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Long non-coding RNA (lncRNA) PGM5P4-AS1 inhibits lung cancer progression by up-regulating leucine zipper tumor suppressor (LZTS3) through sponging microRNA miR-1275

ABSTRACT It is necessary to explore new molecules for the improvement of precise diagnosis and antitumor therapies in lung cancer. LncRNAs (long non-coding RNAs) play an important role in the regulation of cancer cell malignant behavior and tumor development. In this work, we found that a newly discovered lncRNA, lncRNA PGM5P4-AS1, was lower expressed in lung cancer tissues than adjacent tissues. Then, the lncRNA PGM5P4-AS1 was overexpressed or knocked-down in different lung cancer cells, and its effects on the malignant phenotypes were measured by 3-(4, 5-Dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assay, cell cycle assay, wound healing assay, and transwell assay. The results showed that the overexpression of PGM5P4-AS1 inhibited lung cancer cell proliferation, migration, and invasion activities, while these abilities were prominently promoted by the interference of PGM5P4-AS1. Further, the growth of lung cancer tumors in nude mice was also inhibited by PGM5P4-AS1 overexpression. In mechanism, PGM5P4-AS1 has the binding site of miR-1275 and could positively regulate the expression of LZTS3 via sponging miR-1275. In conclusion, PGM5P4-AS1 could be a potential precise diagnosis and therapeutic target biomarker of lung cancer.


Introduction
Lung cancer is considered the most cause of malignant tumor-related death worldwide with a high level of mortality for 18.4% [1]. Lung cancer can be divided into small cell lung carcinoma (SCLC) and non-small cell lung carcinoma (NSCLC), which is further divided into adenocarcinoma (38.5% of all lung carcinoma), large cell carcinoma (2.9% of all lung cancer cases), and squamous cell carcinoma (20% of all lung cancer cases) [2]. Surgery, chemotherapy, and medication are the major therapeutic strategies to treat lung cancer. Many patients relapse and succumb within a short time because of poor treatments and advanced stages of diagnosis [3]. Molecular heterogeneity in lung cancer is one of the reasons for these situations [4,5]. The dysregulated expression of cancer markers has been strongly linked to oncogenes or anti-tumor genes, that participate in carcinogenesis [6,7]. Therefore, it is necessary to extend the potential cancerassociated molecule library to improve the precise diagnosis and antitumor therapies in lung cancer.
Long non-coding RNAs (lncRNAs) are noncoding ribonucleotides that longer than 200 nucleotides. Resembled mRNA, some LncRNAs have 5 -7 m7GPPPN cap and polt A tail, but they still have no function to encode proteins [8][9][10]. Most lncRNAs are active in the nucleus instead of the cytoplasm, and it is logical for lncRNAs to act a pivotal part in biochemical processes and genome activities [11]. LncRNAs can competitively combine with miRNAs as competing endogenous RNAs (ceRNAs) [12]. Studies have proven that many lncRNAs act as tumor suppressors and biomarkers for precise diagnosis, as well as regulate the tumor progression in various cancers [13][14][15]. Zhou et al. [16] found that lncRNA PANDAR had high expression levels in lung cancer, colorectal cancer, renal cell carcinoma, and so forth. The overexpression of PANDAR reduced the growth of tumors and affected mice survival rates and tumor cellular processes.
LncRNA PGM5P4-AS1 is a lncRNA with a length of 950 bp and might be an anti-cancer biomarker in many cancers. For example, Zhang et al. [17] reported that PGM5P4-AS1 was associated with diagnosis and prognosis in hepatocellular carcinoma by bioinformatics analysis. Zhang et al. [18] found that PGM5P4-AS1 might be a potential lncRNA biomarker via the construction of a ceRNAs network in rectal adenocarcinoma. Ouyang et al. [19] demonstrated that PGM5P4-AS1 was down-regulated in breast cancer by bioinformatics analysis and might be related to chemoresistance. However, most of these studies on PGM5P4-AS1 in various cancers are performed by bioinformatics analysis and are rarely verified by experiments. In particular, the effects of PGM5P4-AS1 in lung cancer has not been reported. Through bioinformatics analysis, we found that PGM5P4-AS1 is significantly downregulated in lung adenocarcinoma and lung squamous cell carcinoma; thus, we speculated that PGM5P4-AS1 might play a role in the biological functions of lung cancer.
In the current study, we aimed to explore the effects of PGM5P4-AS1 on lung cancer progression. We found that PGM5P4-AS1 was low expressed in lung cancer tissues by quantitative real-time PCR and then we artificially regulated the expression of PGM5P4-AS1. The modulation of PGM5P4-AS1 influenced the growth of lung cancer xenograft tumors in vivo as well as cell proliferation, migration, and invasion in vitro (measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cell cycle arrest, wound healing, and transwell assay). Further luciferase reporter assay suggested that these effects were related to its regulation on the miR-1275/LZTS3 axis. The evidence revealed that PGM5P4-AS1 could act as a potential therapeutic target biomarker for lung cancer.

Quantitative real-time PCR (qPCR)
Total RNA was extracted by the Total RNA extraction kit (DP419, Tiangen Biotech, China), then the RNA was reversed-transcribed into cDNA with super M-MLV reverse transcriptase (NG212, Tiangen Biotech) . Amplification was performed by 2× Taq PCR MasterMix (KT201, Tiangen Biotech) at the presence of SYBR Green (SY1020, Solarbio, China) as per the users' instructions. The above procedures were set for triplicate for each system. Primers were synthesized by GenScript Co., Ltd, and primer sequences are shown in Table 2. The PCR reaction was performed on Exicycler TM 96 (Bioneer, Korea).

Cell cycle arrest
The serum-starved A549 and SK-MES-1 cells were washed with phosphate buffer saline (PBS) after being transfected for 48 h. Then, cells were fixed with 70% cold ethanol and kept at 4°C for 2 h. PBS was used to wash cells again and resuspend cells. Cells were stained with 25 μL propidium iodide (PI; C1052-2, Beyotime) and incubated with 10 μL RNase (C1052-3, Beyotime) at 37°C for 30 min. siRNAs Note: F, forward; R, reverse. Table 2. qPCR primer sequences.
Note: F, forward; R, reverse. PGM5P4-AS1 inhibited the growth of tumors in nude mice and suppressed the abilities of cell proliferation, migration as well as invasion of lung cancer cells. In mechanism, PGM5P4-AS1 performed antitumor function by up-regulating the expression of LZTS3 via sponging miR-1275.
The cell cycle was detected by the flow cytometer (NovoCyte, ACEA Biosciences, USA).

Xenograft model in nude mice and vector treatment
Male BALB/c nude mice were purchased from Beijing HFK Bioscience Co., Ltd (Beijing, China). Mice feeding and animal experiments followed The Guideline for the Care and Use of Laboratory Animals. SK-MES-1 cells were collected and washed with PBS twice and resuspended with a completed medium. 2 × 10 6 cells were subcutaneously injected into the right armpit of nude mice. PGM5P4-AS1-OE vectors or control vectors (50 μg per mouse) were injected into mice tail vein three times a week after tumors reached the volume of 60-90 mm 3 . Six mice were set for each treatment.

Immunohistochemistry
Tumor sections were incubated with goat serum (SL038, Solarbio) at room temperature for 15 min. Ki67 antibodies (diluted 1:100; A2094, ABclonal, China) were added overnight to the top of the section. The tumor sections we washed twice by PBS and incubated with a goat anti-rabbit IgG (H + L) antibody (diluted 1:500; #31,460, ThermoFisher Scientific) at 37°C for an hour. Then, the DAB Substrate kit (DA1010, Solarbio) was used for color rendering, and HE Staining Kit (G1120, Solarbio) was used to stain again. The positive staining of ki67 was photographed by a microscope (BX53, Olympus, Japan).

Wound healing assay
After 48 h of transfection, cells were cultured with serum-free medium. Equal numbers of cells were planted in cell-cultured plates. Two hundred microliter pipette tips were used to draw a horizontal line when there were 80-90% of cells covered the bottom of the cultured plates. The cells were washed by a serum-free medium to remove the exfoliated cells. Then, the relative position of cells was photographed by a microscope (BX53, Olympus) at 0 h and 24 h, respectively.

Statistics analysis
Statistics were performed by using GraphPad Prism 8.0. Data were presented as mean ± standard deviation. A comparison of two treatments was analyzed with two-side t-tests, and multiple treatments were analyzed with one-way ANOVA. p < 0.05 was suggested statistically significant.

The expression of PGM5P4-AS1 was increased in lung cancer tissues
The bioinformatics analysis showed that PGM5P4-AS1 was lower expressed in lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC) tumor tissues than in normal tissues (p < 0.05; Figure 1(a)). Our qPCR results further confirmed that PGM5P4-AS1 was significantly down-regulated in lung cancer tissues compared to adjacent tissues (0.01±0.01 vs. 0.03±0.01; p < 0.05; Figure 1(b)). We then detected the relative expression levels of PGM5P4-AS1 in several lung cancer cell lines by qPCR. As shown in Figure  1 Figure 1(e)).

PGM5P4-AS1 suppressed the proliferation of lung cancer cells
The proliferation ability of the lung cancer cells was measured by MTT assay. As shown in Figure 2(a), from 24 to 96 h, the results of the MTT assay demonstrated that the interference in PGM5P4-AS1 expression in A549 cells significantly promoted   cell proliferation (p < 0.01). And Figure 2( Figure 2(c)). Meanwhile, the overexpression of PGM5P4-AS1 increased the G1 phase proportion (71.63%±3.61% vs. 58.97%±4.14%) and reduced the S phase proportion (11.88%±1.48% vs. 25.6%±2.88%; Figure 2(d)). These results demonstrated that PGM5P4-AS1 can repress the proliferation of lung cancer cells.

PGM5P4-AS1 inhibited the in vivo growth of lung cancer
The nude mice xenograft tumors assay was employed to evaluate the effects of PGM5P4-AS1 on cancer growth in vivo. The results displayed that the over-expression of PGM5P4-AS1-OE significantly suppressed the growth of the tumor, as evidenced by the reduced tumor volume from 13th to 25th and tumor weight (254.17 mg±39.85 mg vs. 428.50 mg±69.34 mg; p < 0.01, Figure 3(a, c and  d)). The expression of ki67 was detected to evaluate the cell proliferation activity of the tumors. Figure 3 (b) reveals that the expression of ki67 was reduced by the up-regulation of PGM5P4-AS1. And as shown in Figure 3
According to the results of the transwell assay, we found that the invasive activity of the lung

PGM5P4-AS1 decreased the miR-1275 level and promoted the expression of LZTS3
High expression of miR-1275 can promote the cell proliferation and migration of lung cancer cells by targeting the leucine zipper putative tumor suppressor 3 (LZTS3) [20], thus, the expression of miR-1257 and LZTS3 was measured by qPCR. As shown in), down-regulation of PGM5P4-AS1 increased the miR-1257 level and reduced the LZTS3 level Western blotting was carried out to evaluate the expression levels of LZTS3 and its downstream proteins Cyclin D1, matrix metalloproteinase-2 (MMP-2), Bcl-2, and Bcl-xl. As expected, the change of LZTS3 protein level was the same as its mRNA). Further, as revealed in Figure 5(e), the down-regulation of PGM5P4-AS1 resulted in increased protein levels of CyclinD1  Figure 5(f)). These results indicated that PGM5P4-AS1 may promote the expression of the anti-cancer protein LZTS3 by the negative regulation of miR-1275.

Discussion
LncRNAs acts as a pivotal part in genome activities, thereby regulating cellular processes, even the occurrence, and development of malignant cancers. Several papers have delineated that many lncRNAs have a function in the process of lung cancer. Loewen et al. [11] reported that lncRNA HOTAIR was associated with aggressiveness and poor prognosis in lung cancer. LncRNA GAS5 could affect drug resistance in NSCLC [21]. LncRNA H19 could suppress the expression of miR-138 and miR-200a and was related to diseasefree survival (DFS) time [22,23]. Ji et al. [24] demonstrated that LncRNA MALAT1 was identified as a prognostic biomarker and was associated with the survival rate in lung cancer. This study concentrated on the up-expressed PGM5P4-AS1 regulated the cellular processes and the growth of tumors by sponging miR-1275 to up-regulate the expression of LZTS3 in lung cancer.
PGM5P4-AS1, a newly discovered lncRNA, is predicted as a potential biomarker in various cancers. Most studies on PGM5P4-AS1 are performed by bioinformatics analysis and are rarely verified by experiments [17][18][19]. Herein, we found that PGM5P4-AS1 expression was lower in lung cancer tissues than adjacent tissues and we firstly identified its anti-tumor activity in vivo and in vitro. Accumulated evidence show that lncRNAs can interact with miRNA and further regulate its target genes at the post transcription level, which are known as ceRNAs [25,26]. Yang et al. [27] reported that lncRNA LINC01133 inhibited gastric cancer progression via sponging miR-106a-3p to regulate the adenomatous polyposis coli gene expression. Zhao et al. [28] displayed that the knockdown of lncRNA HOTAIR suppressed cell proliferation, metastasis, and apoptosis by affecting the miR-20a-5p/HMGA2 axis. These lncRNArelated ceRNAs are associated with the anti-cancer progression. Thus, we speculated that PGM5P4-AS1 might also affect the malignant phenotypes of lung cancer by the regulation of miRNA and its targets.
Recently, the dysregulation of miR-1275 was proven to affect the development and progression of various cancers. Zheng et al. [29] reported that miR-1275 facilitated the tumorigenesis and metastasis in nasopharyngeal carcinoma by up-regulating integrin β3. Xie et al. [30] found that miR-1275 was associated with radiosensitization in esophageal mediating by Wnt/β-catenin signaling pathway. Ma et al. [31]reported that miRNA-1275 promoted lung cancer cell proliferation and migration via the regulation of FOXK1. Moreover, miR-1275 was proved to be highly expressed in NSCLC and promoted the cellular processes including cell proliferation, migration, and invasion by targeting LZTS3 [20]. In the current study, the results of luciferase reporter assay showed that PGM5P4-AS1 sponged miR-1275. We found that PGM5P4-AS1 overexpression induced blocking of proliferation, migration, and invasion abilities of lung cancer cells were all reversed by miR-1275 mimic. Subsequently, scholars revealed that LZTS3 regulated the process of cell growth and was thought to be a potential tumor suppressor [32]. Liu et al. [33] demonstrated that LZTS3 was predicted to be a potential prognostic biomarker in colorectal cancer by bioinformatics analysis. In the current study, LZTS3 acted as a target of miR-1275 in lung cancer cells. LTZS3 inhibited cell proliferation by down-regulating the cell cycle pathway protein cyclin D1 and suppressed cell migration by down-regulating metastasisrelated protein MMP2.

Conclusions
Our work manifested that PGM5P4-AS1 was low expressed in lung cancer tissues. PGM5P4-AS1 could inhibit lung cancer progression by the upregulation of the LZTS3 expression via sponging miR-1275. PGM5P4-AS1 may be a potential precise diagnosis and therapeutic target biomarker in lung cancer.
4. PGM5P4-AS1 acted as a sponge of miR-1275 in lung cancer cells. 5. LZTS3 acted as a target of miR-1275 in lung cancer cells.