Increased expression of LINC00323 correlates with tumor progression and poor prognosis of gastric cancer

Abstract

BACKGROUD/AIMS:

LINC00323 is a novel lncRNA which has reported to play an important role in the development and recurrence in several cancers. However, the expression and predictive value of LINC00323 in gastric cancer (GC) remain mysterious.

METHODS:

LINC00323 expression in GC tissues and adjacent normal tissues was evaluated by quantitative reverse-transcription PCR (qRT-PCR). The relationship between LINC00323 expression and clinicopathological features and patients’ survival were analyzed. Univariate and multivariate survival analyses were performed.

RESULTS:

LINC00323 expression were found to be significantly increased in GC tissues. High expression of LINC00323 exerted a pro-tumor effect in the late stage of GC development. Kaplan-Meier analysis showed that patients with high LINC00323 were associated with poor overall survival (OS) and progression-free survival (PFS). Moreover, the combination of TNM stage and drinking status better identified GC patient outcome than those of TNM stage alone.

CONCLUSIONS:

Our data showed that LINC00323 overexpression might serve as a novel independent prognostic factor for survival of GC patients, suggesting LINC00323 was a potential biomarker and therapeutic target for GC.

1. Introduction

Gastric cancer (GC) is one of the most common cancer in China and is responsible for an estimated 479,000 new cases and 374,000 deaths in 2020 [1]. China accounted for nearly 50% of all newly diagnosed cases and cancer deaths worldwide, in part due to China’s large population [2]. Due to its frequently advanced stage at diagnosis, the prognosis of advanced GC is dismal, and the 5-year survival rate remains 5% [3]. The most common reason for the poor prognosis is the acquisition of chemoresistance [4]. Therefore, identifying new reliable biomarkers to predict chemoresistance and elucidating its molecular mechanisms in GC patients are essential and urgently required.

Long noncoding RNAs (lncRNAs) were previously regarded as transcriptional noise or junk molecules, larger than 200 nucleotides in size without protein coding capability [5]. However, increasing evidence has highlighted that lncRNAs are implicated in cell proliferation, metastasis, chemoresistance of many cancers through epigenetic modifications [6, 7]. In GC, Zhang et al. [8] reported that lncRNA CRNDE inhibited autophagy flux and attenuated chemoresistance by SRSF6-regulated alternative splicing of PICALM. Recently, a study showed that LOC101929709 promoted GC proliferation and migration via activating PI3K/AKT pathway [9]. As the complicated biological functions in tumor progression, lncRNAs as diagnostic and prognostic biomarkers of GC are attracting considerable attention from investigators [10].

In our previous study, we successfully constructed a cisplatin-resistant-related ceRNA network in GC chemoresistance, and four hub lncRNAs were identified as oncogenes because they were associated with poor survival of GC patients [11]. Among these four lncRNAs, some studies have found that LINC00323 was related to recurrence prognosis of cervical cancer [12], osteosarcoma [13] and breast cancer [14]. However, the expression and predictive value of LINC00323 in GC remain unknown. Therefore, in this study, we investigated the expression of LINC00323 in GC tissues, and analyzed the correlation between LINC00323 and clinicopathological parameters as well as prognosis in GC patients.

2. Materials and methods

2.1. GC tissues collection and medical records

Specimens were obtained from 149 patients were diagnosed with GC at Affiliated Cancer Hospital and Institute of Guangzhou Medical University between 2007 and 2011. Complete past medical history was collected and written informed consent was obtained from all patients or their guardians. The clinical and pathological tumor staging was determined according to the 8${}^{\text{th}}$ edition of the TNM classification of the International Union against Cancer (UICC). The follow-up time ranged from 3 to 107 months with 35 months. Overall survival (OS) was defined as the time from the date of radical surgery to the date of death or last follow-up, and progression-free survival (PFS) time was from the date of radical surgery to the date of relapse, metastasis or last follow-up. The study was reviewed and approved by the medical ethics committee of Affiliated Cancer Hospital and Institute of Guangzhou Medical University.

2.2. RNA preparation and quantitative reverse-transcription PCR (qRT-PCR)

Total RNA was extracted from GC tissues specimens using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). RNA concentration was measured by a NanoDrop 2000c spectrophotometer (ThermoScientific, USA). cDNA was synthesized with the PrimeScript RT Reagent Kit (Takara, Nanjing, China) with random primers. Gene expression was detected by qRT-PCR using the SYBR Green Premix Ex Taq II Kit (Takara, Nanjing, China) on the ABI 7500 real-time PCR system (Applied Biosystems, Foster City, CA, USA). The total RNA levels were normalized with GAPDH. For quantitative real-time PCR (qRT-PCR), the cDNA was quantified in triplicate using SYBR Green Mix (Promega, Madison, WI, USA) on a Light Cycler 480 instrument (Roche, Basel, Switzerland). The relative expression of LINC00323 was calculated using the 2${}^{-\mathrm{\Delta }\mathrm{\Delta }\text{Ct}}$ method and the results were normalized with GAPDH. The following primers were used for qRT-PCR: lncRNA LINC00323 Forward: 5${}^{\prime }$-CTAAACACTGACCTCCCGC-3${}^{\prime }$; Reverse: 5${}^{\prime }$-TTACCACTAACATTTGCTTTGTG-3${}^{\prime }$. GAPDHForward: 5${}^{\prime }$-GTCTCCTCTGACTTCAACAGCG-3${}^{\prime }$; Reverse: 5${}^{\prime }$-ACCACCCTGTTGCTGTAGCCAA-3${}^{\prime }$.

2.3. Statistical analysis

Statistical analyses in this study relied on SPSS version 24.0 statistical software package (SPSS, Chicago, IL, USA) and GraphPad software version 8.0 (San Diego, CA, USA). The significance of the differences between groups was evaluated with Student’s $t$ test, chi-squared test or Fisher’s exact test as appropriate. The Kaplan-Meier method with the log-rank test were used to estimate cancer specific survival curves. Univariate and multivariate Cox proportional hazards regression analyses were performed to identify the potential prognostic biomarkers and clinical factors. Statistical significance was considered to be represented by a value of 0.05.

3. Results

3.1. The expression of LINC00323 is overexpressed in GC tissues

We firstly investigated the expression level of LINC 00323 in GC tissues in the GEPIA database, and the results showed that LINC00323 was highly expressed in GC ( 0.001, Fig. 1A). To further evaluate the expression and clinical significance of this lncRNA in GC, we collected 149 pairs of tumor and adjacent normal tissues from GC patients at our cancer center. The results indicated that LINC00323 level was significantly upregulated in GC tissues compared with their adjacent normal tissues by RT-PCR ($P=$ 0.0001, Fig. 1B). Then, we explored the relationship between LINC00323 expression and clinicopathological features of GC patients. Unfortunately, no significant statistical association was observed between LINC00323 expression and widely recognized clinicopathological variables (Table 1). These results suggested that LINC00323 may function as an oncogene in GC.

Figure 1.

LINC00323 expression was significantly upregulated and predicted poor prognosis of GC patients. (A) Relative expression of LINC00323 in GC tissues from the GEPIA database (http://gepia.cancer-pku.cn/). STAD, Stomach adenocarcinoma. (B) Relative expression of LINC00323 between GC tissues ($n=$ 149) and adjacent normal tissues ($n=$ 24) by qRT-PCR. Results were presented as $\mathrm{\Delta }$cycle threshold ($\mathrm{\Delta }$Ct) in tumor tissues relative to adjacent normal tissues. Error bars: mean $\pm$ SD. (C) Kaplan-Meier method were performed to analyze the prognostic significance of LINC00323 expression in all patients. The P value was determined by a log-rank (mantel-cox) test.

Table 1.

Correlations between LINC00323 expression and clinicopathological features in 149 GC patients

Characteristics	Total (%)	LINC00323 expression		$P$-value
		High (N, %)	Low (N, %)
Gender				0.764
Female	46 (30.9)	22 (47.8)	24 (52.2)
Male	103 (69.1)	52 (50.5)	51 (49.5)
Age (years)				0.618
60	83 (55.7)	40 (48.2)	43 (51.8)
$⩾$ 60	66 (44.3)	34 (51.5)	32 (48.5)
Smoking status				0.564
Never	98 (65.8)	47 (48.0)	51 (52.0)
Ever	51 (34.2)	27 (52.9)	24 (47.1)
Drinking Status				0.183
Never	123 (82.6)	58 (47.2)	65 (52.8)
Ever	26 (17.4)	16 (61.5)	10 (38.5)
Histological grade				0.952
Poorly differentiation	97 (65.1)	48 (49.5)	49 (50.5)
Moderately/Well differentiation	52 (34.9)	26 (50.0)	26 (50.0)
Pathological Pattern				0.682
Adenocarcinoma	125 (83.9)	63 (50.4)	62 (49.6)
Not-simple Adenocarcinoma	24 (16.1)	11 (45.8)	13 (54.2)
Infiltration depth				0.780
Intramucosal/Submucosal/Muscle layer/Subserosa	54 (36.2)	26 (48.1)	28 (51.9)
Serosa or Adjacent structures	95 (63.8)	48 (50.5)	47 (49.5)
Tumor location				0.774
Gastric	132 (88.6)	65 (49.2)	67 (50.8)
Gastric stump/Transgastroesophageal junction	17 (11.4)	9 (52.9)	8 (47.1)
T stage				0.530
T1-T3	52 (24.9)	24 (46.2)	28 (53.8)
T4	97 (65.1)	50 (51.5)	47 (48.5)
N stage				0.566
N0-N1	53 (35.6)	28 (52.8)	25 (47.2)
N2-N3	96 (64.4)	46 (47.9)	50 (52.1)
M stage				0.820
M0	130 (88.4)	65 (50.0)	65 (50.0)
M1	17 (11.6)	8 (47.1)	9 (52.9)
TNM stage				0.738
I	9 (6.0)	6 (66.7)	3 (33.3)
II	36 (24.2)	18 (50.0)	18 (50.0)
III	86 (57.7)	42 (48.8)	44 (51.2)
IV	18 (12.1)	8 (44.4)	10 (55.6)

3.2. LINC00323 overexpression is associated with poor prognosis in GC

To determinate the prognostic value of LINC00323 in this disease, we used Kaplan-Meier methods with log-rank tests. Survival analysis displayed that 5-year OS and 5-year PFS of GC patients with high LINC00323 level were 40.2% and 32.5%, compared with 64.9% and 44.3% of those with low level, respectively (All 0.01, Fig. 1C). Univariate Cox proportional analysis revealed that TNM stage, infiltration depth, and LINC00323 expression were remarkably prognostic predictors for OS and PFS of GC patients. In addition, pathological pattern and tumor location were also prognostic predictors for only OS (All 0.05, Tables 2 and 3). Multivariate Cox regression analysis further showed that LINC00323 expression was an independent risk predictor for OS (HR: 2.419, 95% CI: 1.440–4.061, $P=$ 0.001) and PFS (HR: 1.864, 95% CI: 1.226–2.833, $P=$ 0.004) of GC patients (Tables 2 and 3). Additionally, TNM stage and infiltration depth were also independent predictors of poorer OS and PFS, and tumor location was an independent predictor of only poorer OS in GC patients (Tables 2 and 3). Therefore, these observations imply that LINC00323 is a novel prognostic biomarker for GC patients.

Table 2.

Cox regression analysis of LINC00323 and clinical characteristics associated with OS in GC patients

Clinical variables	Univariate analysis			Multivariate analysis
	HR	95% CI	$P$ value	HR	95% CI	$P$ value
OS
Gender (Male vs. Female)	0.860	0.504–1.465	0.578
Age ( 60 vs. $⩾$ 60)	0.816	0.496–1.344	0.425
Smoking status (Never vs. Ever)	0.734	0.442–1.220	0.233
Drinking status (Never vs. Ever)	0.575	0.321–1.030	0.063
TNM stage (I/II vs. III/IV)	0.232	0.110–0.488	0.001	0.249	0.116–0.534	0.001
Pathological Pattern (Adenocarcinoma vs. Non-simple adenocarcinoma)	0.504	0.282–0.902	0.021	0.700	0.374–1.310	0.265
Infiltration depth (Intramucosal/Submucosal/Muscle layer/Sub- serosa vs. Serosa or Adjacent structures)	0.279	0.145–0.535	0.001	0.382	0.195–0.747	0.005
Histological grade (Poorly differentiation vs. Moderately/ Well differentiation)	1.662	0.951–2.906	0.074
Tumor location (Gastric vs. Transgastro esophageal junction/Gastric stump)	0.433	0.230–0.815	0.009	0.405	0.205–0.802	0.009
LINC00323 expression (High vs. Low)	2.054	1.238–3.409	0.005	2.419	1.440–4.061	0.001

NOTE: OS, overall survival; GC, gastric cancer; TNM, tumour node metastasis; HR, hazard ratio; CI, confidence interval; Bold type indicates 0.05.

Table 3.

Cox regression analysis of LINC00323 and clinical characteristics associated with PFS in GC patients

Clinical variables	Univariate analysis			Multivariate analysis
	HR	95% CI	$P$ value	HR	95% CI	$P$ value
PFS
Gender (Male vs. Female)	0.913	0.584–1.427	0.690
Age ( 60 vs. $⩾$ 60)	1.010	0.666–1.531	0.963
Smoking status (Never vs. Ever)	0.954	0.621–1.465	0.829
Drinking status (Never vs. Ever)	0.877	0.517–1.488	0.627
TNM stage (I/II vs. III/IV)	0.318	0.185–0.548	0.001	0.360	0.206–0.628	0.001
Pathological Pattern (Adenocarcinoma vs. Non-simple adenocarcinoma)	0.688	0.406–1.167	0.166
Infiltration depth (Intramucosal/Submucosal/Muscle layer/Sub- serosa vs. Serosa or Adjacent structures)	0.390	0.241–0.633	0.001	0.497	0.303–0.816	0.006
Histological grade (Poorly differentiation vs. Moderately/ Well differentiation)	1.423	0.907–2.235	0.125
Tumor location (Gastric vs. Transgastro esophageal junction/Gastric stump)	0.621	0.344–1.119	0.113
LINC00323 expression (High vs. Low)	1.724	1.136–2.616	0.010	1.864	1.226–2.833	0.004

NOTE: PFS, progression-free survival; GC, gastric cancer; TNM, tumour node metastasis; HR, hazard ratio; CI, confidence interval; Bold type indicates 0.05.

3.3. LINC00323 overexpression was positively correlated with tumor progression in GC patients

Next, the prognostic value of LINC00323 expression was evaluated in stratified analysis. When patients were stratified by T stage, survival analysis revealed that LINC00323 expression is a prognostic factor for OS ($P=$ 0.014) and PFS ($P=$ 0.042) in patients with T4 stage (Fig. 2B), but not in those with T1-3 (Fig. 2A). Furthermore, we also found that LINC00323 expression was a risk predictor for OS and PFS in patients with N2-3 stage (Fig. 2D), M0 stage (Fig. 2E), adenocarcinoma (AD) (Fig. 2G), deeper tumor invasion (Serosa or Adjacent structures, S or AS) (Fig. 2H), poorly differentiation (Fig. 2I), and tumor originated from stomach (Fig. 2J), but not in those with N0-1 stage (Fig. 2C). In addition, M1 stage patients with high LIN00323 level have only poorer PFS (not OS) than those with low level (Fig. 2F). These data suggested that LINC00323 mainly serves as a potential prognostic biomarker for patients with advanced GC.

Figure 2.

Overexpression of LINC00323 was positively associated with poor outcome of GC patients with different clinicopathological features. (A–F) The survival curve (OS, left panel; PFS, right panel) based on different LINC00323 experssion in GC patients with stage T1-3 and T4 (A and B), patients with stage N0-1 and N2-3 (C and D), patients with stage M0 and M1 (E and F). (G–J) The survival curve (OS, left panel; PFS, right panel) based on different LINC00323 experssion in GC patients with pathological pattern: adenocarcinoma (G), patients with infiltration depth: serosa or adjacent structures (H), patients with poorly differentiation (I) and patients with tumor originated from stomach (J). AD, adenocarcinoma; S or AS, serosa or adjacent structures. The P value was determined by a log-rank (mantel-cox) test.

3.4. Drinking status combined with TNM stage better predicted the outcome of GC patients

It has been well-established that TNM staging system is the key determinant for predicting prognosis and guiding the treatment of cancer patients. However, as shown in Fig. 3A and 3B, TNM staging system in our cohort did not accurately discriminate survival outcomes of GC patients with stage I and II (OS, $P=$ 0.697; PFS, $P=$ 0.931), which was also observed in another study [15]. Therefore, many experts strongly recommend that the find of new biomarkers to improve the discriminatory power of the TNM staging system.

Figure 3.

Drinking status combined with TNM stage better predicted the prognosis of GC patients. (A and B) OS and PFS of all patients were predicted by TNM staging system. (C and D) The survival curve (OS, left panel; PFS, right panel) based on different LINC00323 experssion in GC patients with never smoking or never drinking. (E and F) All patients were stratified into three gourps according to the combined score of TNM stage and drinking status. OS and PFS outcomes of GC patients were predicted by the combined groups. (G and H) Receiver operating characteristic (ROC) analysis compares OS and PFS of GC patients by TNM stage, drinking status and the combined groups. The result shows that the area under the curve (AUC) of TNM$+$drink is the largest among the three predictors.

Since habitual drinking and smoking are adverse prognostic factors for GC [16], patients were stratified into two subgroups by drinking or smoking status. Interestingly, LINC00323 expression is a risk predictor for OS and PFS in GC patients with never smoking and never drinking (Fig. 3C and 3D), but not in those with ever smoking or ever drinking (Fig. S1A and S1B). Therefore, we hypothesized that the combination of smoking or drinking status and TNM staging system may improve the prediction of survival in GC patients. To this end, we established a combined group based on the sum of TNM stage (1 to 4) and drinking status (0 or 1) for every patient. As expected, survival analysis showed that patients with Group 1 (score 1 or 2), Group 2 (score 3) and Group 3 (score 4 or 5) have significantly different 5-year OS rates, 83.7%, 52.6% and 15.5%, respectively (Fig. 3E). Similar predictive accuracy for PFS was observed in patients with different groups (Fig. 3F). Furthermore, we performed receiver operating characteristic (ROC) curve analysis to compare the discriminative power of TNM stage, drinking status and the combination of both. Among three predictors, TNM$+$Drink which yielded AUC values of 0.694 for OS and 0.698 for PFS, remarkably improved the predictive accuracy (Fig. 3G and 3H). Actually, we also established another combined group based on the sum of LINC00323 expression and TNM stage by the above method. However, the AUC values of LINC00323 and TNM$+$LINC00323 did not perform better than that of TNM stage for OS (AUC: 0.572 vs. 0.593 vs. 0.676) and PFS (AUC: 0.511 vs. 0.657 vs. 0.678) (Fig. S1C and S1D). Taken together, these results suggested that drinking status combined with TNM stage obviously improve prognostic prediction for GC patients.

4. Discussion

With the rapid development of next-generation sequencing (NGS) and bioinformatics, thousands of oncogenic and tumor suppressive lncRNAs have been annotated in various cancer, which exert complicated biological functions during tumorigenesis and development [17]. It was now become widely accepted that lncRNAs participate in cancer initiation, progression, apoptosis and chemoresistance, such as GC [18]. And many lncRNAs may become potential tumor biomarkers for tumor diagnosis and treatment [19]. Our previous study found that 71 differentially expressed lncRNAs were identified as oncogenes in GC by bioinformatics [11]. Further analysis showed that LINC00323 overexpression was positively correlated with poor prognosis of GC patients based on the Genomic Data Commons (GDC) data portal [11]. To corroborate this finding, we collected 149 GC tissues from our cancer center. qRT-PCR analysis showed that LINC00323 was significantly upregulated and positively associated with poor survival in GC patients. Our results appear to be similar to previous findings that LINC00323 behaved as an oncogene whose upregulation resulted in increased tumor progression and recurrence in some cancers [12, 13, 14].

In this study, although the correlation between LINC00323 and clinicopathological variables was no statistically significant, stratified analysis demonstrated that LINC00323 maintained its predictive value for patient survival in several subgroups. Consistent with our results, previous reports indicated that TRIM24 or LINC001133 expression was a prognostic factor for OS and DFS in esophageal squamous cell carcinoma (ESCC) patients with different subgroups [20, 21]. These findings suggested that LINC00323 is a novel prognostic biomarker for GC and may be involved in the late stage of GC development.

Several lines of evidences reported that TNM staging system of GC is not adequate for definition of prognosis and therapy [22, 23]. The same problem was observed in our cohort. To address this problem, we constructed a new risk model based on a combination of drinking status and TNM stage, which could provide better predictive accuracy for patient survival than TNM stage alone. We also examined the impact of LINC00323 expression combined with TNM stage on patient outcome. Unfortunately, neither LINC00323 alone nor LINC00323 combined with TNM stage had better sensitivity and specificity than those of TNM stage alone. Therefore, these results suggested that TNM stage and drinking status in combination were potential biomarkers for improving prognostic value for GC patients.

5. Conclusion

In summary, we found for the first time that LINC 00323 expression was upregulated and independently predicted poor outcomes in GC. Furthermore, high expression of LINC00323 was positively associated with the progression of GC patients. Intriguingly, the combination of TNM stage and drinking status better identified GC patient outcome. These results implied that LINC00323 functions as an oncogene and a potential biomarker for diagnosis and prognosis in GC patients.

Authors’ contribution

Conception: author Xian-zi Yang.

Interpretation Or Analysis Of Data: author Si-yu Zhu, Jin-jie Li and Qin Lu.

Preparation Of The Manuscript: all authors.

Revision For Important Intellectual Content: author Xian-zi Yang.

Supervision: author Shu-zhong Cui, Li-si Zeng and Xian-zi Yang.

Patients consent statement

GC tissues collection and medical records.

Specimens were obtained from 149 patients were diagnosed with GC at Affiliated Cancer Hospital and Institute of Guangzhou Medical University between 2007 and 2011. Complete past medical history was collected and written informed consent was obtained from all patients or their guardians. The clinical and pathological tumor staging was determined according to the 8th edition of the TNM classification of the International Union against Cancer (UICC). The follow-up time ranged from 3 to 107 months with 35 months. Overall survival (OS) was defined as the time from the date of radical surgery to the date of death or last follow-up, and progression-free survival (PFS) time was from the date of radical surgery to the date of relapse, metastasis or last follow-up. The study was reviewed and approved by the medical ethics committee of Affiliated Cancer Hospital and Institute of Guangzhou Medical University.

Supplementary data

The supplementary files are available to download from http://dx.doi.org/10.3233/CBM-230031.

Conflict of interest

There is no conflict of interest disclosures from any authors.