BackgroundThyroid cancer is one of the most common endocrine malignancies worldwide, and papillary thyroid cancer (PTC) is the most common pathologic type of thyroid cancer. SQSTM1/p62 activity mediates different biological functions. This study aimed to investigate the effect of SQSTM1/p62, a multifunctional receptor, on biological function and autophagy characteristics in the human PTC cell line TPC-1.MethodsA total of 105 primary PTC samples and matched adjacent normal thyroid tissue samples were obtained to evaluate the expression of p62 in clinical patients. A similar p62 expression pattern was found in PTC cell lines and normal human thyroid follicular epithelial cells. To evaluate the effect of SQSTM1/p62 on TPC-1 cells, we constructed the p62 knockout cell line p62-KO-TPC-1. Cell proliferation, cell cycle, and cell apoptosis were analyzed by colony formation tests, Cell Counting Kit-8 (CCK-8) assays and flow cytometry in vitro. TPC-1 and p62-KO-TPC-1 human PTC cell lines in the logarithmic growth phase were subcutaneously implanted into BALB/c nude mice to verify their proliferation effect in vivo. Furthermore, western blotting and immunohistochemistry (IHC) were used to detect the expression of AKT/AMPK/mTOR signaling pathway-related proteins.ResultsOverall, p62 expression was higher in tumor tissues than in normal tissues in 73 of 105 PTC patients (69.5%). The expression level of p62 in the PTC cell line was higher than that in the normal thyroid cell line. Our data indicated that in vitro, p62 deficiency could decrease the number of colonies, inhibit cell growth and the cell cycle, and induce apoptosis. Tumor xenograft experiments in BALB/c nude mice corroborated these findings. Moreover, the molecular mechanism was explored by western blotting, and we found that the AMPK/AKT/mTOR pathway was involved.ConclusionsThe results indicate that p62 might mediate cell autophagy and apoptosis in TPC-1 cells via the AMPK/AKT/mTOR pathway and could be used as a potential therapeutic approach for PTC.

Prunella vulgaris, a traditional Chinese medicine, has been used to treat various benign and malignant tumours for centuries in China. In our previous studies, Prunella vulgaris extract (PVE) was shown to promote apoptosis in differentiated thyroid cancer (DTC) cells. However, whether other mechanisms are involved in the antitumour effect of PVE in thyroid cancer (TC) cells remains unclear. The present study aimed to investigate the antiproliferative and antimigratory effects of PVE on TC cell lines both in vitro and in vivo. First, the TPC-1 and SW579 human TC cell lines were screened by MTT assay for their high level of sensitivity to PVE. Then, the results of cell growth curve and colony formation assay and cell cycle analyses, wound healing, and migration assays demonstrated that PVE inhibited the proliferation and migration of TPC-1 and SW579 cells. Moreover, the antitumour effect of PVE was verified in a subcutaneous xenotransplanted tumour model. Next, MKI67, PCNA, CTNNB1, and CDH1 were screened by qRT-PCR for their significantly differential expression levels in xenograft tissue with and without PVE treatment, and expression of MKI67, PCNA, and CDH1 was verified by Western blot. Finally, an integrated bioinformatics analysis containing protein-protein interaction network, KEGG pathway, and GO analysis was conducted to explore more potential antitumour mechanisms of PVE. In summary, PVE could inhibit the proliferation and migration of TC cells both in vitro and in vivo, which may have been achieved by modulation of the expression of MKI67, PCNA, and CDH1. These data suggest that PVE has the potential to be developed into a new anticancer drug for the treatment of TC.

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Gene expression valuated by reverse transcription-quantitative PCR (RT-qPCR) are often applied to study the gene function.To obtain accurate and reliable results, the usage of stable reference genes is essential for RT-qPCR analysis. The traditional southern Chinese medicinal herb, Desmodium styracifolium Merr is well known for its remarkable effect on the treatment of urination disturbance, urolithiasis, edema and jaundice. However, there are no ready-made reference genes identified for D. styracifolium. In this study, 13 novel genes retrieved from transcriptome datasets of four different tissues were reported according to the coefficient of variation (CV) and maximum fold change (MFC) of gene expression. The expression stability of currently used Leguminosae ACT6 was compared to the 13 candidate reference genes in different tissues and 7-day-old seedlings under different experimental conditions, which was evaluated by five statistical algorithms (geNorm/NormFinder/ BestKeeper/ΔCT/RefFinder). Our results indicated that the reference gene combinations of PP + UFM1, CCRP4 + BRM and NFD6 + NCLN1 were the most stable reference genes in leaf, stem and root tissues, respectively. The most stable reference gene combination for all tissues was CCRP4 + CUL1. In addition, the most stable reference genes for different experimental conditions were distinct, for instance SMUP1 for MeJA treatment, ERDJ2A + SMUP1 for SA treatment, NCLN1 + ERDJ2A for ABA treatment and SF3B + VAMP721d for salt stress, respectively. Our results lay a foundation for achieving accurate and reliable RT-qPCR results so as to correctly understand the function of genes in D. styracifolium.