Abstract

   The HOXA gene family is associated with various cancer types. However,
   the role of HOXA genes in acute myeloid leukemia (AML) have not been
   comprehensively studied. We compared the transcriptional expression,
   survival data, and network analysis of HOXA-associated signaling
   pathways in patients with AML using the ONCOMINE, GEPIA, LinkedOmics,
   cBioPortal, and Metascape databases. We observed that HOXA2-10 mRNA
   expression levels were significantly upregulated in AML and that high
   HOXA1-10 expression was associated with poor AML patient prognosis. The
   HOXA genes were altered in ~18% of the AML samples, either in terms of
   amplification, deep deletion, or elevated mRNA expression. The
   following pathways were modulated by HOXA gene upregulation:
   GO:0048706: embryonic skeletal system development; R-HSA-5617472:
   activation of HOX genes in anterior hindbrain development during early
   embryogenesis; GO:0060216: definitive hemopoiesis; hsa05202:
   transcriptional mis-regulation in cancer; and GO:0045638: negative
   regulation of myeloid cell differentiation, and they were significantly
   regulated due to alterations affecting the HOXA genes. This study
   identified HOXA3-10 genes as potential AML therapeutic targets and
   prognostic markers.

   Keywords: HOXA gene family, acute myeloid leukemia, transcription
   factor, prognostic value, molecular functions, bioinformatics
   integration analysis

1. Introduction

   Acute myeloid leukemia (AML) is a heterogeneous hematological malignant
   cancer, characterized by the clonal expansion of myeloid blasts in the
   peripheral blood, bone marrow, and/or other tissues. It is the most
   prevalent acute leukemia amongst adults and the leading cause of
   leukemia-associated deaths in the United States each year [[34]1]. An
   estimated 21,450 people will be diagnosed with AML in 2019, and 10,920
   will die of this disease [[35]2]. Reports show that the overall 5-year
   survival rate for AML patients has increased from 16% from 1991–1996 to
   23% from 2003–2008 [[36]3], followed by 28.4% from 2009–2015 [[37]4].
   In fact, about 45% of AML patients show a normal karyotype detected by
   conventional cytogenetics at diagnosis, whilst 97.3% display somatic
   mutations [[38]5]. Therefore, there is an urgent need to identify
   reliable biomarkers, enabling early diagnosis and improved prognosis
   prediction of AML, in addition to developing novel targeted therapeutic
   strategies.

   Mammals possess 39 HOX genes, which are arrayed in four linear clusters
   with 9 to 11 genes per cluster. Based on homologies, the genes are
   assigned to 13 paralogous groups. The HOXA gene family encodes proteins
   that contain the DNA-binding homeobox motif and controls the early
   patterns of embryo segmentation in addition to later developmental
   events [[39]6]. HOX gene expression is typically inhibited in adults,
   although controlled reactivation is required for body repair and
   various homeostatic cellular processes [[40]7,[41]8], including
   hematopoiesis [[42]9], wound healing [[43]10], vascularization
   [[44]10], as well as female reproductive tract development and
   fertility [[45]11].

   The expression of HOX genes is a common feature of AML, which is
   considered a disorder of the HOX pathway, leading to abnormal
   cell-renewal and leukemia development. In AML patients, the expression
   of HOX genes correlates most strongly to the translocation of the MLL
   gene [[46]12]. HOXA9, in particular, has been shown to be a target of
   MLL fusion oncoproteins [[47]13]. Several studies have shown that
   certain AML mutations are related to HOX gene expression and that high
   HOXA gene expression levels are usually accompanied by partial tandem
   MLL duplications [[48]14]. Conversely, when HOX gene expression is low
   or absent, it is typically accompanied by PML-RARA and RUNX1-RUNX1T1
   gene fusions [[49]15,[50]16], as well as mutations of the CEBPA gene
   [[51]17].

   Therefore, the identification of HOXA-mediated oncogenes or tumor
   suppressors as potential mechanisms for predicting biomarkers may
   provide new therapeutic strategies for treating AML. The challenge lays
   in the fact that the differences in transcriptional levels, prognostic
   value, molecular functions, and biological processes for the majority
   of HOXA genes are yet to be elucidated in the context of AML disease.
   In this study, we expanded the knowledge on AML pathology by
   interrogating several universally-acknowledged databases to
   comprehensively analyze the relationship between HOXA subtypes and the
   pathogenesis and progression of AML.

2. Materials and Methods

2.1. Ethics Statement

   This study was approved by the Academic Committee of the Sun Yat-Sen
   University Cancer Center, and it was carried out in accordance with the
   principles of the Helsinki Declaration. All the data analyzed within
   this study were retrieved from published literature, for which we
   confirm that written informed consent was obtained.

2.2. ONCOMINE Database Analysis

   We analyzed HOXA gene transcription levels in different cancers using
   gene expression array datasets within ONCOMINE
   ([52]https://www.oncomine.org), an online cancer microarray database
   [[53]18]. HOXA mRNA expression levels in tumor specimens were compared
   with the levels in normal controls, and P values were obtained using
   the Students’ t-test. The cut-off P value and fold change were defined
   as 0.01 and 2, respectively. Genes co-expressed with HOXA genes were
   analyzed in the Cancer Genome Atlas (TCGA) for Leukemia, using the
   ONCOMINE database. Genes with a correlation value >0.4 were selected
   for further analysis.

2.3. The Gene Expression Profiling Interactive Analysis (GEPIA) Dataset

   GEPIA [[54]19] ([55]http://gepia.cancer-pku.cn) is a newly developed
   interactive web server that enables users to perform expression
   analyses (such as survival and differential analyses) at the subtype
   level. GEPIA can be used to analyze gene and isoform expression by
   performing a comparison to TCGA and GTEx data. As such, we validated
   the differential expression of HOXA genes in AML and healthy donor
   samples.

2.4. The LinkedOmics Dataset

   LinkedOmics [[56]20] ([57]http://www.linkedomics.orglogin.php) is a
   publicly-available portal comprising multiomics data from all 32 TCGA
   cancer types. It can be used to access, analyze, and compare multiomics
   data within and across tumor types. We performed a prognosis analysis
   for the HOXA gene family using the LinkedOmics AML databases.

2.5. TCGA Data and the cBioPortal

   The cBioPortal [[58]21] ([59]http://www.cbioportal.org/) for cancer
   genomics is an open-access, open-source resource designed for the
   interactive exploration of multidimensional cancer genomics datasets.
   It supports and stores data relating to nonsynonymous mutations, DNA
   copy-number, mRNA and microRNA expression, protein-level and
   phosphoprotein level, DNA methylation, and de-identified clinical data.
   The cBioPortal online tool enabled us to calculate the frequency of
   gene alterations, mRNA expression z-scores (RNA Seq V2 RSEM), and HOXA
   family gene correlations.

2.6. Functional Enrichment and Bioinformatics Analysis

   The Metascape [[60]22] ([61]http://metascape.org) online database
   integrates over forty bioinformatics knowledge bases enabling the
   extraction of abundant annotations, as well as the identification of
   enriched pathways and the construction of protein–protein interaction
   networks from lists of gene and protein identifiers. Using the Gene
   Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG)
   tools within Metascape, we analyzed a genes list containing HOXA genes
   to identify the most frequently-altered linked genes.

3. Results

3.1. The Transcriptional Expression of HOXA Genes in Leukemia Patients

   HOXA genes have been identified in the human genome. We used the
   ONCOMINE databases to compare the transcriptional expression of HOXA
   genes within tumorigenic and healthy control samples ([62]Figure 1).
   ONCOMINE analysis revealed that the mRNA expression of HOXA1, HOXA3,
   HOXA4, HOXA5, HOXA6, HOXA7, HOXA9, and HOXA10 was upregulated in
   patients with leukemia ([63]Table 1). The analysis revealed that
   compared to healthy tissues, HOXA1 mRNA was overexpressed 3.45-fold in
   AML [[64]23] and 10.08-fold in acute adult T-cell leukemia/lymphoma
   (ATLL) [[65]24]. In the Haferlach et al. dataset, HOXA3 mRNA expression
   was upregulated in AML (fold change = 2.23) and in pro-B acute
   lymphoblastic leukemia (ALL) (fold change = 3.98), compared to healthy
   controls [[66]25]. Moreover, Haferlach and colleagues showed that HOXA4
   was elevated in AML (fold change = 2.07) compared to the control
   samples [[67]25]. Meanwhile, Andersson et al. reported that HOXA4 was
   overexpressed in pro-B ALL (fold change = 8.08) [[68]26]. In the
   Haferlach et al. dataset [[69]25], the expression of the following HOXA
   genes was higher in the cancerous samples compared to the healthy
   controls: (i) HOXA5 in AML (fold change = 2.52) and pro-B ALL (fold
   change = 4.36); (ii) HOXA6 expression in pro-B ALL (fold change =
   2.01); (iii) HOXA7 expression in AML (fold change = 2.03); (iv) HOXA9
   expression in AML (fold change = 2.99) and pro-B ALL (fold change =
   7.21); and (v) HOXA10 expression in AML (fold change = 2.17) and pro-B
   ALL (fold change = 4.80). Stegmaier et al. reported the elevated
   expression of HOXA5 (fold change = 6.58), HOXA9 (fold change = 53.05),
   and HOXA10 (fold change = 8.48) in AML [[70]27]. In the Choi et al.
   dataset, HOXA10 was upregulated in ATLL with a fold change of 3.95
   [[71]24]. According to the ONCOMINE analysis, no significant
   differences were observed in the mRNA expression levels of HOXA2,
   HOXA11, and HOXA13 between leukemic and healthy control samples.

Figure 1.

   [72]Figure 1
   [73]Open in a new tab

   The transcription levels of HOXA genes in different types of cancers.

Table 1.

   The significant changes in HOXA expression in transcription level
   between different types of leukemia and normal samples (ONCOMINE
   Database).
   Gene Types of leukemia vs. Normal Samples Fold Change P Value t-Test
   Reference
   HOXA1 Acute Myeloid Leukemia vs. Normal 3.451 1.95E-07 9.046 Valk
   [[74]23]
   Acute Adult T-Cell Leukemia/Lymphoma vs. Normal 10.084 3.46E-05 7.407
   Choi [[75]24]
   HOXA2 NA NA NA NA NA
   HOXA3 Acute Myeloid Leukemia vs. Normal 2.230 2.48E-68 19.863 Haferlach
   [[76]25]
   Pro-B Acute Lymphoblastic Leukemia vs. Normal 3.975 8.57E-19 11.883
   Haferlach [[77]25]
   HOXA4 Acute Myeloid Leukemia vs. Normal 8.083 4.21E-06 6.951 Andersson
   [[78]26]
   Pro-B Acute Lymphoblastic Leukemia vs. Normal 2.065 8.86E-16 10.006
   Haferlach [[79]25]
   HOXA5 Acute Myeloid Leukemia vs. Normal 6.584 7.75E-05 5.292 Stegmaier
   [[80]27]
   Acute Myeloid Leukemia vs. Normal 2.522 2.78E-38 14.237 Haferlach
   [[81]25]
   Pro-B Acute Lymphoblastic Leukemia vs. Normal 4.359 1.35E-15 9.826
   Haferlach [[82]25]
   HOXA6 Pro-B Acute Lymphoblastic Leukemia vs. Normal 2.012 2.30E-14
   9.174 Haferlach [[83]25]
   HOXA7 Acute Myeloid Leukemia vs. Normal 2.027 1.40E-67 19.782 Haferlach
   [[84]25]
   HOXA9 Acute Myeloid Leukemia vs. Normal 53.046 3.68E-07 9.445 Stegmaier
   [[85]27]
   Acute Myeloid Leukemia vs. Normal 2.483 2.88E-33 13.493 Haferlach
   [[86]25]
   Acute Myeloid Leukemia vs. Normal 2.986 9.55E-58 17.841 Haferlach
   [[87]25]
   Pro-B Acute Lymphoblastic Leukemia vs. Normal 7.209 1.32E-20 12.798
   Haferlach [[88]25]
   HOXA10 Acute Myeloid Leukemia vs. Normal 8.483 8.82E-06 7.123 Stegmaier
   [[89]27]
   Acute Adult T-Cell Leukemia/Lymphoma vs. Normal 3.946 4.70E-05 4.888
   Choi [[90]24]
   Acute Myeloid Leukemia vs. Normal 2.174 1.30E-40 14.641 Haferlach
   [[91]25]
   Pro-B Acute Lymphoblastic Leukemia vs. Normal 4.802 7.72E-19 11.645
   Haferlach [[92]25]
   HOXA11 NA NA NA NA NA
   HOXA13 NA NA NA NA NA
   [93]Open in a new tab

   Abbreviations: NA, not available.

   We used the GEPIA dataset to compare the mRNA expression levels of HOXA
   genes between leukemic and healthy control samples. The results
   indicated that the expression levels of HOXA2, HOXA3, HOXA4, HOXA5,
   HOXA6, HOXA7, HOXA9, and HOXA10 were significantly increased in human
   AML patients compared to those in normal samples (P < 0.05), whilst the
   expression levels of HOXA1, HOXA11, and HOXA13 were not significantly
   different ([94]Figure 2).

Figure 2.

   [95]Figure 2
   [96]Open in a new tab

   The expression of HOXA genes in acute myeloid leukemia (AML) and
   healthy control samples.

   Next, we studied the role of HOXA genes in the survival of patients
   with AML. Using the LinkedOmics databases, we performed a prognosis
   analysis of HOXA genes in AML patients. The results revealed that the
   increased expression levels of HOXA1, HOXA2, HOXA3, HOXA4, HOXA5,
   HOXA6, HOXA7, HOXA9, and HOXA10 were significantly correlated with poor
   overall survival (OS, P < 0.05) in all of the AML patients ([97]Figure
   3). Therefore, overexpression of HOXA1-10 may represent a poor
   prognostic factor for AML.

Figure 3.

   [98]Figure 3
   [99]Open in a new tab

   The prognostic value of the expression levels of HOXA genes in AML
   patients.

3.2. Genetic Alteration and Correlations of HOXA Genes in AML

   Using the cBioPortal online tool and the ‘TCGA Provisional’ database
   for AML, we obtained information on genetic alterations in HOXA genes
   and identified any correlations between genes. HOXA genes were altered
   in 30/163 (18%) of AML patient samples ([100]Figure 4A). The types of
   genetic alterations included amplification, deep deletion, and mRNA
   overexpression. In addition, cBioPortal was employed to analyze the
   expression of HOXA genes in AML (using mRNA sequencing [RNA-seq]
   version V2 RSEM), and we calculated the interactions between specific
   HOXA genes (including the Pearson’s correlation). The results revealed
   a significant positive correlation between any two HOXA family genes
   members with the exception of HOXA1 and HOXA11 (Pearson = 0.15, P =
   0.0543) ([101]Figure 4B).

Figure 4.

   [102]Figure 4
   [103]Open in a new tab

   Genetic alteration and correlations of HOXA genes in AML. (A) Genetic
   alteration of HOXA genes in AML. (B) Genetic correlations of HOXA genes
   in AML.

3.3. Predicted Functions and Pathway Enrichment Analysis of HOXA Genes in AML
Patients

   Genes that were co-expressed in conjunction with HOXA genes were
   analyzed in the ‘TCGA Leukemia’ dataset using the ONCOMINE database. We
   found that the expression of HOXA genes was positively correlated with
   the upregulation of the following genes: TSLP, HOXB2, HOXB3, HOXB4,
   LOC404266, HOXB6, NKX2-3, PBX3, C10orf140, LOC100271722, LOC100289444,
   MEIS1, CPNE8, LOC400931, SDSL, EMR1, and HOXB7. We subsequently
   compiled a list of the expressed HOXA genes and the most frequently
   altered linked genes, prior to analyzing this gene list using the GO
   and KEGG tools in Metascape ([104]Figure 5A–C). We found that the
   following processes were affected by HOXA gene alterations: GO:0048706:
   embryonic skeletal system development; R-HSA-5617472: activation of HOX
   genes in anterior hindbrain development during early embryogenesis;
   GO:0060216: definitive hemopoiesis, hsa05202: transcriptional
   misregulation in cancer; GO:0030878: thyroid gland development;
   GO:0045638: negative regulation of myeloid cell differentiation;
   GO:0002009: morphogenesis of epithelium; GO:0048872: homeostasis of
   several cell types; and GO:0090596: sensory organ morphogenesis.

Figure 5.

   [105]Figure 5
   [106]Open in a new tab

   The functions of HOXA genes and genes significantly associated with
   HOXA genes alterations. (A) Heatmap of the Gene Ontology (GO) and Kyoto
   Encyclopedia of Genes and Genomes (KEGG) enriched terms colored by
   P-values. (B) Network of GO and KEGG enriched terms colored by P-value.
   (C) Network of GO and KEGG enriched terms colored by clusters.

4. Discussion

   HOX genes belong to a family of transcription factor-encoding genes
   that share a DNA-binding homeobox domain [[107]6]. In addition to their
   role in determining the characteristics of various body segments during
   embryogenesis, HOX genes also control the differentiation and
   regeneration of hematopoietic stem cells and precursor cells. The genes
   of the HOXA cluster, as well as the smaller HOXB family, are especially
   highly transcribed in hematopoietic precursor cells, and their
   expression gradually declines during maturation [[108]28].
   Dysregulation of members of the HOXA gene family has been reported in a
   variety of cancers, including glioblastoma [[109]29], hepatocellular
   carcinoma [[110]30], prostate cancer [[111]31], gastric cancer
   [[112]32], epithelial ovarian cancer [[113]33], breast cancer
   [[114]34], and leukemia [[115]35,[116]36,[117]37,[118]38]. Although the
   role of HOXA genes in the development of several cancers is well
   documented, a bioinformatics analysis of this gene family has not been
   performed in AML patient samples.

   Previous research has shown that AML patients displaying favorable
   cytogenetic characteristics exhibit lower levels of HOX gene
   expression, whilst poor AML prognosis cases are associated with the
   upregulation of these genes [[119]39]. HOXA family members were
   co-expressed with HOXB3, HOXB6, MEIS1, and PBX3 in AML, and the
   expression levels of these genes were highly correlated [[120]39]. The
   same results were observed in our study. Furthermore, we found that the
   expression of HOXA3-10 was elevated in AML samples compared to healthy
   tissues, and a higher expression of HOXA3-10 was significantly
   associated with poor OS in AML. The cBioPortal data indicated
   significant positive correlations between any two HOXA gene members,
   particularly between any two of the HOXA3-10 genes. In addition, we
   found that HOXA gene expression was positively correlated with the
   upregulation of HOXB2, HOXB3, HOXB4, HOXB6, NKX2-3, PBX3, and MEIS1,
   amongst others. This result was consistent with the aforementioned work
   of Drabkin et al. [[121]39].

   It is worth noting that a previous study identified HOXA4 as the only
   prognostic gene in AML, the upregulation of which in patients with
   normal karyotypes was actually associated with longer-term survival
   [[122]40]. This finding is contrary to our results. However, on closer
   inspection of the work, we became aware that only 52 of the 126 AML
   patients (enrolled in the study from 1985 to 1997) were retained for
   the final analyses. Moreover, the study in question did not consider
   FLT3 and MLL duplications, stating that the impact of HOXA4 on survival
   was independent of the white blood cell count and the presence of the
   FLT3-internal tandem duplication (ITD). In addition to the small sample
   size, the work of Grubach et al. failed to consider the prognostic
   stratification of AML due to the limited information available at the
   time of the study [[123]40]. Moreover, AML treatment regimens have
   undergone considerable change since the publication of the study in
   question. In light of this, we adhere to the view that the role of
   HOXA4 in the survival of AML patients remains unclear and we await the
   emergence of further large-scale sample studies involving the HOXA gene
   family.

   Numerous studies describe the functional role of the HOXA gene in the
   pathogenesis of AML. For instance, it has been shown that the KDM3B
   gene exhibits potential tumor-suppressive activity in AML and it
   transcriptionally regulates HOXA1 via retinoic acid response elements
   [[124]41]. A study by Li and colleagues showed that HOXA5 knockout
   significantly inhibits the proliferation of AML cells, whilst shRNA
   down-regulation of HOXA5 may induce apoptosis and overcome drug
   resistance in leukemia cells [[125]42]. In addition, the upregulation
   of HOXA5 through H3K79 methylation may represent a potential mechanism
   for leukemia transformation via the CALM-AF10 fusion gene [[126]43].

   HOXA7 and HOXA9 appear to be required for efficient in vitro myeloid
   immortalization via the replacement of leukemia-associated fusion
   proteins with MLL fusion proteins. The involvement of HOXA7 and HOXA9
   in MLL-mediated transformation suggests that the MLL oncoprotein acts
   as an upstream constitutive activator that promotes myeloid
   transformation through a HOX-dependent mechanism [[127]44].

   Amongst the HOXA genes, HOXA9 is the most studied in AML
   [[128]35,[129]45,[130]46,[131]47]. Using the RNA interference
   technique, HOXA9 has been shown to inhibit MLL rearrangement in
   MLL-rearranged leukemias, suggesting that targeting HOXA9 or its
   downstream signaling partners may present an effective therapeutic
   strategy [[132]46]. Similar to HOXA9, HOXA11 has been implicated in
   leukemia development, exemplified by a patient with juvenile
   myelomonocytic leukemia (JMML) carrying a NUP98-HOXA11 fusion gene
   [[133]48]. In addition, a novel NUP98 partner gene for HOXA13, with an
   expression pattern similar to HOXA9 in leukemic cell lines, implies
   that the NUP98-HOXA13 fusion protein, like the NUP98-HOXA9 fusion
   protein, may play a role in leukemia development via the same mechanism
   [[134]49].

   At present, the functional roles of HOXA genes in AML merit further
   elucidation.

   A large body of evidence has shown that AML, like normal hematopoiesis,
   is initiated and maintained by a subpopulation of leukemic stem cells
   (LSCs) [[135]50]. Studies have shown that NPM-1, ASXL1, and KAT6A are
   the most important upstream regulators of LSCs epigenetic marker genes,
   which mainly regulate HOXA genes, including HOXA5, HOXA6, HOXA7, HOXA9,
   and HOXA10 [[136]51]. All three of these upstream regulators have been
   found to be mutated in AML, and may be driving HOXA gene expressions
   [[137]52]. The work of Jung et al. has implied that the overexpression
   of HOXA genes may be mediated by DNA hypomethylation, a core mechanism
   for LSC activity [[138]51]. Another study has demonstrated that NPM1c
   facilitates HOX/MEIS1 expression and that HOX genes support the
   leukemic state in NPM1-mutant AML [[139]53].

   In this study, we employed GO and KEGG analysis tools to determine how
   the expression of HOXA genes and the most frequently-altered linked
   genes were associated with AML initiation and prognosis. We concluded
   that a few pathways deserved closer attention in the future, including:
   GO:0048706: embryonic skeletal system development; R-HSA-5617472:
   activation of HOX genes in anterior hindbrain development during early
   embryogenesis; GO:0060216: definitive hemopoiesis; hsa05202:
   transcriptional misregulation in cancer; and GO:0045638: negative
   regulation of myeloid cell differentiation. The last pathway has
   attracted our attention.

   Bcor, a repressor of HoxA genes in myeloid cells, plays an
   indispensable role in hematopoiesis by inhibiting myeloid cell
   proliferation and differentiation [[140]54]. Up-regulation of the HOXA
   gene in Bcor deletion may at least partially result in prolonged clonal
   growth of Bcor mutant hematopoietic stem/progenitor cells in
   replantation experiments [[141]54].

   In clinical practice, Venetoclax, a Bcl-2 protein inhibitor of
   apoptosis, has been approved by the FDA for first-line use in AML, in
   combination with azacytidine, decitabine, or low-dose cytarabine.
   Studies have shown that the significant overexpression of specific HOXA
   gene transcripts was detected in samples with high Bcl-2 inhibitor
   sensitivity [[142]55]. Bcl-2 inhibition has been shown to be effective
   in inducing the apoptosis of progenitor cells in patients with
   high-risk myelodysplastic syndrome and secondary AML [[143]56].
   Therefore, the mechanism through which Bcl-2 inhibitors modulate HOXA
   gene expression deserves further investigation.

5. Conclusions

   Our results collectively suggest that the increased expression of
   HOXA3-10 genes may be associated with the occurrence of AML, and that
   the increased expression of HOXA3-10 may serve as a molecular marker to
   identify AML patients with an adverse prognosis. Thus, HOXA3-10 may
   represent a prognostic marker and potential therapeutic target to
   improve the diagnosis and treatment of AML.

Author Contributions

   Conceptualization, S.-L.C., Z.-Y.Q., and Y.L.; methodology, S.-L.C.,
   Z.-Y.Q., and F.H.; formal analysis, S.-L.C., Z.-Y.Q., and Y.W.;
   investigation, S.-L.C., Z.-Y.Q., and Y.-J.D.; writing—original draft
   preparation, S.-L.C. and Z.-Y.Q.; writing—review and editing, S.-L.C.,
   Z.-Y.Q., and Y.L.; supervision, Y.L.; project administration, Y.L.;
   funding acquisition, Y.L.

Funding

   This study was funded by the National Natural Science Foundation of
   China (NSFC Grant No. 81873428) to Yang Liang.

Conflicts of Interest

   The authors declare no conflict of interest.

References