# LINC01426 Triggers Growth and Metastasis of Lung Adenocarcinoma as a Prognostic Indicator

Authors: Youjun Deng; Songhua Cai; Wenyi Liu; Chenglin Yang; Xiaotong Guo
Journal: BioMed Research International
 (2022)

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## Abstract

The vital regulation of abnormally expressed lncRNAs in human cancers has been identified. This study is aimed at illustrating the role of LINC01426 in influencing malignant behaviors of lung adenocarcinoma (LUAD) and the possible mechanism. Differential expressions of LINC01426 in a downloaded profile containing LUAD and normal tissues were analyzed using Gene Expression Profiling Interactive Analysis (GEPIA) database and were reconfirmed in clinical samples collected in our hospital. In addition, LINC01426 level in lung carcinoma cell lines was detected by quantitative real-time polymerase chain reaction (qRT-PCR) as well. The relationship between LINC01426 expression and the age, tumor node metastasis (TNM) staging, lymphatic metastasis, tumor differentiation, and overall survival of LUAD was analyzed. After intervening LINC01426 level in H1299 and PC9 cells, proliferative and metastatic changes were assessed by functional experiments. LINC01426 was upregulated in LUAD tissues and cell lines. Its level was closely linked to TNM staging, lymphatic metastasis, tumor differentiation, and overall survival of LUAD. Knockdown of LINC01426 suppressed proliferative and metastatic abilities in H1299 and PC9 cells. LINC01246 is upregulated in LUAD samples and predicts a poor prognosis. It drives malignant process of LUAD via stimulating proliferative and metastatic abilities.


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## Body

## 1. Introduction

Lung carcinoma is a globally prevalent malignant tumor [1]. Non-small-cell lung carcinoma (NSCLC) and small-cell lung carcinoma (SCLC) are the major subtypes of lung carcinoma. Lung adenocarcinoma (LUAD) is the most common subtype of NSCLC (40%) [2]. Generally speaking, surgical resection is preferred to early stage lung carcinoma. The postoperative 5-year survival of stage I NSCLC is up to 45-65% [3]. LUAD is featured by the high rate of metastasis, and about 90% of LUAD patients die of metastatic lesions, leading to a low 5-year survival (16%) [4]. It is of significance to clarify the molecular mechanism of LUAD progression, thus aiming to improve their survival.lncRNAs cannot be translated to proteins [5]. They are a type of noncoding RNAs containing more than 200 nucleotides [6, 7]. Vast evidences have proven the vital function of lncRNAs in human cancers. It is reported that LINC01116 aggravates the deterioration of epithelial ovarian cancer through mediating cell apoptosis [8]. By activating the miR-4316/ZBTB2 axis, lncRNA CALML3-AS1 induces the malignant progression of bladder cancer [9]. The proliferative and metastatic potentials of breast cancer are stimulated by lncRNA EZR-AS1 via the Wnt signaling [10]. LINC01426, also known as LincRNA-uc002yug.2, is located on human chromosome 21 [11]. Previous studies have reported that LINC01426 is abnormally expressed in esophageal cancer [12] and colorectal carcinoma specimens [13]. It is indicated that LINC01426 may serve as a tumor biomarker. Several previous reports demonstrated the functions of LINC01426 in lung cancer [14–17]. However, the underlying molecular mechanism of LINC01426 in lung adenocarcinoma still remains to be further explored.In the past decades, the involvement of lncRNAs in LUAD progression has been detected [18]. For example, lncRNA GMDS-AS1 suppresses the carcinogenesis of LUAD by regulating the miR-96-5p/CYLD axis [19]. lncRNA DGCR5 drives the growth of LUAD by downregulating hsa-mir-22-3p [20]. Serving as a ceRNA, LINC00665 regulates the AKR1B10-ERK axis via binding miR-98, thus mediating the progression of LUAD [21].LINC01426 is previously identified to be upregulated in LUAD profiling [22]. This study is aimed at uncovering the regulatory effects of LINC01426 on the malignant behaviors of LUAD in vitro and the possible mechanism.


## 2. Methods

### 2.1. GEPIA Dataset Analysis

LUAD-TCGA profile was downloaded fromhttps://portal.GDC.Cancer.gov. Differential expressions of LINC01426 in LUAD tissues (n=483) and normal tissues (n=347) were analyzed using the R project.


### 2.2. Sample Collection

LUAD and adjacent normal tissues were collected in Cancer Hospital and Shenzhen Hospital, Chinese Academy of Medical Sciences and pathologically confirmed. Tumor node metastasis (TNM) staging was defined based on the criteria released by the Union for International Cancer Control (UICC). Samples were frozen in liquid nitrogen and stored at -80°C. Included LUAD patients did not have chemotherapy or radiotherapy and signed informed consent. This study got approval by Ethics Committee of Cancer Hospital and Shenzhen Hospital, Chinese Academy of Medical Sciences, and experimental protocols conformed to the Helsinki Declaration.


### 2.3. Cell Culture

The human bronchial epithelioid cell line (HBE) and lung carcinoma cell lines (A549, H1299, PC9, and H1975) were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were cultured in RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin (GIBCO-BRL; Invitrogen, Carlsbad, CA, USA).


### 2.4. Cell Transfection

Three lines of LINC01426 siRNAs (si-LINC01426-1, si-LINC01426-2, si-LINC01426-3) and negative control (si-NC) were purchased from RiboBio (Guangzhou, China). Cells were grown to 60% of adherence and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).


### 2.5. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Qualified RNA was reversely transcribed into complementary deoxyribose nucleic acid (cDNA) using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). The cDNA was subjected to qRT-PCR using the SYBR Green Master Mix (Applied Biosystems, San Diego, CA, USA). Sequences were as follows: LINC01426: forward: 5′-TCCCGAAGTGCATAGGCAAG-3′, reverse: 5′-CCTGAACAAGTTGGGGAGGA-3′; GAPDH: forward: 5′-TATGATGATATCAAGAGGGTAGT-3′, reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′.


### 2.6. Cell Counting Kit-8 (CCK-8)

Cells were inoculated in a 96-well plate with4×103 cells/well. At 0, 24, 48, 72, and 96 h, absorbance value at 450 nm of each sample was recorded using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) for plotting the viability curves.


### 2.7. 5-Ethynyl-2′-Deoxyuridine (EdU) Assay

Cells were inoculated in 96-well plates with1×104 cells per well. 10 μl of EdU (50 μM) was applied in each well for cell labeling. Two hours later, cells were incubated with 4% methanol for 20 min, followed by PBS washing and incubation with Cell-Light™EdU Apollo®488 (Life Technologies, New York, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclei staining in the dark. Finally, cells were washed in PBS and captured.


### 2.8. Colony Formation Assay

4×102 cells were inoculated in the 6 cm dish and cultivated for 14 days. Visible colonies were fixed in 4% paraformaldehyde for 30 min and stained using 0.5% crystal violet for 30 min. The number of visible colonies was counted.


### 2.9. Wound Healing Assay

Cells were prepared into suspension with1×105 cells/ml and implanted in 6-well plates. Until 80% of cell attachment, an artificial wound was made using a sterilized pipette tip. Cells were washed in phosphate buffered saline (PBS) for 2-3 times and cultured in the medium containing 1% FBS. 24 hours later, wound closure was captured for calculating the percentage of wound healing.


### 2.10. Transwell

Transwell chambers (BD, San Diego, CA, USA) coated with Matrigel were used.4×103 cells were implanted on the top, while 600 μl of medium was applied on the bottom. After 48 h incubation, migratory cells on the bottom were reacted with 15 min methanol and 20 min crystal violet and captured using a microscope. Invasive cells were counted in 10 randomly selected fields per sample. Migratory cell number was similarly detected using Transwell chambers without Matrigel precoating.


### 2.11. Statistical Analyses

Data were expressed asmean±standarddeviation. Data processing was conducted by GraphPad Prism 6 (La Jolla, CA, USA). Each experiment was performed in triplicate. Differences between groups were compared using the Student’s t-test. Kaplan-Meier survival curves were plotted and compared based on LINC01426 level. P<0.05 was considered as statistically significant.


## 2.1. GEPIA Dataset Analysis

LUAD-TCGA profile was downloaded fromhttps://portal.GDC.Cancer.gov. Differential expressions of LINC01426 in LUAD tissues (n=483) and normal tissues (n=347) were analyzed using the R project.


## 2.2. Sample Collection

LUAD and adjacent normal tissues were collected in Cancer Hospital and Shenzhen Hospital, Chinese Academy of Medical Sciences and pathologically confirmed. Tumor node metastasis (TNM) staging was defined based on the criteria released by the Union for International Cancer Control (UICC). Samples were frozen in liquid nitrogen and stored at -80°C. Included LUAD patients did not have chemotherapy or radiotherapy and signed informed consent. This study got approval by Ethics Committee of Cancer Hospital and Shenzhen Hospital, Chinese Academy of Medical Sciences, and experimental protocols conformed to the Helsinki Declaration.


## 2.3. Cell Culture

The human bronchial epithelioid cell line (HBE) and lung carcinoma cell lines (A549, H1299, PC9, and H1975) were purchased from Shanghai Institute of Biochemistry and Cell Biology (Shanghai, China). Cells were cultured in RPMI 1640 containing 10% FBS and 1% penicillin-streptomycin (GIBCO-BRL; Invitrogen, Carlsbad, CA, USA).


## 2.4. Cell Transfection

Three lines of LINC01426 siRNAs (si-LINC01426-1, si-LINC01426-2, si-LINC01426-3) and negative control (si-NC) were purchased from RiboBio (Guangzhou, China). Cells were grown to 60% of adherence and transfected using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA).


## 2.5. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

Qualified RNA was reversely transcribed into complementary deoxyribose nucleic acid (cDNA) using the PrimeScript RT reagent Kit (TaKaRa, Dalian, China). The cDNA was subjected to qRT-PCR using the SYBR Green Master Mix (Applied Biosystems, San Diego, CA, USA). Sequences were as follows: LINC01426: forward: 5′-TCCCGAAGTGCATAGGCAAG-3′, reverse: 5′-CCTGAACAAGTTGGGGAGGA-3′; GAPDH: forward: 5′-TATGATGATATCAAGAGGGTAGT-3′, reverse: 5′-TGTATCCAAACTCATTGTCATAC-3′.


## 2.6. Cell Counting Kit-8 (CCK-8)

Cells were inoculated in a 96-well plate with4×103 cells/well. At 0, 24, 48, 72, and 96 h, absorbance value at 450 nm of each sample was recorded using the CCK-8 kit (Dojindo Laboratories, Kumamoto, Japan) for plotting the viability curves.


## 2.7. 5-Ethynyl-2′-Deoxyuridine (EdU) Assay

Cells were inoculated in 96-well plates with1×104 cells per well. 10 μl of EdU (50 μM) was applied in each well for cell labeling. Two hours later, cells were incubated with 4% methanol for 20 min, followed by PBS washing and incubation with Cell-Light™EdU Apollo®488 (Life Technologies, New York, USA). 4′,6-Diamidino-2-phenylindole (DAPI) was used for nuclei staining in the dark. Finally, cells were washed in PBS and captured.


## 2.8. Colony Formation Assay

4×102 cells were inoculated in the 6 cm dish and cultivated for 14 days. Visible colonies were fixed in 4% paraformaldehyde for 30 min and stained using 0.5% crystal violet for 30 min. The number of visible colonies was counted.


## 2.9. Wound Healing Assay

Cells were prepared into suspension with1×105 cells/ml and implanted in 6-well plates. Until 80% of cell attachment, an artificial wound was made using a sterilized pipette tip. Cells were washed in phosphate buffered saline (PBS) for 2-3 times and cultured in the medium containing 1% FBS. 24 hours later, wound closure was captured for calculating the percentage of wound healing.


## 2.10. Transwell

Transwell chambers (BD, San Diego, CA, USA) coated with Matrigel were used.4×103 cells were implanted on the top, while 600 μl of medium was applied on the bottom. After 48 h incubation, migratory cells on the bottom were reacted with 15 min methanol and 20 min crystal violet and captured using a microscope. Invasive cells were counted in 10 randomly selected fields per sample. Migratory cell number was similarly detected using Transwell chambers without Matrigel precoating.


## 2.11. Statistical Analyses

Data were expressed asmean±standarddeviation. Data processing was conducted by GraphPad Prism 6 (La Jolla, CA, USA). Each experiment was performed in triplicate. Differences between groups were compared using the Student’s t-test. Kaplan-Meier survival curves were plotted and compared based on LINC01426 level. P<0.05 was considered as statistically significant.


## 3. Results

### 3.1. Abnormal Upregulation of LINC01426 in LUAD

Through analyzing LUAD profiling downloaded from GEPIA database, it is found that LINC01426 was highly expressed in LUAD tissues than normal ones (Figure1(a)). Consistently, LINC01426 was upregulated in LUAD tissues collected in our hospital (Figure 1(b)). In addition, compared with the human bronchial epithelioid cell line, LINC01426 level was higher in lung carcinoma cell lines (Figure 1(c)). In the following in vitro experiments, H1299 and PC9 cells were utilized.Figure 1
Abnormal upregulation of LINC01426 in LUAD: (a) differential expressions of LINC01426 in LUAD tissues and normal tissues in the profile downloaded from GEPIA database; (b) LINC01426 levels in LUAD tissues and adjacent normal ones; (c) LINC01426 levels in lung carcinoma cell lines;∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001.
(a)(b)(c)


### 3.2. Correlation between LINC01426 and Clinical Characteristics of LUAD

Clinical data of enrolled LUAD patients were recorded for analyses. No significant difference in LINC01426 level was found between LUAD patients over 60 years and whom younger than 60 years (Figure2(a)). Higher level of LINC01426 was detected in T3-T4 LUAD patients in comparison to T1-T2 patients (Figure 2(b)). LUAD patients with lymphatic metastasis expressed higher level of LINC01426 than nonmetastasis patients (Figure 2(c)). Furthermore, based on tumor differentiation, stage III LUAD patients had higher level of LINC01426 compared with stage I-II patients (Figure 2(d)). Kaplan-Meier curves uncovered poor prognosis in LUAD patients overexpressing LINC01426 (Figure 2(e)). It is indicated that LINC01426 could result in poor prognosis in LUAD.Figure 2
Correlation between LINC01426 and clinical characteristics of LUAD: (a) correlation between LINC01426 and age in LUAD patients; (b) correlation between LINC01426 and TNM staging in LUAD patients; (c) correlation between LINC01426 and lymphatic metastasis in LUAD patients; (d) correlation between LINC01426 and differentiation level in LUAD patients; (e) overall survival in LUAD patients based on LINC01426 level;∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001; ns: no significant difference.
(a)(b)(c)(d)(e)


### 3.3. Knockdown of LINC01426 Attenuated Proliferative Ability in LUAD

To explore biological functions of LINC01426 in LUAD cell behaviors, three lines of LINC01426 siRNAs were constructed. In particular, transfection efficacy of si-LINC01426-1 had the best efficacy, and it was used in the following experiments (Figure3(a)). Knockdown of LINC01426 remarkably decreased viability in H1299 and PC9 cells (Figure 3(b)). Lower colony number was detected in H1299 and PC9 cells transfected with si-LINC01426 than those of controls (Figure 3(c)). Moreover, EdU assay showed that knockdown of LINC01426 reduced EdU-stained cell ratio (Figure 3(d)). It is concluded that LINC01426 was able to stimulate LUAD proliferation.Figure 3
Knockdown of LINC01426 attenuated proliferative ability in LUAD: (a) transfection efficacy of LINC01426 siRNAs in H1299 and PC9 cells; (b) viability in H1299 and PC9 cells transfected with si-LINC01426; (c) clonality in H1299 and PC9 cells transfected with si-LINC01426; (d) EdU-stained cells in H1299 and PC9 cells transfected with si-LINC01426;∗P<0.05, ∗∗P<0.01.
(a)(b)(c)(d)


### 3.4. Knockdown of LINC01426 Attenuated Metastatic Ability in LUAD

Subsequently, migratory ability in LUAD influenced by LINC01426 was assessed by wound healing and Transwell assay. After 24 h cell culture, weakened wound healing capacity was observed in H1299 and PC9 cells with LINC01426 knockdown than those of controls (Figure4(a)). In addition, migratory and invasive cell numbers were reduced by transfection of si-LINC01426 (Figures 4(b) and 4(c)).Figure 4
Knockdown of LINC01426 attenuated metastatic ability in LUAD: (a) distance of wound healing in H1299 and PC9 cells transfected with si-LINC01426; (b) migration in H1299 and PC9 cells transfected with si-LINC01426; (c) invasion in H1299 and PC9 cells transfected with si-LINC01426;∗P<0.05, ∗∗P<0.01; ns: no significant difference.
(a)(b)(c)


## 3.1. Abnormal Upregulation of LINC01426 in LUAD

Through analyzing LUAD profiling downloaded from GEPIA database, it is found that LINC01426 was highly expressed in LUAD tissues than normal ones (Figure1(a)). Consistently, LINC01426 was upregulated in LUAD tissues collected in our hospital (Figure 1(b)). In addition, compared with the human bronchial epithelioid cell line, LINC01426 level was higher in lung carcinoma cell lines (Figure 1(c)). In the following in vitro experiments, H1299 and PC9 cells were utilized.Figure 1
Abnormal upregulation of LINC01426 in LUAD: (a) differential expressions of LINC01426 in LUAD tissues and normal tissues in the profile downloaded from GEPIA database; (b) LINC01426 levels in LUAD tissues and adjacent normal ones; (c) LINC01426 levels in lung carcinoma cell lines;∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001.
(a)(b)(c)


## 3.2. Correlation between LINC01426 and Clinical Characteristics of LUAD

Clinical data of enrolled LUAD patients were recorded for analyses. No significant difference in LINC01426 level was found between LUAD patients over 60 years and whom younger than 60 years (Figure2(a)). Higher level of LINC01426 was detected in T3-T4 LUAD patients in comparison to T1-T2 patients (Figure 2(b)). LUAD patients with lymphatic metastasis expressed higher level of LINC01426 than nonmetastasis patients (Figure 2(c)). Furthermore, based on tumor differentiation, stage III LUAD patients had higher level of LINC01426 compared with stage I-II patients (Figure 2(d)). Kaplan-Meier curves uncovered poor prognosis in LUAD patients overexpressing LINC01426 (Figure 2(e)). It is indicated that LINC01426 could result in poor prognosis in LUAD.Figure 2
Correlation between LINC01426 and clinical characteristics of LUAD: (a) correlation between LINC01426 and age in LUAD patients; (b) correlation between LINC01426 and TNM staging in LUAD patients; (c) correlation between LINC01426 and lymphatic metastasis in LUAD patients; (d) correlation between LINC01426 and differentiation level in LUAD patients; (e) overall survival in LUAD patients based on LINC01426 level;∗P<0.05, ∗∗P<0.01, ∗∗∗P<0.001; ns: no significant difference.
(a)(b)(c)(d)(e)


## 3.3. Knockdown of LINC01426 Attenuated Proliferative Ability in LUAD

To explore biological functions of LINC01426 in LUAD cell behaviors, three lines of LINC01426 siRNAs were constructed. In particular, transfection efficacy of si-LINC01426-1 had the best efficacy, and it was used in the following experiments (Figure3(a)). Knockdown of LINC01426 remarkably decreased viability in H1299 and PC9 cells (Figure 3(b)). Lower colony number was detected in H1299 and PC9 cells transfected with si-LINC01426 than those of controls (Figure 3(c)). Moreover, EdU assay showed that knockdown of LINC01426 reduced EdU-stained cell ratio (Figure 3(d)). It is concluded that LINC01426 was able to stimulate LUAD proliferation.Figure 3
Knockdown of LINC01426 attenuated proliferative ability in LUAD: (a) transfection efficacy of LINC01426 siRNAs in H1299 and PC9 cells; (b) viability in H1299 and PC9 cells transfected with si-LINC01426; (c) clonality in H1299 and PC9 cells transfected with si-LINC01426; (d) EdU-stained cells in H1299 and PC9 cells transfected with si-LINC01426;∗P<0.05, ∗∗P<0.01.
(a)(b)(c)(d)


## 3.4. Knockdown of LINC01426 Attenuated Metastatic Ability in LUAD

Subsequently, migratory ability in LUAD influenced by LINC01426 was assessed by wound healing and Transwell assay. After 24 h cell culture, weakened wound healing capacity was observed in H1299 and PC9 cells with LINC01426 knockdown than those of controls (Figure4(a)). In addition, migratory and invasive cell numbers were reduced by transfection of si-LINC01426 (Figures 4(b) and 4(c)).Figure 4
Knockdown of LINC01426 attenuated metastatic ability in LUAD: (a) distance of wound healing in H1299 and PC9 cells transfected with si-LINC01426; (b) migration in H1299 and PC9 cells transfected with si-LINC01426; (c) invasion in H1299 and PC9 cells transfected with si-LINC01426;∗P<0.05, ∗∗P<0.01; ns: no significant difference.
(a)(b)(c)


## 4. Discussion

According to the latest report released by the International Agency for Research on Cancer, lung carcinoma remains one of the malignant tumors with the highest morbidity and mortality [23]. Due to the high invasiveness and metastasis rates of lung carcinoma, its death cases have accounted for 25% of global cancer deaths [1]. The clinical outcomes of LUAD are far away from satisfy even though medical technologies have been constantly progressed [24]. Potential pathogenic factors for LUAD are diverse, including smoking, air pollution, and aging [25]. Currently, lncRNAs have been considered as risk factors for LUAD, such as lncRNA LOXL1-AS1 [26], LINC00857 [27], and lncRNA HAGLR [28].lncRNAs account for more than 98% of transcripts, and they are transcripts with limited or no protein-encoding functions [29]. lncRNAs are extensively involved in regulating biological activities and pathological processes [30]. Serving as vital regulators, lncRNAs are involved in tumor cell phenotypes [31]. Similar to proteins, biological functions of lncRNAs largely depend on their subcellular distribution. Usually, lncRNAs are responsible for maintaining the nuclear structural integrity. lncRNAs distributed in the nucleus are able to regulate expressions of surrounding genes through interacting with intracellular molecules (i.e., proteins, RNAs, and DNAs) [32, 33]. Very recently, the diagnostic or therapeutic potential of lncRNAs has been well concerned. lncRNA AK021443 stimulates proliferative and migratory abilities of hepatocellular carcinoma through regulating EMT [34]. Proliferative and migratory capacities of papillary thyroid carcinoma are enhanced by lncRNA SNHG12 through the activation of the Wnt signaling [35]. Knockdown of LINC00673 attenuates metastasis in glioma through the PI3K/AKT signaling [36]. LINC01426 is 22,564 bp long, which was initially detected to be highly expressed in esophageal squamous cell carcinoma [12]. It induces the tumorigenesis of esophageal squamous cell carcinoma by promoting alternative splicing of RUNX1 to the shorter RUNX1a subtype. Wang et al. [11] proposed that LINC01426 can be a novel therapeutic target for glioma. Consistently, our findings confirmed the upregulation of LINC01426 in LUAD samples, which was correlated to TNM staging, lymphatic metastasis, tumor differentiation, and overall survival of LUAD. By transfection of si-LINC01426, proliferative and metastatic abilities in H1299 and PC9 cells were suppressed. To sum up, LINC01426 may be an oncogene involved in the progression of LUAD, and it can be utilized as a potential therapeutic target.There are still some obvious limitations in this study. For example, most of the analyses were performed based on the GEPIA dataset. The sample size of the patients collected from our institutions was small. What is more, lack of the in vivo experiments, such as tumorgenesis assay in nude mice, weakened the evidence level of our conclusions. In our future study, we will complete these assays to further explore the role of LINC01426 in the tumor growth of lung cancer.


## 5. Conclusion

LINC01246 is upregulated in LUAD samples and predicts a poor prognosis. It drives malignant process of LUADvia stimulating proliferative and metastatic abilities.



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*Source: 6175053-2022-05-17.xml*