LncRNA MEG8 plays an oncogenic role in hepatocellular carcinoma progression through miR-367-3p/14-3-3ζ/TGFßR1 axis.

Hepatocellular carcinoma (HCC) is one of the most common cancers worldwide, and it carries a poor prognosis. Clarifying the pathologic mechanisms of this disease will be beneficial for the diagnosis and treatment of HCC. LncRNA MEG8 is involved in several tumors but its role in HCC progression remains unknown. This study was designed to explore the role and regulatory mechanisms of MEG8 in HCC progression. MTT, EdU, wound-healing, and transwell assays were employed to analyze the proliferation, migration, and invasion of HCC cells. A luciferase assay was utilized to confirm the predicted binding site. RNA immunoprecipitation and co-immunoprecipitation were employed to verify the binding between MEG8 and miR-367-3p as well as 14-3-3ζ and TGFßR1. Real-time PCR and western blot were employed to detect the expression of interesting genes. Results revealed that MEG8 was increased in HCC tissues and cells, and was correlated with the poor prognosis of HCC patients. Inhibiting MEG8 significantly repressed the HCC cells' ability to proliferate, migrate, and invade. Moreover, MEG8 sponged miR-367-3p to upregulate 14-3-3ζ, the binding of which suppressed TGFßR1 degradation, thereby enhancing TGFß signaling. In conclusion, this work exposed a novel role and regulatory mechanism of MEG8 in HCC and provided new insight into the treatment of HCC.

[Construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice].

OBJECTIVE: To construct the expression vector pLCK-CD69-IRES-EGFP that contains mouse cell surface activation protein CD69 and enhanced green fluorescent protein(EGFP),and to generate CD69 transgenic mice based on this vector. METHODS: First, RNA was extracted from mouse lung tissue and cDNA was synthesized via reverse transcription. PCR primer was designed through the PubMed searching, then mouse CD69 DNA fragment was amplified with PCR. Second, this DNA fragment was subcloned to the pInsulater-LCK-IRES-EGFP plasmid and constructed the transgenic vector after the verification of nucleotide sequence. Third, the expression vector was then transfected into 293 T cells and its expression in 293 T cells was observed under fluorescence microscope. Last, microinjection was performed to transfer the expression vector pLCK-CD69-IRES-EGFP into fertilized eggs, which were implanted into pseudo-pregnant recipient mice. After birth the tail samples of the pups were obtained for the purpose of genotyping to determine the transgenic founders. Fluorescence microscope and flow cytometer were used to measure the expression of CD69 on cells. RESULTS: The construction of the expression vector pLCK-CD69-IRES-EGFP was verified by enzyme digestion and DNA sequencing. The transfected 293 T cell showed expression of the protein under fluorescence microscope. Identification of PCR for the tail tissue of the pups confirmed the present of CD69 transgene and resting lymphocytes demonstrated the expression of CD69. CONCLUSION: The construction of expression vector pLCK-CD69-IRES-EGFP and generation of CD69 transgenic mice have been successfully processed, which lays a foundation of the solid pattern studies in inflammatory diseases.

Shikonin sensitizes wild­type EGFR NSCLC cells to erlotinib and gefitinib therapy.

As patients with non­small cell lung cancer (NSCLC) and wild­type epidermal growth factor receptor (EGFR) are resistant to treatment with erlotinib or gefitinib, potential chemosensitizers are required to potentiate wild­type EGFR NSCLC cells to erlotinib/gefitinib treatment. The present study reported that shikonin could sensitize the anticancer activity of erlotinib/gefitinib in wild­type EGFR NSCLC cells. Furthermore, shikonin could potentiate mitochondrial­mediated apoptosis induced by erlotinib/gefitinib in wild­type EGFR NSCLC cells. In addition, the present study demonstrated that shikonin could induce apoptosis by activating reactive oxygen species (ROS)­mediated endoplasmic reticulum (ER) stress, and that erlotinib/gefitinib may also induce ER stress in wild­type EGFR NSCLC cells; however, shikonin plus erlotinib/gefitinib was more effective in activating ER stress than either agent alone. This indicated that ROS­mediated ER stress may be associated with enhanced mitochondrial apoptosis induced by shikonin plus erlotinib/gefitinib. In addition, shikonin may promote the transition of cytoprotective ER stress­inducing EGFR­tyrosine kinase inhibitor tolerance to apoptosis­promoting ER stress. Furthermore, shikonin may enhance the anti­NSCLC activity of erlotinib/gefitinib in vivo. The data of the present study indicated that shikonin may be a potential sensitizer to enhance the anti­cancer efficacy of erlotinib/gefitinib in wild­type EGFR NSCLC cells resistant to erlotinib/gefitinib treatment.