| Objective To explore the effect of Ghrelin on autophagy of human ovarian cancer cells;next-generation sequencing technology was used to analyze the differentially expressed IncRNAs of human ovarian cancer cells treated with Ghrelin.The differentially expressed IncRNAs related to autophagy were screened and the effect of Ghrelin on their expression was verified.To further clarify the effect of IncRNA LINC00598 on autophagy of human ovarian cancer cells,and to explore whether Ghrelin can affect autophagy of human ovarian cancer cells through LINC00598.Methods ①Cell counting kit-8(CCK-8)was used to detect the effect of Ghrelin on the proliferation of human ovarian cancer cell line SK-OV-3;Western blot was used to detect the expression of Beclin 1 and LC3 Ⅱ in SK-OV-3 cells treated with Ghrelin,Ghrelin receptor inhibitor(D-Lys3-GHRP-6),Ghrelin and receptor inhibitor(Ghrelin+D-Lys3-GHRP-6).②Total RNA was extracted from SK-OV-3 cells of blank control group and Ghrelin group(treated with 600 ng/ml Ghrelin for 24 h)by Trizol method.The differentially expressed lncRNAs in Ghrelin group were screened by next-generation sequencing technology.The target genes were predicted,GO analysis and KEGG pathway analysis were performed.According to the results of bioinformatics analysis,differentially expressed lncRNAs related to autophagy were screened.The expression level of LINC00598 in SK-OV-3 cells of blank control group and Ghrelin group was detected by qRT-PCR.③The LINC00598 overexpression vector(h-LINC00598)and its negative control(pcDNA3.1),LINC00598 small interfering RNA(si-LINC00598)and its negative control(si-NC)were transfected into SK-OV-3 cells by liposome transfection method.After 48 hours,the expression levels of LINC00598,Beclin 1 and LC3 Ⅱ in SK-OV-3 cells were detected by qRT-PCR and Western blot.④SK-OV-3 cells were transfected with h-LINC00598,pcDNA3.1,si-LINC00598 and si-NC by liposome transfection method.SK-OV-3 cells were treated with 600 ng/ml Ghrelin for 24 hours after transfection.The expression levels of Beclin 1 and LC3 Ⅱ in SK-OV-3 cells were detected by Western blot.Results①The results of CCK-8 showed that the survival rate of SK-OV-3 cells was significantly decreased compared with the blank control group after Ghrelin was treated with Ghrelin at different concentrations(400,500,600,700,800 ng/ml)and at different time(24,48,72 h),and the difference was statistically significant(P<0.01).Western blot showed that compared with the blank control group,the expression of LC3 Ⅱ and Beclin 1 in Ghrelin group was significantly increased(P<0.05),while the expression of LC3 II and Beclin1 in D-Lys3-GHRP-6 and Ghrelin+D-Lys3-GHORP-6 group had no significant difference(P>0.05).②The next-generation sequencing results showed that compared with the blank control group,236 lncRNAs(130 up-regulated and 106 down-regulated)and 71 mRNA(66 up-regulated and 5 down-regulated)were differentially expressed in Ghrelin group.qRT-PCR results showed that compared with the blank control group,the expression level of LINC00598 in Ghrelin group was significantly up-regulated(P<0.05).③After 48 hours of transfection of LINC00598 overexpression vector(h-LINC00598)and its negative control(pcDNA3.1)into human ovarian cancer cells,qRT-PCR results showed that compared with the blank control group,the expression level of LINC00598 in h-LINC00598 group was significantly up-regulated(P<0.05),but there was no significant change in the expression level of LINC00598 in pcDNA3.1 group,the difference was not statistically significant(P>0.05).After transfection of LINC00598 siRNA(si-LINC00598)and its negative control(si-NC)into SK-OV-3 cells for 48 h,qRT-PCR results showed that compared with the blank control group,the expression level of LINC00598 in si-LINC00598 group was significantly down regulated(P<0.05),while the expression level of LINC00598 in si-NC group was not significantly changed(P>0.05).Western blot results showed that compared with the blank control group,the protein expression of LC3 Ⅱ and Beclin 1 in si-LINC00598 group was significantly decreased,and the protein expression of LC3Ⅱ and Beclin 1 in h-LINC00598 group was significantly increased,with statistical significance(P<0.05).However,the expression of LC3 Ⅱ and Beclin 1 protein in pcDNA3.1 group and si-NC group had no significant difference(P>0.05).④Western blot results showed that compared with the blank control group,the relative expression of LC3 Ⅱ and Beclinl protein in ghrelin group increased,the relative expression of LC3 Ⅱ and Beclinl protein in ghrelin+h-linc00598 group increased significantly,and the relative expression of LC3 Ⅱ and Beclinl protein in ghrelin+si-linc00598 group decreased significantly,(P<0.05);There was no significant difference in the relative expression of LC3 Ⅱ and Beclinl between pcDNA3.1 group and si-NC group(P>0.05).Conclusion ① Ghrelin can inhibit the proliferation of human ovarian cancer cells;② Ghrelin promotes autophagy of human ovarian cancer cells;③ Ghrelin upregulates the expression of LINC00598 in human ovarian cancer cells;④LINC00598 promotes autophagy of human ovarian cancer cells;⑤Ghrelin promotes autophagy of human ovarian cancer cells through LINC00598. |
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