ABSTRACT
BACKGROUND: Reverse transcription quantitative polymerase chain reaction (RT-qPCR) can accurately detect relative gene expression levels in biological samples. However, widely used reference genes exhibit unstable expression under certain conditions. METHODS AND RESULTS: Here, we compared the expression stability of eight reference genes (RPLP0, RPS18, RPL13, EEF1A1, ß-actin, GAPDH, HPRT1, and TUBB) commonly used in liproxstatin-1 (Lip-1)-treated K562 cells using RNA-sequencing and RT-qPCR. The expression of EEF1A1, ACTB, GAPDH, HPRT1, and TUBB was considerably lower in cells treated with 20 µM Lip-1 than in the control, and GAPDH also showed significant downregulation in the 10 µM Lip-1 group. Meanwhile, when we used geNorm, NormFinder, and BestKeeper to compare expression stability, we found that GAPDH and HPRT1 were the most unstable reference genes among all those tested. Stability analysis yielded very similar results when geNorm or BestKeeper was used but not when NormFinder was used. Specifically, geNorm and BestKeeper identified RPL13 and RPLP0 as the most stable genes under 20 µM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable under 10 µM Lip-1 treatment. TUBB and EEF1A1 were the most stable genes in both treatment groups according to the results obtained using NormFinder. An assumed most stable gene was incorporated into each software to validate the accuracy. The results suggest that NormFinder is not an appropriate algorithm for this study. CONCLUSIONS: Stable reference genes were recognized using geNorm and BestKeeper but not NormFinder. Overall, RPL13 and RPLP0 were the most stable reference genes under 20 µM Lip-1 treatment, whereas RPL13, EEF1A1, and TUBB were the most stable genes under 10 µM Lip-1 treatment.
Subject(s)
Actins , Leukemia , Humans , K562 Cells , Base Sequence , Sequence Analysis, RNA , Hypoxanthine Phosphoribosyltransferase , Neoplasm Proteins , Ribosomal ProteinsABSTRACT
Leukemia is a fatal hematopoietic disorder with a poor prognosis. Drug resistance is inevitable after the longterm use of chemotherapeutic agents. Liproxstatin1, commonly known as a ferroptosis inhibitor, has never been reported to have anticancer effects. In the present study, the antileukemic role of liproxstatin1 in K562 leukemia cells was investigated. Liproxstatin1 inhibited K562 cell proliferation in a dose and timedependent manner. RNA sequencing revealed several pathways that were affected by liproxstatin1, such as the G1/S transition of the mitotic cell cycle and extrinsic or intrinsic apoptotic signaling pathways. The results of flow cytometry indicated that liproxstatin1 arrests the cell cycle at the G1 phase, and even at the G2/M phase. p21WAF1/CIP1, a cyclindependent kinase inhibitor, was upregulated. It was also determined that liproxstatin1 induced BAX and TNFα expression, which was accompanied by cleavage of caspase3 and PARP. The caspase3specific inhibitor zDEVDFMK rescued some of the apoptotic cells. Interestingly, K562 cells were characterized by swelling and plasma membrane rupture when treated with a high concentration of liproxstatin1, which was inconsistent with the typical apoptotic appearance. Thus, it was hypothesized that apoptosismediated pyroptosis occurs during liproxstatin1induced cell death. The expression of the hallmark of pyroptosis, the cleaved Nterminal GSDME, increased. Additionally, it was observed that endoplasmic reticulum stress and autophagy were involved in liproxstatin1induced cell death. Collectively, liproxstatin1 induced cell cycle arrest, apoptosis, and caspase3/GSDMEdependent secondary pyroptosis in K562 leukemia cells, which provides new hope for the treatment of leukemia.