Role And Mechanism Of MANF And Its Interacting Protein PRDX6 In Intrahepatic Cholangiocarcinoma

Intrahepatic cholangiocarcinoma（ICC）is the second most common hepatic malignancy after hepatocellular carcinoma（HCC）and has high malignancy and poor prognosis.It accounts for 10%of the digestive tract tumors and 13%of cancer mortality worldwide.HCC and ICC are both primary liver cancers;however,there are significant differences in their pathogenesis and treatment.One reason for poor prognosis of ICC is unclear pathogenesis,difficult early diagnosis and lack of effective treatment.Mesencephalic astrocyte derived neurotrophic factor（MANF）is a member of a novel neurotrophic factor family.Our previous study found that the expression of MANF is decreased in HCC.Additionally,it inhibits the development of HCC by regulating the NF-κB signaling pathway.In ICC,the expression of MANF is increased,suggesting that it may play different roles in ICC.Studies have reported that the expression level of MANF is negatively correlated with the prognosis of cholangiocarcinoma.It can promote the apoptosis of cholangiocarcinoma cells induced bysorafenib,suggesting that this may be a new strategy for cholangiocarcinoma treatment;however,its mechanism in ICC remains unclear.MANF interacts with peroxiredoxin6（PRDX6）,and may affect its expression.PRDX6 is a member of the prdx protein family,which is associated with GSH peroxidase function.It is found that PRDX6 plays a dual role in promoting and inhibiting cancer in the tumorigenesis.The expression of MANF and PRDX6 is decreased in HCC,and they play an anti-tumor role.However,the role,relationship,and mechanism of these two genes in ICC remains unclear.ObjectiveTo study the role and mechanism of MANF and PRDX6 in ICC.MethodsImmunohistochemistry was used to detect the expression of MANF in 74 cases of ICC cancer and adjacent tissues,and immunofluorescence was used to detect the types of cells expressing MANF in ICC.The correlation between the expression of MANF and its clinicopathological features was analyzed.Lentivirus infection and screening were used to construct stable knockdown ICC cell lines.Western blot was used to verify the knockdown effect.Then the effects of MANF knockdown on the proliferation,colony formation,migration and invasion of ICC cells were detected by MTT assay,plate clone formation,scratch assay,Transwell migration and invasion assay.The tumorigenicity of Hu CCT1 cells after knockdown of MANF gene was detected in nude mice.Thus exploring the role of MANF in the occurrence of ICC.Immunohistochemistry was used to detect the expression of PRDX6 in 74 cases of ICC cancer and adjacent tissues,and immunofluorescence was used to detect the types of cells expressing PRDX6 in ICC.Correlation analysis of PRDX6 expression with its clinicopathological features and MANF expression.TAA induced ICC rat model was used to observe the changes of tumor formation after PRDX6 knockout.RNA sequencing of ICC tumor tissues in knockout group and control group was used to search for differential genes and explore the mechanism of PRDX6 in the occurrence of ICC.Based on the results of yeast two hybrid between MANF and PRDX6,immunofluorescence was used to detect the colocalization of MANF and PRDX6 in ICC tissues,and immunoprecipitation was used to detect the interaction between MANF and PRDX6.q PCR and Western blot detected the effect of knockdown MANF in HCCC9810 and Hu CCT1 cells on PRDX6 expression.The expression of MANF and PRDX6 in the nude mouse tumorigenesis experimental group and the control group was detected,and the effect of knockdown MANF on PRDX6expression was studied.Results1 Expression and clinical significance of MANF in ICCThe results of immunohistochemical staining in 74 cases of ICC liver tissue showed a 93.2%mainly moderate to strong positive rate of MANF protein expression in cancer tissue.Additionally,the positive protein expression of MANF in paracancerous tissue was 31.1%,and typically weak positive.The protein expression of MANF in the cancer tissue group was significantly higher than in the paracancerous group.Immunofluorescence showed that MANF was expressed in cancer cells and macrophages,but not in hepatic stellate cells.Furthermore,the expression of MANF was correlated with the degree of tumor differentiation;poorly differentiated tumor tissue had significantly increased expression of MANF.The expression of MANF was also correlated with the number of tumors present;ICC with?2 tumors had higher expression of MANF.Correspondingly,the expression of MANF was significantly higher in advanced ICC than in early ICC.However,the expression of MANF was not related to age,gender,or other factors.2 Effect of MANF on the malignant behavior of ICC cell linesWe constructed HCCC9810 and Hu CCT1 stably knocked down MANF and vector control groups.The MTT results showed a lower proliferation rate in cells with MANF knockdown when compared with the control group.Compared with the control group,additionally,the numbers of colonies formed in cells with MANF knockdown were significantly reduced.These results suggest that knockdown of MANF can decrease the proliferation of HCCC9810 and Hu CCT1.The scratch test and transwell migration assay showed that migration ability was decreased after knockdown on MANF.Further,the transwell invasion assay showed decreased invasion ability of the two kinds of cells decreased after knockdown of MANF.These results suggested that knockdown of MANF inhibits the migration and invasion of cells.Furthermore,the subcutaneous tumor volume and weight of Hu CCT1 cells transfected with MANF knockdown in nude mice were smaller than those in the control group.Immunohistochemistry showed that there were fewer Ki67 positive cells in the tumor tissue of the knockdown MANF group than that of the control group.This indicated that knockdown of MANF inhibits cell proliferation in nude mice,which,in turn,inhibi ts tumor growth.3 Expression and clinical significance of PRDX6 in ICCImmunohistochemical staining showed a 94.6%moderate and strong positivity rate of PRDX6 protein expression in 74 cases of ICC.By contrast,the positivity rate of PRDX6 protein expression in paracancerous tissues was 38%,and predominantly weakly positive.PRDX6 protein expression was significantly higher in cancer tissue group than in paracancerous group.Immunofluorescence revealed PRDX6 expression in cancer cells and macrophages,but not in hepatic stellate cells.This expression was correlated with the degree of tumor differentiation;poorly differentiated tumor tissue had significantly increased PRDX6 expression.Additionally,this expression was correlated with the number of tumors;cases of ICC with?2 tumors had higher PRDX6 expression.PRDX6expression was not associated with age,gender,lymphatic metastasis and so on.The expression of PRDX6 was correlated with the expression of MANF in cases of ICC;however,the clinical significance and cell localization of MANF and PRDX6 was the same.4 Effects of rat PRDX6 knockout on TAA-induced ICC formationWild type（WT）and PRDX6 knockout(PRDX6^-/-)rats were induced by drinking300 mg/L thioacetamide（TAA）for 27 weeks.The mean number of tumors in WT and PRDX6^-/-rats（n=8）was 157.143±121.136 and 30.375±13.773,respectively.Interestingly,the number of tumors decreased significantly after PRDX6 knockout.Furthermore,the liver:total body weight ratio was decreased in PRDX6^-/-rats.Immunohistochemistry showed low expression of PRDX6 in the knockout group,and positive expression of the ICC marker,CK19,in the two groups.The number of Ki67positive cells in PRDX6^-/-group was significantly decreased.The total section and tumor area was measured under 100?field of vision of HE staining.Ten fields of vision were taken for each sample,and the mean value was calculated to measure the percentage of tumors in the total tumor area.The mean tumor area was significantly decreased in the PRDX6^-/-group（10.651±8.223）,when compared with the WT group（46.169±17.873）.Furthermore,the expression of Ki67 in PRDX6^-/-group was lower than that in WT group by q PCR.These results suggested that PRDX6 knockout inhibited tumor cell proliferation and reduced ICC tumor formation.RNA sequencing analysis revealed 127 and 321 genes that were up-and down-regulated,respectively,after PRDX6 knockout（n=3）.KEGG pathway analysis of the differentially expressed genes between two groups showed that signal transduction pathway genes were most changed,with 47 changed genes.The WNT signaling pathway is the most variable pathway in gene expression.WNT7A,WNT7B,FZD2,Ccnd2 and MMP7 were down-regulated after PRDX6 knockout.The results were confirmed by q PCR and immunohistochemistry.These results suggested that PRDX6promotes the development of ICC by regulating the WNT7A/B signaling pathway.5 MANF regulates its protein level by interacting with PRDX6The interaction between MANF and PRDX6 was revealed via yeast two-hybrid screening.Immunofluorescence showed MANF and PRDX6 colocalization in ICC tissue.Furthermore,immunoprecipitation assay proved the interaction between MANF and PRDX6.Western blot showed decreased protein expression of PRDX6 after the knockdown of MANF in HCCC9810 and Hu CCT1.q PCR revealed that the m RNA expression of PRDX6 was not affected after MANF knockdown.Similarly,the protein expression of MANF and PRDX6 was analyzed in nude mice ICC tissue,which found decreased PRDX6 protein expression after MANF knockdown and the level of m RNA did not change significantly.These results indicated that there is an interaction between MANF and PRDX6 in ICC tissue,where knockdown of MANF reduced the protein expression of PRDX6.Conclusion1 MANF is highly expressed in ICC,and is related to tumor differentiation,tumor number and tumor stage.Knockdown of MANF inhibits the proliferation,migration,invasion of ICC cells and the growth of transplanted tumor in nude mice.2 PRDX6 is highly expressed in ICC cancer tissues,and is related to tumor differentiation,tumor number and tumor stage,and is highly correlated with the expression of MANF in ICC.PRDX6 knockout can reduce the tumor formation in ICC rat model.PRDX6 may promote the development of ICC by regulating WNT7A/B signaling pathway.3 MANF interacts with PRDX6.After knockdown of MANF,the protein expression of PRDX6 was down regulated,and the m RNA did not change significantly,and the mechanism is unclear.