Ghrline Promot the Ovarian Cancer Cell Autophagy by LncRNA linc 00598

Ruixia Bai Inner Mongolia People's Hospital Wanying Song xinganmeng people's hospital Yan Cui Inner Mongolia Medical University Haining Gao Inner Mongolia Medical University Yuxing Zhao Inner Mongolia Medical University Jingkun Lu Inner Mongolia Medical University Xuan Zhang Inner Mongolia Medical University Mei Hong Inner Mongolia Medical University Peng Sun Inner Mongolia Medical University Mingyu Zhang Inner Mongolia Medical University Jiaxian Cui Inner Mongolia Medical University Xiumei Wang Inner Mongolia Medical University Pengwei Zhao (  pengwzhao@126.com ) Inner Mongolia Medical University https://orcid.org/0000-0002-2526-5705


Introduction
Ovarian cancer is the common gynecologic cancer death in worldwide. It threat to women's health (Wu, Gao et al. 2021; Xie, Wang et al. 2021). And a large number of women was died from ovarian cancer each year. The overall 5-year survival rate of ovarian cancer is about 47%. However, in the early stage, the typical symptoms was lacked in the patients of ovarian cancer (Wang, Chen et al. 2021;Wei, Lv et al. 2021). More than 2/3 cancer cases was diagnosed at the advanced stage, and the 5-year survival rate was less than 25%. Hence, the diagnosis and treatment on ovarian cancer gave a big challenge(Wang Ghrelin is one of the endogenous ligands of growth hormone secretagogue receptor, which can promote secretion of growth hormone and is proven to be with the orexigenic and adipogenic effects (Leng, Zhao et al. 2021). Moreover, the positive effect of Ghrelin on metabolism of bone has been observed through regulation of by proliferation ovarian cancer cells ( Cell autophagy can clean out the damaged organelles, proteins in normal (Sun, Hu et al. 2020). However in the apoptotic cells, autophagy can prevent the necrosis, and local in ammation (Meng, Zhou et al. 2020;Cai, An et al. 2021). Hence, in this study, the autophagy effect of ghrelin on the ovarian cancer cell line SK-OV-3 was explored. And the lncRNA which regulate the ghrelin effect SK-OV-3 autophagy was showed.

RNA extraction and sequencing
Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Brie y, mRNA was puri ed from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesize dusing random oligonucleotides and SuperScript II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. The library fragments were puri ed using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were puri ed (AMPure XP system) and quanti ed using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on a HiSeq platform (Illumina) by Seqhealth Technology Co., Ltd., Wuhan, China. Differentially expressed genes were then identi ed by applying a FDR cutoff of 0.05.

Gene function annotation and pathway analysis
Identi cation of enriched KEGG pathways in the upregulated and downregulated gene lists was performed with DAVID (Database for Annotation, Visualization, and Integrated Discovery) v6.8. Pathway analysis was performed using Ingenuity Pathway Analysis (IPA), version to infer the functional roles and relationships of the differentially expressed genes based on the log2 fold-change value of each gene.

Cell transfection
The mimic of miR-378b as well as negative control (NC mimic) were purchased from Invitrogen (Carlsbad, CA, USA). The full length of SNHG10 was ligated in pcDNA3.1 vector (pcDNA3.1SNHG10) was purchased from GenePharma (Shanghai, China) with empty plasmid as negative control (pcDNA3.1). Cell transfection was conducted using Lipofectamine 2000 (Thermo Fisher Scienti c, Waltham, MA, USA).
After 48 h, cells were harvested for following experiments.

Real-Time qPCR analysis
The total RNA was extracted from the SK-OV-3 cells by using the commercial TRIzol reagent (Invitrogen, USA) following the manufacturer's instruction. The quality of the total RNA was determined by agarose electrophoresis, and the RNA was reversely transcribed into complementary DNA (cDNA) by using the TIANScript RT kit (Tiangen Biotech, China). Then, the SYBR Premix Ex Taq TM II Kit (Takara, Japan) was used to determine relative gene expression levels and enrichment. The up-primer sequences for linc00598 is CCTCCCCTACTATCAACATCCC and down primer is TGCCAAGAACGAGCCCTA. The expression levels were normalized by GAPDH (up primer CAATGCCTCCTGCACCACCAACTGC and down primer GCAGTTGGTGGTGCAGGAGGCATTG )

Statistical analysis
Statistical analyses of data were performed using Graphpad Prism 8.0 (GraphPad Software, San Diego, CA, USA). The measurement data were expressed as mean ± standard deviation. Independent sample t test were used in comparisons between groups, and one-way analysis of variance (ANOVA) was used for comparison among multiple groups, followed by Tukey's post hoc tests. Comparison of data between groups at different time points was performed using two-way ANOVA. A p < 0.05 indicated statistically signi cant.

Ghrelin down expression in the ovarian cancer tissues
To obtain the ghrelin expression in the ovarian cancer tissues, the GEPIA database was used to analyse.
The ghrelin expression was lower in the ovarian cancer tissues than the normal tissues (Fig. 1A). And in HPA database ghrelin was lowly expressed in ovarian cancer tissues compared with normal tissues (  P<0.05, Fig. 1B). Hence, the ghrelin was expressed low in the ovarian cancer tissues.

Ghrelin promoted the cell autophagy and inhibited the cell viability in SK-OV-3 cells
To obtain the optimal concentration of ghrelin effect on the SK-OV-3, the SK-OV-3 cells were treated with ghrelin with the concentrations of 400, 500, 600, 700 and 800 ng/ml. Compared with the blank control group (control), the cell survival rate of 400, 500, 600, 700 and 800 ng/ml group decreased at 24 h, 48 h and 72 h (P < 0.01). When SK-OV-3 cells were treated with 600 ng/ml ghrelin for 24 h, 48 h and 72 h, the cell survival rates were around 50%( Fig. 2A). Hence, the ghrelin concentratio of 600 ng/ml was the optimal concentration o and 24 h was the optimal time.
The in uence on the SK-OV-3 cell autophagy by ghrelin was showed by detecting the expression of Beclin-1, LC3 and LC3 (Fig. 2B). The expression of Beclin-1and LC3 in SK-OV-3 cell treated with the 600 ng/ml ghrelin 24h was higher than without ghrelin treated (Fig. 2C&D). To explore the function of ghrelin on the SK-OV-3 cell autophagy further, the D-Lys3-GHRP6, a ghrelin receptor antagonist, was used. The expression of Beclin-1and LC3 in SK-OV-3 cell treated with ghrelin was higher than ghrelin+ D-Lys3-GHRP6 treatment (Fig. 2E-G).
Linc00598 selected as the effecting the SK-OV-3 cells autophagy by ghrelin using RNA-Seq To found the LncRNAs which effected the SK-OV-3 cells autophagy by ghrelin, the SK-OV-3 cells treated with ghrelin was analyzed by RNA-Seq. The differential expression of LncRNA was analyzed by DESeq. The conditions for screening differentially expressed genes were as follows: the multiple of expression difference | log2foldchange | > 1, P < 0.05. Compared with the control group, 236 LncRNAs were differentially expressed (up-regulated 130 and down regulated 106), in the ghrelin treatment group. The volcanic map shows the distribution of LncRNAs, the difference of LncRNAs expression fold and signi cance results (Fig. 3A). In Fig. 3A, the red dot represents the differentially up-regulated LncRNAs, the blue dot represents the differentially down-regulated LncRNAs, and the gray dot represents the non signi cant differentially expressed LncRNAs.
The go enrichment analysis results of differentially expressed LncRNAs are classi ed, according to molecular function MF, biological process BP and cell component CC. The top 10 go term items with the smallest p-value, i.e. the most signi cant enrichment, are selected for display in each go classi cation (Fig. 2B). In the gure 2B, the orange histogram represents the top 10 cell components with the most signi cant enrichment, the green histogram represents the top 10 molecular functions with the most signi cant enrichment, and the blue histogram represents the top 10 biological processes with the most signi cant enrichment. According to the go enrichment results, the enrichment degree is measured by rich factor, FDR value and the number of LncRNA enriched on the GO term. The larger the rich factor, the greater the degree of enrichment. The general value range of FDR is 0-1. The closer is closed to zero, the more signi cant the enrichment. The rst 20 GO term entries with the lowest FDR value,are selected for display (Fig. 2C). Go enrichment analysis showed that these differentially expressed LncRNAs were mainly related to arginine and lysine transmembrane transport, oxidative stress response and developmental process.
The top 20 pathways with the lowest p-value value are displayed, according to the KEGG enrichment analysis the differentially expressed LncRNAs (Fig. 3D). The path in the gure mainly involves four aspects: environmental information processing (orange part), human diseases (green part), metabolism (blue part) and organic systerms (purple part). According to the KEGG enrichment results, the enrichment degree, the top 20 KEGG pathways with the lowest FDR value are selected. (Fig. 3E). KEGG enrichment analysis showed that these differentially expressed LncRNAs were mainly enriched in cytokine receptor signaling pathway, glucagon and insulin signaling pathway and cancer-related signaling pathway.
According to the prediction of target genes and the enrichment analysis results of go and KEGG pathway, the linc00598 was selected as the potential relationship with autophagy, and the linc00598 was differential up-regulation of fold change > 2 times. The expression of linc00598 in SK-OV-3 cells was detected by qPCR. The expression of linc00598 in the ghrelin treated group was higher that blank group.

Linc00598 promote the SK-OV-3 cells autophagy treated by ghrelin
To show the function of linc00598 on the SK-OV-3 cells autophagy, the linc00598 was silenced and overexpressed (Fig. 4A&B). And the expression of Beclin1 and LC3 in the linc00598 silence group (si-linc00598) was lower than the control group and si-NC group (the blank plasmid, Fig. 4C,D,F ). However, in the linc00598 overexpressed group (h-linc00598), the expression of Beclin1 and LC3 was high ( Fig. 4C,E,G).The effect of linc00598 silenced or overexpressed on the SK-OV-3 cells autophagy treated by ghrelin was also explored. The expression of Beclin1 and LC3 in the Ghrelin + Si-linc00598 group was lower than the ghrelin group (Fig. 4A,B,D). And the expression of Beclin1 and LC3 in the Ghrelin + h-linc00598 was higher than ghrelin group (Fig. 4A,C,E).

Discussion
Ovarian cancer has a high mortality rate ( Ghrelin and linc00598 can promote the SK-OV-3 cells autophagy. The linc00598 was silenced and overexpressed in the SK-OV-3 cells. Then the ghrelin was used to effect the SK-OV-3 cells. We found ghrelin mainly through linc00598 to promote the SK-OV-3 cells autophagy. When the linc00598 silenced, ghrelin promote SK-OV-3 cells autophagy was inhibited. And When the linc00598 overexpressed, ghrelin promote SK-OV-3 cells autophagy was inhanced.

Declarations
Ruixia Bai1a, Wanying Song and Pengwei Zhao wrote this article. Xiumei Wang, Pengwei Zhao designed, organized and reviewed this article. All authors have read and agreed to the published version of the manuscript.

Availability of data and materials
All data generated or analyzes during this study are included in this published article.
Ethics approval and consent to participate Not applicable.

Consent for publication
Not applicable.

Declaration of Interest Statement
The authors declare that they have no competing interests. Figure 1 Expression of grhelin in different ovarian cancer tissues. A. The expression levels of ghrelin in different cancer tissues were provided by GEPIA database. B. The expression levels of grhelin in ovarian cancer tissues were provided by HPA database. * P < 0.05 was regarded as statistical signi cance.

Figure 2
In uence of ghrelin on SK-OV-3 Cell viability and the autophagy pathway.A. Effect of ghrelin with 400, 500, 600, 700 and 800 ng / mL on SK-OV-3 Cell viability at 24h, 48h and 72h. B-D Western Blot analysis was conducted to examine the protein levels of Beclin-1, LC3 and LC3 in SK-OV-3 cells after ghrelin added. E-G Western Blot analysis was conducted to examine the protein levels of Beclin-1, LC3 and LC3 in SK-OV-3 cells after ghrelin and D-Lys3-GHRP6 added. Single experiment had 3 repetitions, and * P < 0.05 was regarded as statistical signi cance.  Effect of LINC00598 on the SK-OV-3 cells autophagy pathway. A&B. the expression of LINC00598 in the SK-OV-3 cells after the LINC00598 silenced or overexpressed. C-F. Western Blot analysis was conducted to examine the protein levels of Beclin-1, LC3 and LC3 in SK-OV-3 cells after the LINC00598 silenced or overexpressed. Single experiment had 3 repetitions, and * P < 0.05 was regarded as statistical signi cance.