SPAG5在胃癌细胞恶性增殖中的生物学作用

doi:10.12122/j.issn.1673-4254.2024.08.08

摘要/Abstract

摘要：

目的在胃癌组织及癌旁组织中分析SPAG5的表达情况，通过干扰SPAG5分析其在胃癌细胞生长中的生物学作用。方法结合TCGA分析，免疫组织化学、免疫荧光染色分析SPAG5及MKi67在胃癌及癌旁组织中的表达模式，利用胃癌细胞AGS和MGC803进行体外细胞生物学实验，分别设置空白对照组和干扰组，采用慢病毒干扰，其中空白对照转染shCtrl，干扰组转染shSPAG5。通过Celigo、MTT和克隆形成实验、凋亡检测分析敲减SPAG5基因后对胃癌细胞生长的影响。结果 Proteinatlas数据库、TCGA数据库分析结果发现SPAG5在胃癌中高表达，KM-plot及GEPIA数据库分析SPAG5在肺腺癌、乳腺癌、肝癌、胰腺癌、宫颈癌、膀胱癌中高表达，免疫组化发现SPAG5在胃癌中高表达（P<0.001），免疫组化和免疫荧光共聚焦证明SPAG5与MKi67存在显著相关性（R=0.393，P<0.001）。Real time-PCR及Western blotting结果显示SPAG5在MKN74、BGC823、MGC803、SGC7901和AGS等细胞中表达水平较高（P<0.01）。敲减SPAG5后mRNA和蛋白的表达下降，Celigo、MTT和克隆形成实验结果显示敲减SPAG5抑制胃癌细胞增殖（P<0.01），流式凋亡分析发现敲减SPAG5促进胃癌细胞凋亡（P<0.001）。结论 SPAG5和MKi67在胃癌组织中的表达具有相关性，干扰SPAG5基因可以抑制胃癌细胞的增殖。SPAG5与患者预后相关，可能成为胃癌的潜在标志物。

关键词: SPAG5, 胃癌, MKi67, 细胞增殖

Abstract:

Objective To analyze the expression of SPAG5 in gastric cancer tissues and its regulatory roles in gastric cancer cell growth. Methods TCGA analysis, immunohistochemistry, and immunofluorescence staining were used to analyze the expression patterns of SPAG5 and MKi67 in gastric cancer and adjacent tissues. In gastric cancer AGS and MGC803 cells, the effects of lentivirus-mediated SPAG5 knockdown on cell growth and apoptosis were evaluated using Celigo, MTT, clone formation assays and flow cytometry. Results Proteinatlas and TCGA database analysis suggested that SPAG5 was highly expressed in gastric cancer, and Kaplan-Meier analysis and GEPIA analysis showed high expressions of SPAG 5 in lung adenocarcinoma, breast cancer, hepatocellular carcinoma, pancreatic carcinoma, cervical cancer and bladder carcinoma. Immunohistochemistry revealed that SPAG5 was highly expressed in gastric cancer tissues (P<0.001), and immunofluorescence colocalization analysis demonstrated a significant correlation between SPAG5 and MKI67 (R=0.393, P<0.001). RT-qPCR and Western blotting showed that SPAG5 was highly expressed in MKN74, BGC823, MGC803, SGC7901 and AGS cells. In AGS and MGC803 cells, SPAG5 knockdown significantly inhibited proliferation and promoted apoptosis. Conclusions The expressions of SPAG5 and MKi67 are correlated in gastric cancer tissues, and SPAG5 knockdown inhibits the proliferation of gastric cancer cells. SPAG5 is associated with the prognosis of gastric cancer patients and may serve as a promising biomarker for gastric cancer.

Key words: SPAG5, gastric cancer, MKi67, cell proliferation

庞一丹, 刘雅, 陈思嫒, 张荆雷, 曾今, 潘元明, 安娟. SPAG5在胃癌细胞恶性增殖中的生物学作用[J]. 南方医科大学学报, 2024, 44(8): 1497-1507.

Yidan PANG, Ya LIU, Siai CHEN, Jinglei ZHANG, Jin ZENG, Yuanming PAN, Juan AN. Biological role of SPAG5 in the malignant proliferation of gastric cancer cells[J]. Journal of Southern Medical University, 2024, 44(8): 1497-1507.

图/表 9

表1 引物序列信息

Tab.1 Primer sequences for RT-qPCR

Gene	Upstream primer sequences	Downstream primer sequences	Length (bp)
GAPDH	TGACTTCAACAGCGACACCCA	CACCCTGTTGCTGTAGCCAAA	121
SPAG5	TTGAGGCCCGTTTAGATACCA	GCTTTCCTTGGAGCAATGTAGTT	227

图1 SPAG5表达模式的生物信息分析

Fig.1 Bioinformatic analysis of SPAG5 expression patterns. A:Proteinatlas database analysis showing SPAG5 distribution in both the cell nucleus and cytoplasm. B: TCGA database analysis showing significantly higher expression of SPAG5 in gastric cancer than in normal tissues (*P<0.05). C: Proteinatlas database analysis showing SPAG5 expressions in gastric, rectal adenocarcinoma, esophageal, colon adenocarcinoma, and pancreatic cancer.

图2 KM-plot及GEPIA数据库分析SPAG5在多种实体肿瘤中的表达

Fig.2 Kaplan-Meier plots and GEPIA database analysis of SPAG5 expression in lung adenocarcinoma (A), breast cancer (B), hepatocellular carcinoma (C), pancreatic cancer (D), cervical cancer (E), and bladder cancer (F) and its association with patient survival (*P<0.05).

图 3 免疫组化和生存分析SPAG5

Fig.3 Immunohistochemistry for SPAG5 in gastric and adjacent tissues and survival analysis of SPAG5 expression. A: Immunohistochemistry for detecting expression of SPAG5 in gastric cancer and adjacent tissues (Original magnification: ×100). a: Normal gastric mucosal tissue Negative b: Normal gastric mucosal tissue Positive c: Gastric cancer tissue Negative d: Gastric cancer tissue Positive. B-D: Kaplan-Meier survival analysis of gastric cancer patients with high and low SPAG5 expression using Log-rank test and Breslow test.

表2-1 SPAG5在胃癌及癌旁组织中的表达

Tab.2-1 Expression of SPAG5 in gastric cancer and adjacent tissues

Type	Cases	SPAG5 expression [n (%)]		P
Type	Cases	High	Low	P
Nomal	50	11 (22.0)	39 (78.0)	<0.01
Tumor	122	95 (78.0)	27 (22.0)	<0.01

表2-2 MKi67在胃癌及癌旁组织中的表达

Tab.2-2 Expression of MKi67 in gastric cancer and adjacent tissues

Type	Cases	MKi67 expression [n (%)]		P
Type	Cases	High	Low	P
Nomal	50	27 (54.0)	23 (46.0)	<0.01
Tumor	122	75 (61.5)	47 (38.5)	<0.01

表3 结合临床病理学资料分析SPAG5与MKi67在胃癌组织中的表达相关性

Tab.3 Analysis of the correlation between SPAG5 and MKi67 expressions in gastric cancer tissues

Characteristics	Nomal-SPAG5 expression		Characteristics	Tumor-SPAG5 expression
Characteristics	Low	High	Characteristics	Low	High
Nomal-MKi67^[High]	17	10	Tumor-MKi67^[High]	6	109
Nomal-MKi67^[Low]	22	1	Tumor-MKi67^[Low]	27	30
P	0.006		P	<0.01
Pearson's R	0.393		Pearson's R	0.504

图7 干扰SPAG5后AGS和MGC803细胞的增殖受到显著抑制

Fig.7 Lentivirus-mediated SPAG5 interference inhibits proliferation of AGS and MGC803 cells. A, B: Celigo assay and MTT assays showing inhibition of proliferation of AGS and MGC-803 cells 3 days after lentivirus infection in the experimental group was significantly inhibited. C: Number of clones formed by AGS and MGC-803 cells with SPAG5 knockdown (×100). ***P<0.001.

图8 干扰SPAG5促进AGS和MGC803细胞的细胞凋亡

Fig.8 SPAG5 interference promotes apoptosis in AGS and MGC803 cells. A, B: Scatter plot of flow cytometry after SPAG5 interference in AGS cells and MGC803 cells. C, D: Apoptosis rates in AGS cells and MGC803 cells 5 days after SPAG5 interference (***P<0.001).

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