miR-15a-5p靶向HPSE2促进宫颈癌细胞增殖、迁移和侵袭

doi:10.3877/cma.j.issn.2095-1221.2024.01.002

目的

探讨miR-15a-5p靶向乙酰肝素酶2 （HPSE2）对宫颈癌细胞增殖、迁移和侵袭的影响。

方法

以人宫颈癌细胞系（Hela和SIHA）为研究对象，将miR-15a-5p mimic，miR-15a-5p inhibitor和HPSE2质粒转染进入细胞。分别采用CCK-8检测细胞活力，划痕实验和Transwell实验分别检测细胞迁移和侵袭能力，Western blot检测HPSE2表达，RT-qPCR检测细胞中miR-15a-5p和HPSE2 mRNA水平。两组间比较采用独立样本t检验，多组间比较采用单因素方差分析，组间两两比较采用LSD-t检验。

结果

与人正常宫颈上皮细胞（HcerEpic）比较，宫颈癌细胞Hela、HeRC32和SIHA中miR-15a-5p水平[（3.11 ± 0.32）、（1.51 ± 0.07）、（1.64 ± 0.09）比（1.00 ± 0.05）]升高，而HPSE2 mRNA水平[（0.29 ± 0.04）、（0.45 ± 0.09）、（0.45 ± 0.10）比（1.00 ± 0.05）]降低（P均< 0.05）；与对照比较，转染miR-15a-5p inhibitor抑制宫颈癌细胞Hela和SIHA增殖[72 h：（0.85 ± 0.08）比（1.10 ± 0.12）、（0.76 ± 0.12）比（1.04 ± 0.11）]、迁移[（12.67 ± 2.52）%比（23.00 ± 3.00）%、（14.33 ± 2.52）%比（24.67 ± 2.52）%]和侵袭能力[（59.33 ± 11.24）比（127.00 ± 12.49）、（65.67 ± 14.05）比（136.00 ± 15.52）个]；与对照比较，miR-15a-5p mimic可以进一步抑制宫颈癌细胞Hela和SIHA中HPSE2蛋白表达[（0.61 ± 0.02）比（1.00 ± 0.03）、（0.34 ± 0.05）比（1.00 ± 0.05）]；与miR-15a-5p mimic相比，过表达HPSE2可以增加宫颈癌细胞Hela和SIHA中HPSE2蛋白水平[（2.14 ± 0.13）比（1.00 ± 0.12）、（1.71 ± 0.02）比（1.00 ± 0.03）]；过表达HPSE2可以逆转miR-15a-5p mimic的作用，抑制宫颈癌细胞Hela和SIHA增殖[72 h：（0.83 ± 0.10）比（1.44 ± 0.14）、（0.93 ± 0.06）比（1.25 ± 0.10）]、迁移[（34.00 ± 5.57）%比（43.67 ± 10.41）%、（33.00 ± 3.61）%比（41.00 ± 8.00）%]和侵袭能力[（158.00 ± 13.89）比（260.66 ± 15.26）、（150.67 ± 23.54）比（270.00 ± 12.77）个]。

结论

miR-15a-5p通过调控HPSE2促进宫颈癌细胞增殖、迁移和侵袭。

关键词: 宫颈癌, microRNA, miR-15a-5p, HPSE2

Objective

To investigate the effects of miR-15a-5p targeting heparanase2 (HPSE2) on the proliferation, migration, and invasion of cervical cancer cells.

Methods

Human cervical cancer cell lines (Hela and SIHA) were used as the research objects. miR-15a-5p mimic, miR-15a-5p inhibitor and HPSE2 plasmid were transfected into cells. The cell viability was detected by CCK-8, and the cell migration and invasion ability was detected by scratch test and Transwell assay, respectively. Western blot was used to detect the expression of HPSE2, and RT-qPCR was used to detect the levels of miR-15a-5p and HPSE2 mRNA in cells. Independent sample t-test was used for comparison between two groups, one-way analysis of variance was used for comparison between multiple groups, and LSD-t test was used for pairwise comparison between multiple groups.

Results

Compared with human normal cervical epithelial cells (HcerEpic) , the levels of miR-15a-5p were increased in cervical cancer cells Hela, HeRC32 and SIHA [ (3.11 ± 0.32) , (1.51 ± 0.07) , (1.64 ± 0.09) vs (1.00 ± 0.05) ]. The mRNA level of HPSE2 were decreased [ (0.29 ± 0.04) , (0.45 ± 0.09) , (0.45 ± 0.10) vs (1.00 ± 0.05) ] (all P < 0.05) . Compared with the control, transfection of miR-15a-5p inhibitor inhibited the proliferation [72 h: (0.85 ± 0.08) vs (1.10 ± 0.12) , (0.76 ± 0.12) vs (1.04 ± 0.11) ], migration [ (12.67 ± 2.52) % vs (23.00 ± 3.00) %, (14.33 ± 2.52) % vs (24.67 ± 2.52) %] and invasive ability of cervical cancer cells Hela and SIHA [ (59.33 ± 11.24) vs (127.00 ± 12.49) , (65.67 ± 14.05) vs (136.00 ± 15.52) ]; Compared with the control, miR-15a-5p mimic further inhibited the expression of HPSE2 protein in cervical cancer cells Hela and SIHA [ (0.61 ± 0.02) vs (1.00 ± 0.03) , (0.34 ± 0.05) vs (1.00 ± 0.05) ]. Compared with miR-15a-5p mimic, HPSE2 overexpression increased the protein level of HPSE2 in cervical cancer cells Hela and SIHA [ (2.14 ± 0.13) vs (1.00 ± 0.12) , (1.71 ± 0.02) vs (1.00 ± 0.03) ]. Overexpression of HPSE2 reversed the effect of miR-15a-5p mimic and inhibit the proliferation [72 h: (0.83 ± 0.10) vs (1.44 ± 0.14) , (0.93 ± 0.06) vs (1.25 ± 0.10) ], migration [ (34.00 ± 5.57) % vs (43.67 ± 10.41) %, (33.00 ± 3.61) % vs (41.00 ± 8.00) %] and invasive ability of cervical cancer cells Hela and SIHA [ (158.00 ± 13.89) vs (260.66 ± 15.26) , (150.67 ± 23.54) vs (270.00 ± 12.77) ].

Conclusion

miR-15a-5p promotes the proliferation, migration and invasion of cervical cancer cells by regulating HPSE2.

Key words: Cervical cancer, MicroRNA, miR-15a-5p, HPSE2

表1 引物序列信息

基因	序列（5'→3'）	预期扩增长度（bp）
HPSE2	上游GCTCTGTCTACAGGCAAGGG	387
	下游TGTCTAACAGGCGAGTTTTCAGG
β-actin	上游TGCTGTCCCTGTATGCCTCTG	223
	下游TTGATGTCACGCACGATTTCC
miR-15a-5p	上游UAGCAGCACAUCAUGGUUUACA	105
	下游CTCAACTGGTGTCGTGGA
U6	上游CTCGCTTCGGCAGCACA	102
	下游AACGCTTCACGAATTTGCGT

表1 引物序列信息

基因	序列（5'→3'）	预期扩增长度（bp）
HPSE2	上游GCTCTGTCTACAGGCAAGGG	387
	下游TGTCTAACAGGCGAGTTTTCAGG
β-actin	上游TGCTGTCCCTGTATGCCTCTG	223
	下游TTGATGTCACGCACGATTTCC
miR-15a-5p	上游UAGCAGCACAUCAUGGUUUACA	105
	下游CTCAACTGGTGTCGTGGA
U6	上游CTCGCTTCGGCAGCACA	102
	下游AACGCTTCACGAATTTGCGT

图1 miR-15a-5p在宫颈癌中高表达注：a图为UALCAN数据库检测miR-15a-5p在宫颈癌中的表达；b图为UALCAN数据库检测miR-15a-5p对宫颈癌患者生存的影响；c图为qRT-PCR检测宫颈细胞中miR-15a-5p的表达，与HcerEpi比较，^aP < 0.001

图2 miR-15a-5p抑制剂抑制宫颈癌细胞增殖能力注：a图为qRT-PCR检测抑制剂在Hela和SIHA细胞中的转染效率，与对照比较，^aP < 0.01；b ~ c图为CCK-8检测miR-15a-5p抑制剂对Hela和SIHA细胞增殖的影响，与对照比较，^aP < 0.05

图3 倒置显微镜下观察miR-15a-5p抑制剂抑制宫颈癌细胞迁移与侵袭（结晶紫染色，×200）注：左图为划痕实验检测转染miR-15a-5p12h后Hela迁移能力和Transwell实验评估24h后Hela的侵袭能力；右图为划痕实验检测转染miR-15a-5p12h后SIHA迁移能力和Transwell实验评估24h后SIHA的侵袭能力

表2 miR-15a-5p inhibitor对宫颈癌细胞迁移和侵袭的影响（

± s）

分组	实验次数	细胞迁移率（%）	细胞侵袭数目（个）
Hela+对照	3	23.00 ± 3.00	127.00 ± 12.49
Hela+miR-15a-5p inhibitor	3	12.67 ± 2.52	59.33 ± 11.24
t值		4.571	6.975
P值		0.010	0.002
SIHA+对照	3	24.67 ± 2.52	136.00 ± 15.52
SIHA+miR-15a-5p inhibitor	3	14.33 ± 2.52	65.67 ± 14.05
t值		5.029	5.819
P值		0.007	0.004

表2 miR-15a-5p inhibitor对宫颈癌细胞迁移和侵袭的影响（

± s）

分组	实验次数	细胞迁移率（%）	细胞侵袭数目（个）
Hela+对照	3	23.00 ± 3.00	127.00 ± 12.49
Hela+miR-15a-5p inhibitor	3	12.67 ± 2.52	59.33 ± 11.24
t值		4.571	6.975
P值		0.010	0.002
SIHA+对照	3	24.67 ± 2.52	136.00 ± 15.52
SIHA+miR-15a-5p inhibitor	3	14.33 ± 2.52	65.67 ± 14.05
t值		5.029	5.819
P值		0.007	0.004

图4 生物信息学预测网站预测miR-15a-5p与HPSE2靶向结合位点

图5 miR-15a-5p靶向调控HPSE2注：a图为Western blot检测Hela和SIHA细胞中PIK3R1的表达水平；b ~ c图为双荧光素酶报告基因测定Hela和SIHA细胞中miR-15a-5p和HPSE2之间的靶向关系，^aP < 0.01

表3 miR-15a-5p mimic对宫颈癌细胞中HPSE2蛋白表达的影响（

± s）

分组	实验次数	HPSE2蛋白
Hela+对照	3	1.00 ± 0.03
Hela+miR-15a-5p imimic	3	0.61 ± 0.02
t值		17.990
P值		< 0.001
SIHA+对照	3	1.00 ± 0.05
SIHA+miR-15a-5p mimic	3	0.34 ± 0.05
t值		16.853
P值		< 0.001

表3 miR-15a-5p mimic对宫颈癌细胞中HPSE2蛋白表达的影响（

± s）

分组	实验次数	HPSE2蛋白
Hela+对照	3	1.00 ± 0.03
Hela+miR-15a-5p imimic	3	0.61 ± 0.02
t值		17.990
P值		< 0.001
SIHA+对照	3	1.00 ± 0.05
SIHA+miR-15a-5p mimic	3	0.34 ± 0.05
t值		16.853
P值		< 0.001

图6 HPSE2抑制miR-15a-5p诱导的宫颈癌细胞活性注：a ~ b图为GEPIA和UALCAN数据库分析宫颈癌患者HPSE2表达量；c图为qRT-PCR检测宫颈细胞中HPSE2的表达水平，与HcerEpi比较，^aP < 0.01；d图为Western blot检测转染HPSE2质粒进入细胞后HPSE2蛋白表达；e ~ f图为CCK-8检测HPSE2和miR-15a-5p过表达后对Hela和SIHA增殖的影响，与miR-15a-5p mimic比较，^aP < 0.01

表4 miR-15a-5p mimic和pHPSE2对宫颈癌细胞中HPSE2蛋白的影响（

± s）

分组	实验次数	HPSE2蛋白
Hela+miR-15a-5p mimic	3	1.00 ± 0.12
Hela+miR-15a-5p mimic+pHPSE2	3	2.14 ± 0.13
t值		11.381
P值		< 0.001
SIHA+miR-15a-5p mimic	3	1.00 ± 0.03
SIHA+miR-15a-5p mimic+pHPSE2	3	1.71 ± 0.03
t值		37.082
P值		< 0.001

表4 miR-15a-5p mimic和pHPSE2对宫颈癌细胞中HPSE2蛋白的影响（

± s）

分组	实验次数	HPSE2蛋白
Hela+miR-15a-5p mimic	3	1.00 ± 0.12
Hela+miR-15a-5p mimic+pHPSE2	3	2.14 ± 0.13
t值		11.381
P值		< 0.001
SIHA+miR-15a-5p mimic	3	1.00 ± 0.03
SIHA+miR-15a-5p mimic+pHPSE2	3	1.71 ± 0.03
t值		37.082
P值		< 0.001

图7 HPSE2抑制miR-15a-5p诱导的宫颈癌细胞迁移与侵袭（结晶紫染色，×200）注：左图为划痕实验检测转染miR-15a-5p mimic 12 h后Hela的迁移能力和Transwell实验评估转染miR-15a-5p mimic 24 h后Hela的侵袭能力；右图为划痕实验检测转染miR-15a-5p mimic 12 h后SIHA的迁移能力和Transwell实验评估转染miR-15a-5p mimic 24 h后SIHA的侵袭能力

表5 miR-15a-5p/ HPSE2调控通路对宫颈癌细胞迁移和侵袭的影响（

± s）

分组	实验次数	细胞迁移率（%）	细胞侵袭数目（个）
Hela+对照	3	23.67 ± 4.16	107.67 ± 19.55
Hela+miR-15a-5p mimic	3	43.67 ± 10.41^a	260.66 ± 15.26^a
Hela+miR-15a-5p mimic+pHPSE2	3	34.00 ± 5.57^a	158.00 ± 13.89^a
F值		4.571	6.975
P值		0.010	0.002
SIHA+对照	3	25.67 ± 3.21	95.00 ± 12.12
SIHA+miR-15a-5p mimic	3	41.00 ± 8.00^a	270.00 ± 12.77^a
SIHA+miR-15a-5p mimic+pHPSE2	3	33.00 ± 3.61^ab	150.67 ± 23.54^ab
F值		101.257	60.452
P值		< 0.001	< 0.001

表5 miR-15a-5p/ HPSE2调控通路对宫颈癌细胞迁移和侵袭的影响（

± s）

分组	实验次数	细胞迁移率（%）	细胞侵袭数目（个）
Hela+对照	3	23.67 ± 4.16	107.67 ± 19.55
Hela+miR-15a-5p mimic	3	43.67 ± 10.41^a	260.66 ± 15.26^a
Hela+miR-15a-5p mimic+pHPSE2	3	34.00 ± 5.57^a	158.00 ± 13.89^a
F值		4.571	6.975
P值		0.010	0.002
SIHA+对照	3	25.67 ± 3.21	95.00 ± 12.12
SIHA+miR-15a-5p mimic	3	41.00 ± 8.00^a	270.00 ± 12.77^a
SIHA+miR-15a-5p mimic+pHPSE2	3	33.00 ± 3.61^ab	150.67 ± 23.54^ab
F值		101.257	60.452
P值		< 0.001	< 0.001

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miR-15a-5p promotes the proliferation, migration and invasion of cervical cancer cells by targeting HPSE2

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miR-15a-5p promotes the proliferation, migration and invasion of cervical cancer cells by targeting HPSE2