hsa_circ_0090923在前列腺癌中的表达及其对前列腺癌细胞增殖和迁移的调控

doi:10.3877/cma.j.issn.1674-3253.2023.03.016

目的

探究雄激素受体基因来源的环状RNA hsa_circ_0090923在前列腺癌中的表达水平及其对前列腺癌细胞增殖和迁移能力的影响。

方法

通过circBase，RjunBase和MiOncoCirc数据库分析hsa_circ_0090923在前列腺癌中表达的情况。使用qPCR，RT-PCR以及Sanger测序等方法检测hsa_circ_0090923在前列腺上皮细胞（PNT1A）和前列腺癌细胞（LNCaP，22RV1，C4-2）中的表达。应用环状RNA过表达技术，在前列腺癌细胞过表达hsa_circ_0090923，进而采用CCK-8，EdU细胞成像和细胞划痕实验检测hsa_circ_0090923对前列腺癌细胞的增殖和迁移的影响。采用CircInteractome数据库预测与hsa_circ_0090923结合的miRNA。利用DIANA-miTED数据库分析miRNA在前列腺癌组织中的表达水平。通过双荧光素酶实验证明hsa-miR-940与hsa_circ_0090923结合。

结果

hsa_circ_0090923由雄激素受体基因的第2号外显子形成，它在前列腺癌组织中特异表达，且在前列腺癌细胞中高表达。过表达hsa_circ_0090923促进前列腺癌细胞的增殖和迁移。CircInteractome数据库分析显示hsa_circ_0090923可能通过与miRNA（hsa-miR-633，hsa-miR-637，hsa-miR-940）相互作用从而促进肿瘤的进展。

结论

hsa_circ_0090923在前列腺癌中高表达，并促进前列腺癌细胞的增殖和迁移，有望成为前列腺癌诊断和治疗的新靶点。

关键词: 前列腺肿瘤, 雄激素受体, 基因, 测序, 靶点, 数据库, RNA, 增殖, 迁移

Objective

To investigate the expression level of the androgen receptor gene-derived circular RNA (hsa_circ_0090923) in prostate cancer and its effect on the proliferation and migration of prostate cancer cells.

Methods

The expression of hsa_circ_0090923 in prostate cancer was analyzed via circBase, RjunBase and MiOncoCirc databases. The expression of hsa_circ_0090923 in prostate epithelial cells (PNT1A) and prostate cancer cells (LNCaP, 22RV1, C4-2) was detected by qPCR, RT-PCR and Sanger sequencing. After over-expression of hsa_circ_0090923, the ability of cell proliferation and migration was detected using CCK-8 assay, EdU cell imaging and cell scratch assay. The miRNAs binding to hsa_circ_0090923 were predicted using CircInteractome database.

Results

The gene of hsa_circ_0090923 was formed by exon 2 of the androgen receptor gene, which was specifically expressed in prostate cancer tissues, and was highly expressed in prostate cancer cells. Over-expression of hsa_circ_0090923 promotes proliferation and migration of prostate cancer cells. CircInteractome database analysis showed that hsa_circ_0090923 may promote tumor progression by interacting with miRNAs (hsa-miR-633, hsa-miR-637, hsa-miR-940). The expression levels of miRNAs in prostate cancer tissues were analyzed using DIANA-miTED database. And the binding of hsa-miR-940 to hsa_circ_0090923 was demonstrated by dual luciferase assay.

Conclusions

hsa_circ_0090923 is highly expressed in prostate cancer and promotes the proliferation and migration of prostate cancer cells. It is expected to become a new target for the diagnosis and treatment of prostate cancer.

Key words: Prostate cancer, Androgen receptor, Gene, Sequence, Target, Detabase, RNA, Proliferation, Migration

图1 hsa_circ_0090923在前列腺癌细胞中的表达注：a示hsa_circ_0090923由AR基因的第2号外显子通过反向剪接而形成；b示RT-PCR产物sanger法测序证明hsa_circ_0090923在前列腺癌细胞中的表达，且环化位点与circBase上注释序列吻合；c为MiOncoCirc数据库在线分析显示hsa_circ_0090923在前列腺癌中特异性表达（BRCA：乳腺浸润癌；PAAD：胰腺癌；PRAD：前列腺腺癌；SARC：肉瘤）；d为hsa_circ_0090923在前列腺癌（PRAD）与其他肿瘤组织（Non-PRAD）中的表达水平；e为hsa_circ_0090923在前列腺上皮细胞（PNT1A）以及前列腺癌细胞中的相对表达水平（LNCaP，22RV1，C4-2）

表1 雄激素受体(AR)基因来源的circRNA在肿瘤组织中的表达情况

环化位点位置	环化位点编号	Circbase编号	基因	肿瘤例数	长度(nt)
chrX:67686009\|67711689:+	AR_BS003	无	AR	49	25680
chrX:67643255\|67686126:+	AR_BS001	无	AR	46	42871
chrX:67643255\|67643407:+	AR_BS005	hsa_circ_0090923	AR	39	152
chrX:67711401\|67711689:+	AR_BS002	无	AR	36	288
chrX:67643255\|67711689:+	AR_BS006	无	AR	11	68434
chrX:67680719\|67686126:+	AR_BS004	无	AR	5	5407
chrX:67711401\|67722984:+	AR_BS10	无	AR	3	11583
chrX:67711401\|67717622:+	AR_BS009	无	AR	3	6221
chrX:67643255\|67722984:+	AR_BS011	无	AR	1	79729
chrX:67686009\|67686126:+	AR_BS008	hsa_circ_0090925	AR	1	117
chrX:67643255\|67717622:+	AR_BS007	无	AR	1	74367
chrX:66863097\|66937464:+	无	hsa_circ_0090924	AR	0	702
chrX:66788682\|66950461:+	无	hsa_circ_0090922	AR	0	8107

表1 雄激素受体(AR)基因来源的circRNA在肿瘤组织中的表达情况

环化位点位置	环化位点编号	Circbase编号	基因	肿瘤例数	长度(nt)
chrX:67686009\|67711689:+	AR_BS003	无	AR	49	25680
chrX:67643255\|67686126:+	AR_BS001	无	AR	46	42871
chrX:67643255\|67643407:+	AR_BS005	hsa_circ_0090923	AR	39	152
chrX:67711401\|67711689:+	AR_BS002	无	AR	36	288
chrX:67643255\|67711689:+	AR_BS006	无	AR	11	68434
chrX:67680719\|67686126:+	AR_BS004	无	AR	5	5407
chrX:67711401\|67722984:+	AR_BS10	无	AR	3	11583
chrX:67711401\|67717622:+	AR_BS009	无	AR	3	6221
chrX:67643255\|67722984:+	AR_BS011	无	AR	1	79729
chrX:67686009\|67686126:+	AR_BS008	hsa_circ_0090925	AR	1	117
chrX:67643255\|67717622:+	AR_BS007	无	AR	1	74367
chrX:66863097\|66937464:+	无	hsa_circ_0090924	AR	0	702
chrX:66788682\|66950461:+	无	hsa_circ_0090922	AR	0	8107

图2 前列腺癌细胞环状RNA hsa_circ_0090923过表达效果验证注：Vector为空载质粒pCD25-ciR；a为Vector质粒以及OE-hsa_circ_0090923质粒示意图，其中OE-hsa_circ_0090923质粒转染细胞后能产生hsa_circ_0090923；b为qPCR检测22RV1细胞分别转染Vector质粒以及OE-hsa_circ_0090923质粒后hsa_circ_0090923的表达水平，其中OE-hsa_circ_0090923质粒能在22RV1细胞中过表达hsa_circ_0090923；c为qPCR检测C4-2细胞分别转染Vector质粒以及OE-hsa_circ_0090923质粒后hsa_circ_0090923的表达水平，其中OE-hsa_circ_0090923质粒能在C4-2细胞中过表达hsa_circ_0090923

图3 hsa_circ_0090923促进前列腺癌细胞的增殖注：Vector为对照组空载质粒pCD25-ciR；a为CCK-8细胞增殖实验显示过表达hsa_circ_0090923后，22RV1细胞增殖活性显著升高；b为EdU细胞成像显示过表达hsa_circ_0090923后22RV1细胞的DNA复制较对照组活跃；c为CCK-8细胞增殖实验显示过表达hsa_circ_0090923后，C4-2细胞增殖活性显著升高；d为EdU细胞成像显示过表达hsa_circ_0090923后，C4-2细胞的DNA复制较对照组活跃

图4 hsa_circ_0090923促进前列腺癌细胞的迁移注：细胞划痕实验显示，过表达hsa_circ_0090923后，在24 h以及48 h 22RV1细胞划痕的愈合程度较对照组显著增加，提示hsa_circ_0090923促进前列腺癌细胞的迁移能力

图5 hsa_circ_0090923与hsa-miR-940等miRNA相互作用注：a为CircInteractome分析软件显示4个miRNA分子与hsa_circ_0090923相互作用；b为hsa-miR-940，hsa-miR-515-5p，hsa-miR-633，hsa-miR-637在前列腺癌组织中的表达水平，其中hsa-miR-940表达水平在这4中miRNA中最高。c为双荧光素酶报告基因显示，hsa-miR-940与hsa_circ_0090923结合

表2 与hsa_circ_0090923相互作用的miRNA分子

circRNA Mirbase ID	circRNA(上)-miRNA(下)配对
hsa_circ_0090923(5'...3')
hsa-miR-515-5p(3'...5')
hsa_circ_0090923(5'...3')
hsa-miR-633(3'...5')
hsa_circ_0090923(5'...3')
hsa-miR-637(3'...5')
hsa_circ_0090923(5'...3')
hsa-miR-940(3'...5')

表2 与hsa_circ_0090923相互作用的miRNA分子

circRNA Mirbase ID	circRNA(上)-miRNA(下)配对
hsa_circ_0090923(5'...3')
hsa-miR-515-5p(3'...5')
hsa_circ_0090923(5'...3')
hsa-miR-633(3'...5')
hsa_circ_0090923(5'...3')
hsa-miR-637(3'...5')
hsa_circ_0090923(5'...3')
hsa-miR-940(3'...5')

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Expression of hsa_circ_0090923 in prostate cancer and its effect on the proliferation and migration of prostate cancer cells

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Expression of hsa_circ_0090923 in prostate cancer and its effect on the proliferation and migration of prostate cancer cells