Effect of Yinhuotang on Lung Adenocarcinoma Cell Proliferation via Regulating Sphingosine-1-phosphate Receptor 5 Signaling Pathway
Objective:This study aimed to investigate the effect of Yinhuotang-containing serum on the proliferation of lung adeno-carcinoma cells via regulating the sphingosine-1-phosphate receptor 5/Kirsten rat sarcoma/rapid accelerated fibrosarcoma(S1 PR5/KRAS/RAF)signaling pathway.Methods:The Yinhuotang-containing serum was prepared.Different concentrations of drug-contai-ning serum were applied to intervene in human non-small lung cancer cells(A549,H1299,and PC9)for 24 h to determine the opti-mal inhibitory concentration.The optimal inhibitory concentration was used to intervene in three lung adenocarcinoma cells for 24 h,48 h,and 72 h,so as to determine the optimal inhibitory time and the most sensitive cell line.Lentiviral transfection was used to construct lung adenocarcinoma cell lines stably overexpressing S1PR5,and the experiments were divided into the following groups:OE NC group(blank serum+S1PR5 overexpression null group),OE S1PR5 group(blank serum+S1PR5 overexpression group),YHT+OE NC group(Yinhuotang-containing serum+S1PR5 overexpression null group),and YHT+OE S1PR5 group(Yinhuotang-containing serum+S1PR5 overexpression null group).Quantitative reverse transcription polymerase chain reaction(qRT-PCR)and Western blot(WB)methods were used to detect the expression of S1PR5/KRAS/RAF signaling pathway messen-ger RNA(mRNA)and proteins.Results:Cell counting kit-8(CCK-8)results showed that 10％Yinhuotang-containing serum had the most significant inhibitory effect on A549,H1299,and PC9 cells at 24 h(P＜0.05,P＜0.01),but there was no obvious time-dependence when 10％Yinhuotang-containing serum was used for intervention,so 24 h of action of 10％Yinhuotang-containing se-rum was chosen as the intervention condition.Because the highest inhibition rate of PC9 cells was observed in the Yinhuotang-con-taining serum,PC9 cells were selected for the mechanism verification.qRT-PCR results showed that the S1PR5 overexpression group was able to up-regulate the expression level of S1PR5 mRNA(P＜0.05),and that Yinhuotang was able to reduce the expres-sion of S1PR5,KRAS,and Ras-related C3 botulinum toxin substrate 1(RAC1)(P＜0.05).WB results showed that the protein ex-pression of S1PR5,KRAS,p-RAF1,mitogen-activated extracellular signal-regulated kinase1(p-MEK1),phosphatidylinositol-3-ki-nase(p-PI3K),and RAC1 in the S1PR5 overexpression group was significantly elevated(P＜0.05).After the intervention of Yin-huotang,the protein expression of S1PR5,KRAS,p-RAF1,extracellular signal regulated kinase1/2(p-ERK1/2),and RAC1 was significantly reduced(P＜0.05).Conclusion:Yinhuotang-containing serum inhibits the proliferation of lung adenocarcinoma cells through down-regulation of the S1PR5/KRAS/RAF signaling pathway.
Sphingosine-1-phosphate receptor 5Kirsten rat sarcomaRapid accelerated fibrosarcomaYinhuotangDrug-contai-ning serumLentiviral transfectionProliferationLung adenocarcinoma
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