Abstract
Chemoresistance remains the uppermost disincentive for cancer treatment on account of many genetic and epigenetic alterations. Long non-coding RNAs (lncRNAs) are emerging players in promoting cancer initiation and progression. However, the regulation and function in chemoresistance are largely unknown. Herein, we identified ARHGAP5-AS1 as a lncRNA upregulated in chemoresistant gastric cancer cells and its knockdown reversed chemoresistance. Meanwhile, high ARHGAP5-AS1 expression was associated with poor prognosis of gastric cancer patients. Intriguingly, its abundance is affected by autophagy and SQSTM1 is responsible for transporting ARHGAP5-AS1 to autophagosomes. Inhibition of autophagy in chemoresistant cells, thus, resulted in the upregulation of ARHGAP5-AS1. In turn, it activated the transcription of ARHGAP5 in the nucleus by directly interacting with ARHGAP5 promoter. Interestingly, ARHGAP5-AS1 also stabilized ARHGAP5 mRNA in the cytoplasm by recruiting METTL3 to stimulate m6A modification of ARHGAP5 mRNA. As a result, ARHGAP5 was upregulated to promote chemoresistance and its upregulation was also associated with poor prognosis in gastric cancer. In summary, impaired autophagic degradation of lncRNA ARHGAP5-AS1 in chemoresistant cancer cells promoted chemoresistance. It can activate the transcription of ARHGAP5 in the nucleus and stimulate m6A modification of ARHGAP5 mRNA to stabilize ARHGAP5 mRNA in the cytoplasm by recruiting METTL3. Therefore, targeting ARHGAP5-AS1/ARHGAP5 axis might be a promising strategy to overcome chemoresistance in gastric cancer.
Highlights
Despite of significant advances, cancers such as gastric cancer remains one of the uppermost causes of mortality[1,2]
As the RNA-binding protein HuR was important to regulate the stability of mRNA27,28, we explored if ARHGAP5-AS1 can regulate the stability of ARHGAP5 mRNA through affecting its binding to HuR
Increasing evidence confirmed the involvement of deregulated Long non-coding RNAs (lncRNAs) in the pathogenesis of various diseases including cancers
Summary
Antibodies, and chemicals Human gastric cancer cell lines SGC7901 and BGC823 were all purchased from the Type Culture Collection of the Chinese Academy of Sciences (Shanghai, China). RNA extraction and quantitative real-time PCR Total RNAs were isolated using the Trizol reagent (Invitrogen) and concentrations were quantified using NanoDrop 2000 (Wilmington, DE, USA), followed with DNase I digestion and reverse transcribed by random primers to generate cDNA templates strictly according to the manufacturer’s instructions (Thermofisher Scientific Inc., Shanghai, China). ADM concentration determination About 5 × 105 cells were seeded overnight in six-well plate and transfected with given siRNAs or plasmids for 48 h, and incubated with doxorubicin hydrochloride (ADM, 5 μg/mL) at 37 °C for 2 h. Afterwards, about 500 ng quantified RNAs were yielded to incubate with 50 μL Streptavidin T1 magnetic beads and washed five times, reverse transcriptase-mediated cDNA synthesis on beads should be immediately performed to be further used for qRT-PCR to detect nascent-transcribed RNAs of interested. Differences were regarded as significant when p < 0.05
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