Limitations in the separation ability of conventional liquid chromatography system remains a challenge in developing a versatile method for simultaneously determining both hydrophilic and lipophilic constituents in herbal medicines (HMs). To measure compounds covering a broad polarity span in HMs, we developed a directly-coupled reversed-phase and hydrophilic interaction liquid chromatography-tandem mass spectrometry system. Samples were firstly separated according to lipophilicity by using a C18 column. Utilizing a T-piece as connector, the eluent was then pumped into an amide column to get further separation that mainly based on the hydrogen bonding effects. Dan-Qi pair, an extensively used herb-combined prescription in China, was selected to test the practicability and performance of the established system. A total of 27 components, containing 9 hydrophilic and 18 lipophilic constituents, were simultaneously determined using a schedule multiple reaction monitoring method in 15 min. Up to 69.9% content could be monitored in one injection in Dan-Qi pair extract, showing a significant advantage over previous methods. The proposed method was expected to benefit the controllability of herbal medicines.

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BACKGROUND Hepatocellular carcinoma (HCC) exhibits high invasiveness and mortality rates, and the molecular mechanisms of HCC have gained increasing research interest. The abnormal DNA damage response has long been recognized as one of the important factors for tumor occurrence and development. Recent studies have shown the potential of the protein RING finger and WD repeat domain 3 (RFWD3) that positively regulates p53 stability in response to DNA damage as a therapeutic target in cancers. AIM To investigate the relationship between HCC and RFWD3 in vitro and in vivo and explored the underlying molecular signalling transduction pathways. METHODS RFWD3 gene expression was analyzed in HCC tissues and adjacent normal tissues. Lentivirus was used to stably knockdown RFWD3 expression in HCC cell lines. After verifying the silencing efficiency, Celigo/cell cycle/apoptosis and MTT assays were used to evaluate cell proliferation and apoptosis. Subsequently, cell migration and invasion were assessed by wound healing and transwell assays. In addition, transduced cells were implanted subcutaneously and injected into the tail vein of nude mice to observe tumor growth and metastasis. Next, we used lentiviral-mediated rescue of RFWD3 shRNA to verify the phenotype. Finally, the microarray, ingenuity pathway analysis, and western blot analysis were used to analyze the regulatory network underlying HCC. RESULTS Compared with adjacent tissues, RFWD3 expression levels were significantly higher in clinical HCC tissues and correlated with tumor size and TNM stage ( P < 0.05), which indicated a poor prognosis state. RFWD3 silencing in BEL-7404 and HCC-LM3 cells increased apoptosis, decreased growth, and inhibited the migration in shRNAi cells compared with those in shCtrl cells ( P < 0.05). Furthermore, the in vitro results were supported by the findings of the in vivo experiments with the reduction of tumor cell invasion and migration. Moreover, the rescue of RFWD3 shRNAi resulted in the resumption of invasion and metastasis in HCC cell lines. Finally, gene expression profiling and subsequent experimental verification revealed that RFWD3 might influence the proliferation and metastasis of HCC via the Wnt/β-catenin signalling pathway. CONCLUSION We provide evidence for the expression and function of RFWD3 in HCC. RFWD3 affects the prognosis, proliferation, invasion, and metastasis of HCC by regulating the Wnt/β-catenin signalling pathway.