholangiocarcinoma cells infected with blank control and infected with RPL34-shrNA were collected,preserved by Trizol,and sent to the gene detection platform.(3)The Affymetrix? Human genome U219 microarray was used to detect the changes in gene expression after infection with RPL34.>1.5 or <0.67 is defined as the baseline for determining up-regulated or downregulated genes.Gene ontology and KEGG pathway analysis to determine the signaling pathways and/or genes that are affected by Gene knockout.(4)Select some key genes for Western blotting validation and TMAs+IHC clinical validation and statistical analysis.(5)Plasmids of full-length BCAS2 were constructed,and BCAS2 was overexpressed in RPL34 knockout cells.Cell proliferation was detected by CCK8 and invasion ability was detected by Transwell.Results:(1)After RPL34 knockdown,E-cadherin expression was up-regulated,suggesting that RPL34 promotes the invasion of perihilar cholangiocarcinoma cells by influencing EMT.However,there was no significant change in the metabolism-related indexes.(2)Gene chip results showed that 160 genes were significantly changed after the endogenous RPL34 was knocked down.Functional classification showed that the top eight signaling pathways were: potassium ion transmembrane transport,nucleosome assembly,cardiac conduction,cellular protein metabolism,protein catabolic,nonhomologous protein entry into double-chain fracture repair,telomerase telomere maintenance and messenger RNA splice,etc.The first down-regulated differential genes include:SETD2(SET domain containing 2)(Fold change=-2.20,P=0.043)、COA6(cytochrome c oxidase assembly factor 6)(Fold change=-2.05,P=0.035)、TSEN15(tRNA splicing endonuclease subunit 15)(Fold change=-1.94,P=0.011)、BCAS2(breast carcinoma amplified sequence 2)(Fold change=-1.94,P=0.039)和 HNRNPLL(heterogeneous nuclear ribonucleoprotein L-like)(Fold change=-1.92,P=0.00029).After bioinformatic analysis,it was found that BCAS2 and HNRNPLL genes were related to cancer.(3)The RPL34 regulatory protein BCAS2,which is localized in the nucleus and cytoplasm,is weakly expressed in non-tumor epithelium and strongly expressed in tumor tissues,with an expression rate of 76.0%(92/121)in tumors.Its overexpression is positively correlated with tumor differentiation.Compared with patients with negative BCAS2 expression,patients with positive BCAS2 had shorter recurrence-free survival time(1.69 years vs 3.14 years,P=0.012).(4)The proliferation and invasion of perihilar cholangiocarcinoma cells decreased after RPL34 knockdown,but the proliferation and invasion of perihilar cholangiocarcinoma cells recovered after BCAS2 overexpression.It suggests that BCAS2 can partially reverse the biological behavior abnormalities caused by RPL34 knockdown.Conclusion: This part of the research on the mechanism more clearly reveals the RPL34 role in perihilar cholangiocarcinoma cell proliferation and the potential mechanisms: RPL34 may promote tumor invasion and growth through EMT.Gene analysis indicated that that knockdown of RPL34 may increase invasion and growth of perihilar cholangiocarcinoma cells by affecting nuclear translation,transcription,splicing and assembly.The RPL34-related protein BCAS2 is abnormally expressed in hilar cholangiocarcinoma,and its overexpression is closely related to the malignant biological behavior and poor prognosis of cholangiocarcinoma.The overexpression of BCAS2 can reverse the decline in cell proliferation and invasion ability caused by the knockdown of RPL34,suggesting that RPL34 may play a role through BCAS2.SummaryRPL34 is highly expressed in perihilar cholangiocarcinoma and promotes the invasion and growth of pCCA cells.Gene chip detection,bioinformatics analysis and protein western blot validation suggest that RPL34 may promote the invasive growth of pCCA through EMT and various signaling pathways.High throughput screening found that the related protein BCAS2 abnormal expression in pCCA,overexpression of BCAS2 can reverse the decline of cell proliferation and invasion ability caused by knockdown RPL34,suggesting that BCAS2 is a possible downstream molecule of RPL34.RPL34 is expected to be a tumor marker to predict the recurrence and prognosis of perihilar cholangiocarcinoma,and targeting RPL34 mediated signaling pathways may be a potential therapeutic strategy for pCCA.