The Mechanism Of MiR-143-3p Modulating Cell Proliferation In Gallbladder Cancer

ObjectiveWe identified miRNAs differentially expressed in GBC by microarray,and performed preliminary study on the biological role and molecular mechanism of specific miRNA in the GBC development process,which established effective diagnosis and therapeutic targets for treatment of gallbladder cancer.Methods(1)To identify critical miRNA for further study,miRNAs in 4 pairs of GBC tissues and corresponding non-tumor tissues were screened using Agilent human miRNA array.The expressions of miR-143-3p were detected in 45 pairs of GBC tissues and corresponding non-tumor tissues by RT-PCR to validate the consequence of miRNA array,and its relationships with clinical pathological features as well as the prognosis of patients with gallbladder cancer were analyzed.The survival analysis was performed by Kaplan-Meier method and Cox regression analysis.(2)The biological functions of miR-143-3p on cell proliferation were evaluated using assays both in vitro and in vivo.(3)Using bioinformatics software to predict the downstream target genes of miR-143-3p.We combined dual-luciferase reporter gene and western blot to identify the target gene of miR-143-3p in GBC.The expressions of MYBL2 were detected in 65 pairs of GBC tissues and cholecystitis tissues by immunohistochemical(IHC)assays.The survival analysis of MYBL2 was performed by Kaplan-Meier method and Cox regression analysis in GBC patients.The effects of MYBL2 on cell proliferation and DNA synthesis were evaluated using Cell Counting Kit-8 assay(CCK-8),colony formation,and 5-ethynyl-2’-deoxyuridine(EdU)retention assay,flowcytometry analysis,western blot,and a xenograft model of GBC cells in nude mice.Ki-67 and MYBL2 expression were evaluated in xenograft tumors through IHC detection.Rescue expremients were performed to further assess the relationship between miR-143-3p and MYBL2.Results(1)Based on miRNA microarray and RT-PCR,The expressions of miR-143-3p in gallbladder cancer tissues was significantly lower.The expressions of miR-143-3p in gallbladder cancer was respectively related to the depth of tumor invasion(P = 0.035),and TNM stage(P = 0.035).The lower expression of miR-143-3p survived significantly shorter than those with higher(P = 0.002).Multivariate analysis with the Cox regression models showed that miR-143-3p expression was an independent prognostic factors of postoperative survival in patients with gallbladder cancer(P = 0.019).(2)The high miR-143-3p expression could inhibit the gallbladder cancer cell proliferation both in vitro and in vivo.(3)MYBL2 was the tareget gene of miR-143-3p.(4)MYBL2 may serve as an oncogene in GBC pathogenesis,which promotes gallbladder cancer cell proliferation.Negative modulation of MYBL2 expression by miR-143-3p was essential for miR-143-3p inhibited GBC cell proliferation.Conclusions(1)The expressions of miR-143-3p in gallbladder cancer tissues was significantly lower,which was associated with the poor prognosis of GBC patients.miR-143-3p expression was an independent prognostic factors of postoperative survival in patients with gallbladder cancer,which might be a potiential diagnosis and therapeutic targets for treatment of gallbladder cancer.(2)MYBL2 may serve as an oncogene in GBC pathogenesis,which promotes gallbladder cancer cell proliferation through the regulation of the cell cycle at the S and G2/M phase transitions.Negative modulation of MYBL2 expression by miR-143-3p was essential for miR-143-3p inhibited GBC cell proliferation.