SPARCL1表达下调对胆囊癌吉西他滨耐药的作用机制及临床意义

和华; 刘少朋; 刘海潮; 白明辉

doi:10.16766/j.cnki.issn.1674-4152.002674

SPARCL1表达下调对胆囊癌吉西他滨耐药的作用机制及临床意义

doi: 10.16766/j.cnki.issn.1674-4152.002674

郑州大学附属洛阳中心医院肝胆胰脾及疝外科，河南洛阳 471000

基金项目:

河南省卫生健康委员会联合共建项目 LHGJ20191194

洛阳市科技医疗卫生项目 1820003A

详细信息

通讯作者:
刘少朋, E-mail: 1281733563@qq.com

中图分类号: R735.8 R730.43
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- 被引次数: 0
出版历程
- 收稿日期: 2021-05-15
- 网络出版日期: 2022-11-30

The mechanism and clinical significance of SPARCL1 down-regulation expression on gemcitabine resistance in gallbladder cancer

Department of Hepatobiliary and Pancreatic Surgery, Luoyang Central Hospital Affiliated to Zhengzhou University, Luoyang, Henan 471000, China

摘要

摘要: 目的研究富含半胱氨酸酸性分泌型糖蛋白类似物1(SPARCL1)在胆囊癌中的作用。方法将人胆囊癌吉西他滨耐药细胞株GBC-SD/GEM分为空白对照组和SPARCL1过表达转染组。检测转染后SPARCL1表达、各细胞增殖能力及基质金属蛋白酶-9(MMP-9)、波形蛋白(VIM)、纤维连接蛋白1(FN1)表达；检测2014年1月—2017年1月在洛阳中心医院行手术治疗的50例胆囊癌(吉西他滨耐药组、敏感组)及癌旁组织中SPARCL1表达，分析其与患者临床病理特征及预后间的关系。结果转染组SPARCL1基因相对表达量(29.12±1.10)及蛋白相对表达量(23.08±2.15)均显著高于空白对照组(3.34±0.98、2.58±0.71，均P＜0.05)。转染组细胞增殖数及MMP-9、VIM、FN1相对表达量分别为33.09±12.11、1.69±0.75、1.78±0.43、1.62±0.31，均低于对照组(327.15±9.28、3.81±0.78、4.12±0.24、4.69±0.63，均P＜0.05)。耐药组、敏感组及癌旁组织中SPARCL1表达量分别为5.65±2.01、15.02±1.17、28.46±2.53，组间差异有统计学意义(P＜0.05)。SPARCL1表达与性别、年龄、病理类型、肿瘤大小、CA19-9、CEA和肿瘤分期无相关性(均P＞0.05)，而与淋巴结转移和肿瘤分化程度有相关性(均P＜0.05)。耐药组患者中位生存期明显低于敏感组(14.77个月vs. 27.28个月)。结论 SPARCL1在胆囊癌中低表达，且与吉西他滨化疗耐药及预后相关。
- 富含半胱氨酸酸性分泌型糖蛋白类似物1 /
- 胆囊癌 /
- 吉西他滨 /
- 耐药 /
- 预后
Abstract: Objective To examine the role of cysteine rich acidic secretory glycoprotein analogue 1 (SPARCL1) in gallbladder cancer. Methods Gemcitabine resistant human gallbladder carcinoma cell line GBC-SD/GEM cell line was divided into blank control and SPARCL1 overexpression transfection groups. The expression levels of SPARCL1, matrix metalloproteinase-9 (MMP-9), vimentin (VIM) and fibronectin 1 (FN1), and the proliferation ability of each cell was detected after transfection. The expression of SPARCL1 in 50 cases of gallbladder cancer (gemcitabine-resistant group and-sensitive group) and paracancerous tissues operated in Luoyang Central Hospital from January 2014 to January 2017 were detected, and its relationship with the clinicopathologic features and prognoses of patients were analysed. Results The expression of SPARCL1 gene (29.12±1.10) and SPARCL1 protein (23.08±2.15) in the transfection group were significantly higher than that in the control group respectively (3.34±0.98, 2.58±0.71, all P < 0.05). The proliferation capacities and expression levels of MMP-9, VIM and FN1 in the SPARCL1 transfection group were 33.09±12.11, 1.69±0.75, 1.78±0.43 and 1.62±0.31, which were all significantly lower than those in the control group (327.15±9.28, 3.81±0.78, 4.12±0.24, 4.69±0.63, all P < 0.05). The expression of SPARCL1 in gemcitabine resistance group, sensitive group and paracancerous tissues were 5.65±2.01, 15.02±1.17 and 28.46±2.53, respectively, the difference between groups was significant (P < 0.05). SPARCL1 expression was independent of gender, age, pathological type, tumour size, CA19-9, CEA and tumour stage (all P > 0.05), but significantly correlated with lymph node metastasis and tumour differentiation (all P < 0.05). Median survival time of patients in the gemcitabine-resistant group was significantly lower than that of the sensitive group (14.77 months vs. 27.28 months). Conclusion SPARCL1 is poorly expressed in gallbladder cancer and is associated with gemcitabine chemotherapy resistance and prognosis.
- Cysteine rich acidic secretory glycoprotein analogue 1 /
- Gallbladder cancer /
- Gemcitabine /
- Drug resistance /
- Prognosis

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图 1 SPARCL1对GBC-SD/GEM细胞克隆的影响(n=10)

注：A为空白对照组; B为SPARCL1转染组; C为2组细胞克隆形成数比较, ^aP＜0.01。

Figure 1. Effect of SPARCL1 on GBC-SD/GEM cell clones(n=10)

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图 2 转染后细胞内MMP-9、VIM、FN1表达

注：A为蛋白印迹实验；B为2组蛋白相对表达量比较，^aP＜0.01。

Figure 2. Expression of MMP-9, VIM and FN1 in cells after transfection

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图 3 SPARCL1在胆囊癌组织及癌旁组织中的表达(HE染色，×200)

注：A为敏感组，B为耐药组，C为癌旁正常组织。

Figure 3. Expression of SPARCL1 in gallbladder carcinoma tissues and adjacent tissues(HE dyed, ×200)

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图 4 吉西他滨耐药组患者与敏感组患者的生存期比较(n=50)

Figure 4. Comparison of survival time of patients in gemcitabine resistant group and sensitive group(n=50)

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表 1 实时免疫荧光引物序列

Table 1. Real-time immunofluorescence primer sequence

基因	引物序列
SPARCL1	上游：TTGGCTCCTGGTGTTAGTTC
	下游：ATCCTGCTCTTGGTTTCCTT
内参GADPH	上游：TGACTTCAACAGCGACACCCA
	下游：CACCCTGTTGCTGTAGCCAAA

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表 2 不同组别中SPARCL1的表达(例)

Table 2. Expression of SPARCL1 in different groups(cases)

组别	例数	SPARCL1
组别	例数	阳性	阴性
耐药组	29	3	26
敏感组	21	10	11
癌旁正常组织	50	43	7
注：3组比较，χ²=43.394，P＜0.001。

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表 3 SPARCL1表达与胆囊癌临床病理因素的相关性(例)

Table 3. Correlation between SPARCL1 expression and clinicopathological factors in gallbladder carcinoma(cases)

临床病理因素	类别	例数	SPARCL1		χ²值	P值
临床病理因素	类别	例数	阳性	阴性	χ²值	P值
性别	男性	27	7	20	＜0.001	0.990
	女性	23	6	17
年龄(岁)^[11]	≥55	17	3	14	0.934	0.334
	＜55	33	10	23
肿瘤直径(cm)^[12]	≥2	9	2	7	0.081	0.775
	＜2	41	11	30
Nevin分期(Ⅱ~Ⅳ)^[13]	Ⅱ、Ⅲ	43	12	31	0.581	0.446
	Ⅳ	7	1	6
病理类型	腺癌	41	12	29	1.265	0.261
	腺鳞癌	9	1	8
CA19-9(kU/L)	≥37	15	4	11	0.005	0.944
	＜37	35	9	26
CEA(μg/L)	≥5	28	8	20	0.219	0.640
	＜5	22	5	17
分化程度	高分化	22	12	10	16.638	＜0.001
	中、低分化	28	1	27
淋巴结转移	有	15	1	14	4.163	0.041
	无	35	12	23

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表 4 胆囊癌预后的Cox多因素分析结果

Table 4. Cox multivariate analysis of prognosis of gallbladder carcinoma

变量	例数	OS(月)	B	SE	Wald χ²	P值	HR值	95% CI
有淋巴结转移	15	13	0.524	1.833	6.661	0.005	0.493	0.231~0.882
高分化	22	23	0.924	0.085	0.285	0.697	1.272	0.688~3.101
SPARCL1阳性表达	23	34	0.515	0.659	7.522	0.001	0.619	0.313~0.899
注：变量赋值方法如下，淋巴结转移，1=有转移，2=无转移；分化程度，1=高分化，2=中低分化；SPARCL1，1=阳性，2=阴性。OS为总生存期(overall survival)。

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参考文献(27)

[1]	李福利, 夏咸军, 刘洪, 等. 高尔基磷酸化蛋白3在胆囊癌组织中表达的临床意义及其与血管生成的关系[J]. 中国普通外科杂志, 2020, 29(2): 190-197. https://www.cnki.com.cn/Article/CJFDTOTAL-ZPWZ202002014.htm LI F L, XIA X J, LIU H, et al. Clinical significance of expression of Golgi phosphorylation protein 3 in gallbladder carcinoma tissue and its association with angiogenesis[J]. Chinese Journal of General Surgery, 2020, 29(2): 190-197. https://www.cnki.com.cn/Article/CJFDTOTAL-ZPWZ202002014.htm
[2]	章娴, 陈一兴, 孙菁, 等. 胆囊癌根治术后复发因素分析及放疗靶区指导[J]. 中国临床医学, 2020, 27(3): 380-385. https://www.cnki.com.cn/Article/CJFDTOTAL-LCYX202003010.htm ZANG X, CHEN Y X, SUN J, et al. Prognostic factors of locoregional failure of gallbladder cancer after radical resection: guidance for adjuvant radiotherapy[J]. Chinese Journal Of Clinical Medicine, 2020, 27(3): 380-385. https://www.cnki.com.cn/Article/CJFDTOTAL-LCYX202003010.htm
[3]	SHEN C Y, YIN Y, CHEN H J, et al. Secreted protein acidic and rich in cysteine-like 1 suppresses metastasis in gastric stromal tumors[J]. BMC Gastroenterol, 2018, 18(1): 105. doi: 10.1186/s12876-018-0833-8
[4]	WANG H Y, FENG C, LU M X, et al. Integrative single-cell transcriptome analysis reveals a subpopulation of fibroblasts associated with favorable prognosis of liver cancer patients[J]. Transl Oncol, 2021, 14(1): 100981. DOI: 10.1016/j.tranon.2020.100981.
[5]	EVILORIA K, MUNASINGHE A, ASHER S, et al. A holistic approach to dissecting SPARC family protein complexity reveals FSTL-1 as an inhibitor of pancreatic cancer cell growth[J]. Sci Rep, 2016, 25(6): 37839. DOI: 10.1038/srep37839.
[6]	KLINGLER A, REGENSBURGER D, TENKERIAN C, et al. Species-, organ- and cell-type-dependent expression of SPARCL1 in human and mouse tissues[J]. PLoS One, 2020, 21: 15(5): e0233422. DOI: 10.1371/journal.pone.0233422.
[7]	蔡香雪, 李婷, 韦露薇, 等. 抑癌基因SPARCL1低表达对卵巢癌耐药和临床预后的影响[J]. 现代妇产科进展, 2020, 29(4): 256-262. https://www.cnki.com.cn/Article/CJFDTOTAL-XDFC202004006.htm CAI X X, LI T, WEI L W, et al. Low expression of SPARCL1 contribute to cisplatin resistance and predict poor prognosis in ovarian cancer[J]. Progress in Obstetrics and Gynecology, 2020, 29(4): 256-262. https://www.cnki.com.cn/Article/CJFDTOTAL-XDFC202004006.htm
[8]	施骅, 潘俊娣, 叶斌. 替吉奥联合卡培他滨对胆囊癌患者炎性因子和淋巴细胞功能的影响[J]. 中华全科医学, 2017, 15(6): 954-956. doi: 10.16766/j.cnki.issn.1674-4152.2017.06.013 SHI H, PAN J D, YE B. The influence of tegafur combined with capecitabine on inflammatory factors and lymphocyte function of patients with gallbladder cancer[J]. Chinese Journal of General Practice, 2017, 15(6): 954-956. doi: 10.16766/j.cnki.issn.1674-4152.2017.06.013
[9]	GONG K, GONG Z J, LU P X, et al. PLAC8 overexpression correlates with PD-L1 upregulation and acquired resistance to chemotherapies in gallbladder carcinoma[J]. Biochem Biophys Res Commun, 2019, 27, 516(3): 983-990.
[10]	张淦梅. 肿瘤病理诊断中特殊染色联合免疫组化技术的应用效果及检测阳性率评价[J]. 临床检验杂志(电子版), 2019, 8(4): 262-263. https://www.cnki.com.cn/Article/CJFDTOTAL-LNJI201904221.htm ZHANG G M. Application effect and positive rate evaluation of special staining combined with immunohistochemistry in tumor pathological diagnosis[J]. Clinical Laboratory Journal(Electronic Edition), 2019, 8(4): 262-263. https://www.cnki.com.cn/Article/CJFDTOTAL-LNJI201904221.htm
[11]	刘杰, 陈万青, 郑荣寿, 等. 2013年中国胆囊癌发病与死亡分析[J]. 中国肿瘤, 2018, 27(3): 161-166. https://www.cnki.com.cn/Article/CJFDTOTAL-ZHLU201803001.htm LIU J, CHEN W Q, ZHENG R S, et al. Incidence and Mortality of Gallbladder Cancer in China, 2013[J]. China Cancer, 2018, 27(3): 161-166. https://www.cnki.com.cn/Article/CJFDTOTAL-ZHLU201803001.htm
[12]	焦丹, 柴凤霞, 邢程, 等. 胆囊癌的临床病理特点及淋巴转移的临床病理危险因素分析[J]. 中国普通外科杂志, 2019, 28(8): 983-988. https://www.cnki.com.cn/Article/CJFDTOTAL-ZPWZ201908014.htm JIAO D, CHAI F X, XING C, et al. Analysis of clinicopathologic characteristics of gallbladder cancer and clinicopathologic risk factors for lymphatic metastasis[J]. Chinese Journal of General Surgery, 2019, 28(8): 983-988. https://www.cnki.com.cn/Article/CJFDTOTAL-ZPWZ201908014.htm
[13]	YAN S D, WANG Y Y, CHEN X, et al. Clinical analysis of 15 cases of gallbladder neuroendocrine carcinoma and comparison with gallbladder adenocarcinoma using a propensity score matching[J]. Cancer Manag Res, 2020, 26(12): 1437-1446.
[14]	WANG Y X, LIU S Y, Yan Y Q, et al. SPARCL1 promotes C2C12 cell differentiation via BMP7-mediated BMP/TGF-β cell signaling pathway[J]. Cell Death Dis, 2019, 10(11): 852.
[15]	LIU X J, ZHAO J, LUAN X R, et al. SPARCL1 impedes trophoblast migration and invasion by down-regulating ERK phosphorylation and AP-1 production and altering EMT-related molecule expression[J]. Placenta, 2020(89): 33-41.
[16]	王雨欣. SPARCL1调节C2C12细胞分化的机制研究[D]. 哈尔滨: 东北农业大学, 2020. WANG Y X. Mechanism of SPARCL1 regulating differentiation of C2C12 cells[D]. Harbin: Northeast Agricultural University, 2020.
[17]	LUO H L, CHIANG P H, HUANG C C, et al. Methylation of SPARCL1 is associated with oncologic outcome of advanced upper urinary tract urothelial carcinoma[J]. Int J Mol Sci, 2019, 20(7): 1653.
[18]	ZHAO S J, JIANG Y Q, XU N W, et al. SPARCL1 suppresses osteosarcoma metastasis and recruits macrophages by activation of canonical WNT/β-catenin signaling through stabilization of the WNT-receptor complex[J]. Oncogene, 2018, 37(8): 1049-1061.
[19]	陈都, 钱诚. 乳腺癌肿瘤微环境中间质细胞介导的化疗耐药性研究进展[J]. 实用肿瘤学杂志, 2020, 34(4): 382-386. https://www.cnki.com.cn/Article/CJFDTOTAL-SYZL202004020.htm CHEN D, QIAN C. Research progress of mesenchymal cell-mediated chemotherapy resistance in tumor microenvironment of breast cancer[J]. Practical Oncology Journal, 2020, 34(4): 382-386. https://www.cnki.com.cn/Article/CJFDTOTAL-SYZL202004020.htm
[20]	李婷. 抑癌基因SPARCL1的表达调控机制及其在卵巢癌顺铂耐药细胞中的功能研究[D]. 南宁: 广西医科大学, 2018. LI T. Regulation mechanism of tumor suppressor gene SPARCL1 and its function in cisplatin-resistant ovarian cancer cells[D]. Nanning: Guangxi Medical University, 2018.
[21]	钱俊峰, 徐芳, 王洪源, 等. 血清CYFRA21-1、MMP-9及TTF-1水平在诊断肺恶性肿瘤患者中的临床价值[J]. 中华全科医学, 2019, 17(6): 951-954. doi: 10.16766/j.cnki.issn.1674-4152.000833 QIAN J F, XU F, WANG H Y, et al. Clinical value of CYFRA21-1, MMP-9 and TTF-1 levels in differentiating benign from malignant lung tumors[J]. Chinese Journal of General Practice, 2019, 17(6): 951-954. doi: 10.16766/j.cnki.issn.1674-4152.000833
[22]	李静, 刘琴, 柯锦. miR-582-5p通过靶向SOCS1基因调节皮肤黑色素瘤细胞迁移、侵袭和EMT的机制研究[J]. 河北医药, 2021, 43(2): 165-170. https://www.cnki.com.cn/Article/CJFDTOTAL-HBYZ202102001.htm LI J, LIU Q, KE J. The effects of miR-582-5p in regulating the migration, invasion and EMT of melanoma cells of skin by targeting SOCS1 gene[J]. Hebei Medical Journal, 2021, 43(2): 165-170. https://www.cnki.com.cn/Article/CJFDTOTAL-HBYZ202102001.htm
[23]	AMANDA M, KHALISHA M, FATEMIA M, et al. Fibronectin acts as a molecular switch to determine SPARC function in pancreatic cancer[J]. Cancer Lett, 2020(477): 88-96.
[24]	虞阳. SPARCL1在肝门部胆管癌中的表达和作用机制研究[D]. 上海: 第二军医大学, 2017. YU Y. Expression and mechanism of SPARCL1 in hilar cholangiocarcinoma[D]. Shanghai: Naval Medical University, 2017.
[25]	王进, 余晓凡, 欧阳楠, 等. 甲基化调控SLIT3和SPARCL1基因在吸烟致肺腺癌中的表达及其对患者预后的影响[J]. 中华医学杂志, 2019, 99(20): 1553-1557. WANG J, YU X F, OUYANG N, et al. Expression and prognosis effect of methylation-regulated SLIT3 and SPARCL1 genes in smoking-related lung adenocarcinoma[J]. National Medical Journal of China, 2019, 99(20): 1553-1557.
[26]	GONG Y B, FAN X H. MiR-539-3p promotes the progression of epithelial ovarian cancer by targeting SPARCL1[J]. Eur Rev Med Pharmacol Sci, 2019, 23(6): 2366-2373.
[27]	周源. Sparcl1基因在非小细胞肺癌组织中的表达与临床意义及生物学功能[D]. 沈阳: 中国医科大学, 2018. ZHOU Y. Expression, clinical significance and biological function of Sparcl1 gene in non-small cell lung cancer[D]. Shenyang: China Medical University, 2018.