mir-21a-5p promote the progress of inammation after Traumatic Spinal Cord Injury via up-regulating neurotoxic reactive astrocyte (A1) polarization by inhibiting CNTF/STAT3/Nkrf pathway

Reactive astrocytes play an important role in Traumatic Spinal Cord Injury (TSCI). Interestingly, naive astrocytes can easily transform into neurotoxic reactive astrocytes(A1s) when inammatory stimulation occurs. Previous researches have reported that miR-21a-5p is involved in the regulation of various stages of Spinal Cord Injury (SCI). However, it is not clear whether miR-21a-5p affected the polarization of reactive astrocytes. The purpose of our study was to detect the effects and mechanism of miR-21a-5p in the induction of neurotoxic reactive astrocytes (A1s) formation. locus 1; S100a10: synthase; GAPDH: glyceraldehyde 3-phosphate dehydrogenase; DAPI: 4’,6-diamidino-2-phenylindole; NFκB: nuclear factor-κB; Nkrf: NF-κB repressing factor; qRT-PCR: quantitative real-time polymerase chain reaction; ELISA: Enzyme-linked immunosorbent assay; ChIP: chromatin immunoprecipitation. Cntfr α, normalized by GAPDH. Western blotting was used to detect the protein level of CNTFR α, normalized by β-actin. (D). Prediction of targeting sequence between miR-21a-5p and Cntfr; Dual-luciferase reporter assays were performed to determine the targeting sequence of miR-21a-5p and Cntfr α. (E). Astrocyte lysate used for RNA pulldown assay, then tested the expression of miR-21a-5p with qRT-PCR, relative levels of miR-21a-5p were normalized by Input. (F). miR-21 inhibitor was used to down-regulate the expression of miR-21a-5p in astrocytes, and then naive astrocytes were induced into A1s. The expression of Cntfr α was detected by qRT-PCR and normalized by GAPDH. (G, H). Astrocytes were transfected with miR-21a-5p mimic, inhibitor, and negative control, then treated with CNTF for 0, 15 30, 60 minutes, western blotting was used to detect the expression of p-STAT3 and STAT3, β-actin. The results were analyzed by Image J, GraphPad, and SPSS. The data are expressed in terms of mean ±SD, n=3. *P <0.05, **P <0.01, ***P <0.001, ****p <0.0001.


Background
Traumatic Spinal Cord Injury (TSCI), the most serious damage after spinal traumatic injury, usually leads to lifelong disability due to the destroyed continuity of the spinal cord [1][2][3]. Previous studies have been reported that the cost of life-long treatment per patient is at least $1.1 million in the United States [4,5]. However, there is still no effective treatment for TSCI at present. However, there is still no effective treatment for TSCI at present. Amanda Phuong Tran et.al reported that neuroin ammation was associated with the weakening axonal regenerative activity, leading to the poor recovery of injured tissue in the early stage of TSCI, which might in uence the prognosis of TSCI [6].
MicroRNA, a kind of small non-coding RNA containing 19-25 nucleotides [40], can down-regulate the expression of mRNA via targeting the 3'UTR [40,41]. Recently, several studies have reported that microRNA might be necessary for the recovery of SCI [42,43]. Signi cantly, miR-21a-5p regulates the formation of glial scars [44] and brosis [45,46], inhibiting the axon regeneration after SCI [47] as we previous described. In addition, miR-21a-5p was also con rmed to promote the polarization of A2s [18] in Ischemic Spinal Cord Injury (ISCI). Nevertheless, it is still unclear whether miR-21a-5p exerted a key role in regulating the induction of A1s after TSCI.
In this study, we aim to explore the relationship between miR-21a-5p and the polarization of reactive astrocytes, and detect possible mechanism in TSCI. Our study provides a prospective viewpoint of the polarization of reactive astrocytes, offering possible solutions for TSCI repairment. Mice were anesthetized with 3% pentobarbital (30 mg/kg, i.p.), then T8-T10 laminectomy was performed to expose the spinal cord. TSCI model was completed by 68099 precision percussion (RWD Life Science, Shenzhen, China; 1m/s speed, 2 mm depth, 1 second dwell time). The success of the model was con rmed by tail spasms and retraction-like utters of the legs.

Materials And
Animal experiments a. Mice were divided into sham and TSCI. The sham and 3 days post-TSCI mice spinal cord tissues were used for gene chip assay to detect the differential expression of mRNA.
3 days after TSCI, mice were anesthetized with 3% pentobarbital, then spinal cord tissues were carefully removed after transcardial perfusion. For staining use, the tissues were xed overnight with 4% paraformaldehyde at 4℃. Then, after dehydrated in xylene and gradient alcohol solutions, the tissues were embedded in para n. For gene chip analysis, qRT-PCR, and western blotting use, the sample was quickly placed in a frozen tube and put in liquid nitrogen, or directly extracted total RNA or protein.

mRNA gene chip assays
The tissues were extracted from sham and 3 days post-TSCI mice and frozen in liquid nitrogen. Gene chip assays were performed by Genechem Co. (Shanghai, China).
Primary mouse astrocytes were extracted from 1-3 days C57BL/6, the brain and spinal cord were dissected layer by layer; the brain and spinal cord were separated; removed the meninges and blood vessels, and the brain and spinal cord were extracted. After being fragmented, tissues were digested with trypsin (Solarbio) at 37 ℃ for 15 minutes. And the cell suspension was centrifuged and re-suspended in the cell culture solution. An 1h pre-plating step was performed to further remove broblast. The unattached astrocytes were planted in a 100mm dish. The 3rd-generation astrocytes were used for researches.
RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR) TRIzol TM (Accurate Biology, Hunan, China) was used to extract total RNA from astrocytes or spinal cord tissue. The SpectraMax®QuickDrop TM spectrophotometer was used for detecting the concentration of total RNA. The total RNA was stored at -80℃ or directly used.
Mir-X TM miRNA First-Strand Synthesis Kit (Takara, Dalian, China) and miR-21a-5p primer (Takara) were used to detect the expression level of miR-21a-5p, and U6 was used as an endogenous control to normalize the results. The 1000ng total RNA was prepared for reverse transcription using Exo M-MLV RT Kit with gDNA Clean for qPCR II (Accurate Biology). We performed qRT-PCR with the SYBR®Green Premix Pro Taq HS qPCR Kit (Accurate Biology) at LightCycler®480II Fast Real-Time PCR System (Roche, Switzerland). GAPDH was used as an endogenous control to normalize the results. The results were calculated with the value 2 -ΔΔCT . The primer pairs used are in Table. 1.

Protein extraction and western blotting
Astrocytes or spinal cord tissue were lysed by RIPA (Solarbio) with 1% PMSF (Beyotime Biotechnology, Shanghai, China) and 1% Phosphatase Inhibitor Cocktail 100X (CWBio, Beijing, China). The concentration of protein was measured by BCA detection kit (Beyotime Biotechnology). The protein was stored at -80 ℃ or directly used.
The protein samples were separated in SDS-PAGE gel (Beyotime Biotechnology) and transferred to polyvinylidene di uoride membranes (Millipore, USA). After blocking in 5% Skim milk powder (BioFroxx, Germany) at room temperature for 1 hour, membranes were incubated with primary antibodies at 4℃ overnight. The next day, after washing with PBST, membranes were incubated with secondary antibodies at room temperature for 1h. Finally, images were detected by FluorChem M (ProteinSimple, USA).
Antibodies used were as follows:

Enzyme-linked immunosorbent assay (ELISA)
The concentration of IL-1β released by A1s was detected by Enzyme-linked immunosorbent assay (ELISA) for quantitative detection of mouse IL-1β Kit (Invitrogen, ThermoFisher Scienti c). The experimental operation was performed according to the protocol of the kit. The colorimetric optical density (OD) was measured by SpectraMax®i3x enzyme labeling instrument (Molecular Devices, USA).
Dual-luciferase reporter assay HEK 293t cells were planted in a 24-well plate. For the construction of plasmids, WT or MUT Cntfr α 3'UTR fragments were inserted into PmirGLO Dual-Luciferase miRNA Target Expression Vector (BioSune, Jinan, China). Then, plasmids, miR-21a-5p mimic, inhibitor, and negative control were transfected into HEK 293t cells. After 48 hours, added 1 × PLA cell Lysis Buffer and shaken to lyse the cells at room temperature for 15min, then collected the cell lysate. 20μl cell lysate was added to a dedicated 96-well plate, then 100μl LARII and 100μl Stop & Glo®Reagent were added in turns by the Centro XS 3 LB 960 (Berthold, Germany) and MikroWin software to detect the re y luciferase activities and Renilla luciferase activities respectively. We completed this experiment using the Promega Dual-Luciferase system (Promega, Madison, USA). The difference of luciferase activity between re y and Renilla was calculated and analyzed.

RNA pull-down assay
Mus-Cntfr in pcDNA3.1 (+), antisense-MUT-Cntfr in pcDNA3.1 (+), and pcDNA3.1 (+) were linearized with restriction enzymes, then used for in vitro transcription. In in vitro transcription, we used the MEGAscript T7 Kit (Ambion, Thermo Fisher Scienti c, Shanghai, China) and biotin 16 UTP (Ambion, Thermo Fisher Scienti c, Shanghai, China) for biotin-labeled RNA transcripts. MEGA clear Kits (Ambion, Thermo Fisher Scienti c, Shanghai, China) were used for puri cation in vitro. 3μg of biotinylated RNA was heated at 90°C for 5 min and then laid at room temperature for 30 min, then cooled to 4 °C. RNA was mixed with 1 mg protein extracted from astrocytes, incubated with shaking at room temperature for 3 h. Each binding reaction was added with 60μl streptavidin agarose beads (Invitrogen, Thermo Fisher Scienti c, Shanghai, China) and incubated with a rolling shaker at room temperature for 2h. Then, a qRT-PCR assay was used to detect the expression of miR-21a-5p in pull-down RNA.

Immuno uorescence
For astrocytes, the cells planted in a 24-well plate were washed with PBS 3 times, treated with 4% paraformaldehyde (Solarbio) for 15 min, 0.5% Triton X-100 penetrated for 10 min, and then blocked with 10% normal goat serum (Solarbio) for 1 hour. Then, cells were incubated with primary antibody at 4℃ overnight. The next day, samples were incubated with secondary antibodies for 30min at room temperature. Finally, Antifade Mounting Medium with DAPI (Beyotime Biotechnology) was used for sealing.
The procedure of staining was the same as that was performed in cells.
An upright uorescence microscope (Olympus, Tokyo, Japan) was used for obtaining images.
Antibodies used were as follows:

Statistical analysis
GraphPad Prism v8.0 software (La Jolla, CA, USA) and SPSS v22.0 software (IBM, Chicago, IL, USA) was used for statistical analysis. Differences between two groups were analyzed by Student's t-test. Data were presented as mean ± standard deviation (SD). One-way analysis of variance (ANOVA) with Bonferroni's tests was performed for comparisons between multiple groups. P < 0.05 showed statistical signi cance.

Results
The expression of miRNA and mRNA in A1 reactive astrocytes induced by TSCI To determine the expression of miR-21a-5p after TSCI, we performed a qRT-PCR analysis between the sham operation and 3-days post-TSCI. The expression of miR-21a-5p after TSCI was signi cantly increased compared with the sham group (Fig. 1A). Immuno uorescence staining showed the increased expression of C3, an A1s marker, in GFAP + cells after TSCI (Fig. 1B), indicating the polarization of A1s. To verify the effect of IL-1α (3 ng/ml), TNF-α (30 ng/ml) and C1q (400 ng/ml) on inducing A1s, qRT-PCR was used to detect the expression of mRNA. As shown in Fig. 1C, the expression of C3, Serping1, H2d1 were up-regulated and S100a10 was down-regulated in A1 model group. Meanwhile, the expression of miR-21a-5p was also up-regulated in A1s (Fig. 1D), which indicated that miR-21a-5p may affect the induction of A1s.
To verify the targeting gene of miR-21a-5p, a gene chip assay was performed between the 3-d post-TSCI and sham operation groups. Furthermore, bioinformatics analysis indicated that Cntfr α, Epha4, Pitx2, or Abcd2 might exist a binding sequence of miR-21a-5p (Fig. 1E). qRT-PCR further demonstrated the mRNAs level of Cntfr α, Epha4, Pitx2 were all decreased in 3-days post-TSCI group (Fig. 1F, Supplementary Fig.   S1A-C), suggesting the possibility of them being the targeting gene of miR-21a-5p.
Dual-luciferase reporter assay was performed to verify the binding of miR-21a-5p and Cntfr α. The luciferase activity of Cntfr α-WT was decreased in the miR-21a-5p mimic group but increased in the miR-21a-5p inhibitor group. However, there was no statistical difference in luciferase activities of Cntfr α-MUT between miR-21a-5p mimic and inhibitor groups (Fig. 2D). And then, RNA pulldown assay was used to further verify the binding condition of Cntfr α to miR-21a-5p in astrocytes (Fig. 2E). To sum up, miR-21a-5p decreased the Cntfr α expression by targeting 3'UTR.
CNTFR is a speci c receptor of ciliary neurotrophic factor (CNTF), which can activate the STAT3 signal pathway. To con rm the in uence of miR-21a-5p on the activation of CNTF/STAT3 pathway, miR-21a-5p mimic and inhibitor were transfected into astrocytes. Western blotting showed that the phosphorylation of STAT3 was enhanced in the miR-21a-5p overexpression group (Fig. 2G) but weakened in a miR-21a-5p knockdown group (Fig. 2H).
CNTF inhibited the induction of A1s via promoting STAT3/Nkrf pathway To con rm the effect of CNTF/CNTFR α on the polarization of relative astrocytes, astrocytes were pretreated with CNTF for 24 hours. The results of qRT-PCR, western blotting, and ELISA assay showed that CNTF signi cantly decreased iNOS and IL-1β expression in A1s (Fig. 3A, G, H). Moreover, CNTF reduced C3, Serping1, H2d1 expression but up-regulated S100a10 expression in A1s (Fig. 3C-F). Western blotting also showed the same results in C3 and S100a10 protein levels (Fig. 3H). Meanwhile, immuno uorescence staining showed high C3 expression and low S100a10 expression in GFAP + cells in A1s, which reversed by CNTF (Fig. 3I-J). Furthermore, native astrocytes treated with CNTF exhibited slightly low of A1s markers expression and high A2s marker expression. Those data con rmed that CNTF could exert an anti-in ammatory effect by inhibiting the induction of A1s.
Since CNTF activates STAT3 signal pathway, S3I-201 (10 µM) was used to repress the activation of STAT3 pathway (DMSO used as a control group). As shown in Fig. 4A-D, S3I-201 treatment signi cantly increased C3, Serping1, and H2d1 expression but decreased S100a10. Those results suggested that CNTF inhibited the induction of A1s through the STAT3 signal pathway. But how transcription factor STAT3 plays a role in A1s induction was still unclear.
Known that the NF-kB signaling pathway plays an important role in the induction of A1s and NF-κB repressing factor (Nkrf) can effectively inhibit the NF-kB pathway, we speculated that Nkrf might affect the induction of A1s. As shown in Fig. 4E, pre-treating with CNTF signi cantly reserved the low Nkrf expression in A1s, and this effect was restrained in the S3I-201 treatment group. And then, we detected whether Nkrf expression could be promoted by the transcription factor STAT3. ChIP assay further con rmed that the Nkrf promoter was signi cantly enriched in p-STAT3, which demonstrated the important role of STAT3 in Nkrf expression (Fig. 4F).
In conclusion, these data strongly con rmed that CNTF inhibits the induction of A1s through STAT3/Nkrf pathway.
MiR-21a-5p promoted the induction of A1s by suppressing the effect of CNTF To verify whether miR-21a-5p affected the function of CNTF, miR-21 mimic and inhibitor were transfected into astrocytes, and then pre-treated with CNTF. The inhibition effect of CNTF in A1s induction was signi cantly weakened after treating with miR-21a-5p mimic (Fig. 5A-D). Meanwhile, the effect of CNTF was enhanced with the transfection of miR-21a-5p inhibitor (Fig. 5E-H). Immuno uorescence staining showed that the effect of CNTF on decreasing C3 expression and increasing S100a10 expression in GFAP + cells was enhanced after up-regulating miR-21a-5p (Fig. 5I-J) but weakened in miR-21a-5p knockdown group (Fig. 5K-L). In addition, miR-21a-5p overexpression slightly increased A1s markers without CNTF pre-treatment while down-regulating miR-21a-5p slightly decreased A1s markers and increased A2s markers. Those data illustrated that miR-21a-5p could repress the inhibitory effect of CNTF in A1s induction.
To determine whether miR-21a-5p inhibited the function of CNTF through targeting Cntfr α, we transfected Cntfr α siRNA into A1s with the treatment of miR-21a-5p knockdown. Firstly, we chose the siRNA contained the highest transfection e ciency through qRT-PCR and western blotting assay (Fig. 6A-B). As shown in Fig. 6C, cells transfected with Cntfr α siRNA showed a lower level of Cntfr α compared to those co-transfected with miR-21a-5p inhibitor. Signi cantly, qRT-PCR showed that the miR-21a-5p knockdown enhanced the inhibitory effect of CNTF on A1s induction, which was reversed by Cntfr α down-regulation (Fig. 6D-G). Immuno uorescence staining further con rmed that co-transfection with miR-21a-5p inhibitor and Cntfr α siRNA exhibited increased C3 expression and decreased S100a10 expression in GFAP + cells compared with cells treated with miR-21a-5p inhibitor (Fig. 6H-I). Meanwhile, we also detected the level of A1/A2 markers after cells transfected with Cntfr siRNA to clarify the function of Cntfr α whether affected by miR-21a-5p. Importantly, qRT-PCR and immuno uorescence staining showed higher A1s markers expression and lower A2s markers compared with the down-regulation of miR-21a-5p group.

MiR-21 promoted A1s induction through down-regulating Cntfr α in vivo
To con rm whether miR-21a-5p regulated the polarization of relative astrocytes in vivo, antagomir-21 was used to regulate the expression of miR-21a-5p in mice. Cntfr α siRNA was used to regulate the expression of Cntfr α in mice. As shown in Fig. 7A-B, the expression of miR-21a-5p in the antagomir-21 and antamir-21 + Cntfr α siRNA group was decreased while the expression of Cntfr α was increased in antagomir-21 group, which effect can be abolished by Cntfr α knockdown. Signi cantly, qRT-PCR assay showed the decreased C3, Serping1, H2d1 expression and increased S100a10 after treatment with antagomir-21 which can be reversed by Cntfr α siRNA treatment (Fig. 7C-F), indicating that miR-21a-5p could promote the A1s induction via decreasing Cntfr α in vivo. In the early stage of TSCI, the stimulation caused by ischemic injury might affect the polarization of reactive astrocytes in addition to neuroin ammation as there was higher S100a10 expression in the NC TSCI group compared with the sham group (Fig. 7F).
Subsequently, western blotting showed the phosphorylation of STAT3 was signi cantly increased by antagomir-21 treatment but decreased after treatment with Cntfr α siRNA after TSCI (Fig. 7G), which demonstrated that miR-21a-5p could affect STAT3 signal pathway through down-regulating Cntfr α after TSCI.
Those data con rmed that miR-21a-5p promotes the induction of A1s through down-regulating Cntfr α after TSCI in vivo.

Discussion
Up to now, there is no effective treatment for axon regeneration, which is the main reason leading to poor prognosis of TSCI [1]. Promoting axonal regeneration and anti-in ammation are often used to treat TSCI in clinic [3]. Thus, it makes sense to regulate the alteration of reactive astrocytes from A1s to A2s, and then suppress in ammation for TSCI recovery.
In our study, we con rm for the rst time that miR-21a-5p could promote the induction of A1 reactive astrocytes via CNTF/STAT3/Nkrf pathway after Traumatic Spinal Cord Injury (Fig. 8). Liddelow et al. reported that IL-1α, TNF-α and C1q released by microglia might convert reactive astrocytes into a neurotoxic state in CNS disease [16]. Our study con rmed that A1s induction could be simulated by TSCI model in vivo. And astrocytes were transferred into A1s via treating with IL-1α, TNF-α and C1q in vitro.
Based on this, the aim of our study was to explore key molecules regulated A1s polarization.
Our previous studies described that miR-21a-5p regulated glial scar formation [44] and inhibited the polarization of astrocytes to A2s in ISCI [18]. However, the effect of miR-21a-5p on neurotoxic reactive astrocytes (A1s) in TSCI has not been cleared. Interestingly, we found miR-21a-5p was up-regulated in A1s both in vivo and in vitro. Thus, we considered that miR-21a-5p may be an important factor for the polarization of relative astrocyte in TSCI.
Concerning the potential mechanism of the miR-21a-5p-related reactive astrocytes polarization, gene chip assay and the bioinformatics analysis were used to explore targeting genes that could mediate the polarization of reactive astrocytes. Subsequently, we found four genes may exist binding site of miR-21a-5p. Upon further analysis, Cntfr α was con rmed to be targeted by miR-21a-5p. CNTFR α is a speci c receptor of ciliary neurotrophic factor (CNTF), which was expressed in neuro, microglia, and astrocyte.
Furthermore, it would activate the classical STAT3 signal pathway via binding with CNTF. Interestingly, when astrocytes were treated with CNTF after up-regulating miR-21a-5p, STAT3 signal pathway was weakened. Therefore, miR-21a-5p could down-regulate CNTF/STAT3 pathway and might be essential for regulating the induction of A1s. Nevertheless, it is still unclear how CNTF/STAT3 regulated the polarization of relative astrocytes. Previous researches con rmed the positive effect of CNTF on reactive astrogliosis [23,[28][29][30] and M2 macrophages induction through activating classical STAT3 signal pathway [33]. Considering the important role of STAT3 in regulating the polarization of reactive astrocytes, we speculated that CNTF might regulate the polarization of reactive astrocytes from A1s to A2s by activating STAT3 signal pathway. In our study, we demonstrated that CNTF could down-regulate A1s markers and up-regulate A2s marker in vitro, which would be inhibited by repressing the activation of STAT3. But it is still unclear how transcription factor STAT3 affects A1s induction. NF-κB signal pathway is important for the polarization of A1 reactive astrocytes [15,35,36]. Moreover, NF-κB repressing factor (Nkrf), a speci c inhibitor of NF-κB signaling pathway [48][49][50], might be related to the induction of M1 microglia and release the microglia-induced neuroin ammation [51]. However, the effect of Nkrf in reactive astrocytes is still unclear. Signi cantly, we found that CNTF could regulate the expression of Nkrf via promoting the transcription of STAT3, which was con rmed by qRT-PCR and ChIP assay. These data revealed the novel mechanism that CNTF regulated A1s polarization through STAT3/Nkrf pathway.
As miR-21a-5p was a key upstream molecule of CNTFR α, it was of great signi cance to verify whether miR-21a-5p could affect the function of CNTF/CNTFR α. Our data showed the effect of CNTF on A1s was strongly enhanced after down-regulating the expression of miR-21a-5p. Importantly, effects after downregulating miR-21a-5p were reduced when Cntfr α was knocked down. Down-regulating miR-21a-5p could also markedly regulate the polarization of reactive astrocytes which would reverse by Cntfr α knockdown in mice TSCI model. Interestingly, we also found that A1/A2 markers were slightly affected by miR-21a-5p without the treatment of CNTF in A1s. Further study would be performed to detect the mechanism. Thus, miR-21a-5p will be a key factor for targeted treatment of TSCI in the future.
Taken together, we con rmed that astrocyte-mediated neuroin ammation was regulated by miR-21a-5p. It is an innovative point of our research to explore the effect of miR-21a-5p on astrocytic neuroin ammation after TSCI from another perspective. In addition, regulating miR-21a-5p could improve the environment for neuro regeneration. However, our study only referred to the expression of A1/A2 marker genes, but did not detect other astrocytic functions such as activity, migration and effects on neurons, which will be con rmed in our future study. And then, we demonstrated that CNTF inhibited A1s polarization through directly activating Nkrf. Is there any stimulating factor that directly promotes the alteration of reactive astrocytes to A2 induced by miR-21a-5p/CNTF/STAT3? We will further clarify it in future.
There is plenty of evidence to show that it is necessary for the recovery of TSCI to promote the alteration of reactive astrocytes from A1s to A2s. However, it remains to be detected whether other factors affect the alteration of A1/A2 reactive astrocytes. Are there only two types of reactive astrocytes? These problems will be solved through Single-cell sequencing, proteomics or other studies.

Conclusions
Collectively, our study con rmed that miR-21a-5p could promote the induction of A1s through Cntfr α/STAT3/Nkrf axis after TSCI, which may provide prospective sight for the reactive astrocytes alteration and promote the development of TSCI recovery.

Declarations
The transformation of naive astrocytes into A1s was induced by IL-1α, TNF-α, and C1q. (C). qRT-PCR was used to detect the mRNA expression of C3, Serping1, H2D1, S100a10, GAPDH was used for normalization. (D). qRT-PCR was used to detect the expression of miR-21a-5p, U6 was used for normalization. (E). Heat map of mRNA that signi cantly changed in 3d post-TSCI group and bioinformatics analysis for choosing targeting genes of miR-21a-5p. (F). qRT-PCR was used to detect the expression of Cntfr α between sham group and 3d post-TSCI group, GAPDH was used to normalize it. The results were analyzed by GraphPad and SPSS. The data are expressed in terms of mean ±SD, n=3. *P <0.05, **P <0.01, ***P <0.001, ****p <0.0001. Figure 2 miR-21a-5p downregulated the expression of Cntfr α by targeting 3'UTR. miR-21a-5p mimic, inhibitor, and negative control were transfected into astrocytes. (A, B). qRT-PCR was used to detect the mRNA level of normalized by β-actin. (D). Prediction of targeting sequence between miR-21a-5p and Cntfr; Dualluciferase reporter assays were performed to determine the targeting sequence of miR-21a-5p and Cntfr α.