Honokiol Inhibit Cancer Cell Stemness Of Human Urinary Bladder Cancer Through Upregulation MiR-145 Expression Level

ObjectiveInvestigate the molecule mechanisms of honokiol inhibiting the sternness of the bladder cancer stem cell (BCSC)MethodMy thesis is composed of two parts. In the first part of my study, several approaches including MTT assay, ALDEFLUOR experiments and real-time fluorescence quantitative PCR were used to investigate the effects of honokiol on the survival and growth of BCSCs. The BCSC cell growth curve under honokiol treatment was measured using MTT assay. The inhibition effects on BCSC and the related genetic expression of stem cell were detected by ALDEFLUOR experiments and real-time fluorescence quantitative PCR. Furthermore, the inhibition effect of honokiol on bladder cancer was verified using in vivo mice model, in which the nude mice were bearing tumor. At the same time, the potential toxicity on hepatic, spleen and kidney was measured.meanwhile detectIn the second part of my study, I focued on the molecular mechanisms underlying honokiol inhibiting the stemness of the BCSC. MiRNA array analysis was used to screen the downstream effectors related to honokiol, miR-145 was found as one of the targets of honokiol, and was verified using mice tumor tissue and the T24 and 5637 bladder cancer cell lines. We found that miR-145 suppressed the sternness of T24 cells using ALDEFOUR test. And then, the mechanisms of honokiol regulating the miR-145 expression were further investigated by chromatin immunoprecipitation assay, bioinformatics methods for searching the downstream target of the miRNA-145, and followed by the verification of report plasmids.ResultsExperiments in vitro show that honokiol significantly inhibited bladder cancer stem cell survival in a dose dependent manner. The survival rates of T24 cells are 82.1%、54.2%,4.2% under the treatment of 4.8 μg/ml,9.6 μg/ml and 19.2 μg/ml of honokiol. The survival rates of 5637 cells are 78.2%,54.3% and17.2% under the treatment of 4.8 μg/ml,9.6 μg/ml,19.2 μg/ml of honokiol. Honokiol markedly inhibited sternness of the bladder cancer stem cells, after treatment of 9.6μg/ml honokiol, with the ALDHHigh percentage of T24 bladder cancer cell reduced from 8.4% in vehicle treatmetn group to 4.1% in the experimental group, whereas the ALDHHigh percentage of 5637 is 8.7% in vehicle treatmetn group to 3.4% in the experimental group. With the treatment of honokiol, the expression of sternness related gene, such as EZH2, POU5F1, NANOG ALDH1al also decreased significantly, compared with vehicle control group.The in vivo experiment of tumor xenograft in nude mice with T24 cancer cells indicated that honokiol is effective to inhibit the tumor growth. Under the treatment of honokiol at the dose of 80 mg/kg for 35 days, the tumor volumes in treatment group was 244.5±1.2 mm3, significantly smaller than that in control group 1240.1±2.1 mm3, the weight of tumor was 0.2±0.1g compared to the wight of control group is 0.8±0.2g. The toxicity on liver, spleen and kidney was not detected.The cancer-related microRNA PCR array analysis indicated that, with the influence of honokiol,the expression of miR-145 in the cancer cell was significantly increased, which is consistent with the conclusion of the quantitative PCR with the mice tumor issues and the T24 and 5637 line of bladder cancer cells. Under the influence of miR-145 mimic on the T24 cells, the ALDHHigh percentage of tumor cells decreased remarkably compared to the control group, with the reduction of sternness-associated gene expression. Furthermore, ChIP results revealed that trimethylation level of the histone H3K27 on promoter of miR-145 was significantly decreased after the treatment of honokiol, so as to the EZH2 recruitment to the miR-145 promoter. To further identify the downstream target of the miR-145, we found a conservative recognition sequence in the 3’UTR of the sternness related gene KLF4 through bioinformatics methods. By cloning the 3’UTR of KLF4 into reporter plasmid pCHECK2, we co-transfect the wild-type and mutant KLF33’UTR-luciferase plasmid and miR-145 into the cells. The luciferase activity of KLF43’UTR is reduced by the miR-145, whereas mutant plasmid lost its repsonse to miR-145, which indicates that KLF4 is the downstream target of the miR145. Consistently, overexpression of KLF4 increase cancer cell stemness. Co-transfection of KLF4 plasmid results in the reversion of miR-145 induced cancer stem cell depletion.ConclusionHonokiol acts on the bladder cancer stem cell, causing the trimethylation of histone H3-K27 decreased, which down-regulated the inhibition effect to the miR-145 expression.miR-145 suppressed the translation of the KLF4, leading to the decreased expression of KLF4.The expression of stemness gene regulated by KLF4 reduced, thus suppressed the stemness of the cancer stem cell. In this study we elucidate the signaling pathway by which honokiol acts on the stemness of bladder cancer cell,(honokio1→H327me3→miR-145→KLF4→CSC).It provides theoretical basis for the clinical use of honokiol against bladder tumor and offers new targets and inspired ideas of the development of new drugs.