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Abstract

BACKGROUND:

Nuclear pore membrane protein 121 (POM121) is a novel biomarker involved in tumorigenesis and metastasis. However, little is known about the role of POM121 in non-small-cell lung cancer (NSCLC).

OBJECTIVE:

The aim of this study was to detect the expression of POM121 in NSCLC and its relationship with clinicopathologic feature and cell biological behavior, and explore the underlying mechanisms.

METHODS:

The expression of POM121 in NSCLC tissues and para-carcinoma tissues was compared by quantitative real-time PCR and immunohistochemistry analysis. The relationship between POM121 protein and clinicopathological characteristics in NSCLC was investigated. Roles of POM121 in NSCLC cells were investigated by CCK-8 assay, clone formation assay, transwell migration and invasion assay, and in vivo experiments. Variations of signaling pathways were determined by qRT-PCR and Western blot.

RESULTS:

The POM121 expression in NSCLC tissues was significantly higher than that in para-carcinoma tissues, both at the mRNA and protein level. The POM121 expression was related to sex, advanced differentiation, tumor diameter, lymph node metastases, distant metastases, American Joint Committee on Cancer (AJCC) stage, venous invasion, and perineural invasion in NSCLC. Kaplan-Meier analysis indicated that NSCLC patients with high POM121 expression had poor overall survival. Downregulation of POM121 inhibited cell proliferation, clone formation, migration and invasion. TGF- $\beta$ /SMAD and PI3K/AKT pathways were involved in POM121-induced functional changes in NSCLC cells.

CONCLUSION:

POM121 plays an oncogenic role in NSCLC through TGF- $\beta$ /SMAD and PI3K/AKT pathways. POM121 expression is a potential independent prognostic factor for NSCLC.

Keywords

POM121 non-small-cell lung cancer prognosis TGF-β/SMAD PI3K/AKT

1. Introduction

Lung cancer is the most frequently diagnosed cancer worldwide and the leading cause of cancer death among males [1]. Over half of lung cancer patients are declared to be in advanced stage at initial diagnosis because of the atypical symptoms that present in the early stages [2]. The 5-year survival rate is 26.1% for patients with regional disease and only 3.9% for patients with distant metastases [3]. Non-small-cell lung cancer (NSCLC) accounts for 80%–85% of lung cancers [4]. Thus, it is necessary to identify new sensitive and specific biomarkers of NSCLC.

NPCs serve a vital role in maintaining normal cellular functions through regulating cellular signaling between the cytoplasm and nucleus [5]. Dysregulation of NUPs can trigger abnormal nucleocytoplasmic transport, resulting in mislocalization of transcription factors and tumor suppression proteins [6]. As a member of NUPs, nuclear pore membrane protein 121 (POM121) gene is located on human chromosome 7q 11.23 [7]. POM121 protein is 121 kDa, composed of a single transmembrane domain and two peptide domains extending into the endoplasmic reticulum and the cytoplasm [8, 9]. POM121 and NUP107-160 complex is involved in inner/outer nuclear membrane fusion events, which are required for nuclear envelope formation [10]. POM121 is also essential for nuclear pore complex (NPC) assembly [11, 12]. Replacement of POM121 was connected with defective nuclear transport, aberrant cytoplasmic membrane stacks, and decreased cell viability [13]. Guo et al. reported that POM121 enabled efficient HIV-1 pre-integration complex nuclear import during KPNB1-dependent cargo nuclear transportation [14]. Rodriguez-Bravo et al. demonstrated the important role of POM121 in prostate cancer aggressiveness [15]. but the association between POM121 and NSCLC remains unknown.

To explore the role of POM121 in NSCLC, POM121 expression in NSCLC tissues was detected, and associations between POM121 expression and pathological characteristics were analyzed. The function and mechanism of POM121 in regulating proliferation and metastasis of NSCLC were also explored in vitro and vivo. Combined with the above results, POM121 has the potential to be a therapeutic target for NSCLC patients.

2. Materials and methods

2.1 Tissue samples and clinical data

Formalin-fixed, paraffin-embedded lung tissue samples were collected from 857 patients whichincluded 796 NSCLC tissues, 61 small-cell lung cancer (SCLC) tissues, and 212 normal lung tissues. All samples were obtained from patients who were histologically diagnosed at Bayannur Hospital between 2005 and 2012 and at Nanjing First Hospital from 2008 to 2013. None of the patients enrolled in this study received chemotherapy, immunotherapy, or radiation therapy before surgery or biopsy. The clinicopathological features of tissue donors included age, sex, tumor size, lymph node metastases, distance metastasis, histological type, differentiation grade, histological type, AJCC stage, and venous invasion. OS was defined as the period from initial diagnosis to death (or last follow-up). The project was approved by the Human Ethics Committee of Bayannur Hospital (BSYY2019217), and all patients participating in the study signed informed consent.

2.2 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

POM121 mRNA expression was detected in 31 pairs of NSCLC tissues and matched normal lung tissues. Total RNA was extracted from tissues using Trizol reagent (#15596026, ThermoFisher, Waltham, MA, USA) according to the manufacturer’s instructions. DNase was utilized to get rid of all traces of genomic DNA. Extracted RNA was transcribed into cDNA using the SuperScript IV First-Strand Synthesis System (#18091050, ThermoFisher). qRT-PCR was performed using SYBR Select Master Mix (#4472920, ThermoFisher) on an ABI StepOne Plus Real-Time PCR System using the following protocol: 95 ${}^{\circ}$ C for 10 min;40 cycles of amplification at 95 ${}^{\circ}$ C for 15 s and 56 ${}^{\circ}$ C for 1 min; 95 ${}^{\circ}$ C for 15 s, 60 ${}^{\circ}$ C for 1 min. Results were normalized to GAPDH and all experiments were repeated in triplicate.

2.3 Tissue microarrays (TMA) and immunohistochemistry analysis (IHC)

Tissue specimens were shaped in special molds and fixed with 4% paraformaldehyde. After embedding in paraffin, tissue cores were collected to construct tissue microarrays. Blocks were sectioned to a thickness of 4 $\mu$ m onto slides. Tissue sections were deparaffinized in xylene and rehydrated in an alcohol gradient, then incubated in 3% H ${}_{2}$ O ${}_{2}$ to inhibit endogenous peroxidase activity. Tissues were heated in 0.01 M citrate buffer (pH 6.0) to retrieve antigens, then incubated overnight at room temperature with anti-human POM121 rabbit polyclonal antibody (1:200; #15645-1-AP, Proteintech, Manchester, UK). Staining was observed and scored independently by two pathologists in a blind manner. The semi-quantitative H-score method was performed to quantify POM121 protein expression. Staining intensity scores were categorized as follows: 0, negative; 1, weakly positive; 2, moderately positive; and 3, strongly positive. The final staining score was the percentage of positive cells staining for POM121 multiplied by the respective intensity score. The minimum possible final staining score was 0 (no staining), and the maximum possible score was 300 (100% of cells with a staining intensity score of 3) [16]. The level of POM121 protein expression was defined by the $\chi$ -tile software program. Scores under 105 were considered as low or no expression, while others were defined as high expression.

2.4 Cell lines and cell culture

Human NSCLC cell lines A549 and HCC827 were obtained from NHC Key Laboratory of antibody technique (Nanjing Medical University). The medium was RPMI 1640 with 10% fetal bovine serum and 1% double antibody. The cells were cultured in a incubator of 5% CO ${}_{2}$ at 37 ${{}^{\circ}}$ C.

2.5 Western blot

The total protein was extracted with RIPA lysate (#15596018, Invitrogen, USA) and heated at 95 ${{}^{\circ}}$ C for 10 min with the loading buffer. The protein samples were subjected to polyacrylamide gel electrophoresis and transferred to PVDF membrane. Then the membrane was sealed with 5% skim milk at room temperature for 2 h, incubated with primary antibody at 4 ${{}^{\circ}}$ C (1:1000) overnight. After rinse with PBST, the membrane was incubated secondary antibody (1:2000) for 1 h. The SuperSignal exposure solution was covered on the film surface, and ChemiDoc XRS ${}^{+}$ system was used to detect protein bands. The above experiment was repeated for three times.

2.6 CCK-8 proliferation experiment

Cells of each group were planted in 96-well plates at a density of $1\times 10^{4}$ / well. The original medium was replaced by 10 $\mu$ L CCK-8 reagent (#CK04, Dojindo, Japan) and 90 $\mu$ L medium at 24, 48, 72, and 96 h respectively. Cells were incubated in darkness at 37 ${}^{\circ}$ C for 3 h. The absorption value of each well at the wavelength of 450 nm were detected by a microplate microscope.

2.7 Clone formation assay

The cells were re-inoculated into 6-well plates with 1 $\times$ 10 ${}^{3}$ cells per well. After 12–14 days of culture, 4% paraformaldehyde was added to the 6-well plate to fix cells at room temperature for 20 min, and then 0.5% crystal violet solution was added for 20 min at room temperature. The cells were washed with pre-cooled PBS at 4 ${{}^{\circ}}$ C, and then photographed for analysis.

2.8 Transwell migration and invasion assay

Migration and invasion assays were conducted using transwell chambers without and with Matrigel respectively. Cells (5 $\times$ 10 ${}^{4}$ ) were planted in each transwell chamber with 300 $\mu$ L serum-free medium. The chamber was placed in a 12-well plate, and 900 $\mu$ L medium containing 20%FBS was added into each well. 24 h later, the upper surface of the chamber membrane was wiped gently with a cotton swab. The cell membrane was fixed with methanol for 30 min and soaked in 0.1% crystal violet solution for 30 min. After wash with water, cells were photographed and counted.

2.9 Tumor formation experiment in nude mice

A549 cells transfected with shControl and shPOM121 were prepared into cell suspension with a density of 1 $\times$ 10 ${}^{8}$ cells/ml, and 100 $\mu$ L suspension was injected into the armpit of each nude mice. Tumor size was measured every 3 days, tumor volume $=$ longest diameter $\times$ shortest diameter ${}^{2}$ /2. Five weeks later, the nude mice were sacrificed, and subcutaneous tumors were obtusely separated.

Figure 1.

POM121 expression in NSCLC tissues. (A) POM121 mRNA levels in the 31 NSCLC tissues (T) were higher than that in matched normal tissues (N). (B) Positive rates of high POM121 in NSCLC tissues and SCLC tissues were higher than that in normal tissues. (C) Representative images of POM121 protein in lung tissues. (a) Non-small-cell squamous carcinoma with high POM121 expression (staining score $=$ 280). (b) Non-small-cell adenocarcinoma with high POM121 expression (staining score $=$ 220). (c) Small cell lung cancer with high POM121 expression (staining score $=$ 190). (d) Normal lung tissues with no POM121 expression (staining score $=$ 0). (Original views $\times$ 4, bar $=$ 500 $\mu$ m; enlarged views $\times$ 40, bar $=$ 50 $\mu$ m). * $P<$ 0.05.

2.10 Statistical analysis

Statistical analyses were performed using SPSS version 18.0 software (SPSS Inc., USA). Measurement data were expressed as mean $\pm$ standard deviation. Comparisons between two groups were evaluated using Student’s $t$ -test or chi-square test. The Pearson $\chi^{2}$ test was performed to analyze correlations between POM121 expression and clinicopathological features. OS and disease-free survival were evaluated by Kaplan-Meier analysis and log-rank test. Univariate and multivariate analyses were performed by a Cox proportional hazards regression model. In order to adjust the influence of other variables on the variables POM121 expression, other variables related to patient survival in univariate analysis and the variables POM121 expression were put into multivariate analysis. Statistical significance was set as $P<$ 0.05.

3. Results

3.1 POM121 is overexpressed in NSCLC

POM121 mRNA expression levels were detected in 31pairs of NSCLC tissues and matched normal lung tissues by qRT-PCR. POM121 mRNA expression in NSCLC tissues was higher than that in matched normal lung tissues ( $P<$ 0.01, Fig. 1A).

To detect POM121 protein expression in lung cancer tissues and normal lung tissues, IHC assays were performed. Results showed that POM121 protein was located in the nuclear membrane and was overexpressed in cancerous tissues (Fig. 1B). High POM121 expression was more prevalent in NSCLC tissues (60.07%, 502/796) than that in normal lung tissues (22.17%, 47/212), consistent with the comparison between SCLC tissues and normal tissues (Fig. 1C).

3.2 POM121 expression is connected with clinical characteristics in NSCLC

The correlation between POM121 expression and pathological parameters in NSCLC patients was analyzed. POM121 expression was significantly associated with sex ( $\chi^{2}=$ 6.398, $P=$ 0.011), differentiation ( $\chi^{2}=$ 17.862, $P=$ 0.000), tumor diameter ( $\chi^{2}=$ 47.589, $P=$ 0.000), lymph node metastases ( $\chi^{2}=$ 6.938, $P=$ 0.031), distant metastases ( $\chi^{2}=$ 5.273, $P=$ 0.022), AJCC stage ( $\chi^{2}=$ 47.028, $P=$ 0.000), venous invasion ( $\chi^{2}=$ 9.6535, $P=$ 0.002), and perineural invasion ( $\chi^{2}=$ 15.940, $P=$ 0.000). However, there was no significant difference between POM121 expression and age, smoking, histological type, preoperative CEA, preoperative CA125, or preoperative CyFRA21-1 (Table 1).

Table 1
Association of POM1121expression with clinicopathological characteristics in NSCLC patients

Characteristics	$n$	POM121 expression (%)		Pearson $\chi^{2}$	$P$ value
		Low or no (%)	High (%)
Total	796	502 (63.07)	294 (36.93)
Gender
Male	530	318 (60.00)	212 (40.00)	6.398	0.011*
Female	266	184 (69.17)	82 (30.83)
Age
$\geqslant$ 60	322	210 (65.22)	112 (34.78)	1.075	0.300
$<$ 60	474	292 (61.60)	182 (38.40)
Smoking
Yes	446	281 (63.00)	165 (37.00)	0.002	0.968
No	350	221 (63.14)	129 (36.86)
Histological type
Adenocarcinnoma	362	229 (63.26)	133 (36.74)	5.091	0.278
Squamous cell carcinoma	276	162 (58.70)	114 (41.30)
Adenosquamous carcinoma	142	98 (69.01)	44 (30.99)
Undifferentiated carcinoma	12	7 (58.33)	5 (41.67)
Large cell carcinoma	4	2 (50.00)	2 (50.00)
Differentiation				17.862	0.000*
Well	80	36 (45.00)	44 (55.00)
Moderate	508	317 (62.40)	191 (37.60)
Poor	208	149 (71.63)	59 (28.37)
Tumor diameter				47.589	0.000*
Tis	21	9 (42.86)	12 (57.14)
T1	337	231 (68.55)	106 (31.45)
T2	302	152 (50.33)	150 (49.67)
T3	100	81 (81.00)	19 (19.00)
T4	36	29 (80.56)	7 (19.44)
Lymph node metastases				6.938	0.031*
N0	476	307 (64.50)	169 (35.50)
N1	186	103 (55.38)	83 (44.62)
N2	134	92 (68.66)	42 (31.34)
Distant metastases				5.273	0.022*
M0	774	483 (42.55)	291 (57.45)
M1	22	19	3
AJCC stage				47.028	0.000*
0	21	3 (14.29)	18 (85.71)
I	331	208 (62.84)	123 (37.16)
II	250	139 (55.60)	111 (44.40)
III	172	134 (77.91)	38 (22.09)
IV	22	18 (81.82)	4 (18.18)
Venous invasion
Negative	729	448 (61.45)	281 (38.55)	9.6535	0.002*
Positive	67	54 (80.60)	13 (19.40)
Perineural invasion
Negative	724	441 (60.91)	283 (39.09)	15.940	0.000*
Positive	72	61 (84.72)	11 (15.28)
Preoperative CEA, ng/ml
$<$ 5	308	180 (58.44)	128 (41.56)	5.898	0.052
$\geqslant$ 5	471	313 (66.45)	158 (33.55)
Unknown	17	9 (52.94)	8 (47.06)
Preoperative CA125, ng/ml
$<$ 20	417	275 (65.95)	142 (34.05)	3.132	0.209
$\geqslant$ 20	362	217 (59.94)	145 (40.06)
Unknown	17	10 (58.82)	7 (41.18)
Preoperative CyFRA21-1, ng/ml
$<$ 3.3	448	291 (64.96)	157 (35.04)	3.764	0.152
$\geqslant$ 3.3	319	195 (61.13)	124 (38.87)
Unknown	29	16 (55.17)	13 (44.83)

3.3 High POM121 expression is associated with poor prognosis in NSCLC patients

Prognostic factors for NSCLC patients were detected by both univariate and multivariate analyses. POM121 expression was significantly associated with the OS of NSCLC patients in univariate analysis, as was smoking, differentiation, tumor diameter, distant metastasis, and AJCC stage. In multivariate analysis, independent prognostic factors for OS included high POM121 expression (HR $=$ 0.612; 95% CI 0.519–0.722; $P=$ 0.000), differentiation (HR $=$ 1.343; 95% CI, 1.180–1.528; $P=$ 0.000), and AJCC stage (HR $=$ 1.545; 95% CI, 1.265–1.886; $P=$ 0.000) (Table 2). Kaplan-Meier survival curves confirmed that high POM121 expression was significantly correlated with poor OS, as well as advanced AJCC stage, differentiation, tumor diameter, lymph node metastases, and distant metastasis (Fig. 2).

Table 2
Univariate and multivariate analysis of prognostic factors for overall survival in lung cancer

	Univariate analysis			Multivariate analysis
	HR	$P$ -value	95%CI	HR	$P$ -value	95%CI
POM121 expression
High vs low or no	0.640	0.000*	0.548 0.748	0.612	0.000*	0.519 0.722
Age (year)
$<$ 60 vs $\geqslant$ 60	0.914	0.272	0.780 1.073
Gender
Male vs female	0.909	0.219	0.780 1.058
Smoking
Yes vs no	1.300	0.001*	1.113 1.518
Histological type
Adenocarcinnoma vs squamous cell carcinoma vs adenosquamous carcinoma vs undifferentiated carcinoma vs large cell carcinoma	0.943	0.234	0.855 1.039
Differentiation
Well vs moderate vs poor	1.386	0.000*	1.219 1.575	1.343	0.000*	1.180 1.528
Tumor disameter
Tis $+$ T1 $+$ T2 vs T3 $+$ T4	1.314	0.006*	1.081 1.598
Lymph node metastasis
N0 vs N1 $+$ N2	1.181	0.487	0.739 1.887
Distant metastasis
M0 vs M1	1.660	0.000*	1.425 1.934
AJCC stage
0 $+$ I $+$ II vs III $+$ IV	1.478	0.000*	1.245 1.755	1.545	0.000*	1.265 1.886
Venous invasion
Negative vs positive	0.726	0.029	0.546 0.967	0.476	0.000*	0.324 0.698
Perineural invasion
Negative vs positive	0.731	0.026*	0.555 0.964
Preoperative CEA, ng/ml
$<$ 5 vs $\geqslant$ 5	0.716	0.000*	0.619 0.828
Preoperative CA125,ng/ml
$<$ 20 vs $\geqslant$ 20	0.701	0.000*	0.607 0.810
Preoperative CyFRA21-1, ng/ml
$<$ 3.3 vs $\geqslant$ 3.3	0.777	0.000*	0.677 0.890

HR, Hazard ratio; CI, confdence interval. * $P<$ 0.05.

Figure 2.

Survival curves of NSCLC patients using the Kaplan-Meier method and the log-rank test. (A) Overall survival curves for patients with low POM121 expression (blue line) and patients with high POM121 expression (green line). (B) Overall survival curves by TNM stage, TNM 0, I and II (blue line), TNM III and IV (green line). (C) Overall survival curves for tumor size, Tis, T1 and T2 (blue line), T3 and T4 (green line). (D) Overall survival curves for patients with well differentiation (blue line), patients with moderate differentiation (green line) and patients with poor differentiation (yellow line). (E) Overall survival curves by lymph node metastases, N0 (blue line), N1 (green line). (F) Overall survival curves by distant metastases, M0 (blue line), M1 (green line).

3.4 POM121 plays an oncogenic role in NSCLC cells

In further investigation of the functions of POM121 on cell biological process, shRNA-POM121 was constructed and transfected into A549 and HCC827 cell lines. POM121 protein expressions in both cell lines were reduced after transfection (Fig. 3A). In contrast to the control group, shPOM121 significantly inhibited the cell proliferation (Fig. 3B), clone formation (Fig. 3C), migration (Fig. 3D) and invasion (Fig. 3E).

Figure 3.

POM121 plays an oncogenic role in NSCLC cells. (A) A549 and HCC 827 cells showed a significant decrease in protein level after shPOM121 transfection. (B) POM121 downregulation significantly inhibited the proliferation of both cell lines. (C) A significant decrease in cell anchorage-dependent growth was detected after POM121 knockdown. (D, E) Decreased POM121 expression impaired abilities of migration (D) and invasion (E) of NSCLC cells. (F) Downregulation of POM121 inhibited the tumor growth in the xenograft mouse model.

Subcutaneous transplanted models in nude mice were constructed to explore the modulating effect of POM121 in vivo. Downregulation of POM121 reduced tumor growth within 27 days (Fig. 3F). Compared with the control group, the mice in shPOM121 group generated tumors with lessened size and weight (Fig. 3F).

3.5 POM121 inhibition blocks the TGF-

\beta

and AKT signaling pathway

In exploration of the mechanism by which POM121 promotes the malignant biological behaviors in NSCLC, 343 proteins correlated with POM121 were determined in UALCAN dataset (data not shown). Signaling pathway enrichment analysis of these proteins showed that POM121 may affect ubiquitin mediated proteolysis, cell cycle, RNA transport, TGF- $\beta$ signaling pathway and so on (Fig. 4A). Thereinto, TGF- $\beta$ signaling pathway is an important regulator contributing to cancer initiation and progression [17]. The results of qPCR and Western blot confirmed that shPOM121 significantly decreased the mRNA and protein expression of TGF- $\beta$ 1, Smad4, P-PI3K and P-AKT, and increased the expression of Smad7 (Fig. 4B–C).

Figure 4.

POM121 promotes carcinogenesis through TGF- $\beta$ /SMAD and PI3K/AKT pathways. (A) Gene set enrichment analysis indicated the association between POM121 and TGF- $\beta$ signaling pathway. (B) qPCR showed significant suppression of TGF- $\beta$ /SMAD pathway and phosphor-PI3K/AKT in shPOM121 cells compared with the control. (C) Western blot showed significant suppression of TGF- $\beta$ /SMAD pathway and phosphor-PI3K/AKT in shPOM121 cells compared with the control.

4. Discussion

In this study, qRT-PCR and IHC were performed to detect POM121 mRNA and protein levels in NSCLC tissues. POM121 mRNA levels were much higher in carcinomatous tissues than that in matched para-carcinoma tissues. As was the case with mRNA, a larger proportion of high POM121 protein expression in cancerous tissues (60.07%) was confirmed compared with normal lung tissues (22.17%). High expression of POM121 has also been confirmed in colorectal cancer [18], laryngeal cancer [19], and oral squamous cell carcinoma [20]. Our results suggested that POM121 is overexpressed in NSCLC tissues, which is consistent with previous studies.

The relationship between POM121 and clinicopathological features of NSCLC patients was analyzed according to 796 NSCLC tissue samples and their associated clinical data. Increased POM121 expression was found to be significantly associated with advanced differentiation, lymph node metastases, distant metastases, and venous invasion. Multivariate cox regression analysis and Kaplan-Meier survival curves confirmed high POM121 expression as a negative prognostic factor for NSCLC patients. These results suggested that POM121 may play a critical role in the progression and metastasis of NSCLC, and NSCLC patients with high POM121 expression had poor OS rates.

So far, investigations to explore the function of POM121 in human cancers are few. Veronica et al. demonstrated that POM121 promotes tumorigenesis, proliferation, and drug-resistance in prostate cancer cells [15]. In this study, modulating effect of POM121 on NSCLC was detected in vitro and vivo. After POM121 downregulation, both A549 and HCC827 cells presented weakened abilities of cell proliferation, migration and invasion. Subcutaneously implanted tumor model also proved negative effect of shPOM121 on NSCLC cell growth. These results confirmed that POM121 inhibition suppressed proliferation, migration and invasion of NSCLC cells, which means POM121 could contribute to NSCLC proliferation and metastasis.

TGF- $\beta$ is a cytokine that can regulate cell differentiation, proliferation, apoptosis, angiogenesis and immune response. Almost all cells can synthesize, secrete and be regulated by TGF- $\beta$ [21]. Through TGF- $\beta$ 1/2 receptors, TGF- $\beta$ recruits and activates transcription factors Smad2 and Smad3 which in turn form dimers or trimers with SMAD4 proteins, and then transfer signal into the nucleus [22]. On the one hand, TGF- $\beta$ can inhibit the growth of epithelial cells [23], and on the other hand, it can also promote the malignant biological behavior of primary tumor cells [24, 25]. Research shows that TGF- $\beta$ promotes the occurrence of EMT by activating a variety of transcription regulators, such as Twist 1, Twist 2, Snail 1 and Snail 2, resulting in enhanced cell migration and invasiveness [26, 27]. In this study, it was confirmed that POM121 silencing inhibited the expressions of TGF- $\beta$ 1 and Smad4, and increased the level of Smad7, revealing that POM121 could promote the migration and invasion of NSCLC cells through TGF- $\beta$ signaling pathway.

Beside SMAD-mediated signaling, the TGF- $\beta$ can also activate other intracellular pathways such as PI3K/ AKT pathway [28]. PI3K/Akt is a classical pathway promoting NSCLC cell proliferation [29]. This study provided the evidence that POM121 inhibition in NSCLC cells could suppress the phosphorylation of PI3K/Akt pathway, which accounts for negative effects of POM121 downregulation on proliferation in vitro and vivo.

In conclusion, the study confirmed that POM121 knockdown could weaken proliferation, migration and invasion of NSCLC through TGF- $\beta$ /SMAD and PI3K/AKT pathways. The study provides a new perspective for exploring the role of POM121 in NSCLC and a new theoretical basis for the treatment strategies for NSCLC patients.

Footnotes

Acknowledgments

We thank H. Nikki March, PhD, from LiwenBianji, Edanz Editing China (www.liwenbianji.cn/ac), for editing the English text of a draft of this manuscript. This study is supported by National Natural Science Foundation of China (82060438), Natural Science Foundation of Inner Mongolia Autonomous Region (2020MS08002), Inner Mongolia Autonomous Region Science and Technology Plan Project (2020GG0273), and Doctoral Research Station of Bayannur (BYZ2019-1-2).

Author contributions

Conception: Lirong Guan, Kun Zhao

Interpretation or analysis of data: Lirong Guan, Lingli Zhang, Tengqi Wang, Lizhou Jia, Ning Zhang

Preparation of the manuscript: Lirong Guan, Lingli Zhang, Ning Zhang, Huishan Yan

Revision for important intellectual content: Tengqi Wang, Lizhou Jia

Supervision: Kun Zhao

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POM121 promotes proliferation and metastasis in non-small-cell lung cancer through TGF- β /SMAD and PI3K/AKT pathways

Abstract

BACKGROUND:

OBJECTIVE:

METHODS:

RESULTS:

CONCLUSION:

Keywords

1. Introduction

2. Materials and methods

2.1 Tissue samples and clinical data

2.2 RNA extraction and quantitative real-time polymerase chain reaction (qRT-PCR)

2.3 Tissue microarrays (TMA) and immunohistochemistry analysis (IHC)

2.4 Cell lines and cell culture

2.5 Western blot

2.6 CCK-8 proliferation experiment

2.7 Clone formation assay

2.8 Transwell migration and invasion assay

2.9 Tumor formation experiment in nude mice

3. Results

3.1 POM121 is overexpressed in NSCLC

3.2 POM121 expression is connected with clinical characteristics in NSCLC

Table 1 Association of POM1121expression with clinicopathological characteristics in NSCLC patients

Table 2 Univariate and multivariate analysis of prognostic factors for overall survival in lung cancer

Footnotes

Acknowledgments

Author contributions

References

Table 1
Association of POM1121expression with clinicopathological characteristics in NSCLC patients

Table 2
Univariate and multivariate analysis of prognostic factors for overall survival in lung cancer