The Study On The Role And Mechanism Of OLFM4 In The Sensitivity Of Gallbladder Cancer To Cisplatin

Objectives:Gallbladder cancer is rare but the prognosis is extremely poor,and early diagnosis is difficult.Surgery is the only effective treatment.Gallbladder cancer is not sensitive to radiotherapy and chemotherapy,and there is no effective biotherapy.Patients treated with combined chemotherapy and surgery have advantages in survival to some extent.Cisplatin is the first-line chemotherapy drug used in gallbladder cancer,but the drug resistance of gallbladder cancer makes the treatment ineffective.Therefore,it is necessary to find biomarkers for early diagnosis or prognosis markers for gallbladder cancer and targets to improve the chemosensitivity,thus providing an essential reference for the diagnosis and treatment of gallbladder cancer.OLFM4 is an important factor in the tumorigenesis and development of gastric cancer,colon cancer,and gallbladder cancer,and can regulate biological functions such as cell proliferation,adhesion,and apoptosis.Its high expression in gallbladder cancer is associated with poor prognosis in patients.OLFM4 may be an observation indicator of tumor tumorigenesis and development,and it also can be used as a promising prognostic factor for patients.Most importantly,OLFM4 is related to tumor chemoresistance,but the mechanism is unclear.In this study,we will explore the potential and mechanism of OLFM4 as a multifunctional role factor in gallbladder cancer,scilicet:exploring the role of OLFM4 in both predict functions on the prognosis and a sensitizing target for chemotherapy.Methods:Section 1:We collected 5 pairs of fresh gallbladder cancer and para carcinoma tissue and analyzed differential gene expression between gallbladder cancer and adjacent tissue by Agilent mRNA expression profiling sequencing,and bioinformatics analyses were performed to identify differential signaling pathways in gallbladder cancer;GSEA analysis was used to identify key genes.OLFM4 was selected as the target gene of the study,the expression of OLFM4 in clinical samples was detected by immunohistochemistry in gallbladder cancer tissue microarray,and the association between the expression of OLFM4 and the survival of patients was analyzed.Section 2:We applied CCK-8,clone formation assay,Boyden Chamber assay,flow cytometry,Western blot,and other experimental methods to verify the biological functions of OLFM4 in gallbladder cancer with the usage of GBC-SD cell line and nude mice.Section 3:CCK-8 was used to detect cell viability after cisplatin treatment,flow cytometry was applied to detect cell apoptosis after cisplatin treatment,Caspase-3 activity detection kit was used to measure Caspase-3 activity in cisplatin-treated cells,Western blot was used to determine changes in apoptotic proteins,and Co-IP was used to verified protein-protein interaction,the effect of cisplatin treatment in vivo was verified in tumor-bearing nude mice,the cell apoptosis in tumor tissue was detected by TUNEL staining,and the pathological features of tumor tissue after cisplatin treatment was observed by HE staining.We used the above methods to verify the effect and mechanism of OLFM4 sensitizing gallbladder cancer to cisplatin treatment in vivo and in vitro.Results:Section 1:Agilent mRNA expression profiling sequencing results showed that OLFM4 was significantly up-regulated in gallbladder cancer.In the TCGA database,OLFM4 was significantly overexpressed in cancer tissues of cholangiocarcinoma,and was associated with poor prognosis;high expression of OLFM4 was found in clinical gallbladder cancer samples,and the high expression was associated with poor prognosis in gallbladder cancer patients.The overall survival of patients with high OLFM4 expression was lower than that of patients with low OLFM4 expression.Section 2:Knockdown of OLFM4 inhibited in vitro and in vivo cell proliferation and invasion of gallbladder cancer cells.Section 3:The IC50 value of cells to cisplatin decreased after OLFM4 depletion.The apoptosis of cells in OLFM4-knockdown group was significantly increased after cisplatin treatment compared with the control group.WB detection of cells in OLFM4-knockdown group after cisplatin treatment showed that the expression levels of apoptosis-related proteins were higher than those in the control group.RNA-seq sequencing showed that knockdown of OLFM4 could down-regulate the apoptosis inhibitor ARL6IP1(ARMER),thereby increasing the sensitivity of cells to cisplatin.Co-IP result showed that OLFM4 and ARL6IP1 did not have direct protein interaction,and in cisplatin-treated tumor-bearing nude mice,compared with the control group,the tumor growth of the OLFM4-knockdown group was inhibited,and the tumor volume was significantly reduced;and TUNEL staining found that there were significantly more apoptotic cells in the tumor tissue,HE showed that tumor cells in tumor tissue were significantly reduced and were replaced by fibrous tissue.Conclusions:Section 1:Patients with high expression of OLFM4 have a poor prognosis,and OLFM4 can be used as a prognostic indicator for patients with gallbladder cancer.Section 2:OLFM4 can affect the biological function of gallbladder cancer in proliferation and invasion.Section 3:Knockdown of OLFM4 in gallbladder cancer cells can increase the sensitivity of cells to cisplatin by regulating the ARL6IP1/Caspase-3 axis,and it can synergize with the effect of cisplatin to increase cell apoptosis,proving that OLFM4 can act as a therapeutic target for cisplatin sensitization.