The MYCN/miR-20a-5p/NFKBIB/NF-κB signaling axis promotes the proliferation, invasion and migration of neuroblastoma

doi:10.21203/rs.3.rs-2996752/v1

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The MYCN/miR-20a-5p/NFKBIB/NF-κB signaling axis promotes the proliferation, invasion and migration of neuroblastoma

https://doi.org/10.21203/rs.3.rs-2996752/v1

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Purpose

To investigate the effect of MYCN on the proliferation, invasion and migration of NB and its molecular mechanism.

Methods

We first compared the expression level of MYCN mRNA in NB cells by PCR. Next, the expression level of miR-20a-5p in NB cells was detected by PCR. Then, cell counting kit-8, wound healing and transwell assays were used to determine the ability of proliferation, migration and invasion of NB cells. Both cells were infected with miR-20a-5p lentivirus particles, and then the infection efficiency was detected by PCR. Repeat the above cell function test to determine the proliferation, migration, invasion and apoptosis of NB cells after overexpression or low expression of miR-20a-5p. Bioinformatics analysis was used to predict the binding sites of miR-20a-5p and NFKBIB. A double luciferase reporting assay was used to verify the targeting relationship between miR-20a-5p and NFIBIB. WB confirmed that miR-20a-5p could activate the NF- κB pathway.

Results

MYCN and miR-20a-5p can promote the proliferation, invasion and migration of NB cells, and inhibit NFKBIB by upregulating miR-20a-5p, thereby activating the NF- κB pathway.

Conclusion

In this study, we speculate that the MYCN/miR-20a-5p/NFKBIB/NF- κ B signaling axis can promote the proliferation, invasion and migration of NB cells.

Neuroblastoma (NB) is a solid tumor in children [1]that easily occurs in the adrenal gland, and its degree of malignancy is high, accounting for 12% of malignant tumors in children[2]. Although NB has the characteristics of self-limitation, it easily causes the death of children because of its rapid development [3]. Many studies have shown that oncogenes are extensively involved in the occurrence and development of tumors through posttranscriptional regulation.An in-depth understanding of oncogenes will help reveal the molecular mechanism of NB development and metastasis. Aamplification of the oncogene MYCN exists in 20%-30% of NB cases and is related to the degree of malignancy and poor prognosis[4]. Its transcriptional regulatory factor N-myc can regulate the expression of many kinds of miRNAs, and these miRNAs can also control the progression of NB and are related to the degree of malignancy of NB [5].

Persistent inflammatory stimulation can induce the occurrence of NB, and the activation of the nuclear factor κ B (NF-κB) pathway plays an essential role in NB [6]. The NF-κB signaling pathway activated by tumor necrosis factor α (TNF-α) is regulated by many genes[6, 7]. Noncoding microRNAs (miRNAs) are involved in the above regulatory process, among which the miR-17-92 gene cluster controlled by the MYCN gene can promote the proliferation, invasion and metastasis of NB [8, 9]. In the early stage of this experiment, bioinformatics analysis was used to predict that the target of miR-20a-5p encoded by the miR-17-92 gene cluster was NFKBIB, and NFKBIB could be coupled with NF-κB protein to inhibit the activation of the NF-κB pathway.We attempted to study the correlation among the oncogenes MYCN, miR-20a-5p and the NF-κB pathway in NB and explore their effects on the proliferation, invasion and migration of NB cells and the underlying mechanism.

1. Cell culture

The human NB-cell lines SH-SY5Y and SK-N-BE(2) were purchased from Wuhan Punosai Life Technology (Wuhan, China). Cells were cultured in Dulbecco’s modified Eagle medium (DMEM) (BI, Biological Industries, Israel) supplemented with 12% fetal bovine serum (FBS) (Gibco,Thermo Fisher Scientific, Waltham, MA) in a humidified atmosphere at 37°C with 5% CO2. In all experiments, cancer cells subcultured fewer 5–6 times were used.

2. Construction of a stably transfected cell line by cell transfection and screening

2.1 Lentivirus-transfected cells

SH-SY5Y and SK-N-BE(2) cells in the logarithmic growth phase were inoculated in 6-well plates and cultured at 37°C, 5% CO2 and saturated humidity for 24 hours. When the cell density reached 50–70%, the cells were used for lentivirus infection. According to the titer information of the samples provided by Gikegiin, the virus, liquid A or P, and serum-free DMEM/F12 medium were added to the 6-well plates successively and then cultured for 48 h and 72 h. miR-20a-5p overexpression and knockdown lentivirus vectors were purchased from Shanghai Gik Kiin Technology (Shanghai, China)

2.2 Screening and establishment of stably transfected cell lines

SH-SY5Y and SK-N-BE(2) cells in the logarithmic growth phase were inoculated with 8×10⁴ cells in a 12-well plate, 37℃, 5% CO2, and saturated humidity incubator for 24 h. When the cell density reached 70% ~ 80%, 6 µg/ml puromycin (the best screening concentration according to the preexperiment) and antibiotic-free DMEM/F12 medium containing 12% fetal bovine serum were added to continue the culture. The fresh puromycin medium was changed once a day to remove the dead cells that were not successfully infected. After continuous screening for one week, stable cell lines were obtained and cryopreserved after expanded culture.

3. Quantitative reverse transcription-PCR

Total RNA extraction, cDNA synthesis and qRT-PCR were performed according to the instructions of TRIzol, TransScript ®Uni One-Step gDNA Removal and cDNA Synthesis SuperMix and PerfectStart ®Green qPCR SuperMix kits, respectively. MiR-20a-5p and NFKBIB primers were purchased from Shanghai Shengong Bioengineering Co(Shanghai, China). β-actin was used as the endogenous control for mRNA or miRNA detection, and the fold change was calculated by the comparative quantitative method (2^−ΔΔCt). Related primer sequences are listed in Supplementary Table 1.

4. Cell counting kit-8 assay

The cell suspensions of each group (1×10⁴ cells/ml) were inoculated into 96-well plates (100 µl per well) and 3 replicate wells. Cell counting kit-8 solution (10 µL per well) was added at 0 h, 24 h, 48 h and 72 h and incubated in the incubator for two h. An enzyme labeling instrument determined the absorbance of each well at 450 nm .

5. Wound healing assay

The cells of each group in the logarithmic growth phase were inoculated with 1×10⁵ cells in a 24-well plate at 37℃, 5% CO2 and saturated humidity for 24 hours, and three straight scratches were made along the bottom of the culture plate with a 100 µl sterile transfer gun. After washing with PBS 3 times, serum-free medium was added and cultured in the incubator. The cells were observed by fluorescence microscopy at 0 h and 24 h. The closure of the gap was imaged at designated time intervals under a 200× field, and the wound closure speed was evaluated using ImageJ software.

5. Transwell assay

Matrigel (approximately 50 µL per well) was precoated on the Transwell chamber, and it was placed at 37°C overnight to solidify. The next day, the Transwell chamber was removed from the incubator and hydrated with serum-free DMEM-F12 medium for standby. A total 2×10⁵ cells in 200 µL serum-free DMEM-F12 were reseeded into the top of the insert of a Transwell chamber (Corning Inc, Corning, NY) while 600 µL medium with 12% FBS was loaded into the well below. After 24 h of incubation, invasive cells that passed through the filter were fixed with 0.1% paraformaldehyde (Solarbio Science & Technology Co., Ltd, Beijing, China) and stained with 0.1% crystal violet solution (Solarbio Science & Technology Co.Ltd).Finally, the cells on the surface of the upper chamber were wiped off with a cotton swab, and the total number of cells that migrated to the back of the Transwell chamber was counted under an inverted microscope.The cells that passed through the filter were imaged at 200× magnification in six random fields and measured using ImageJ software .

6. Flow Cytometry

A BD PE Annexin V Apoptosis Detection Kit (BD Biosciences, NJ) was employed for the apoptosis assay. Cells from each group in the logarithmic growth phase were taken, digested, centrifuged and finally washed twice with PBS.The cells of each group were resuspended in binding buffer and adjusted to a final concentration of 4 × 10⁵ / mL. The two cell lines were set to blank (no staining solution), single staining (5 µL PE Annexin V or 7-AAD staining solution) and double staining (5 µL Annexin PE staining solution and 5 µL 7-AAD staining solution added successively) groups of cells. The cells were incubated at room temperature without light for 15 min,after which cell apoptosis was evaluated on a flow cytometer (BD Biosciences, NJ) and quantified by BD FACSDiva 8.0.1 software.

7. Luciferase reporter assay

The DNA fragment containing the wild-type or mutant 3'-UTR of NFKBIB was inserted into the pmir-GLO plasmid (MACHEREY-NAGEL, Shanghai,China). HEK-293T cells were cotransfected with plasmids containing h-NFKBIB-3'-UTR wild-type or mutant fragments and miR-20a-5p mimics or miRA using Lipofectamine 3000. After 48 h of cotransfection, a double luciferase reporter gene analysis system (Promega Corporation, Madison, WI) was used for analysis. The ratio of firefly luciferase signal to sea kidney luciferase signal was calculated to evaluate the binding of miR-20a-5p and NFKBIB.

8. Western blotting

Cell proteins of each group were extracted with lysate containing protease inhibitor (1:10), and the concentration of total protein was determined by the BCA protein assay kit (Thermo Fisher Scientific, Inc.). Equal amounts of protein(20 µL) were separated by 10% or 15% sodium dodecyl sulfate—polyacrylamide gel electrophoresis (SDS—PAGE) and transferred onto polyvinylidene difluoride (PVDF) membranes (Thermo Fisher Scientific, Inc.Membranes were incubated with primary antibodies at 4°C overnight and then with secondary antibodies at room temperature for 2 h. After washing with TBS-Tween (TBST) three times, the PVDF was soaked in the developer for 60 seconds,and signals were detected using a ChemiDoc™ imaging system (Bio-Rad Laboratories, Inc. The following antibodies were purchased: anti-NFKBIB (1:1000, #15519S, Cell Signaling Technology), anti-NF-κB (1:1000, #8242S, Cell Signaling Technology), anti-β-actin(1:2000, #20536, Protein Technology,Co.,Ltd,Shanghai,China)and the secondary antibody anti-rabbit IgG (1:2000, #SA00001 Protein Technology,Co,Ltd,Shanghai,China).

8. Statistical analyses

The results are expressed as the mean ± SD of at least three independent experiments.Statistical analyses were performed using IBM SPSS Statistics version 22.0 (SPSS Inc., Chicago, IL) and GraphPad Prism 8.0 (GraphPad Software Inc., La Jolla, CA). Data are presented as the mean ± standard deviation. Significant differences were calculated by Student’s t test. A P value < 0.05 was considered statistically significant.

1. To verify the role of MYCN and miR-20a-5p in neuroblastoma.

In order to reveal the role of MYCN and miR-20-5p in NB, the expression of MYCN mRNA in SK-N-BE (2) cells and SH-SY5Y cells was measured by qRT-PCR. The results showed that the expression of MYCN mRNA in SK-N-BE (2) cells was significantly higher than that in SH-SY5Y cells (t(df) = 5.089(4), p = 0.007,Fig. 1A). The MYCN gene was amplified in SK-N-BE (2) cells. Next, qRT-PCR was used to detect the expression of miR-20-5p in SK-N-BE (2) cells with MYCN gene amplification and SH-SY5Y cells without MYCN gene amplification. The results showed that the expression of miR-20-5p in SK-N-BE (2) cells was significantly higher than that in SH-SY5Y cells (t (df) = 4.04 (4), p = 0.016, Fig. 1C).In SK-N-BE (2) cells, after overexpressing and knocking down miR-20a-5p, we found that the change in miR-20a-5p mRNA expression was accompanied by a change in MYCN mRNA (t(df) = 2.987(4),p = 0.04; t(df) = 4.485(4),p = 0.0084;Fig. 1B). The proliferation, invasion and migration of the two cell lines were detected by cell counting kit-8 assays, transwell invasion assays and wound healing assays.The proliferation (t (df) = 2.684 (4), p = 0.004, Fig. 1D), invasion (t (df) = 6.182 (4), p = 0.003, Fig. 1E) and migration (t (df) = 4.690 (4), p = 0.009, Fig. 1F) of SK-N-BE (2) cells with MYCN gene amplification were statistically significantly increased compared with SH-SY5Y cells.The above results further verify the role of MYCN and miR-20a-5p in NB at the cellular level, indicating that MYCN and miR-20a-5p can enhance the proliferation, invasion and metastasis of NB.

2. Detection of miR-20a-5p expression after transfection by qRT–PCR

The overexpressed and depressed lentivirus particles miR-20a-5p and its control group lentivirus were infected with the SK-N-BE(2) cell line and SH-SY5Y cell line, respectively. After 72 hours, the expression rate of green fluorescent protein in each group was more than 80% (Fig. 2A and 2B).After successful transfection, puromycin was used to screen the cell lines with stable overexpression and knockdown of miR-20a-5p for qRT-PCR detection.The results of qRT-PCR detection showed that the expression level of miR-20a-5p in SK-N-BE (2) cells was significantly higher than that in the control group(t(df) = 2.837(4), p = 0.045, Fig. 2C),and the expression level after knockdown of miR-20a-5p was significantly lower than that in the control group (t (df) = 3.015 (4), p = 0.038, Fig. 2C).In SH-SY5Y cells, the expression level of miR-20a-5p was significantly higher than that of the control group (t(df) = 3.578(4),p = 0.023, Fig. 2D), the expression level after knocking down miR-20a-5p was significantly lower than that in the control group(t(df) = 11.12(4), p = 0.0004, Fig. 2D). The above results show that our stable overexpression and stable knockout of miR-20a-5pin the NB cell line was successful.

3. miR-20a-5p promotes the proliferation, invasion, migration and apoptosis of NB.

Transwell invasion assays showed that the number of invaded SK-N-BE (2) cells increased significantly after overexpression of miR-20a-5p (t (df) = 10.78 (4), p = 0.004, Fig. 3C). After knocking down miR-20a-5p, the invasiveness of invaded SK-N-BE (2) cells was significantly lower than that of short hairpin negative control group (sh NC) (t (df) = 15.01 (4), p = 0.001, Fig. 3C). After overexpression of miR-20a-5p in SH-SY5Y cells, the number of invaded cells also increased significantly (t (df) = 32.33 (4), p < 0.0001). Figure 3D). After knockdown miR-20a-5p, the invasiveness of SH-SY5Y cells was significantly lower than that of short hairpin negative control group (sh NC) (t (df) = 9.525 (4), p = 0.0007, Fig. 3C). Among them, the proliferation ability of SH-SY5Y cells increased significantly after overexpression of miR-20a-5p (t (df) = 35.08 (4), p < 0.0001), while that of SK-N-BE (2) cells decreased significantly after knockdown miR-20a-5p (t (df) = 16.06 (4), p < 0.0001). These results suggest that miR-20a-5p can promote the invasion of NB cells.

Wound healing experiments showed that after overexpression of miR-20a-5p, the healing rate of SK-N-BE (2) cells was (72.00 ± 4.50)%, while that of oe NC cells was (54.12 ± 1.56)%. There was a significant difference (t (df) = 5.493 (4), p = 0.005, Fig. 3C). After knocking down miR-20a-5p, the healing rate of SK-N-BE (2) cells was (43.60 ± 2.04)%, while that of the sh NC group was (52.15 ± 0.87)%. There was a significant difference (t(df) = 6.864 (4), p = 0.002). Figure 3E). After overexpression of miR-20a-5p, the healing rate of SH-SY5Y cells was (24.33 ± 1.24)%, while that of oe NC cells was (44.18 ± 1.16)% (t (df) = 6.127 (4), p = 0.0036, Fig. 3F). After knocking down miR-20a-5p in SH-SY5Y cells, the healing rate was (24.33 ± 1.24)%, and that in sh NC group was (43.3 ± 2.72)%. There was significant difference (t (df) = 12.85,p = 0.0002, Fig. 3F). The migration ability of SH-SY5Y cells was significantly enhanced after overexpression of miR-20a-5p (t (df) = 24.277 (4), p < 0.0001, Fig. 3F). However, the migration ability of SK-N-BE (2) cells decreased significantly after miR-20a-5p knockdown(t (df) = 8.042 (4), p = 0.001). The above results indicate that miR-20a-5p regulates the migration of NB cells.

Flow cytometry showed that the early apoptosis rates of SK-N-BE (2) and SH-SY5Y cells increased significantly after knockdown of miR-20a-5p. The results showed that miR-20a-5p significantly increased the viability of NB cells (t (df) = 7.143 (4), p = 0.002 Fig. 4A ; t(df) = 10.45,p = 0.0005, Fig. 4B).

After overexpression or downregulation of miR-20a-5p in SK-N-BE (2) cells, the results of cell counting kit-8 assay showed that there was significant difference in cell proliferation between the oe miR-20a-5p group and the oe NC group at 24 h, 48 h and 72 h (t (df) = 2.435 (3), p = 0.02.Fig.C ). There were significant differences in cell proliferation in the short-hair R-20a-5p (shR-20a-5p) group at 24 h, 48 h and 72 h (t (df) = 2.630 (3), p = 0.009, Fig. 4.E). After overexpression or downregulation of miR-20a-5p in SH-SY5Y cell line, except for 0 h, at 24 h, 48 h and 72 h, the results of the cell counting kit-8 assay showed that there was significant difference in cell proliferation between the oe miR-20a-5p group and the oe NC group (t (df) = 2.032 (3), p = 0.0029,Fig. D). There was a significant difference in cell proliferation in the short-hair R-20a-5p (shR-20a-5p) group (t (df) = 3.272 (3), p = 0.006, Fig.F). The results showed that the proliferation ability of NB cells was enhanced after overexpression of miR-20a-5p, and decreased after downregulation of miR-20a-5p. Notably, overexpression of miR-20a-5p can significantly enhanced the proliferation of SH-SY5Y cells, while knockdown of miR-20a-5p can significantly reduced the proliferation of SK-N-BE (2) cells (t(df) = 2.123 (3), p = 0.0004, Fig. 4G). It is suggested that miR-20a-5p plays an important role in regulating the proliferation of NB.

4. miR-20a-5p activates the NF- κB pathway through downregulation of NFKBIB in NB

Based on the prediction of miR-20a-5p targets by bioinformatics analysis, we found that NFKBIB is a hypothetical target (Fig. 5.A) of miR-20a-5p. Then double luciferase experiment showed that overexpression of miR-20a-5p decreased the luciferase activity of NFKBIB, while the luciferase activity of other groups changed little, indicating that there was a specific interaction between miR-20a-5p and NFKBIB (t (df) = 22.76(6), p = 2, Fig. 5.B). Subsequently, we measured by qRT-PCR that the overexpression of miR-20a-5p in NB cells was accompanied by the downregulation of NFKBIB expression (t (df) = 3.876 (4), pendant 0.01, Fig. 5C; t (df) = 18.64 (4), p < 0.0001), and the downregulation of miR-20a-5p was accompanied by the upregulation of NFKBIB expression (t (df) = 3.876 (4), p = 0.017, Fig. 5C);t (df) = 3.724 (4), p = 0.02, Fig. 5.D).

The expression of NFKBIB and NF- κB in the two cell lines was detected by Western blotting. The results showed that the expression level of NFKBIB in SK-N-BE (2) cells with MYCN amplification was significantly lower than that in SH-SY5Y cells without amplification (t (df) = 9.256 (4), p = 0.0008, Fig. 5E.a), and the expression level of NF-κB in SH-SY5Y cells without MYCN gene mplification was significantly higher than that in SH-SY5Y cells without MYCN gene amplification (t (df) = 2.975 (4), p = 0.04, Fig. 5E). In SK-N-BE (2) cells, the overexpression of miR-20a-5p was upregulated with the expression of NF- κ B (t (df) = 10.39 (6), p < 0.0001, Fig. 5F.b) and the expression of NFKBIB was downregulated (t (df) = 4.173 (6), paired 0.059, Fig. 5F.a). After knocking down miR-20a-5p, the expression of NFKBIB was significantly downregulated (t (df) = 3.699 (6), p = 0.0101 Fig. 5F.a), and the expression of NF- κ B was significantly up-regulated (t (df) = 5.258 (6), p = 0.019, Fig. 5F.b). In SH-SY5Y cells, miR-20a-5p overexpression was accompanied by up-regulation of NF- κB expression (t(df) = 10.48 (6), p < 0.0001, Fig. 5G.b) and down-regulation of NFKBIB expression (t(df) = 6.397 (6), p = 0.0007). Figure 5G.a). After knocking down miR-20a-5p, the expression of NFKBIB was significantly downregulated (t (df) = 6.609 (6), p = 0.0006, Fig. 5G.a), and the expression of NF- κB was significantly upregulated (t (df) = 2.496 (6), p = 0.0468). Figure 5G.b). These results suggest that miR-20a-5p can downregulate the expression of NFKBIB in NB and activate NF- κ B pathway.

MYCN is a member of the oncogene family MYC, and its transcription factor N-myc is generally only expressed in tumors of neurogenic origin, such as NB, and gliomas[10, 11]. In vivo experiments have confirmed that overexpression of the MYCN gene in transgenic mice can induce NB[12]. Some scholars have found that there is a correlation between the miR-17-92 gene cluster and MYCN gene amplification at the tissue and cell levels. The increased expression of these miRNAs can promote tumor cell proliferation, induce tumor angiogenesis and inhibit tumor apoptosis[13, 14]. In the NB samples amplified by MYCN, the upregulation of MYCN occurred simultaneously with the severe increase in the expression of miR-17-92 cluster[15]. This is consistent with our results that the expression of MYCN mRNA in SK-BE-2 cells with MYCN amplification is significantly higher than that in SH-SY5Y cells without MYCN amplification. In addition, it was found that the members of the miR-17-92 cluster were tumor cells significantly upregulated in MYCN-amplified NB[16]. The results of Chen ¹⁸ showed that the expression level of miR-17-92 cluster in MYCN-amplified cells SK-N-BE (2) cells was significantly higher than that in MYCN-unamplified SH-SY5Y cells. This is also consistent with our results. In this study, the expression of miR-20a-5p in SK-N-BE (2) cell line with MYCN amplification was significantly higher than that in the SH-SY5Y cell line without MYCN amplification. We further compared the proliferation, invasion and migration ability of the SK-N-BE (2) cell line and SH-SY5Y cell line, and found that the proliferation, invasion and migration ability of SK-N-BE (2) cells amplified by MYCN was significantly stronger than that of MYCN nonamplified SH-SY5Y cells.

miR-20a-5p is a member of the miR-17-92 gene cluster[17]. Generally, as an oncogene, it plays an important role in the occurrence and development of many tumors[18]. In breast cancer, miR-20a-5p affects the growth, migration, invasion and apoptosis of breast cancer cells through the LncRNAHOTAIR/miR-20a-5p/HMGA2 axis[19]. In bladder cancer, miR-20a-5p negatively regulates NR4A3 to promote cancer cell metastasis, and ultimately affects the progression of bladder cancer[20]. In this study, after overexpressing and knocking down miR-20a-5p in SK-N-BE (2) cells with MYCN amplification, we found that the change in miR-20a-5p RNA expression was accompanied by the change of MYCN mRNA. Therefore, we think that MYCN may promote the proliferation, invasion and migration of NB cells by increasing the expression of miR-20a-5p. The difference is that Yu et al.[21] found that miR-20a-5p can inhibit autophagy of NB cells through negative regulation of ATG7 in NB, thus inhibiting the proliferation of NB cells and promoting apoptosis. This may be caused by the two sides of autophagy, the heterogeneity of the tumor and the complexity of the proliferative signal network.

It has been confirmed that the NF- κ B pathway enhances the invasive ability of neuroblastoma cells by regulating the expression of its downstream protein CXCRA[22], and lentivirus-mediated siRNA inhibits neuroblastoma invasion and metastasis by downregulating the expression of NF- κB downstream genes CXCR4 and VEGF[23]. NFKBIB is a member of the suppressor protein family of NF-κ B transcription factors. When this pathway is not activated, NFKBIB binds NF- κB through its C-terminal specific anchor protein repeat sequence and covers the nuclear localization sequence to prevent NF- κB transfer into the nucleus ²⁵. To further study the correlation between the oncogene miR-20a-5p and the NF- κB pathway in NB, we found that NFKBIB is a hypothetical target of miR-20a-5p by bioinformatics methods, and then a double luciferase report experiment was used to verify that the direct target of miR-20a-5p was NFKBIB. Then through qRT-PCR and Western Blotting, it was found that miR-20a-5p could downregulate the expression of NFKBIB and activate the NF κB pathway in NB cells. Thus, the oncogene MYCN in NB may promote the expression of miR-20a-5p, and then activate the NF- κB pathway after phosphorylation inactivation of the protein IKB- β expressed by the miR-20a-5p target gene NFKBIB, and promote the proliferation, invasion and migration of NB.

Through this study, we speculate that MYCN/miR-20a-5p/NFKBIB/NF- κB axis can promote the proliferation, invasion and metastasis of NB, but the mechanism of MYCN gene amplification in NB is still unknown. In future experiments, we will continue to explore the downstream mechanism of this regulatory axis, and further explore the possible role of this regulatory axis in tumor cells and the tumor microenvironment, further clarify the mechanism of MYCN gene amplification, and provide new predictive molecules and therapeutic targets for basic research and clinical treatment of NB.

This study found that the oncogene MYCN may promote the expression of miR-20a-5p in NB, thereby inactivating the IkB expressed by the target gene miR-20a-5p- β protein, activating NFr- κ B pathway, promote the proliferation, invasion and migration of NB. On this basis, we speculate that the MYCN/miR-20a-5p/NFKBIB/NF- κB axis can promote the proliferation, invasion and migration of NB. This study, further investigate the effects of NF- κB uncontrolled inflammation related tumors regulated by the pathway and provide a new idea.

Availability of data and materials

All data analyzed during this study are included in this article.

Acknowledgements

Not applicable.

Authors' contributions

WL and AM conceived the project, analyzed the data, and wrote the manuscript. AM, WL, YW, GM, HT,SZ and HL performed the experiments and analyzed the data. AY and WL contributed equally to this study. All authors contributed to the article and approved the submitted version.

Declaration of Conflicting Interests

Conflict of interest The authors have no confict of interest and no financial issues to disclose.

Funding

This work was financially supported by the 2020 Xinjiang Uygur Autonomous Region Science and Technology Aid Plan for Xinjiang（2020E0267）.

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The MYCN/miR-20a-5p/NFKBIB/NF-κB signaling axis promotes the proliferation, invasion and migration of neuroblastoma

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Abstract

Purpose

Methods

Results

Conclusion

Figures

Introduction

Materials and methods

1. Cell culture

2. Construction of a stably transfected cell line by cell transfection and screening

3. Quantitative reverse transcription-PCR

4. Cell counting kit-8 assay

5. Wound healing assay

5. Transwell assay

6. Flow Cytometry

7. Luciferase reporter assay

8. Western blotting

8. Statistical analyses

Result

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

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