The Regulatory Mechanism Of LncRNA-ncRuPAR On Gastric Cancer And The Intervention Effect Of ZiYinHuaTan Prescription

Objective: We found the expression level of lncRNA-ncRuPAR in gastric cancer tissues was significantly lower than that in adjacent tissues,and was closely related to the degree of gastric cancer invasion,lymph node metastasis,distant metastasis,tumor size,and TNM stage In the previous study.The aim of this study is to explore the relationship between ncRuPAR and the effects of proliferation,invasion,metastasis and apoptosis on gastric cancer and its possible mechanism of actionthrough a series of in vitro and in vivo experiments,as well as to verify the intervention effect of ZiYinHuaTan prescription on the proliferation of gastric cancer cells HGC27 and MGC803 and its possible mechanism.In order to deepen the understanding of the molecular mechanism of gastric cancer and provide data support for explaining the possible mechanism of ZiYinHuaTanPrescription.Methods:(1)The expression of ncRuPAR in four gastric cancer cell lines AGS,HGC27,MKN45 and MGC803 were detected by qPCR.(2)HGC27 was infected with LV-NCRUPAR(15034-1)and LV-NCRUPAR-CON respectively to construct lncRNA-ncRuPAR overexpression cell line HGC27-ncRuPAR and control cell line HGC27-Empty Vector;while MGC803 was infected with LV-NCRUPAR-RNAi and LV NCRUPAR-RNAi-CON respectively to construct lncRNA-ncRuPAR interference cell line MGC803-ncRuPAR-RNAi and control cell line MGC803-Empty Vector.After puromycin 2μg/mL selection for 72 h,stable lentivirus-infected cells were established.qPCR detects the expression of ncRuPAR in each cell line.(3)The effects of ncRuPAR on the proliferation of gastric cancer cells HGC27 and MGC803 were detected by CCK-8 assay.The effects of lncRNA-ncRuPAR on the cycle and apoptosis of gastric cancer cells HGC27 and MGC803 were analyzed by flow cytometry.The effects of ncRuPAR on the migration of gastric cancer cells HGC27 and MGC803 were observed by cell scratch test.Transwell experiments were performed to observe the effect of ncRuPAR on the invasive ability of gastric cancer cells HGC27 and MGC803.(4)HGC27-ncRuPAR,HGC27-Empty Vector and HGC27-Control cell lines were subcutaneously injected in nude mice respectively to observe tumor volume and weight of nude mice in each group,and to compare the effect of ncRuPAR on tumor growth in nude mice.(5)Affymetrix gene expression microarray technique was used to screen differentially expressed genes between HGC27-ncRuPAR and HGC27-Empty Vector.And IPA analysis was performed on the differential gene results of chip analysis.Then we investigated the possible pathways that lncRNA-ncRuPAR might influence the proliferation and apoptosis of gastric cancer cells.The effect of ncRuPAR on the expression of PAR-1 and PI3K/Akt signaling pathway and CyclinD1 and Bcl-2 proteins in HGC27 and MGC803 cells was detected by Western Blot assay.(6)HGC27 and MGC803 were treated by ZiYinHuaTan presciription.Then the cell proliferation was detected by CCK-8 kits to observe the effect of ZiYinHuaTan presciription on the proliferation of gastric cancer cells.The effects of ZiYinHuaTan presciription on the cycle and apoptosis of gastric cancer cells HGC27 and MGC803 were analyzed by flow cytometry qPCR was used to detect the effect of ZiyinHuatan presciription on ncRuPAR expression in gastric cancer cells to observe whether ZiyinHuatan presciription can regulate the expression of ncRuPAR.Western blot was used to validate the expression of PAR-1 protein and PI3K/Akt signaling pathway and CyclinD1 and Bcl-2 proteins on HGC27 and MGC803 after being treated by ZiYinHuaTan presciriptionto explore the possible mechanism of ZiYinHuaTan prescription.Results:(1)qPCR results showed that: the expression of ncRuPARwas low in HGC27 and high in MGC803.(2)HGC27 was selected to construct ncRuPAR overexpression stable strain and MGC803 was selected to construct ncRuPAR interference stable strain.qPCR validated that ncRuPAR overexpression and interference stable strains were successfully constructed.(3)The results of CCK-8 and flow cytometry showed that the overexpression of lncRNA-ncRuPAR blocked the cell cycle of HGC27 in G1 phase,inhibited the proliferation of gastric cancer cells and promoted the apoptosis of gastric cancer cells,while the interference of ncRuPAR expression inhibited MGC803 cells proliferation,and inhibited apoptosis.The results of scratch and transwell experiments showed that ncRuPAR had no significant effect on the migration and invasion of gastric cancer cells HGC27 and MGC803.(4)In vivo experiments showed that tumor formation in nude mice was slowed down,and the tumor volume was reduced in HGC27-ncRuPAR group compared with the control group.(5)Western blot showed that lncRNA-ncRuPAR inhibited PAR-1 protein expression and the activation of PI3K/Akt signaling pathway and reduced CyclinD1 expression.(6)The results of CCK-8 and flow cytometry showed that theZiYinHuaTan prescription can inhibited the proliferation of gastric cancer cells and promoted the apoptosis of gastric cancer cells.qPCR showed that ZiYinHuaTanprescriptioncould increase the expression of ncRuPAR in HGC27 and MGC803.Western blot showed that ZiYinHuaTtan Prescription could inhibit the activation of PI3K/Akt signaling pathway and decrease the expression of CyclinD1 and Bcl-2.Conclusion:ncRuPAR can inhibit the proliferation and promote the apoptosis of gastric cancer cells by down-regulating the expression of PAR-1 and inhibiting the PI3K/Akt pathway.The up-regulation of ncRuPAR expression and inhibition of PI3K/Akt pathway are possible mechanisms of traditional Chinese medicine ZiYinHuaTan prescription in inhibiting the proliferation of tumor cellsand promoted the apoptosis of gastric cancer cells.