Method for researching influence of triptolide on proliferation of multiple myeloma cells by regulating miR-381 / HMGB1

Abstract

The invention discloses a method for researching the influence of triptolide on the proliferation of multiple myeloma cells by regulating miR-381 / HMGB1 (High Mobility Group Box 1), and relates to the technical field of biology. Research results of the research method show that miR-381 methylation silencing in RPMI8226 cells is an important reason for promoting cell proliferation, TPL inhibits proliferation of the RPMI8226 cells by reversing a miR-381 methylation modification state, inducing and restoring expression and regulating HMGB1 genes in a targeted manner, and the research lays a theoretical basis for exploring an anti-MM action mechanism of TPL.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for studying the effect of triptolide on regulating miR-381 / HMGB1 on the proliferation of multiple myeloma cells. Background Art

[0002] miRNA is a type of non-coding RNA that targets and regulates the expression of target genes at the post-transcriptional level by complementary matching with the 3'-UTR non-coding region of the target gene. During the development of tumors, miRNA participates in the biological behaviors of tumor cell proliferation, differentiation, apoptosis, migration, and angiogenesis by regulating oncogenes and tumor suppressor genes. Studies on the role of miR-381 in the occurrence of different tumors have shown that miR-381 can play the role of oncogenes or tumor suppressor genes in different tumor tissues. Its specific selection mechanism is still unclear, which also suggests that the expression of miR-381 may be cell and tissue specific. In glioma cells, miR-381 is often highly expressed, resulting in a weakened inhibitory effect on the MEK / ERK and AKT signaling pathways, thereby promoting cell proliferation. In colon cancer tissues, miR-381 expression is significantly downregulated, and its reduced expression is related to the clinical stage, degree of differentiation, depth of infiltration, and distant metastasis of colon cancer. It is a potential biomarker for colon cancer.

[0003] Epigenetic silencing of tumor suppressor genes is an important mechanism leading to tumorigenesis, and abnormal methylation of the promoter bases of miRNA genes with tumor suppressor function leading to downregulation of expression is also an important factor causing tumorigenesis. In esophageal cancer cells, the promoter of miR-375 gene is methylated, resulting in low expression, while methylation does not occur in normal human mucosal tissues. In MM studies, abnormal epigenetic modification of non-coding RNA is closely related to the proliferation, apoptosis and angiogenesis of MM cells.

[0004] DNA methylation often occurs on the cytosine-phosphate-guanine (CpG) structure and is one of the causes of gene inactivation. Analysis of the upstream sequence of miR-381 found that its upstream promoter region contains CpG structure, which provides a molecular basis for the occurrence of miR-381 methylation.

[0005] Tripterygium wilfordii is a local medicinal material produced in Fujian. TPL is an epoxy diterpene lactone compound isolated from the root bark of Tripterygium wilfordii. It has pharmacological effects such as anti-inflammatory and immunomodulatory in clinical practice. Experimental studies have found that TPL has the effect of inhibiting proliferation and promoting apoptosis of various blood tumor cells. Previous studies have also confirmed that TPL can inhibit the proliferation of RPMI8226 cells and induce apoptosis by regulating DNA and histone methylation, as well as the endoplasmic reticulum stress pathway, and is a drug with significant anti-MM effects. In this study, after applying TPL to RPMI8226 cells, it was found that the methylation level of miR-381 in RPMI8226 cells decreased and its expression was upregulated, and the cell proliferation activity decreased, further confirming that TPL inhibited the proliferation of RPMI8226 cells and reversed the methylation modification of miR-381. Studies have found that in renal cancer, miR-381 can cooperate with miR-424 to inhibit the proliferation of renal cancer cells and promote their apoptosis, as well as regulate cell cycle changes. In lung cancer, miR-381 affects the proliferation, invasion and metastasis of lung cancer cells by regulating the target gene inhibitor of differentiation protein-1 (ID-1).

[0006] High mobility group protein B1 (HMGB1) is a highly conserved nuclear protein. Under normal circumstances, HMGB1 mainly exists in the endoplasmic reticulum and Golgi apparatus and binds to DNA, maintains the stability of nucleosome structure and participates in gene recombination and replication. However, HMGB1 is often upregulated in tumor tissues and is closely related to poor prognosis. Evidence shows that the PI3K / AKT pathway activated by HMGB1 plays an important role in thyroid tumors. HMGB1 expression is elevated in papillary thyroid cancer cells, and miR-101-3p can target and regulate the decrease of HMGB1 expression level, thereby inhibiting the proliferation, invasion and migration of papillary thyroid cancer cells.

[0007] Abnormal epigenetic modification is one of the important mechanisms of multiple myeloma (MM). In previous studies, our research group found that methylation modification of DNA, histones and microRNA (miRNA) is widely involved in the malignant proliferation and apoptosis inhibition of MM cells. In addition, it has been confirmed that triptolide (TPL) can induce apoptosis of various blood tumor cells by reversing epigenetic modification.

[0008] With the deepening of research on miRNA, the correlation between miR-381 and tumorigenesis has increasingly attracted the attention of researchers. Studies have found that the methylation level of miR-381 in MM patients is significantly higher than that in healthy subjects, and the expression of miR-381 in MM cells increases after demethylation treatment, but the molecular mechanism of miR-381 regulated by methylation in MM has not been reported. High mobility group protein B1 (HMGB1) is an evolutionarily conserved protein that is ubiquitous in eukaryotic cells. Many studies have confirmed that HMGB1 is expressed at an elevated level in tumor cells such as colon cancer, breast cancer, melanoma, liver cancer, and gastric cancer. In MM cells, long non-coding RNA inhibits MM cell apoptosis by upregulating HMGB1 expression, thereby promoting cancer. Summary of the invention

[0009] In view of this, the embodiments of the present invention provide a method for studying the effect of triptolide on the proliferation of multiple myeloma cells by regulating miR-381 / HMGB1, and further explore the effect of TPL on the proliferation of MM cells through the epigenetic modification and regulation mechanism of miR-381 / HMGB1.

[0010] To achieve the above object, the present invention mainly provides the following technical solutions:

[0011] On the one hand, an embodiment of the present invention provides a method for studying the effect of triptolide on regulating miR-381 / HMGB1 on the proliferation of multiple myeloma cells, the method comprising the following steps:

[0012] The method comprises the following steps:

[0013] Step 1: Cell culture and experimental grouping;

[0014] Step 2: Detect cell proliferation activity by MTT method;

[0015] Step 3, bisulfite sequencing (BSP) was used to detect the methylation level of miR-381;

[0016] Step 4: Real-time fluorescence quantitative RT-PCR to detect the expression level of miR-381;

[0017] Step 5, using bioinformatics online software (http: / / www.targetscan.org) to predict the target gene relationship between miR-381 and HMGB1;

[0018] Step 6, Western blot detection of CREB1 protein expression level;

[0019] Step 7: Statistical analysis.

[0020] Further, in step 1, the MTT method was used to detect different control groups, namely blank control group, TPL group, miR-381 mimics group, miR-381 mimics NC group, miR-381 inhibitor group, miR-381 inhibitor NC group, small interfering RNA-HMGB1 transfection group and small interfering RNA-HMGB1 transfection NC group.

[0021] Further, in step 5, the online software is Target Scan.

[0022] Furthermore, in step 7, SPSS 20.0 statistical software was used to analyze the experimental data. The measured data were expressed as mean ± standard deviation. One-way analysis of variance was used to compare the data between groups, and the t-test was used to compare the data between the two groups. P < 0.05 was considered to be statistically significant.

[0023] Furthermore, the research results of the research method are as follows: after TPL acted on RPMI 8226 cells for 48 hours, the MTT results showed that the proliferation activity of cells in the TPL group decreased, and the difference was statistically significant compared with the OD value of cells in the Control group.

[0024] Furthermore, the research results of the research method are as follows: the cell proliferation inhibition rate in the TPL group is 56.6%.

[0025] Furthermore, the research results of the research method are as follows: the real-time fluorescence quantitative RT-PCR results showed that the expression level of miR-381 in the TPL group was increased, and the Western blot results showed that the expression level of HMGB1 in the TPL group was significantly decreased.

[0026] Furthermore, the research results of the research method are as follows: after transfection of cells with miR-381 inhibitor, MTT results showed that the proliferation activity of cells in the miR-381 inhibitor group was further increased, and Western blot results showed that the expression level of HMGB1 in the miR-381 inhibitor group was significantly upregulated. After transfection of cells with miR-381 mimics, MTT results showed that the proliferation activity of cells in the miR-381 mimics group was reduced, and Western blot results showed that the expression level of HMGB1 in the miR-381 mimics group was reduced.

[0027] Furthermore, the research results of the research method are as follows: HMGB1 is a potential target gene of miR-381 as predicted by the target gene prediction analysis of the Target Scan (http: / / www.targetscan.org) online software, and the luciferase activity experiment shows that the luciferase activity of the miR-381 mimic transfection group is significantly reduced.

[0028] Furthermore, the research results of the research method are as follows: after Si-HMGB1 transfection of cells, MTT results showed that the proliferation activity of cells in the Si-HMGB1 group was reduced, and Western blot results showed that the expression level of HMGB1 in the Si-HMGB1 group was significantly reduced.

[0029] Compared with the prior art, the present invention has the following beneficial effects:

[0030] The above research method of the present invention was used to construct a cell model of miR-381 silencing and miR-381 overexpression, observe the effect of miR-381 on the expression of HMGB1 in RPMI8226 cells, further verify the downstream targets of miR-381, and silence HMGB1 expression by using RNA interference technology to observe its effect on the anti-cancer effect of miR-381. The experimental results showed that after transfection of RPMI8226 cells with miR-381 inhibitors or miR-381 mimics, HMGB1 expression showed an up-regulated or down-regulated trend, and the proliferation activity of cells was enhanced or weakened. At the same time, interfering with the expression of HMGB1 can significantly inhibit cell proliferation. These results confirm that miR-381 can inhibit the proliferation of RPMI8226 cells by inhibiting the expression of the target gene HMGB1. The results of this research method showed that methylation silencing of miR-381 in RPMI8226 cells is an important reason for promoting cell proliferation. TPL inhibits the proliferation of RPMI8226 cells by reversing the methylation modification state of miR-381, inducing restoration of expression, and targeting the regulation of the HMGB1 gene. This study laid a theoretical foundation for exploring the anti-MM mechanism of TPL. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 is the effect of TPL on miR-381 methylation and its expression in RPMI;

[0032] Figure 2 is the effect of TPL on HMGB1 protein expression in RPMI 8226;

[0033] Figure 3 is the effect of miR-381 inhibitor on HMGB1 protein expression in RPMI 8226;

[0034] Figure 4 is the effect of miR-381 mimics on HMGB1 protein expression in RPMI 8226;

[0035] Figure 5 The relationship between miR-381 and HMGB1 was predicted by bioinformatics;

[0036] Figure 6The luciferase assay confirmed the targeting relationship between miR-381 and HMGB1, P < 0.05 compared with the control group and P < 0.05 compared with the miR-381 mimic control group.

[0037] Figure 7 is the effect of Si-HMGB1 on HMGB1 protein expression in RPMI 8226. DETAILED DESCRIPTION

[0038] In order to further explain the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, technical solutions, features and effects of the present invention are described in detail below with preferred embodiments. The specific features, structures, or characteristics of the multiple embodiments in the following description can be combined in any suitable form.

[0039] Materials and Methods

[0040] 1. Main Reagents

[0041] Triptolide (molecular formula: C20H24O6), 1 mg / ml propylene glycol was used for sample preparation before the test, and the sample was diluted to the required concentration with culture medium during the test; MTT reagent was a product of Sigma; Lipofectamine 2000 and TRIzol reagent were products of Invitrogen; 10% fetal bovine serum was a product of Hangzhou Sijiqing Company; RPMI1640 culture medium was a product of Gibco; DNA sulfurization kit and DNA purification kit were products of Promega; Mir-X TM miRNAFirst-Strand Synthesis Kit, Prime Script RT Master Mix reverse transcription kit and SYBRAdvantage qPCRPremix are all products of Takara; miR-381 mimics (miR-381 mimics), miR-381 inhibitors (miR-381 inhibitors), small interfering RNA-HMGB1 (Si-HMGB1) and their controls are products of Guangzhou Ruibo Biotechnology Co., Ltd.; HMGB1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) monoclonal antibodies are products of Abcam.

[0042] 2. Cell culture and experimental grouping

[0043] Multiple myeloma RPMI 8226 cell line was a cryopreserved cell line of our center. After routine thawing, the cells were cultured in an incubator at 37°C, 5% CO2 and saturated humidity. The cells in different treatment groups were divided into a blank control group (Control) containing only cell culture medium; a drug-treated group (TPL) with 80 nmol / L of triptolide; a miR-381 overexpression group (miR-381 mimic) transfected with miR-381 mimic, and a micro-miR-381 mimic transfection control group (miR-381 mimic NC); a miR-381 expression inhibition group (miR-381 inhibitor) transfected with a miR-381 inhibitor, and a micro-miR-381 mimic transfection control group (miR-381 inhibitor NC); a HMGB1 expression inhibition group (Si-HMGB1) transfected with small interfering RNA-HMGB1, and a small interfering RNA-HMGB1 transfection control group (Si-HMGB1 NC).

[0044] Example 1 Transfection of miR-381 mimics, miR-381 inhibitors and Si-HMGB1

[0045] RPMI 8226 cells in the logarithmic growth phase were taken, the cell density was adjusted to 5×104 / ml, and inoculated into 6-well culture plates. According to the instructions of micrONTM miRNA mimics, micrOFFTM miRNA inhibitors and siRNA products, miR-381 mimics, miR-381 inhibitors and Si-HMGB1 lyophilized powder were diluted to a final concentration of 50nmol / L, and cells were transiently transfected using Lipofectamine2000.

[0046] Example 2 MTT method to detect cell proliferation activity

[0047] Take RPMI8226 cells in the logarithmic growth phase, adjust the cell density to 2×108 / ml, and inoculate them in a 96-well plate. Add 20μL of 5g / L MTT reagent to each well of the cells in the different intervention groups and culture them for 4h, then add 100μL of dimethyl sulfoxide. Detect the absorbance value (OD value) of each well at a wavelength of 570nm on an ELISA reader, and calculate the cell proliferation inhibition rate. Proliferation inhibition rate = (OD value of the control group - OD value of the TPL group) / OD value of the control group × 100%

[0048] Example 3 Bisulfite sequencing (BSP) to detect the methylation level of miR-381

[0049] DNA bisulfite modification and purification were performed according to the DNA sulfonation kit operating procedures. A 25 μl reaction system was used, and the reaction conditions were 95°C pre-denaturation for 10 min, 94°C denaturation for 30 s, 55°C annealing for 57 s, 72°C extension for 1 min, 37 cycles, and finally 72°C extension for 6 min. The BSP amplification product was purified and recovered and then TA cloned and sequenced. The methylation status of each CpG site was analyzed. Ten clones were measured for each sample and the methylation positive rate of its CpG site was calculated. The BSP primer sequence and sequencing of miR-381 were completed by Shanghai Shenggong Biotechnology Co., Ltd.

[0050] Example 4 Real-time fluorescence quantitative RT-PCR detection of miR-381 expression level

[0051] Total RNA was extracted by Trizol method. Reverse transcription kit was used for cDNA synthesis. SYBR Advantage qPCR Premix kit was used for gene PCR amplification. A 25 μL reaction system was used, ddH2O 8.5 μL, SYBR 12.5 μL, primer 2 μL, cDNA 2 μL, reaction conditions were 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing for 30 s, 40 cycles, and finally 72℃ extension for 5 min.

[0052] The upstream primer of miR-381 was 5′-AGTCTATACAAGGCAAGCTCTC-3′, and the downstream primer was 3′-CACTTCCTCAGCACTTGTTGGTAT-5′. U6 was used as the internal reference, and 2-ΔΔCt was used to analyze the relative expression of the gene.

[0053] Example 5 Prediction and verification of the targeting relationship between miR-381 and HMGB1

[0054] The target gene prediction was performed using the bioinformatics online software Target Scan (http: / / www.targetscan.org). The nucleotides including the binding sequence were selected as the insertion sequence in the 3'-UTR sequence of HMGB1, and SpeⅠ and HindⅢ were introduced at the 5' end of the upstream and downstream primers, respectively. The pmiR-HMGB1 plasmid constructed by the miR-381 mimic and the luciferase reporter gene plasmid pmiR-report vector was transiently transfected into RPMI8226 cells using Lipofectamine 2000, and pRL-TK was used as the control plasmid vector. Luciferase activity was detected 24 hours after transfection. The validation analysis was compared with the ratio of firefly luciferase activity to Renilla luciferase activity.

[0055] Example 6 Detection of HMGB1 protein expression level by Western blot

[0056] The total protein of each group of cells was extracted and quantified by the BCA method. After separation by 10% SDS-PAGE gel electrophoresis, the protein was electrotransferred to a cellulose acetate membrane for 1.5 h, blocked with 5% skim milk powder for 2 h, and incubated with mouse anti-human HMGB1 monoclonal antibody and mouse anti-human GAPDH monoclonal antibody at 4°C overnight. After washing, the membranes were incubated with horseradish peroxidase-labeled anti-mouse secondary antibodies for 1 h, and chemiluminescent reagents were added for exposure, development, photography and grayscale analysis of protein bands in a dark room using image analysis software.

[0057] 3. Statistical Analysis

[0058] SPSS 20.0 statistical software was used for experimental data analysis. The measurement data were expressed as mean ± standard deviation. One-way analysis of variance was used to compare data between groups, and t-test was used to compare data between two groups. P < 0.05 was considered statistically significant.

[0059] IV. Results

[0060] 1. Effect of TPL on RPMI 8226 cell proliferation, miR-381 methylation, and miR-381 and HMGB1 expression After 48 hours of TPL treatment on RPMI 8226 cells, MTT results showed that the proliferation activity of cells in the TPL group decreased, and the difference was statistically significant compared with the OD value of cells in the Control group (0.36±0.06 vs. 0.83±0.05, t=11.29, P<0.05). The cell proliferation inhibition rate in the TPL group was 56.6%. BSP results showed that the methylation rate of miR-381 in the Control group was (73.28±8.75)%, while that in the TPL group was (28.41±3.67)%, and the difference between the two groups was statistically significant (t=8.20, P<0.05). The results of real-time fluorescence quantitative RT-PCR showed that the expression level of miR-381 in the TPL group was increased compared with that in the Control group (5.10±0.25 vs. 1, t=28.68, P<0.05), and the difference was statistically significant ( Figure 1 Western blot results showed that the expression level of HMGB1 in the TPL group was significantly decreased compared with that in the Control group (0.16±0.02 vs. 0.86±0.05, t=22.83, P<0.05), and the difference was statistically significant ( Figure 2 ).

[0061] 2. Effects of miR-381 inhibition on multiple myeloma cell proliferation and HMGB1 protein expression

[0062] After transfection of cells with miR-381 inhibitor, MTT results showed that the proliferation activity of cells in the miR-381 inhibitor group was further increased, and the difference was statistically significant compared with the cell OD value of the control group (2.11±0.12 vs. 0.78±0.03, t=17.87, P<0.05). Western blot results showed that the expression level of HMGB1 in the miR-381 inhibitor group was significantly upregulated, and the difference was statistically significant compared with the blank control group (0.87±0.01 vs. 0.54±0.03, t=20.89, P<0.05). Figure 3 ).

[0063] 3. Effect of miR-381 overexpression on multiple myeloma cell proliferation and HMGB1 protein expression

[0064] After transfection of cells with miR-381 mimics, MTT results showed that the proliferation activity of cells in the miR-381 mimics group was reduced, and the difference was statistically significant compared with the cell OD value of the blank control group (0.18±0.03 vs. 1.08±0.04, t=32.16, P<0.05). Western blot results showed that the expression level of HMGB1 in the miR-381 mimics group was reduced, and the difference was statistically significant compared with the blank control group (0.20±0.02 vs. 1.13±0.07, t=22.49, P<0.05). Figure 4 ).

[0065] 4. Luciferase reporter gene assay to verify the targeting relationship between miR-381 and HMGB1

[0066] Target gene prediction analysis using Target Scan (http: / / www.targetscan.org) online software revealed that the 3'-UTR base sequence of HMGB1 had a matching continuous region with the 5' end region of miR-381 ( Figure 5 ), suggesting that HMGB1 is a potential target gene of miR-381. Luciferase activity experiments showed that the luciferase activity of the miR-381mimic (microRNA-381 mimic) transfection group was significantly reduced, compared with the blank control group and the miR-381mimic transfection control group (0.34±0.04 vs. 1.12±0.06, t=19.74, P<0.05; 0.34±0.04 vs. 1.13±0.08, t=15.14, P<0.05), the differences were statistically significant ( Figure 6 ).

[0067] 5. Effects of Si-HMGB1 interference on multiple myeloma cell proliferation and HMGB1 protein expression

[0068] After Si-HMGB1 transfection, MTT results showed that the proliferation activity of cells in the Si-HMGB1 group was reduced, and the cell OD value was compared with that in the blank control group (0.17±0.01 vs. 0.87±0.03, t=37.90, P<0.05), with a statistically significant difference. The cell proliferation inhibition rate in the Si-HMGB1 group was 75.4%. Western blot results showed that the expression level of HMGB1 in the Si-HMGB1 group was significantly reduced, and the difference was statistically significant compared with that in the blank control group (0.18±0.03 vs. 1.07±0.11, t=14.00, P<0.05). Figure 7 ).

[0069] V. Conclusion

[0070] Previous studies have found that miR-203 in RPMI8226 cells is silenced due to DNA methylation modification, resulting in abnormal cell proliferation and apoptosis inhibition. The mechanism is that miR-203 methylation mediates targeted regulation of CREB1 gene expression, leading to malignant biological characteristics of RPMI8226 cells, and miR-203 demethylation can reverse the above effects. However, the epigenetic modification mechanism of miR-381 methylation in MM has rarely been reported.

[0071] In this study, BSP and real-time fluorescence quantitative RT-PCR detection revealed that miR-381 was highly methylated and lowly expressed in RPMI8226 cells, suggesting that miR-381 was methylated and silenced in RPMI8226 cells and that miR-381 may play a role as a tumor suppressor gene in the occurrence and development of MM.

[0072] In this study, we analyzed the potential target genes of miR-381 using the miRNA target gene prediction software Target Scan and found that the 5' end region of miR-381 could target and bind to the 3'-UTR region base sequence of HMGB1, and verified the specific binding between the two using the dual luciferase reporter gene analysis system. Therefore, we speculate that the inhibitory effect of TPL on RPMI8226 cell proliferation is achieved by regulating the miR-381 / HMGB1 signal modified by DNA methylation.

[0073] For matters not covered in the embodiments of the present invention, those skilled in the art can select from the prior art.

[0074] The above disclosure is only a specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art can easily think of changes or substitutions within the technical scope disclosed by the present invention, which should be included in the protection scope of the present invention. Therefore, the protection scope of the present invention should be based on the protection scope of the above claims.

Claims

1. A research method for the effect of triptolide on regulating miR-381 / HMGB1 on the proliferation of multiple myeloma cells, characterized in that: The method comprises the following steps: Step 1: Cell culture and experimental grouping; Step 2: Detect cell proliferation activity by MTT method; Step 3, detecting the methylation level of miR-381 by bisulfite sequencing; Step 4: Real-time fluorescence quantitative RT-PCR to detect the expression level of miR-381; Step 5: Use bioinformatics online software to predict the target gene relationship between miR-381 and HMGB1; Step 6, Western blot detection of CREB1 protein expression level; Step 7: Statistical analysis.

2. The method for studying the effect of triptolide on regulating miR-381 / HMGB1 on the proliferation of multiple myeloma cells according to claim 1, characterized in that: In step 1, the MTT method was used to detect different control groups, namely blank control group, TPL group, miR-381 mimics group, miR-381 mimics NC group, miR-381 inhibitor group, miR-381 inhibitor NC group, small interfering RNA-HMGB1 transfection group and small interfering RNA-HMGB1 transfection NC group.

3. The research method of claim 1, wherein the effect of triptolide on the proliferation of multiple myeloma cells by regulating miR-381 / HMGB1 is characterized in that: In step 5, the online software is Target Scan.

4. The method for studying the effect of triptolide on regulating miR-381 / HMGB1 on the proliferation of multiple myeloma cells according to claim 1, characterized in that: In step 7, SPSS 20.0 statistical software was used to analyze the experimental data. The measured data were expressed as mean ± standard deviation. One-way analysis of variance was used to compare the data between groups, and the t-test was used to compare the data between the two groups. P < 0.05 was considered to be statistically significant.

5. The research method of claim 1, wherein the effect of triptolide on the proliferation of multiple myeloma cells by regulating miR-381 / HMGB1 is characterized in that: The research results of the research method are as follows: after TPL acted on RPMI 8226 cells for 48 hours, the MTT results showed that the proliferation activity of cells in the TPL group decreased, and the difference was statistically significant compared with the OD value of cells in the Control group.

6. The research method of claim 1, wherein the effect of triptolide on the proliferation of multiple myeloma cells by regulating miR-381 / HMGB1 is characterized in that: The research results of the research method are as follows: the cell proliferation inhibition rate in the TPL group is 56.6%.

7. The method for studying the effect of triptolide on regulating miR-381 / HMGB1 on the proliferation of multiple myeloma cells according to claim 1, characterized in that: The research results of the research method are as follows: the real-time fluorescence quantitative RT-PCR results showed that the expression level of miR-381 in the TPL group was increased, and the Western blot results showed that the expression level of HMGB1 in the TPL group was decreased.

8. The method for studying the effect of triptolide on regulating miR-381 / HMGB1 on the proliferation of multiple myeloma cells according to claim 1, characterized in that: The research results of the research method are as follows: after transfection of cells with miR-381 inhibitor, MTT results showed that the proliferation activity of cells in the miR-381 inhibitor group was further increased, and Western blot results showed that the expression level of HMGB1 in the miR-381 inhibitor group was upregulated. After transfection of cells with miR-381 mimics, MTT results showed that the proliferation activity of cells in the miR-381 mimics group was reduced, and Western blot results showed that the expression level of HMGB1 in the miR-381 mimics group was reduced.

9. The method for studying the effect of triptolide on regulating miR-381 / HMGB1 on the proliferation of multiple myeloma cells according to claim 1, characterized in that: The research results of the research method are as follows: HMGB1 is a potential target gene of miR-381 as predicted by the Target Scan online software, and the luciferase activity experiment shows that the luciferase activity of the miR-381 mimic transfection group is reduced.

10. The research method of claim 1, wherein triptolide regulates the effect of miR-381 / HMGB1 on the proliferation of multiple myeloma cells, characterized in that: The research results of the research method are as follows: after Si-HMGB1 transfected cells, MTT results showed that the cell proliferation activity of the Si-HMGB1 group was reduced, and Western blot results showed that the HMGB1 expression level of the Si-HMGB1 group was reduced.

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