Impacts of CircBACH1 on proliferation, apoptosis and chemosensitivity of multiple myeloma cells by regulating miR-140-5p /MDM2 axis

doi:10.11958/20230126

Abstract

Abstract:

Objective To investigate the effect of CircBACH1 on proliferation, apoptosis and chemotherapy sensitivity of multiple myeloma (MM) cells and the regulatory mechanism on miR-140-5p/MDM2 axis during this process. Methods Bone marrow plasma cells were collected from the MM patient group and normal control group. Human MM cell line U266 cells were divided into the untransfected group, the si-NC group, the si1-CircBACH1 group, the si2-CircBACH1 group, the si-CircBACH1+anti-miR-NC group and the si-CircBACH1+anti-miR-140-5p group. The mRNA expressions of CircBACH1, miR-140-5p and mouse double microsoma 2 (MDM2) were detected by real-time quantitative PCR. Cell proliferation activity was detected by CCK-8 assay. Flow cytometry was used to detect apoptosis and sensitivity to bortezomib. The expression levels of B-cell lymphofactor 2 (Bcl-2), Bcl-2 associated X protein (Bax) and cleaved caspase -3 were detected by Western blot assay. Dual luciferase reporter gene assay was used to verify the targeting relationship of CircBACH1, miR-140-5p and MDM2. Results Compared with the normal control group, the mRNA expression levels of CircBACH1 and MDM2 were increased in bone marrow plasma cells and U266 cells in the MM group, while the expression of miR-140-5p was decreased (P<0.05). Compared with the control group and the si-NC group, the mRNA expression of CircBACH1 and MDM2, cell proliferation activity and Bcl-2 protein expression were decreased in the transfected si-CircBACH1 group. The expression of miR-140-5p, cell apoptosis rate, chemotherapy sensitivity, cleaved caspase-3 and Bax expressions were increased (P<0.05). In the anti-miR-140-5p supplementation experiment, the effect of si-CircBACH1 on MM cells was reversed, and the expression of MDM2 was increased (P<0.05). Dual luciferase gene reporting experiment verified the targeting relationship between CircBACH1 and MDM2 and miR-140-5p. Conclusion Knock down of CircBACH1 can up-regulate the expression of miR-140-5p, down-regulate the expression of MDM2, inhibit the proliferation of MM cells, promote the apoptosis of MM cells and improve the chemosensitivity.

Key words: multiple myeloma, cell proliferation, apoptosis, CircBACH1, miR-140-5p, mouse double minute 2, chemosensitivity

CLC Number:

R733.3

Figures/Tables 8

References 18

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基因名称	引物序列（5′→3′）	产物大小/bp
CircBACH1	上游：GCTGTCGCAAGAGAAAACTTGA	198
CircBACH1	下游：AAACTCCACACATTTGCACACT	198
MDM2	上游：CAGTAGCAGTGAATCTACAGGGA	139
MDM2	下游：CTGATCCAACCAATCACCTGAAT	139
GAPDH	上游：CAGAACATCATCCCTGCCTCTAC	216
GAPDH	下游：TTGAAGTCAGAGGAGACCACCTG	216
miR-140-5p	上游：CAGTGGTTTTACCCTA	142
miR-140-5p	下游：GTGCAGGGTCCGAGGT	142
U6	上游：CTCGCTTCGGCAGCACA	89
U6	下游：AACGCTTCACGAATTTGCG	89

基因名称	引物序列（5′→3′）	产物大小/bp
CircBACH1	上游：GCTGTCGCAAGAGAAAACTTGA	198
CircBACH1	下游：AAACTCCACACATTTGCACACT	198
MDM2	上游：CAGTAGCAGTGAATCTACAGGGA	139
MDM2	下游：CTGATCCAACCAATCACCTGAAT	139
GAPDH	上游：CAGAACATCATCCCTGCCTCTAC	216
GAPDH	下游：TTGAAGTCAGAGGAGACCACCTG	216
miR-140-5p	上游：CAGTGGTTTTACCCTA	142
miR-140-5p	下游：GTGCAGGGTCCGAGGT	142
U6	上游：CTCGCTTCGGCAGCACA	89
U6	下游：AACGCTTCACGAATTTGCG	89

组别	CircBACH1	miR-140-5p	MDM2
正常对照组	1.02±0.07	0.98±0.06	0.96±0.05
MM组	1.97±0.15^a	0.37±0.03^a	1.61±0.11^a
U266细胞	2.01±0.12^a	0.34±0.03^a	1.68±0.10^a
F	405.689^＊＊	1304.333^＊＊	346.024^＊＊

组别	CircBACH1	miR-140-5p	MDM2
正常对照组	1.02±0.07	0.98±0.06	0.96±0.05
MM组	1.97±0.15^a	0.37±0.03^a	1.61±0.11^a
U266细胞	2.01±0.12^a	0.34±0.03^a	1.68±0.10^a
F	405.689^＊＊	1304.333^＊＊	346.024^＊＊

组别	CircBACH1	miR-140-5p	MDM2
Control组	1.00±0.00	1.00±0.00	1.00±0.00
si-NC组	0.98±0.05	1.02±0.05	1.01±0.06
si-CircBACH1组	0.46±0.03^ab	1.93±0.13^ab	0.39±0.02^ab
si-CircBACH1+anti- miR-NC组	0.44±0.02	1.91±0.14	0.41±0.03
si-CircBACH1+anti- miR-140-5p组	0.46±0.03	1.32±0.08^c	0.77±0.05^c
F	276.128^＊＊	69.823^＊＊	187.459^＊＊