A study on mechanism of GOLM1 regulating PI3K/AKT/mTOR signaling pathway to promote proliferation, invasion and migration of lung adenocarcinoma cells

doi:10.19401/j.cnki.1007-3639.2022.03.003

Abstract

Abstract:

Background and purpose: Golgi membrane protein 1 (GOLM1) plays the role of an oncogene in lung adenocarcinoma (LUAD), however, the effects of GOLM1 on the proliferation, invasion and migration of LUAD cells and its mechanism are not clear yet. This study investigated the effects of GOLM1 on the proliferation, invasion and migration of LUAD cells and its mechanism of action. Methods: We selected cancer tissues and corresponding paracancerous tissue specimens from 90 LUAD patients who underwent surgical resection in Karamay Central Hospital from April 2019 to April 2021. The expression of GOLM1 protein in LUAD tissues and paracancerous tissues was detected by immunohistochemistry, and the relationship between GOLM1 protein expression and clinicopathological characteristics of LUAD tissues was analyzed. Western blot was used to detect the expression of GOLM1 protein in human lung epithelial cells BEAS-2B and human lung adenocarcinoma H460, A549, PG49 and H1299 cells. Lung adenocarcinoma A549 cells in logarithmic growth stage were randomly divided into blank group (cells not transfected), GOLM1 small interfering RNA negative control (si-NC) group (cells transfected with si-NC), GOLM1 small interfering RNA (si-GOLM1) group (cells transfected with si-GOLM1), insulin-like growth factor-1 (IGF-1) group [10 μmol/L phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/mammalian target of rapamycin (mTOR) signaling pathway activator IGF-1 for 30 min] and si-GOLM1+IGF-1 group (after treatment with 10 μmol/L IGF-1 for 30 min, si-GOLM1 was transfected). Cell counting kit-8 method was used to detect cell proliferation in each group of lung adenocarcinoma A549 cells. Scratch test was used to detect cell migration in each group of lung adenocarcinoma A549 cells. Transwell experiment was used to detect cell invasion in each group of lung adenocarcinoma A549 cells. Western blot was used to detect the expressions of GOLM1 and PI3K/AKT/mTOR signaling pathway related proteins in each group of lung adenocarcinoma A549 cells. Xenograft model was constructed by subcutaneously injecting A549 cells on the right side of BALB/c nude mice, which were divided into: nude mice blank group, nude mice si-NC group, nude mice si-GOLM1 group, nude mice IGF-1 group, nude mice si-GOLM1+IGF-1 group, with 6 mice in each group. The nude mice were sacrificed six weeks after injection, the tumor was collected, and the weight and volume of the tumor were measured. Results: The results of immunohistochemistry showed that the positive expression rate of GOLM1 protein was significantly higher in LUAD tissues than in adjacent tissues (P<0.05). The expression of GOLM1 protein was significantly correlated with the degree of tumor differentiation, lymph node metastasis, and clinical stage (P<0.05), but not significantly correlated with gender, age and smoking status of LUAD patients (P>0.05). Compared with BEAS-2B cells, the relative expression level of GOLM1 protein in human lung adenocarcinoma H460, A549, PG49 and H1299 cells was significantly increased (P<0.05), and the relative expression level of GOLM1 protein in A549 cells was the highest. Therefore, A549 cells were selected for subsequent experiments. Compared with the blank group and the si-NC group, OD value, scratch healing rate, number of invaded cells, GOLM1, p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR protein relative expression levels in the lung adenocarcinoma A549 cells of the si-GOLM1 group were significantly reduced (P<0.05). In the lung adenocarcinoma A549 cells of the IGF-1 group, there was no significant change in GOLM1 protein, and the other corresponding indicators were significantly increased (P<0.05). Compared with the si-GOLM1 group, the OD value, scratch healing rate, number of invaded cells, p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR protein relative expression levels of lung adenocarcinoma A549 cells in the si-GOLM1+IGF-1 group were significantly increased (P<0.05), and there was no significant difference in GOLM1 protein relative expression level (P>0.05). Compared with the nude mice blank group and nude mice si-NC group, the mass and volume of transplanted tumors in the nude mice si-GOLM1 group were significantly reduced, while the mass and volume of transplanted tumors in the nude mice IGF-1 group were significantly increased (P<0.05). Compared with the nude mice si-GOLM1 group, the mass and volume of transplanted tumors in the nude mice si-GOLM1+IGF-1 group were significantly increased (P<0.05). Conclusion: Silencing GOLM1 gene can inhibit the activation of PI3K/AKT/mTOR signaling pathway, thereby inhibiting the proliferation, migration and invasion of lung adenocarcinoma A549 cells.

Key words: Golgi membrane protein 1, Lung adenocarcinoma, Cell proliferation, Cell invasion, Cell migration, Phosphatidylinositol 3-kinase/protein kinase B/mammalian target of rapamycin pathway

CLC Number:

R734.2

ZHU Haipeng, HU Jun, JIANG Min, CAI Ruonan, WANG Junqiao, LI Li. A study on mechanism of GOLM1 regulating PI3K/AKT/mTOR signaling pathway to promote proliferation, invasion and migration of lung adenocarcinoma cells[J]. China Oncology, 2022, 32(3): 207-217.

Figures/Tables 12

Fig. 1

Tab. 1

Fig. 2

Tab. 2

Tab. 3

Fig. 3

Fig. 4

Tab. 4

Fig. 5

Tab. 5

Fig. 6

Tab. 6

References 21

[1]	LUO C, LEI M, ZHANG Y, et al. Systematic construction and validation of an immune prognostic model for lung adenocarcinoma[J]. J Cell Mol Med, 2020, 24(2): 1233-1244. doi: 10.1111/jcmm.v24.2
[2]	KLECZKO E K, KWAK J W, SCHENK E L, et al. Targeting the complement pathway as a therapeutic strategy in lung cancer[J]. Front Immunol, 2019, 10: 954. doi: 10.3389/fimmu.2019.00954
[3]	YAN J L, ZHOU B H, GUO L, et al. GOLM1 upregulates expression of PD-L1 through EGFR/STAT3 pathway in hepatocellular carcinoma[J]. Am J Cancer Res, 2020, 10(11): 3705-3720.
[4]	郑兴, 吴兆红, 陈岗东, 等. miRNA27a靶向GOLM1对非小细胞肺癌生物行为学的影响[J]. 中国免疫学杂志, 2020, 36(1): 52-56.
	ZHENG X, WU Z H, CHEN G D, et al. Effect of miRNA27a targeting GOLM1 on biobehavioral of non-small cell lung cancer[J]. Chin J Immunol, 2020, 36(1): 52-56.
[5]	曾建昌, 刘艳, 杨俊, 等. 复方苦参注射液联合奥希替尼介导PI3K/AKT/mTOR信号通路在肺腺癌细胞H1975中的作用机制[J]. 中国免疫学杂志, 2021, 37(10): 1191-1195.
	ZENG J C, LIU Y, YANG J, et al. Mechanism of compound sophora flavescens injection combined with osimertinib in lung adenocarcinoma cell H1975 via PI3K/AKT/mTOR signaling pathway[J]. Chin J Immunol, 2021, 37(10): 1191-1195.
[6]	王永贵. SLC25A22在骨肉瘤组织中的表达及其与预后的相关性[J]. 现代肿瘤医学, 2021, 29(17): 3093-3096.
	WANG Y G. Expression of SLC25A22 in osteosarcoma and its correlation with prognosis[J]. J Mod Oncol, 2021, 29(17): 3093-3096.
[7]	RONG L, LI Z D, LENG X, et al. Salidroside induces apoptosis and protective autophagy in human gastric cancer AGS cells through the PI3K/AKT/mTOR pathway[J]. Biomed Pharmacother, 2020, 122: 109726. doi: 10.1016/j.biopha.2019.109726
[8]	WANG Y Q, HUANG J, WU Q, et al. Downregulation of breast cancer resistance protein by long-term fractionated radiotherapy sensitizes lung adenocarcinoma to SN-38[J]. Invest New Drugs, 2021, 39(2): 458-468. doi: 10.1007/s10637-020-01003-3
[9]	ZHANG R, ZHU Z, SHEN W Z, et al. Golgi membrane protein 1 (GOLM1) promotes growth and metastasis of breast cancer cells via regulating matrix metalloproteinase-13 (MMP13)[J]. Med Sci Monit, 2019, 25: 847-855. doi: 10.12659/MSM.911667
[10]	YE Q H, ZHU W W, ZHANG J B, et al. GOLM1 modulates EGFR/RTK cell-surface recycling to drive hepatocellular carcinoma metastasis[J]. Cancer Cell, 2016, 30(3): 444-458. doi: 10.1016/j.ccell.2016.07.017
[11]	翟维佳, 封全灵, 张慧芳, 等. GOLM1对宫颈癌上皮间质转化的影响及其与临床病理特征的关系[J]. 肿瘤学杂志, 2019, 25(11): 976-979.
	ZHAI W J, FENG Q L, ZHANG H F, et al. Effect of GOLM1 expression on epithelial-mesenchymal transition and its relationship with clinicopathological features of cervical cancer[J]. J Chin Oncol, 2019, 25(11): 976-979.
[12]	余展鹏, 江雁琼. 基于数据挖掘分析GOLM1基因在前列腺癌中的表达及其临床意义[J]. 中国癌症防治杂志, 2019, 11(2): 158-162.
	YU Z P, JIANG Y Q. Analysis of GOLM1 gene expression in prostate cancer and its effect on prognosis based on data mining[J]. Chin J Oncol Prev Treat, 2019, 11(2): 158-162.
[13]	ARUNA, LI L M. Overexpression of Golgi membrane protein 1 promotes non-small-cell carcinoma aggressiveness by regulating the matrix metallopeptidase 13[J]. Am J Cancer Res, 2018, 8(3): 551-565.
[14]	ZHANG S K, GE W M, ZOU G Y, et al. MiR-382 targets GOLM1 to inhibit metastasis of hepatocellular carcinoma and its down-regulation predicts a poor survival[J]. Am J Cancer Res, 2018, 8(1): 120-131.
[15]	YANG L Q, LUO P C, SONG Q, et al. DNMT1/miR-200a/GOLM1 signaling pathway regulates lung adenocarcinoma cells proliferation[J]. Biomedecine Pharmacother, 2018, 99: 839-847.
[16]	CIRONE M. Cancer cells dysregulate PI3K/AKT/mTOR pathway activation to ensure their survival and proliferation: mimicking them is a smart strategy of gammaherpesviruses[J]. Crit Rev Biochem Mol Biol, 2021, 56(5): 500-509. doi: 10.1080/10409238.2021.1934811
[17]	GU Z H, YOU Z X, YANG Y C, et al. Inhibition of MicroRNA miR-101-3p on prostate cancer progression by regulating cullin 4B (CUL4B) and PI3K/AKT/mTOR signaling pathways[J]. Bioengineered, 2021, 12(1): 4719-4735. doi: 10.1080/21655979.2021.1949513
[18]	DENG L, WU X R, ZHU X J, et al. Combination effect of curcumin with docetaxel on the PI3K/AKT/mTOR pathway to induce autophagy and apoptosis in esophageal squamous cell carcinoma[J]. Am J Transl Res, 2021, 13(1): 57-72.
[19]	炎士珂, 冯婧文, 昌娇, 等. FGFC1经PI3K/AKT/mTOR途径抑制非小细胞肺癌细胞增殖和迁移[J]. 中国生物化学与分子生物学报, 2021, 37(8): 1069-1077.
	YAN S K, FENG J W, CHANG J, et al. FGFC1 inhibits proliferation and migration of non-small cell lung cancer cells via the PI3K/AKT/mTOR signaling pathway[J]. Chin J Biochem Mol Biol, 2021, 37(8): 1069-1077.
[20]	孟东雪, 郭玉荣, 罗斌军, 等. 扶正抑瘤汤通过PI3K/AKT/mTOR信号通路抑制非小细胞肺癌增殖、凋亡及自噬[J]. 中国癌症防治杂志, 2021, 13(2): 177-182.
	MENG D X, GUO Y R, LUO B J, et al. Fuzheng Yiliu decoction inhibits the proliferation, apoptosis and autophagy of non-small cell lung cancer through PI3K/AKT/mTOR signaling pathway[J]. Chin J Oncol Prev Treat, 2021, 13(2): 177-182.
[21]	丁志丹, 方泽民, 王旭广, 等. 贝母素乙调控PI3K/AKT/mTOR通路减缓上皮-间质转化进程抑制人肺癌A549细胞侵袭及迁移的研究[J]. 中草药, 2019, 50(6): 1382-1387.
	DING Z D, FANG Z M, WANG X G, et al. Inhibition of peiminine on invasion and migration of human lung cancer A549 cells by decreasing epithelial-mesenchymal transition process via PI3K/AKT/mTOR pathway[J]. Chin Tradit Herb Drugs, 2019, 50(6): 1382-1387.

Characteristic	Case n	GOLM1		χ²	P value
Characteristic	Case n	Negative	Positive	χ²	P value
Gender				0.436	0.509
Male	47	9 (19.15)	38 (80.85)
Female	43	6 (13.95)	37 (86.05)
Age/year				0.009	0.925
<60	41	7 (17.07)	34 (82.93)
≥60	49	8 (16.33)	41 (83.67)
Smoking				0.010	0.921
Yes	31	5 (16.13)	26 (83.87)
No	59	10 (16.95)	49 (83.05)
Degree of differentiation				9.681	0.002
Medium/low differentiation	56	4 (7.14)	52 (92.86)
High differentiation	34	11 (32.35)	23 (67.65)
Lymph node metastasis				21.056	<0.001
Yes	34	8 (23.53)	26 (76.47)
No	56	41 (73.21)	15 (26.79)
Clinical stage				6.828	0.009
Ⅰ/Ⅱ	59	28 (47.46)	31 (52.54)
Ⅲ	31	6 (19.35)	25 (80.65)

Group	GOLM1/GAPDH
BEAS-2B cell	0.17±0.02
H460 cell	0.95±0.06^a
A549 cell	1.53±0.13^a
PG49 cell	1.13±0.10^a
H1299 cell	0.67±0.04^a
F value	239.446
P value	<0.001

Group	D value
Group	0 h	24 h	48 h
Blank group	0.24±0.02	0.54±0.03	0.95±0.07
si-NC group	0.26±0.04	0.52±0.04	0.92±0.09
si-GOLM1 group	0.23±0.02	0.26±0.02^ab	0.39±0.03^ab
IGF-1 group	0.25±0.04	0.81±0.06^abc	1.25±0.12^abc
si-GOLM1+IGF-1 group	0.22±0.03	0.59±0.03^c	0.89±0.06^c
F value	1.531	156.203	90.188
P value	0.224	<0.001	<0.001

Group	Scratch healing rate/%	Number of invasion cells
Blank group	46.25±3.08	65.86±4.28
si-NC group	44.73±3.23	68.34±3.98
si-GOLM1 group	18.65±1.79^ab	29.06±2.55^ab
IGF-1 group	79.62±5.62^abc	112.39±7.87^abc
si-GOLM1+IGF-1 group	52.27±3.35^c	75.66±4.43^c
F value	215.315	216.280
P value	<0.001	<0.001

Group	GOLM1/GAPDH	p-PI3K/PI3K	p-AKT/AKT	p-mTOR/mTOR
Blank group	1.51±0.12	0.45±0.03	0.68±0.04	0.79±0.05
si-NC group	1.52±0.14	0.48±0.02	0.71±0.05	0.78±0.06
si-GOLM1 group	0.63±0.04^ab	0.15±0.01^ab	0.18±0.01^ab	0.25±0.03^ab
IGF-1 group	1.56±0.11^c	0.83±0.05^abc	1.08±0.06^abc	1.21±0.11^abc
si-GOLM1+IGF-1 group	0.65±0.05^ab	0.51±0.02^c	0.72±0.05^c	0.83±0.06^c
F value	142.249	406.605	300.233	154.652
P value	<0.001	<0.001	<0.001	<0.001