Recombinant human granulocyte colony stimulating factor (rhG-CSF) participates in the progression of implantation via the hsa_circ_0001550-miRNA-mRNA interaction network

Abstract Inadequate endometrial receptivity is a key factor affecting the successful implantation of embryos. Recombinant human granulocyte colony stimulating factor (rhG-CSF) can increase endometrial thickness and improve the outcomes of assisted reproductive technologies (ARTs). In this preliminary study, the function and possible molecular mechanisms of recombinant human granulocyte colony stimulating factor (rhG-CSF) which affects endometrial receptivity and implantation in human Embryonic Stem Cells (hESCs) were investigated. The cell viability of endometrial stromal cells treated with rhG-CSF 0.5 ng/ml for 24 h was significantly increased. Moreover, the expression of hsa_circ_0001550 was downregulated in endometrial stromal cells treated with rhG-CSF. Furthermore, the hsa_circ_0001550-miRNA-mRNA network was constructed and the downstream target genes (including 4 miRNAs and 117 mRNAs) of hsa_circ_0001550 were mainly involved in the cAMP and calcium signalling pathways, which play important roles in regulating endometrial receptivity and embryo implantation. We conclude that rhG-CSF participates in the regulation of embryo implantation by regulating the hsa_circ_0001550-miRNA-mRNA interaction network.


Introduction
Endometrial receptivity is a critical factor that affects the success of embryo implantation (Nazari et al., 2020;L. Wang et al., 2020), and is defined as the state in which the embryo locates, adheres, and invades the endometrium resulting in implantation (Lessey & Young, 2019).Almost 70% of IVF pregnancy failure is caused by endometrial factors (Margalioth et al., 2006) and improvements in the endometrial receptivity and pregnancy outcomes of IVF-ET patients are clearly required.
Several studies have reported that recombinant human granulocyte colony stimulating factor (rhG-CSF) can improve endometrial receptivity (Margalioth et al., 2006) and affect pregnancy outcomes (Jain et al., 2018) by improving endometrial blood flow (Myles et al., 2017) and increasing endometrial thickness (Lee et al., 2016;Y. Li et al., 2014).At present, most of the studies on rhG-CSF have focussed on outcomes, in terms of clinical pregnancy (Arefi et al., 2018;Jain et al., 2018;Kalem et al., 2020) and its specific mechanism of action is unclear.
CircRNAs, a newly discovered class of non-coding RNAs with a closed circular structure, which tolerate exonuclease cleavage, are abundantly present in the eukaryotic transcriptome (S.Wang et al., 2019).Possible roles of circRNA have been reported in follicular maturation (Cheng et al., 2017), the occurrence of polycystic ovary syndrome (PCOS) (Che et al., 2019;J. Liu et al., 2020), endometrial receptivity (L.Liu et al., 2017;Shen et al., 2019), ovarian endometriosis (Hu et al., 2019;X. X. Xu et al., 2018), mouse embryo development (Lin et al., 2016), and the development from pre-receptive to receptive phases in the goat endometrium (Song et al., 2019).All these studies have shown that circRNAs play key roles in reproduction and are essential for embryo implantation.
hsa_circ_0001550 is encoded at chr5：167915606-167921655, which includes 534 base sequences (formed of exons 2-5); the host gene symbol is RARS.Although hsa_circ_0001550 has been reported in previous studies (D.Xu et al., 2020), we have reported that the expression of hsa_circ_0001550 increases significantly in patients with repeated implantation failure (RIF) compared with a normal group (L.Liu et al., 2017).However, the specific mechanism of its function remains unknown.In addition, most studies have shown that an increase in endometrial molecular markers is related to the successful implantation of embryos (L. Liu et al., 2017;G. Xu et al., 2019).
This study aimed to evaluate the potential mechanism of rhG-CSF in implantation; specifically, the biofunction of hsa_circ_0001550 in the progression of embryo implantation using human Embryonic Stem Cells (hESCs) as a model system, which were treated with rhG-CSF.

Samples and cell culture
Ethical approval for tissue collection was provided by the Institutional Ethics Committee of the First Hospital of Lanzhou University (Lanzhou, China).Endometrial biopsies were obtained using an endometrial sampler (Endocell) from patients undergoing hysteroscopy who were confirmed not to have endometrial pathological changes.The endometrial samples of six patients (for tissue extraction of RNA and cell culture) were taken from the endometrial proliferative stage (the 5th to 10th day of the menstrual cycle).The secretory phase endometrial tissues (for tissue extraction of RNA) were from three patients, while two cases had no transplantable embryos and one case cancelled their cycle of transplantation for personal reasons.All patients gave informed consent.
Endometrial specimens were washed with PBS three times and twice the volume of 0.1% IV collagenase added (Solarbio, China), followed by incubation at 37 C for 30 min to 1 h.After the tissue was completely digested, the same volume of complete medium was added to terminate digestion.The cell suspension was filtered through 100 and 40 lm cell sieves sequentially (Wei et al., 2018), then centrifuged at 204 g for 5 min.The supernatant was discarded, and the appropriate volume of medium was added to precipitate and inoculate a petri dish.hESCs were cultured in DMEM/Nutrient Mixture F-12 (DMEM/F-12) containing 1% (v/v) penicillin/streptomycin and 10% (v/v) FBS (Hyclone, USA).The cells were cultured in a 5% CO 2 incubator at 37 C.After one passage, cells were >99% pure as determined by immunostaining for vimentin (Wei et al., 2018).

Total RNA isolation and quality control
Total RNA from cells was isolated using Total RNA Extractor (Sangon Biotech) following the manufacturer's instructions.A NanoDrop ND-2000 instrument (NanoDrop Technologies) was used to determine the quantity and quality of total RNA samples for all experimental requirements (concentration >100 ng/ll; 260/280:1.8-2.0).

Reverse-transcription and real-time quantitative PCR (qRT-PCR)
cDNA was synthesized using qRT-PCR with TB Green Premix Ex TaqII (Takara, Bio, USA) and ACTB was used as an internal control.The primers were synthesized by Sangon Biotech (primers sequences are shown in Table 1).Thermocycling conditions included programme segment 1: 95 C, 10 min; programme segment 2: 95 C, 10s followed by 60 C, 1min; programme segment 3: 95 C, 10s; 60 C, 1min and 95 C, 15s.Specificity was confirmed by a single peak on the melting curves.The relative expression levels of circRNA were normalized to ACTB.All experiments were repeated three times.

CCK8 assay
A cell suspension was obtained by the digestion of hESCs with 0.25% trypsin in the logarithmic growth period.The cell density was adjusted to 5 Â 10 4 /ml, and 100 ll of the cell suspension was added to a 96well plate, then placed in a 37 C and 5% CO 2 incubator.After the cells had grown to 50-60%, the original culture medium was discarded and 100 ll DMEM/F-12 culture medium containing different concentrations of rhG-CSF (0, 0.5, 1, 5 and 10 ng/ml) was added.Each concentration was set up with three wells and repeated three times.After treating with the different concentrations of rhG-CSF for 24, 48 and 72 h, the original culture medium was discarded and 100 ll of DMEM/F-12 medium containing 10 ll of enhanced CCK8 was added to each well while avoiding bubbles.The 96-well plate was placed in an incubator and cultured for 2 h.After 2 h, the absorbance at 450 nm was detected using a Benchmark Plus microplate spectrometer.Cell viability was determined using an enhanced cell counting kit-8 assay (enhanced CCK8, Bio, China) based on published procedures.Cell viability ¼ (OD450 experimental well À OD450 blank well)/(OD450 control well À OD450 blank well) Â 100%.The experiment was repeated independently three times.

Bioinformatic analyses
CircBank (http://www.circbank.cn/index.html)and circRNA interactome (https://circinteractome.nia.nih.gov/) were used to predict the target miRNAs of hsa_circ_0001550.Arraystar's custom-made miRNA target prediction software was used to predict the target gene of these miRNAs.The interaction network was depicted using Cytoscape (cytoscape.org)software, which preliminarily accounted for the interactions of hsa_circ_0001550-miRNAs-mRNAs with infertility.GO analyses using the DAVID (https://david.ncifcrf.gov/home.jsp)online database were used to annotate and account for the biological process, cellular components and molecular function of these genes.The top six ranked GO terms of biological process, cellular component and molecular function were analyzed.The results of the GO analysis were analyzed statistically using a hypergeometric test with a p-value <0.05 as the screening standard.KEGG pathway enrichment was performed using the online software KOBAS 3.0 (kobas.cbi.pku.edu.cn) to explore the biological function and signalling pathways of the targeted genes in depth.A hypergeometric test was provided by the KOBAS 3.0 online software and the screening threshold was p-value <0.01.

Statistical analysis
Data are expressed as mean ± SEM and the means were compared using a two-tailed, unpaired Student's t-test.p-Value < 0.05 was considered significant.Statistical analyses were performed using GraphPad Prism 8.0 (GraphPad).

Results
The influence on endometrial stromal cell proliferation with different concentrations of rhG-CSF As shown in Figure 1(A), the proliferation of hESCs was promoted in 0.5, 1 and 5 ng/ml rhG-CSF.The cell viability of hESCs was increased significantly when treated with rhG-CSF 0.5 ng/ml (p < 0.0001).Therefore, rhG-CSF 0.5 ng/ml was selected as the optimal concentration on hESCs.
Next, the optimal timing of 0.5 ng/ml rhG-CSF on hESCs was investigated (Figure 1(B)).It was found that rhG-CSF at 0.5 ng/ml for 24, 48 and 72 h promoted cell proliferation.However, when hESCs were treated with rhG-CSF for 24 h, cell viability was increased significantly (p < 0.0001).The concentration of 0.5 ng/ml rhG-CSF treated with hESCs for 24 h was therefore selected for subsequent experiments.
The expression of hsa_circ_0001550 in endometrial tissues, and hESCs treatment with rhG-CSF Phylogenetic analysis using the UCSC Genome Browser (http://genome.ucsc.edu/)revealed that hsa_circ_0001550 is positioned at 5q34 (167913463-167946309).The transcript information of the mother gene, RARS, shown in Figure 2, is highly conserved among mammals, suggesting an important function of hsa_circ_0001550.The linear and circular structure diagrams of hsa_circ_0001550 were drawn by PowerPoint according to the hsa_circ_ 0001550 structure (Figure 2).
In order to study the molecular function of hsa_circ_ 0001550 in endometrial tissues, the expression of hsa_circ_0001550 was examined both in the secretory and proliferative phases.As shown in Figure 3(A), the expression of hsa_circ_0001550 was significantly reduced in the endometrium during the mid-secretory phase.The expression of hsa_circ_0001550 in cells was therefore studied further and found to be significantly lower in hESCs treated with 0.5 ng/ml G-CSF compared with the control group (p < 0.05, Figure 3(B)).Nevertheless, the expression of RARS showed no difference compared to the control group (p > 0.05, Figure 3(C)).

Prediction of target miRNAs and mRNAs
In order to investigate the correlation between circRNAs and miRNAs, the target miRNAs of hsa_circ_ 0001550 were predicted using circBank (http://www.circbank.cn/index.html)and the circRNA interactome (https://circinteractome.nia.nih.gov/)online database.These included 49 and 18 target miRNAs respectively.Four common miRNAs were screened from two databases, which were named hsa-miR-1231, hsa-miR-1290, hsa-miR-578 and hsa-miR-634 (Figure 4(A)).To understand the biological function of these miRNAs, the mRNA targets of hsa-miR-1231, hsa-miR-1290, hsa-miR-578 and hsa-miR-634 were predicted via TargerScan Human (http://www.targetscan.org/vert_72/) in which there are a total of 10,091 targeted mRNA (Supplementary Table 1) and miRDB (http://www.mirdb.org/index.html),from which 2,238 mRNAs were obtained (Supplementary Table 2).Meanwhile, we downloaded the datasets GSE103465 (Supplementary Table 3) and GSE92324 (Supplementary Table 4) from the GEO database, which are related to endometrial receptivity.Based on p-values < 0.05, 3578 targeted genes were chosen from GSE103465 [Supplementary Table 3(a)] and 4907 mRNAs were chosen from GSE92324 [Supplementary Table 4(a)].Eventually, 117 target mRNAs (Supplementary Table 5) of hsa-miR-1231, hsa-miR-1290, hsa-miR-578 and hsa-miR-634 were chosen from these four datasets (Figure 4(B)).Next, the hsa_circ_0001550 -miRNA-mRNA network was constructed to clarify gene regulatory relationships in patients with low endometrial receptivity and 117 target genes were selected from the four databases for follow-up analysis.The 117 target genes were screened according to the chip data of GSE103465 and GSE92324.In the two databases, 41 mRNAs were confirmed as upregulated (labelled in red) and 67 mRNAs were downregulated in the RIF group (labelled in green) compared.There were nine genes whose expression was inconsistent in the two databases, indicated in blue (Figure 5).

Enrichment analysis of hsa_circ_0001550targeted genes
GO analyses were performed in order to understand the function of the hypothetical 117 targeted genes using the DAVID online database (https://david.ncifcrf.gov/summary.jsp).The results are shown in Supplementary Table 6.The top six ranked GO terms (p-value < 0.05, biological process, cellular component and molecular function) were selected to illustrate the results (Figure 6).The common target genes of hsa-miR-1231, hsa-miR-1290, hsa-miR-578 and hsa-miR-634 are involved in the regulation of translation, ion transmembrane transport, angiogenesis, etc.These target genes are mainly located in the plasma membrane and cell-cell adherens junctions.In the aspect of molecular function, hsa_circ_0001550 target genes play important roles in regulating the progress of protein binding, Rac GTPase binding and cadherin binding involved in cell-cell adhesion, which may play critical roles in the regulation of embryo implantation (Y.Li et al., 2021).
Assuming a p-value <0.01 as the screening standard, 45 KEGG enrichment pathways were related to the predicted mRNAs using the KOBAS 3.0 online    database (http://kobas.cbi.pku.edu.cn/kobas3) and the results shown in Supplementary Table 7.The top 10 significantly enriched KEGG pathways were as follows: apelin signalling pathway, calcium signalling pathway, cAMP signalling pathway, relaxin signalling pathway, cGMP-PKG signalling pathway, tight junction, salivary secretion, Alzheimer's disease, pathways in cancer and focal adhesion (Figure 7).The calcium signalling pathway (Figure 8(A)) and cAMP signalling pathway (Figure 8(B)) were the most critical enriched pathways related to endometrial receptivity and embryo implantation.

Discussion
Defective endometrial receptivity continues to be an important factor leading to the failure of embryo implantation (Achache et al., 2010).Various gene abnormalities have been reported to participate in the regulation of endometrial receptivity (Hashimoto et al., 2017;Shi et al., 2017).As a routine, intrauterine perfusion (Eftekhar et al., 2016) and subcutaneous injection (Eftekhar et al., 2016) of rhG-CSF have been widely used in clinical treatment.A recent clinical study (Kamath et al., 2020) demonstrated that the rhG-CSF has a positive effect on the outcomes of IVF-ET patients, which can improve endometrial receptivity by increasing endometrial thickness (Miralaei et al., 2019) and improve pregnancy outcomes in patients with RIF (Kalem et al., 2020;Zeyneloglu et al., 2020).However, the molecular biological function of rhG-CSF is still uncertain.rhG-CSF has been reported to have positive effects on oocyte maturation (W€ urfel, 2015), follicular development (H.Wang et al., 2005), endometrial vascular remodelling (Rahmati et al., 2014) and the process of decidualization in ESCs (Tanaka et al., 2000).Salmassi et al. proposed that the level of G-CSF in serum and follicular fluid can predict IVF outcomes (Salmassi et al., 2005).In the present study, we have shown that the cell viability of hESCs increased significantly when treated with rhG-CSF at a concentration of 0.5 ng/ml for 24 h (Figure 1), which indicated that rhG-CSF significantly promoted the proliferation of endometrial stromal cells.Moreover, we have investigated the detailed molecular pathway by which rhG-CSF promotes endometrial stromal cell proliferation.In a previous study, we reported that hsa_circ_0001550 is differentially expressed in patients with RIF compared with normal patients (2017).However, the role of hsa_circ_0001550 in endometrial receptivity has not been explored.
The secretory phase changes in the endometrium occur 6-10 days after ovulation; the endometrium at this stage has the ability to accept embryos.In this study, we concluded that hsa_circ_0001550 was significantly under-expressed in the endometrium at the mid-secretory phase (Figure 3(A)), indicating that hsa_circ_0001550 leads to low endometrial receptivity.However, hsa_circ_0001550 was significantly decreased in the hESCs treated with rhG-CSF (Figure 3(B)), indicating that hsa_circ_0001550 could play an important role during the progression of embryo implantation or serve as a novel biomarker for female infertility related disorders generally.Moreover, the expression of RARS was unchanged in cells treated with rhG-CSF (Figure 3(C)).Arginyl-tRNA synthetase (ArgRS) is a translation product of the RARS gene, which is the mother gene of hsa_circ_0001550, and is an important enzyme in protein biosynthesis.Its main function is to participate in tRNA acylation, which belongs to the first class of ArgRS.The role of ArgRS in mammalian translation and cell growth (Kyriacou & Deutscher, 2008); processes closely related to the embryo implantation process, has been reported.
To understand the molecular function of hsa_circ_ 0001550, in more detail potential target miRNAs were predicted (Figure 4(A)).The four miRNAs (hsa-miR-1231, hsa-miR-1290, hsa-miR-578 and hsa-miR-634) have not previously been reported to play a role in implantation.Wang et al. found that miR-1231 is downregulated in prostate cancer (Y.Wang et al., 2020) and differential expression of hsa-miR-1290 is found in the serum of pregnant women with Down's syndrome (Zbucka-Kretowska et al., 2019) and gestational diabetes (Lamadrid-Romero et al., 2018).A further study concluded that hsa-miR-634 might play important roles in the pathogenesis of obesity (J.Li et al., 2015) and can decrease cell proliferation and induce apoptosis in cervical cancer cells by targeting the mTOR signalling pathway (Cong et al., 2016).A further study by Chen et al. concluded that CircZFR as an miR-578 sponge promotes breast cancer progression (Z.Chen et al., 2020).These results suggest that the role of the predicted miRNAs in endometrial receptivity and the progression of implantation needs to be studied further.
In addition, we predicted the targeted mRNAs of these four miRNAs (Figure 4(B)) and the hsa_ circ_0001550-miRNA-mRNA network was constructed (Figure 5).
Among the downstream mRNAs, several targets were previously demonstrated to be associated with endometrial receptivity.Segal et al. established a mouse ART model dagger and confirmed that VEGFA plays an important role in the establishment of early pregnancy (Segal et al., 2020).Moreover, CD38 may be involved in maintaining the uterine immune microenvironment (Khan et al., 2019).HOXB3 has been reported to maintain transcriptional regulation during embryo implantation (Chan et al., 2010) and AKAP2 is involved in the dynamic regulation of PKA localization during oocyte and early embryo development (Webb et al., 2008).
In order to explore the possible molecular function and mechanism of hsa_circ_0001550 in more depth, we performed a GO analysis of targeted mRNAs and the top six GO terms are listed in Figure 6.Moreover, the KEGG pathways were analyzed and the top 10 are shown in Figure 7. GTPase activating proteins regulate glucose metabolism and actin cytoskeleton dynamics, which are crucial for cell-to-tissue-scale behaviour (Denk-Lobnig & Martin, 2019).Meanwhile, Rho-GTPase can regulate multiple cellular functions by regulating cAMP levels (X.Xu et al., 2016) and the adhesion molecule cadherin plays a crucial role in the regulation of embryo implantation (Donaghay & Lessey, 2007) and endometrial receptivity (Zhou et al., 2020), is expressed in the endometrium of patients during the 'implantation window' (Maia-Filho et al., 2015).Bellati et al. showed that the expression of cadherins was significantly decreased in the endometrium of patients with primary infertility and recurrent pregnancy loss (Bellati et al., 2019) and infertile patients with hydrosalpinx had lower E-cadherin and N-cadherin levels in the endometrium (Poncelet et al., 2010).In the present study, the function and possible molecular pathways affecting endometrial receptivity and implantation of rhG-CSF in hESCs were investigated.Meanwhile, hsa_circ_0001550, as a new biomarker, was found to be differentially expressed in the implantation window.We showed that rhG-CSF affected downstream target genes by regulating the expression of hsa_circ_0001550 and could be involved in the regulation of the calcium (Figure 8(A)) and cAMP (Figure 8(B)) signalling pathways.
As one of the most ubiquitous second messengers in eukaryotic cells, Ca 2þ is typically representative of cell signal transduction and intercellular communication (M.Wang et al., 2018).In our study, six downstream target genes (CD38, PLCB1, PTGER3, ADRB1, EDNRB and ATP2B4) of hsa_circ_0001550 are involved in the Ca 2þ signalling pathway (Figure 8(A), the locations of the target genes are marked in red).As can be seen, except for PCLB1, which mediates the expression of IP3R protein, the remaining five target genes are all involved in intracellular and extracellular signalling.CD38 is also involved in prolactin secretion and the ATP conduction pathway.These biological pathways are a prerequisite for the maintenance of endometrial receptivity (Azkargorta et al., 2018) and the Ca 2þ signalling pathway is involved in the angiogenesis process and improves endometrial receptivity (Munaron & Fiorio Pla, 2009).A further study showed that changes in intracellular Ca 2þ levels may participate in the decidualization of hESCs and regulate endometrial receptivity (Sohn et al., 2019).Thus, hsa_-circ_0001550 participates in Ca 2þ signalling through the regulation of the downstream target genes.
As shown in Figure 8(B), there were six downstream target genes (ADRB1, FOS, RRAS2, PTGER3, T1AM1 and ATP2B4) involved in the cAMP signalling pathway (the locations of the target genes are marked in red).Among them, RRAS2 mediates cell proliferation and apoptosis regulation, T1AM1 is involved in endothelial cytoskeleton changes in the downstream pathways, and ATP2B4 is involved in the phosphorylation process by PKA.These physiological processes are all involved in the regulation of endometrial receptivity.A previous study reported that cAMP activation is essential for the maintenance of pregnancy during the luteal phase (Casarini et al., 2016) which is the optimal period of endometrial receptivity.In addition, the decidualization of endometrial stromal cells during the secretory phase is important in the establishment of endometrial receptivity with cAMP a key regulatory molecule of the decidualization process (Yeo et al., 2007).Another study found that the decidualization process of hESCs was blocked in the presence of cAMP inhibitors (H.Chen et al., 2017).
Among these enriched target genes, ADRB1, PTGER3 and ATP2B are involved in both Ca 2þ and cAMP signalling, maintaining endometrial receptivity through their interaction.The Ca 2þ and cAMP pathways are therefore potentially related to the establishment of endometrial receptivity and embryo implantation.Our research has provided new evidence for the role of rhG-CSF in improving the success of embryo implantation but did not reveal the specific targeted molecular mechanism However, our preliminary observations using a bioinformatics approach indicate that rhG-CSF may regulate the hsa_circ_0001550-miRNA-mRNA network, which affects endometrial receptivity and embryo implantation.We have confirmed the overexpression efficiency and detected cellular viability (unpublished data) using the up-regulation of hsa_circ_0001550 expression by an adenovirus, showing that overexpression of hsa_circ_ 0001550 inhibits the proliferation of endometrial stromal cells.The effects of hsa_circ_0001550 on the cell cycle and apoptosis are still under further investigation.Future studies will examine the effect of implantation on rhG-CSF and hsa_circ_0001550, to provide us with stronger evidence.

Figure 1 .
Figure 1.The influence on endometrial stromal cell proliferation.(A) The cell viability analysis after treatment with rhG-CSF at the indicated concentrations for 24 h.(B) The cell viability analysis after treatment with rhG-CSF at the indicated time for 0 ng/ml and 0.5 ng/ml.Data are presented as the mean ± SEM from three independent experiments.ÃÃ p < 0.01; ÃÃÃ p < 0.005; ÃÃÃÃ p < 0.0001; ns p > 0.05.

Figure 2 .
Figure 2. Graphical views showing multi-species comparisons of hsa_circ_0001550 using UCSC genome browser.The conservation scores were indicated by the blue peaks.

Figure 3 .
Figure 3. (A) The expression of hsa_circ_0001550 in the endometrial proliferation and secretory phases.(B) The expression of hsa_circ_0001550 in the hESCs treated with rhG-CSF at 0.5 ng/ml.(C) The expression of RARS in the hESCs treated with rhG-CSF at 0.5 ng/ml.Data are presented as the mean ± SEM from three independent experiments.ÃÃ p < 0.01; ÃÃÃ p < 0.005; ns p > 0.05.

Figure 4 .
Figure 4. Target gene prediction of hsa_circ_0001550.(A) The targeted miRNAs of hsa_circ_0001550.(B) The targeted mRNAs of miRNA.

Figure 8 .
Figure 8. Signal pathway diagram of downstream target gene of hsa_circ_0001550.(A) Calcium signalling pathway diagram; (B) cAMP signal pathway diagram.