CircSMARCA5通过miR-4295/PTEN轴调控糖酵解抑制胃癌细胞增殖和侵袭

doi:10.19401/j.cnki.1007-3639.2022.04.002

中国癌症杂志 ›› 2022, Vol. 32 ›› Issue (4): 298-308.doi: 10.19401/j.cnki.1007-3639.2022.04.002

CircSMARCA5通过miR-4295/PTEN轴调控糖酵解抑制胃癌细胞增殖和侵袭

蔡娟¹()(), 陈志强², 左学良³()()

1.皖南医学院第一附属医院（弋矶山医院）肿瘤内科,重大疾病非编码RNA转化研究安徽普通高校重点实验室,安徽芜湖 241001
2.南京医科大学第一附属医院肝胆中心,江苏南京 210029
3.皖南医学院第一附属医院（弋矶山医院）胃肠外科,安徽芜湖 241001

收稿日期:2021-11-20 修回日期:2022-03-01 出版日期:2022-04-30 发布日期:2022-05-07
通信作者: 左学良 E-mail:caijuan1987@yeah.net;zuoxueliang0202@126.com
作者简介:蔡娟（ORCID: https://orcid.org/0000-0001-7996-2525）,博士,主治医师 E-mail: caijuan1987@yeah.net
基金资助:
国家自然科学基金(82103293);国家自然科学基金(82172651);国家自然科学基金(82002556);安徽省自然科学基金(1908085QH332);安徽省自然科学基金(2108085MH287);安徽省重点研究与开发计划(202104j07020019);安徽省高校优秀青年人才支持计划(gxyq2021257);皖南医学院弋矶山医院科研能力“高峰”培育计划(GF2019J08);皖南医学院弋矶山医院科研能力“高峰”培育计划(GF2019G17)

CircSMARCA5 inhibits glycolysis and suppresses proliferation and invasion of gastric cancer cells through miR-4295/PTEN axis

CAI Juan¹()(), CHEN Zhiqiang², ZUO Xueliang³()()

1. Department of Oncology, Key Laboratory of Non-coding RNA Transformation Research of Anhui Higher Education Institution, The First Affiliated Hospital, Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China
2. Hepatobiliary Center, The First Affiliated Hospital of Nanjing Medical University, Nanjing 210029, Jiangsu Province, China
3. Department of Gastrointestinal Surgery, The First Affiliated Hospital, Yijishan Hospital of Wannan Medical College, Wuhu 241001, Anhui Province, China

Received:2021-11-20 Revised:2022-03-01 Published:2022-04-30 Online:2022-05-07
Contact: ZUO Xueliang E-mail:caijuan1987@yeah.net;zuoxueliang0202@126.com

摘要/Abstract

摘要：

背景与目的：胃癌是常见的消化系统恶性肿瘤。作者既往研究发现circSMARCA5在胃癌中表达降低并能够抑制胃癌进展,但其具体机制目前仍不清楚。本研究探究circSMARCA5抑制胃癌细胞增殖和侵袭的分子机制。方法：采用细胞计数试剂盒-8（cell counting kit-8,CCK-8）和transwell实验检测过表达circSMARCA5对胃癌细胞增殖和侵袭能力的影响。通过检测细胞外酸化率、葡萄糖摄取水平和乳酸生成量,分析过表达circSMARCA5对胃癌细胞糖酵解的影响。采用实时荧光定量聚合酶链式反应（real-time fluorescent quantitative polymerase chain reaction,RTFQ-PCR）检测circSMARCA5、miR-4295和PTEN的基因表达,采用蛋白质印迹法（Western blot）检测GLUT1和LDHA的蛋白水平。建立BACB/c裸小鼠皮下移植瘤模型,观察过表达circSMARCA5对移植瘤生长的影响,利用免疫组织化学方法检测两组皮下瘤中GLUT1、LDHA的表达水平及Ki-67增殖指数。通过双荧光素酶报告基因实验、Pearson相关性分析和RNA免疫沉淀（RNA immunoprecipitation,RIP）实验检测circSMARCA5与miR-4295、miR-4295及PTEN的靶向调控关系。结果：过表达circSMARCA5能够抑制胃癌细胞增殖和侵袭。CircSMARCA5过表达组细胞的糖酵解速率、糖酵解能力值、葡萄糖摄取水平和乳酸生成量均低于对照组。此外,过表达circSMARCA5能够抑制裸小鼠皮下移植瘤的生长。进一步研究发现,circSMARCA5可发挥分子海绵作用下调miR-4295表达,而miR-4295通过与PTEN mRNA的3′-UTR结合抑制PTEN表达。在circSMARCA5过表达组细胞中上调miR-4295或下调PTEN表达可部分逆转circSMARCA5对胃癌细胞增殖、侵袭和糖酵解的影响。结论：CircSMARCA5通过竞争性结合miR-4295上调PTEN表达,调控细胞糖酵解,从而抑制胃癌细胞的增殖和侵袭。

关键词: 胃肿瘤, 环状RNA, miR-4295, PTEN, 糖酵解, 侵袭

Abstract:

Background and purpose: Gastric cancer is one of the most common malignant tumors of digestive system. Previous study demonstrated that circSMARCA5 was downregulated and could function as a tumor suppressor in gastric cancer. However, the molecular mechanism has not yet been documented. This study aimed to investigate the effects of circSMARCA5 on the proliferation and invasion of gastric cancer cells and their molecular mechanisms. Methods: Cell counting kit-8 (CCK-8) assays and transwell assays were performed to examine the cell proliferative and invasive abilities, respectively. The effect of circSMARCA5 on gastric cancer cell glycolysis was assessed by detecting the extracellular acidification rate, glucose uptake level and lactate production. Real-time fluorescent quantitative polymerase chain reaction (RTFQ-PCR) assay was performed to detect the expression levels of circSMARCA5, miR-4295 and PTEN. The levels of GLUT1 and LDHA were measured by Western blot assay. The transplanted xenograft model in nude mice was established, and the effects of circSMARCA5 on the tumor growth were observed. Immunohistochemistry assays were performed to examine the expression levels of GLUT1, LDHA and Ki-67 proliferation index in xenograft tumors. Dual luciferase reporter gene assays, Pearson's correlation analysis and RNA immunoprecipitation (RIP) assays were used to confirm the targeting relationship of circSMARCA5 and miR-4295 as well as miR-4295 and PTEN. Results: CircSMARCA5 overexpression inhibited the proliferation and invasion of gastric cancer cells. Compared to the control group, the glycolysis rate, glycolysis capacity, glucose uptake and lactate production in the circSMARCA5-overexpresing group were significantly decreased. In addition, nude mouse transplanted xenograft assays showed that the volume and the weight of the tumors in the circSMARCA5-overexpressing group were lower compared with the control group. Further studies indicated that circSMARCA5 acted as a molecular sponge to inhibit the expression of miR-4295. Besides, miR-4295 could inhibit the expression of PTEN by binding with the 3'-UTR of PTEN mRNA. Rescue experiments by upregulating miR-4295 or downregulating PTEN expression in the circSMARCA5-overexpressing gastric cancer cells were performed, and the results showed that upregulation of miR-4295 or PTEN knockdown could abolish the inhibitory effects of circSMARCA5 overexpression on cell proliferation, invasion and glycolysis. Conclusion: CircSMARCA5 suppresses the proliferation and invasion of gastric cancer cells by targeting miR-4295, increasing the expression level of PTEN and subsequently regulating glycolysis.

Key words: Stomach neoplasms, Circular RNA, miR-4295, PTEN, Glycolysis, Invasion

中图分类号:

R735.2

蔡娟, 陈志强, 左学良. CircSMARCA5通过miR-4295/PTEN轴调控糖酵解抑制胃癌细胞增殖和侵袭[J]. 中国癌症杂志, 2022, 32(4): 298-308.

CAI Juan, CHEN Zhiqiang, ZUO Xueliang. CircSMARCA5 inhibits glycolysis and suppresses proliferation and invasion of gastric cancer cells through miR-4295/PTEN axis[J]. China Oncology, 2022, 32(4): 298-308.

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Clinicopathological features	Case n (%)	Clinicopathological features	Case n (%)
Age/year		Lymph node metastasis
<60	21 (35.0)	Negative	22 (36.7)
≥60	39 (65.0)	Positive	38 (63.3)
Gender		Vascular invasion
Female	16 (26.7)	No	29 (48.3)
Male	44 (73.3)	Yes	31 (51.7)
Tumor size D/cm		TNM staging
<5	41 (68.3)	Ⅰ/Ⅱ	24 (40.0)
≥5	19 (31.7)	Ⅲ	36 (60.0)
Differentiation
Well/Moderate	28 (46.7)
Poor	32 (53.3)

CircSMARCA5通过miR-4295/PTEN轴调控糖酵解抑制胃癌细胞增殖和侵袭

CircSMARCA5 inhibits glycolysis and suppresses proliferation and invasion of gastric cancer cells through miR-4295/PTEN axis

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