AL365181.3 as a novel prognostic biomarker for lung adenocarcinoma

doi:10.21203/rs.3.rs-4855196/v1

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AL365181.3 as a novel prognostic biomarker for lung adenocarcinoma

https://doi.org/10.21203/rs.3.rs-4855196/v1

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As a lncRNA, AL365181.3 is aberrantly expressed in multiple cancer types, including lung adenocarcinoma (LUAD). However, the biological process underlying the ability of AL365181.3 to promote the progression of LUAD is unclear. Here, the pancancer expression level of AL365181.3 was analyzed using the TCGA and GTEx databases, as well as its clinical characteristics and prognostic value. Finally, the in vitro and in vivo biological functions of AL365181.3 in LUAD were revealed by using various functional assays. We found that AL365181.3 was significantly more highly expressed in many types of cancer tissues, including LUAD tissues, than in adjacent normal tissues. LUAD patients with high AL365181.3 expression had poor prognoses. Functional enrichment analyses indicated that AL365181.3 is involved in the regulation of metabolism, MAPK signaling and other tumor regulatory signaling pathways. Finally, we found that knockdown of AL365181.3 reduced the proliferation and migratory capacity of LUAD cells, and knockdown of AL365181.3 resulted in a reduced in vivo tumorigenic capacity of LUAD cells. These findings provide a comprehensive understanding of the role of AL365181.3 in LUAD.

Biological sciences/Cancer/Lung cancer/Non small cell lung cancer

Biological sciences/Computational biology and bioinformatics/Genome informatics

AL365181.3

LUAD

prognostic marker

proliferation

migration

Lung cancer is a leading cause of cancer-related mortality worldwide[1], and its incidence and mortality rates have been increasing annually. Lung cancer has an insidious onset, and most lung cancer patients have already developed distant metastases by the time of diagnosis, limiting the typical 5-year survival rate of individuals with lung cancer to no more than 25%[2]. Among these subtypes, lung adenocarcinoma (LUAD) is the most common molecular subtype of non-small cell lung cancer (NSCLC), and its proportion of NSCLC is gradually increasing to almost 50% of lung cancers[3]. Although considerable progress has been made in the past several decades in various treatments for lung cancer[4], the morbidity and mortality of LUAD remain high[5]. Hence, there is an immediate need for more effective markers for LUAD prevention and therapeutic targets for improved diagnosis and treatment.

A kind of noncoding RNA molecule longer than 200 nucleotides that does not have an open reading frame to encode proteins is known as a long noncoding RNA (lncRNA)[6], and these molecules play a critical regulatory role in the development of cancers[7]. The rich variety of lncRNAs, their spatial and temporal specific expression and diverse regulatory mechanisms make them unique diagnostic and prognostic markers and molecular targets. Recent studies on the functions, mechanisms of action and clinical significance of lncRNAs in various tumors are increasing annually. LncRNAs have been demonstrated to be involved in the regulation of tumor cell proliferation, apoptosis, cell cycle progression, invasion, metastasis, tumor stem cell stemness maintenance, the tumor microenvironment and metabolism and play important roles in tumor development and resistance to radiotherapy[8–11]. Recent studies have identified a series of lncRNAs, such as iron death-associated lncRNAs[12, 13], scorch death-associated lncRNAs[14, 15] and methylation-driven lncRNAs, as prognostic markers for LUAD[16]. These studies suggest that lncRNAs not only are potential biomarkers for the clinical treatment of LUAD but are also expected to be targets for molecularly targeted therapies in LUAD, with important clinical translational value. Consequently, the study of the mechanism of lncRNA-related molecules has become a hotspot of research in the field of oncology.

In this investigation, we integrated and analyzed the transcriptome sequencing data of the TCGA cohort and successfully identified many new DEGs, and the lncRNA AL365181.3 was one of these DEGs. However, the possible role and therapeutic value of AL365181.3 in LUAD are unclear. In the present investigation, we showed that in patients with LUAD, abnormal expression of AL365181.3 was linked to a poor clinical outcome. Moreover, we mined TCGA data to identify the downstream targets of AL365181.3 and to determine the potential regulatory signaling pathways involved in LUAD. Finally, CCK8, colony formation, wound healing and Transwell assays were used to confirm the biological function of AL365181.3 in LUAD progression. In conclusion, our findings suggest a potential role for AL365181.3 in regulating tumor development and in the diagnostic assessment of LUAD.

Analysis of clinical information, prognosis, and expression status

The tumor AL365181.3 expression, prognosis, and clinical information were analyzed using the following databases: TCGA, Kaplan‒Meier plotter[17], and GEPIA[18].

Receiver operating characteristic (ROC) curve analysis

We utilized the pROC function from the R package to determine the area under the curve (AUC) of the receiver operating characteristic (ROC) curves produced by screening signature genes.

Cell culture conditions

Purchased from the Chinese Academy of Sciences Cell Bank, H1299 and A549 cells were grown in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin at 37°C and 5% CO₂.

Transfection

In 6-well plates, the cells were incubated with serum-free medium, which was changed at 70% confluence. siRNA and the transfection reagent LipofectamineTM 3000 were mixed at the recommended ratios, the transfection complex was usually added dropwise to the 6-well plates, and the dishes were gently shaken to ensure homogeneous mixing; after 4–6 h, the complete medium was replaced. After 24–48 hours, subsequent experiments were carried out. The siRNAs for AL365181.3-1 (si-AL365181.3-1#1: 5’- GCAAGAGAAUUGAAGGUUAGACUAC-3’; si-AL365181.3-1#2: 5’- CUAAUAGUUACUGAUGUCUACCUGA-3’) were synthesized by Gene Pharma (Shanghai, China).

qRT‒PCR

Total RNA was extracted using TRIzol reagent. cDNA was subsequently reverse transcribed. We performed the amplification reaction according to the instructions provided in the Green qPCR Supermix on top of the trans starter. In addition, we quantified mRNA expression using the 2^−ΔΔCt method. The following are primer sequences: q-AL365181.3-F: AGGTCTCTCTCTCTCTCT, q-AL365181.3-R: CCCTAAGGCCCTGCTTATATTG; q-18S-F: CTTCGGGGCTTCGGGG; and q-18S-R: CATAGGAATCCTTCTGACC.

CCK8, colony formation assay

Cell viability and growth were measured in 96-well plates with a CCK8 assay. Sample cells were inoculated in 96-well plates and incubated with CCK8 solution at a fixed time point for 1 h. The absorbance was measured at 450 nm with a spectrophotometer. For the colony formation experiments, the cells were inoculated in 6-well plates and cultured for 10 to 14 days. The solidified cells were stained and then photographed.

Apoptosis analysis

A FITC Annexin V Apoptosis Detection Kit I was used to detect apoptosis. After being disrupted by EDTA-free trypsin and rinsed with ice-cold PBS, the cells were incubated with PI and Alexa Fluor 488 annexin V for 15 minutes. Afterward, the cells were examined using a NovoCyte flow cytometer.

Wound healing assay

A monolayer of cells was scratched along a straight line to form a wound. To determine the gap width, the isolated cells were cleaned with PBS, and images of the scratches were taken at predetermined intervals.

Transwell migration and invasion assays

In transwell experiments, ^5×104 cells were inoculated in the upper chamber of a small chamber containing serum-free medium, and medium containing 15% FBS was added to the lower chamber. Cells that crossed the membrane of the chambers were counted after 24 h of incubation.

Xenograft model studies

Female BALB/c nude mice aged 4 weeks were obtained from GemPharmatech Technology (Jiangsu, China). A total of 4 × 10⁶ A549 cells were injected into the left flank of each mouse. Six injections of either si-NC or siRNA (20 nmol) were given to eight mice at intervals of three days using 0.1 mL of saline buffer. Xenograft models were split into two groups (n = 4) after fifteen days, when the tumors had grown to a size of approximately 5 mm by 5 mm. After being anesthetized with isoflurane, the tumor-bearing mice were sacrificed via cervical dislocation on day 30. The tumors from the xenografts were removed, weighed, and imaged with a camera.

IHC

Following the methods described earlier, immunohistochemistry (IHC) was performed [19]. For an overnight incubation at 4°C, primary antibodies against Ki67 (sc-23900, Santa Cruz) were applied to the xenograft sections of nude mice. The secondary antibody was then administered.

Statistical analysis

The two sets of data were compared for significance using Student’s t test. A P value less than 0.05 was considered to indicate statistical significance. *P < 0.05; **P < 0.01; ***P < 0.001.

Molecular characteristics analysis

According to the lnCAR database, AL365181.3 is an intronless transcript of 3222 nucleotides (nt)[20]. Figure S1A shows the genomic information for AL365181.3. AL365181.3 is located at chr1:156641666.156644887. According to the online database, AL365181.3 did not exhibit coding potential (Fig. S1B-C). Furthermore, we found that LUAD cells contain AL365181.3, which is located primarily in the cytoplasm (Fig. S1D).

The expression of AL365181.3 varies in human tumors

To validate the involvement of AL365181.3 in regulating human tumor development, we verified the RNA expression pattern of AL365181.3 in a variety of cancers and showed that the expression level of AL365181.3 varied greatly among different tumors, with AL365181.3 RNA being significantly highly expressed in 15 cancers in the TCGA database (Fig. 1A). We also confirmed that AL365181.3 was highly expressed in 13 paired adjacent normal tissues (Fig. 1B). These findings suggest that AL365181.3 can play a procancer role in different types of tumors.

Prognostic value of AL365181.3 in human tumors

AL365181.3 expression differs among different types of cancer, so its prognostic value in human cancer was investigated. We found that the expression level of AL365181.3 was related to the overall survival (OS) time of patients with four kinds of tumors (Fig. 2A), disease-specific survival (DSS) of patients with 4 kinds of tumors (Fig. 2B) and progression-free interval (PFI) of patients with 8 kinds of tumors (Fig. 2C).

ROC curve analysis of AL365181.3 in human tumors

We investigated whether AL365181.3 can serve as a biomarker for human tumors. According to the ROC curve analysis, AL365181.3 could be used to diagnose 20 types of tumors with high sensitivity and specificity (AUC > 0.75) (Fig. S2A-E).

AL365181.3 is highly expressed in LUAD tissues and is correlated with adverse clinical parameters in LUAD patients

Using AL365181.3 expression data from TCGA, we examined AL365181.3 expression in LUAD, and we found that AL365181.3 was more highly expressed in LUAD (Fig. 3A-B). Moreover, Gene Expression Omnibus (GEO) data provided similar results (Fig. 3C-D). We also examined the clinical relevance of AL365181.3 in LUAD using the TCGA LUAD dataset. The pathological stage and TNM stage were significantly correlated with AL365181.3 expression (Fig. 3E-H, Table 1). Importantly, in LUAD patients with many clinical features, including TNM stage, pathological stage, residual tumor, cancer location, anatomic neoplasm subdivision, age, smoking status, and number of peak years smoked, those with high AL365181.3 expression had significantly worse OS (Fig. 3I-R).

Table 1

Correlation of AL365181.3 expression with clinicopathologic features in LUAD patients.
Characteristics	Low expression of AL365181.3	High expression of AL365181.3	p
n	269	270
Pathologic T stage, n (%)			0.009
T1&T2	244 (45.5%)	224 (41.8%)
T3&T4	24 (4.5%)	44 (8.2%)
Pathologic N stage, n (%)			0.007
N0	187 (35.8%)	163 (31.2%)
N1&N2&N3	71 (13.6%)	102 (19.5%)
Pathologic M stage, n (%)			0.066
M0	186 (47.7%)	179 (45.9%)
M1	8 (2.1%)	17 (4.4%)
Pathologic stage, n (%)			< 0.001
Stage I	172 (32.4%)	124 (23.4%)
Stage II	47 (8.9%)	78 (14.7%)
Stage III&Stage IV	47 (8.9%)	63 (11.9%)

A nomogram was also constructed using the TCGA-LUAD cohort to predict OS, DSS, and the PFI. The prognostic indicators in the nomogram included AL365181.3 expression and pathological stage (Fig. 4A–C). LUAD OS, DSS, and the PFI were reliably predicted by the nomogram based on calibration curves (Fig. 4D–F). In conclusion, AL365181.3 can be used as a sensitive diagnostic indicator and can be used as a promising biomarker for LUAD.

Analysis of the AL365181.3-related signaling pathways in LUAD

Using the “clusterProfiler” R package, we performed functional annotation of the AL365181.3-associated differentially expressed genes (DEGs) in LUAD patients to elucidate the mechanism of the effect of AL365181.3, and 418 DEGs (mRNAs and lncRNAs) were identified with threshold values of |log2-fold change (FC)|>2 and adjusted p value < 0.05; these genes included 29 upregulated and 282 downregulated lncRNAs and 71 upregulated and 36 downregulated mRNAs (Fig. S3A–B). The KEGG enrichment results showed that the DEGs were involved mainly in pentose and glucuronate interconversions, steroid hormone biosynthesis, ascorbate and aldarate metabolism, drug metabolism-cytochrome P450, and metabolism of xenobiotics by cytochrome P450 (Fig. S3C). Next, we performed Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of the GEO dataset. The results revealed that AL365181.3 is involved in the regulation of metabolic metabolism, MAPK signaling and other tumor regulatory signaling pathways (Fig. S3D-E).

GSEA indicated that AL365181.3 was involved mainly in arginine and proline metabolism, phenylalanine metabolism and other pathways, other enzymes involved in drug metabolism, fructose and mannose metabolism, glycosaminoglycan degradation, the pentose phosphate pathway, the metabolism of cytochromes to xenobiotics, linoleic acid metabolism, the pentose phosphate pathway, nicotinic acid and nicotinamide metabolism, phenylalanine metabolism, retinol metabolism, ascorbic and aldehyde metabolism, and steroid hormone biosynthesis and tyrosine metabolism (Fig. 5).

The enrichment of hallmark genes indicated the following: early estrogen response, late estrogen response, glycolysis, heme metabolism, reactive oxygen species pathway, xenobiotic metabolism, and pancreatic beta cells (Fig. 6). These findings strongly demonstrated that AL365181.3 is involved mainly in the regulation of LUAD metabolism-related pathways.

AL365181.3 knockdown inhibits the proliferation of LUAD cells in vitro

The above results suggest that AL365181.3 expression is significantly upregulated in LUAD tissues and that AL365181.3 may affect the progression of LUAD. To investigate the biological function of AL365181.3 in LUAD, two siRNA sequences targeting AL365181.3, named siRNA-AL365181.3#1 and siRNA-AL365181.3#2, were designed and synthesized, and random nonsense siRNA sequences were selected as negative controls. The target cells were transfected separately by RNAi, and the knockdown efficiency of the transfected cell lines was examined via qRT‒PCR analysis (Fig. 7A). CCK8 and cell colony formation assays confirmed that in both A549 and H1299 cells, AL365181.3 knockdown reduced the proliferative capacity (Fig. 7B-C). In addition, knocking down AL365181.3 enhanced LUAD cell apoptosis (Fig. 7D).

AL365181.3 knockdown inhibits the migration and invasion of LUAD cells

The effect of AL365181.3 on the metastasis of LUAD cells was subsequently confirmed. Transwell and wound healing assays demonstrated that cell migration was markedly inhibited by downregulation of AL365181.3 expression (Fig. 7E-F). In conclusion, these results demonstrated that AL365181.3 has an essential function in regulating the migratory capacity of LUAD cells.

AL365181.3 knockdown inhibits LUAD cell xenograft tumor growth

Using a xenograft tumor model, we examined the effect of AL365181.3 on LUAF in vivo. Beginning on day 15, the mice were injected with NC or AL365181.3 siRNA every three days, and on day 30, they were euthanized. Compared to those of the NC cells, the tumors of the A549 cells grown with lower AL365181.3 concentrations grew at a significantly slower rate (Fig. 8A-C). Compared with those in the NC group, the weights of the si-AL365181.3#2-treated xenograft tumors were lower (Fig. 8D). qPCR revealed that AL365181.3 levels were significantly lower in si-AL365181.3-injected xenograft tumors than in control tumors (Fig. 8E). Compared with those in the NC group, the injection of si-AL365181.3#2 into xenograft tumors resulted in a decrease in the level of Ki67 expression, as observed through IHC staining (Fig. 8F). These results suggest that AL365181.3 could contribute to the oncogenic impact of LUAD.

LncRNAs are characterized by abundant species and spatial and temporal specificity; are widely involved in the regulation of cell apoptosis, immunity and other physiological and pathological processes; and have unique advantages as disease markers and therapeutic targets, which are of great value in clinical diagnosis and prognosis assessment[21]. In tandem with the advancements of bioinformatics and sequencing technologies, research on lncRNAs has also been increasing. Studies have shown that lncRNAs not only have potential as diagnostic and prognostic markers for LUAD but also play important regulatory roles in malignant phenotypes, such as LUAD proliferation, invasion, metastasis and chemoresistance. For example, the lncRNA DARS-AS1 enhances LUAD cell growth and metastasis through the miR-188-5p/KLF12 axis[22]. The lncRNA GAS6-AS1 inhibits LUAD progression and reprogramming of glucose metabolism by suppressing E2F1-mediated expression of the glucose transporter protein GLUT1[23]. The m6A-regulated lncRNA LCAT3 plays a pro-oncogenic role in LUAD by binding to FUBP1 to activate c-MYC[24]. The lncRNA SNHG7 enhances doxorubicin resistance in LUAD cells by inducing autophagy and macrophage M2 polarization[25]. Through epigenetic suppression of LATS2 expression, the m6A methyltransferase METTL3-induced lncRNA SNHG17 enhances LUAD gefitinib resistance[26]. These studies suggest that lncRNAs have significant clinical translational value and may be useful as biomarkers in the clinical diagnosis of LUAD.

In our study, we analyzed AL365181.3 in human cancers and found that the level of AL365181.3 varied greatly among different tumors, and AL365181.3 RNA was significantly upregulated in 15 cancers in the TCGA database; moreover, AL365181.3 was increased in 13 cancers compared to paired adjacent normal tissues. Further studies revealed that AL365181.3 expression was increased in LUAD tissue specimens and that high AL365181.3 expression was negatively correlated with LUAD pathological stage and TNM stage, suggesting that AL365181.3 expression may be related to LUAD progression. Previous studies revealed that lncRNAs have clinical predictive value in many tumors[27–30]. At present, we found that high AL365181.3 expression had a significant detrimental effect on OS and DSS in LUAD patients. Furthermore, the ROC curve demonstrated an AUC greater than 0.75 for AL365181.3. These findings imply that AL365181.3 could function as a diagnostic and prognostic LUAD biomarker.

To date, no studies have focused on the function of AL365181.3. In this study, we explored the regulatory mechanism of AL365181.3 in LUAD development. Functional enrichment analysis indicated that AL365181.3 is involved mainly in the regulation of metabolism, MAPK signaling and other tumor regulatory signaling pathways. Finally, we also showed that AL365181.3 knockdown significantly reduced LUAD cell proliferation and metastasis. This finding has enhanced our understanding of the relationship between LUAD and AL365181.3. However, there are still some limitations. However, the main molecular mechanism through which AL365181.3 affects the development of LUAD needs to be further investigated.

This discovery offers the first proof of the biological role and clinical importance of AL365181.3 in LUAD. Improved LUAD survival was closely correlated with a decreased AL365181.3 expression. AL365181.3 knockdown also inhibited LUAD cell proliferation and migration and in vivo tumorigenicity. For individuals with LUAD, AL365181.3 is a potential biomarker for both diagnosis and prognosis.

Ethics approval and consent to participate

This study was authorized by the Institutional Ethics Committees of the Shanghai University of Medicine & Health Sciences Affiliated Zhoupu Hospital (Approval notice: 2023-C-225-E01).

Animal experiment statement

Animal experiments were conducted according to the Health Guide for the Care.

Consent for publication

Not applicable.

Data availability

Data is provided within the manuscript or supplementary information files.

Competing interests

The authors declare no competing interests.

Funding

This research was supported by the Shanghai Pudong New Area Zhoupu Hospital discipline construction introduction talent project (ZP-XK-2019B-1), and the Medical Discipline Construction Project of Pudong Health Committee of Shanghai (PWZzb2022-26).

Authors contribution

Conceptualization, Qiang Wang; methodology, Xiaoying Liu; formal analysis, Xiaoying Liu and Jinlong Liu; writing—original draft preparation, Xiaoying Liu; investigation: Xiaoying Liu, Yingou Zeng, and Di Qiao; methodology: Xiaoying Liu and Jinlong Liu; writing—review and editing, Qiang Wang, Xiaoying Liu, Yingou Zeng, and Di Qiao.

Acknowledgments

We thank Bullet Edits Limited for the linguistic editing and proofreading of the manuscript. In addition, we sincerely thank the public databases, including TCGA and GTEx for providing the comprehensive data, and all bioinformatics tool for data analysis.

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AL365181.3 as a novel prognostic biomarker for lung adenocarcinoma

Status:

Version 1

Abstract

Figures

Introduction

Materials and methods

Analysis of clinical information, prognosis, and expression status

Receiver operating characteristic (ROC) curve analysis

Cell culture conditions

Transfection

qRT‒PCR

CCK8, colony formation assay

Apoptosis analysis

Wound healing assay

Transwell migration and invasion assays

Xenograft model studies

IHC

Statistical analysis

Results

Molecular characteristics analysis

The expression of AL365181.3 varies in human tumors

Prognostic value of AL365181.3 in human tumors

ROC curve analysis of AL365181.3 in human tumors

Analysis of the AL365181.3-related signaling pathways in LUAD

AL365181.3 knockdown inhibits the proliferation of LUAD cells in vitro

AL365181.3 knockdown inhibits the migration and invasion of LUAD cells

AL365181.3 knockdown inhibits LUAD cell xenograft tumor growth

Discussion

Conclusion

Declarations

References

Additional Declarations

Supplementary Files

Status:

Version 1