Microarray Analysis of Potential Biomarkers for brachial plexus avulsion caused neuropathic pain

Nerve injury-induced neuropathic pain remains a challenging clinical problem due to a lack of satisfactory treatment. Pain after BPA (Brachial Plexus Avulsion) is resistant to most traditional pain relief treatments due to the lack of understanding of the cellular or molecular mechanism of pain development. The present study aimed to investigate the expression of mRNA in the brachial plexus avulsion neuropathic pain model and analyze biological functions. Sprague-Dawley rats were treated with complete brachial plexus avulsion. An animal behavior test was carried out to distinguish the pain group from the control group. In this study, a microarray mRNA assay and reverse transcriptase quantitative polymerase chain reaction (RT-PCR) was conducted. The whole blood was collected from two groups for Microarray mRNA analysis. The predicted mRNA targets were studied by gene ontology analysis and pathway analysis. The PIK3CB, HRAS, and JUN genes were verified by RT-PCR. In total, differentially expressed genes(DEGs) were identified between individuals with or without neuropathic pain (case and control), and A biological processes were enriched. We identified 3 targeted mRNAs, including PIK3CB, HRAS, and JUN, which may be potential biomarkers for BPA-caused NP. The results showed that PIK3CB, HRAS, and JUN gene expression was increased in the control group but decreased in the neuropathic pain group. The PIK3CB gene was part of the Neurotrophin signaling pathway. The function of the HRAS gene was synergetic in the aspect of axon guidance and the Neurotrophin signaling pathway. The JUN gene participates in axon regeneration. These results suggest that PIK3CB, HRAS, and JUN genes might become potential biomarkers for the prediction of and new targets for the prevention and treatment of neuropathic pain after BPA. These findings indicate that mRNA expression changes in the blood may play an important role in the development of NP after BPA, which is of theoretical and clinical importance for future research and clinical-treatment strategies.

biological functions. Sprague-Dawley rats were treated with complete brachial plexus avulsion. An animal behavior test was carried out to distinguish the pain group from the control group. In this study, a microarray mRNA assay and reverse transcriptase quantitative polymerase chain reaction (RT-PCR) was conducted. The whole blood was collected from two groups for Microarray mRNA analysis. The predicted mRNA targets were studied by gene ontology analysis and pathway analysis. The PIK3CB, HRAS, and JUN genes were verified by RT-PCR. In total, differentially expressed genes(DEGs) were identified between individuals with or without neuropathic pain (case and control), and A biological processes were enriched. We identified 3 targeted mRNAs, including PIK3CB, HRAS, and JUN, which may be potential biomarkers for BPAcaused NP. The results showed that PIK3CB, HRAS, and JUN gene expression was increased in the control group but decreased in the neuropathic pain group. The PIK3CB gene was part of the Neurotrophin signaling pathway. The function of the HRAS gene was synergetic in the aspect of axon guidance and the Neurotrophin signaling pathway. The JUN gene participates in axon regeneration. These results suggest that PIK3CB, HRAS, and JUN genes might become potential biomarkers for the prediction of and new targets for the prevention and treatment of neuropathic pain after BPA. These findings indicate that mRNA expression changes in the blood may play an important role in the development of NP

Introduction
Nerve injury-induced neuropathic pain remains an intractable disease due to a lack of satisfactory treatment [9]. In 2017, Palma Ciaramitaro et al. [1]  hyperalgesia, and persistent pain, which is often difficult to cure [10]. The pain may be manifested as burning or pressure. Pain after BPA is resistant to most pain relief treatments the exact molecular mechanisms responsible for this pathology remain unknown [3]. Previous studies demonstrated that the mRNA plays a key role in the development and maintenance of neuropathic pain [11][12][13].
. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. ; https://doi.org/10.1101/2021.08.05.455327 doi: bioRxiv preprint Some authors proved that c-Jun plays a vital role in the survival of ventral horn motoneurons in adult mice [4]. At present, there is no literature to prove that the HRAS gene is related to neuropathic pain caused by brachial plexus injury. Previous studies have shown that PIK3CB influences the early development of neuropathy in sensory neurons [2]. The spinal cord plays an important role in the process of central sensitization [14]. Furthermore, we aimed to investigating the mRNA changes of neuropathic pain caused by the brachial plexus avulsion model, thereby providing a novel insight into the mechanism of neuropathic pain.

Animals
This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of

Surgery procedure
All surgical procedures were performed after anesthesia induced by a 1% sodium pentobarbital solution (40 mg/kg body weight). Place the rat prone on a sterilized pad with the head oriented away from the surgeon and the right forepaw abducted and extended. Use the fingertips to locate the clavicle. With a scalpel, make a 1.5 cm horizontal incision in the skin under the clavicle 2 mm. Use micro-dissecting scissors to separate the skin from the superficial fascia, exposing the pectoralis major muscle.
The pectoralis major muscle was cut paralleled with the muscle fibers to expose the brachial plexus, leaving the cephalic vein intact. The subclavian vessels were located and the upper, middle and lower trunks were dissected. In the complete brachial plexus avulsion (BPA) group (n = 20), the upper, middle, and lower trunk were grasped with forceps and haul out from the spinal cord. The tissue layers were then brought together, and the skin was closed with 4-0 silk sutures (Ethicon), as described previously [7].
. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. to the left forepaw. The threshold was the lowest force that evoked a withdrawal response. Each filament was applied five times. When rats showed at least two withdrawal responses to a filament, the bending force of the filament was defined as the withdrawal threshold.
Cold allodynia: Cold allodynia was assessed by an acetone spray test as described by Choi et al. [8]. 250 μl acetone was squirted onto the surface of the paw. Neuropathy rats frequently responded with a withdrawal that was clearly exaggerated in amplitude and duration. The withdrawal responses were assessed on a scale of 3-0 points: 3 points, a vigorous response in which the rat licked the paw; 2 points, a response in which the paw has elevated the paw; 1 point, a response in which the paw had little or no weight born on it and 0 points, the paw was not moved [5].

mRNA microarray
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The whole blood was collected from the rats. Total RNA was extracted from whole blood using a QIAamp RNA blood mini kit (Qiagen) per the manufacture's instruction. Purified total RNA for each strain used in Affymetrix GeneChip assays (Affymetrix GeneChip Rat Gene 1.0). Microarrays were processed using an Agilent GeneArray Scanner with Affymetrix Microarray Suite version 5.0.0.032 software.

Reverse transcription-quantitative polymerase chain reaction (RT-PCR) assay
RNA was reverse transcribed into cDNA using Takara (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. ; https://doi.org/10.1101/2021.08.05.455327 doi: bioRxiv preprint as a housekeeping gene. The relative expression of genes was calculated using the 2 −∆∆Ct method.

Bioinformatic evaluation.
GO analysis was applied to analyze the function of the expression genes according to the Gene Ontology, which is the crucial function of NCBI that can organize genes into hierarchical classification and uncover the gene network on the basis of biological process and molecular function.
Pathway analysis was applied to find out the significant pathway of the differential genes according to KEGG, Biocarta, and Reactome. Still, we turn to Fisher's exact test and test to select the most significant pathway, and the threshold of significance was decided by P-value and FDR. The enrichment Re was calculated like the equation above.

Statistical analysis
The random variance model t-test was adopted to filter the differinitially expressed mRNAs between the control and pain groups using GraphPad 5.0. Following the significance analysis 2  . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. ; and false discovery rate analysis, differentially expressed genes were selected according to their P-values. P<0.05 was considered to indicate a statistically significant difference.

mRNA microarray
Animals exhibiting significant decreases in the pain threshold(mechanical threshold decreases from 15 g pre-surgery to 8 g post-surgery and allodynia score increases from 0 presurgery to 2-3 post-surgery) were placed in the NP(Neuropathic Pain) group. There were 10 rats do the BPA surgery and 6 rats had neuropathic pain. There were 6 rats in the NP group. The sham-operated animals whose brachial plexus was just dissected but not used were assigned to the control group. There were 10 rats in the control in the control group.
. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021.  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. ; https://doi.org/10.1101/2021.08.05.455327 doi: bioRxiv preprint the enriched terms were about inflammatory processes involved in protein modification and regulation of biological processes.
The result was similar to the GO analysis.
To further investigate the functions of DEGs, we did a KEGG pathway analysis. The top 20 pathways were shown in table 3.
The most significantly enriched pathways were Metabolic pathways, antigen processing and presentation, and Herpes simplex infection.

PCR verification
We aimed to explore the mechanism of neuropathic pain after brachial plexus avulsion and find nerve-related mRNAs.
Therefore, we mainly focused on the differentially expressed genes(DEGs) dysregulated only in the neuropathic pain groups.
The three nerve-related mRNAs (Pik3cb, Hras, and Jun genes) exhibited decreased expression in the neuropathic pain group.
To validate the microarray results, RT-qPCR was performed for Pik3cb, Hras, and Jun genes. It was found that the relative expression of 3 mRNA among them was significantly altered, which coincided with the results of the microarray (Figure 1 ).

Bioinformatics analysis of the diff-reg mRNA
The Pik3cb, Hras, and Jun genes were intersection genes, which were involved in neuropathic pain according to GO and pathway analyses. The results showed that Pik3cb, Hras, and Jun gene expression was high in the control group but was low in the neuropathic pain group. The function of the Hras gene was synergetic in the aspect of axon guidance and the Neurotrophin signaling pathway. The Jun gene function was axon regeneration. The low expression of two genes in the neuropathic pain group was revealed that neuropathic pain is unfavorable for nerve . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. ; https://doi.org/10.1101/2021.08.05.455327 doi: bioRxiv preprint regeneration.

Discussion
Brachial Plexus Avulsion(BPA) has been demonstrated to be a polygenic disease and its pathogenic mechanism is associated with changes in many genes. In this study, we have used microarray to identify differentially expressed genes(DEGs) and activated signaling pathways in association with BPAinduced neuropathic pain(NP) in a rat BPA model. We showed that Jun, HRAS, and PIK3B were the nerve-related downregulated DEG.
Our results are consistent with that of previous studies [7].
Although the precise roles of the three marker genes in BPA-. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021 [15,17].
We found that three genes expressed decreased and were related to nerve regeneration. Some authors proved that the downregulation of c-Jun gene expression is not conducive to the survival of motoneurons. HRAS might serve specific roles in the development and maintenance of nervous tissues [6]. In our study, the Metabolic signaling pathway and Phagosome signaling pathway are involved in BPA, which play a very important role in BPA-induced NP. In the peripheral nervous system, recent studies suggested that the nerve-related gene plays an important role in neuropathic pain after spinal cord injury [16]. Some authors suggested that there is a high possibility of neuropathic pain caused by nerve damage [18,19]. The transcriptome changed play an important role in neuropathic pain [13]. So our research is meaningful and feasible. Ji-An Yang et al [20] proved that Jun is . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. In summary, our studies indicated that Jun, HRAS, and PIK3B might serve a significant role in neuropathic pain and nerve regeneration. we demonstrated that microarray. The three nerve-related genes were downregulated in the spinal cord in NP rats after brachial plexus avulsion. Furthermore, KEGG analysis found that Metabolic pathways with significance were identified. These results strongly suggest that neuropathic pain . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. ; https://doi.org/10.1101/2021.08.05.455327 doi: bioRxiv preprint may attenuate nerve regeneration via inhibition of neurotrophin signaling pathway and axon guidance pathway, which is of theoretical and clinical importance for future research and clinical-treatment strategies. Several limitations should be acknowledged in our study. First, the sample size was relatively small. Besides, the results were all base on a rat model. In the future, we will perform some more in-depth studies around nerverelated genes.

Conflicts of interest
The authors declare no conflict of interest  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted August 5, 2021. ; https://doi.org/10.1101/2021.08.05.455327 doi: bioRxiv preprint