LncRNA PITPNA‐AS1/miR‐223‐3p/PTN axis regulates malignant progression and stemness in lung squamous cell carcinoma

Abstract Background Long noncoding RNAs (lncRNAs) are a kind of molecule that cannot code proteins, and their expression is dysregulated in diversified cancers. LncRNA PITPNA‐AS1 has been shown to act as a tumor promoter in a variety of malignancies, but its function and regulatory mechanisms in lung squamous cell carcinoma (LUSC) are yet unknown. Methods The mRNA and protein expression of genes were examined by RT‐qPCR, western blot, and IHC assay. The cell proliferation, migration, invasion, and stemness were detected through CCK‐8, colony formation, Transwell and spheroid formation assays. The CD44+ and CD166+‐positive cells were detected through flow cytometry. The binding ability among genes through luciferase reporter and RNA pull‐down assays. The tumor growth was detected through in vivo nude mice assay. Results The lncRNA PITPNA‐AS1 had increased expression in LUSC and was linked to a poor prognosis. In LUSC, PITPNA‐AS1 also enhanced cell proliferation, migration, invasion, and stemness. This mechanistic investigation showed that PITPNA‐AS1 absorbed miR‐223‐3p and that miR‐223‐3p targeted PTN. MiR‐223‐3p inhibition or PTN overexpression might reverse the inhibitory effects of PITPNA‐AS1 suppression on LUSC progression, as demonstrated by rescue experiments. In addition, the PITPNA‐AS1/miR‐223‐3p/PTN axis accelerated tumor development in vivo. Conclusions It is the first time we investigated the potential role and ceRNA regulatory mechanism of PITPNA‐AS1 in LUSC. The data disclosed that PITPNA‐AS1 upregulated PTN through sponging miR‐223‐3p to enhance the onset and progression of LUSC. These findings suggested the ceRNA axis may serve as a promising therapeutic biomarker for LUSC patients.


| INTRODUC TI ON
Lung cancer is one of the leading causes of cancer-related mortality globally. 1 Large cell carcinoma, adenocarcinoma, neuroendocrine carcinoma, and squamous cell carcinoma are all types of non-small cell lung cancer (NSCLC), which accounts for 80-85% of all lung malignancies. 2 Lung squamous cell carcinoma (LUSC) is a well-known form of NSCLC with a greater recurrence risk. 3,4 As a result, identifying molecular biomarkers for LUSC carcinogenesis is paramount.
Long noncoding RNAs (lncRNAs) are RNA molecules with a length of more than 200 nucleotides but no ability to code for proteins. 5,6 Many studies have shown that lncRNAs are important regulators of gene expression and have a role in the oncogenesis and development of many cancers. For example, via modulating TTN expression, the lncRNA TTN-AS1 speeds up the carcinogenesis and spread of cutaneous melanoma. 7 Furthermore, lncRNA ZFAS1 absorbs miR-892b to regulate LPAR1 and aids carcinogenesis in nasopharyngeal cancer. 8 FEZF1-AS1 is a lncRNA that stimulates the Wnt pathway, which promotes gastric cancer carcinogenesis. 9 The role of lncRNA in LUSC is now the subject of a multitude of research studies. The lncRNA FAM201A, for example, affects LUSC development by altering ABCE1 expression. 10 Furthermore, the lncRNA HULC promotes LUSC development by upregulating PTPRO. 11 To speed up LUSC development, the lncRNA NNT-AS1 targets the miR-22/FOXM1 axis. 12 In addition, the lncRNA SNHG1 interacts with TAp63 to modulate ZEB1 expression and exacerbates LUSC metastases. 13 LncRNA PITPNA antisense RNA 1 (PITPNA-AS1) has been identified as a new lncRNA that has a role in the progression of a variety of malignancies. In triple-negative breast cancer, PITPNA-AS1 has been shown to have an oncogenic role by targeting the miR-520d-5p/DDX54 axis. 14 PITPNA-AS1 also absorbs miR-129-5p to control UNC5B and accelerates papillary thyroid tumorigenesis. 15 We found that PITPNA-AS1 has a higher expression level and exacerbates LUSC cell proliferation and migration through interacting with TAF15 to stabilize HMGB3. 16 In LUSC, the crucial ceRNA regulation mechanism of PITPNA-AS1 remains to be further explored.
MicroRNAs (miRNAs) are tiny RNA molecules that may control gene expression by interacting with the 3′-UTR of target genes mRNAs. 17 KY-2018-052-01). Liquid nitrogen was used to freeze the samples, which were then kept at −80 °C.

| RT-qPCR
Total RNA was extracted from LUSC cells or tissues via TRIzol reagent (Invitrogen), and the cDNA was synthesized using the Primescript RT Reagent (TaKaRa). RT-qPCR analysis was performed using SYBR®Premix Ex Taq™ Reagent (TaKaRa) through StepOne Plus Real-Time PCR system (Applied Biosystems). GAPDH or U6 was, respectively, utilized to be lncRNA/mRNA and miRNA internal controls. The fold change in mRNA expression was calculated through the 2 −ΔΔCt method.

| Cell transfection
The LUSC cells were cultured on 6-well plates and then transfected
LUSC cells were cultured on the 96-well plate, and then CCK-8 solution was added at 0, 24, 48, and 72 h. Finally, the absorbance at 450 nm wavelength was measured.

| Colony formation assay
LUSC cells were seeded onto 6-well plates. After 2 weeks, the cells were fixed with ethanol and stained with crystal violet (Beyotime). The colonies were then examined under the microscope.

| Transwell assay
Transwell chamber ([8.0 μm pore size; EMD Millipore]) with (or without) Matrigel (Becton Dickinson) was applied for the assessment of invasion (or migration) of LUSC cells. The cells in serumfree medium were placed in the upper chamber, while the lower chamber was filled with medium supplemented with 10% bovine calf serum. Methanol was used to fix the cells after 48 hours, and crystal violet (0.1%) was employed to stain the cells. Finally, the cells that had migrated or invaded were examined under the microscope.
The spheres were then observed under the microscope.

| Flow cytometry assay (CD44 and CD166)
The CD44 and CD166 antibodies were acquired from Beijing biosynthesis biotechnology CO., LTD. The LUSC cells were maintained in a serum-free medium. After 1 week, the population of CD44 + and CD166 + -positive cells was detected through flow cytometry.

| In vivo assay
The nude BALB/c mice (6-week-old, 22-25 g) were obtained from the Charles River. The animal experiments were approved by the Ethics Committee of Beijing Tiantan Hospital, Capital Medical University.
The LUSC cells were injected subcutaneously into the right lower limbs of mice. Every week, the size of the tumor was measured. After the mice had been euthanized, the volume and weight of tumors were measured. SPSS 22.0 software was applied to perform statistical analysis. The Student's t test (for two groups) or the one-way ANOVA (more than two groups) was used for statistical comparison. Kaplan-Meier analysis and the log-rank test were utilized to assess survival curves. The P-value <0.05 was defined as statistically significant. All the data were expressed as the mean ± standard deviation (SD) of 3 replicates.

| DISCUSS ION
lncRNAs have received a lot of attention in recent years, and they have played a pivotal role in various malignancies, notably LUSC. [10][11][12][13][14] PITPNA-AS1 is a novel lncRNA that has been investigated in LUSC to accelerate tumorigenesis. 16 However, the ceRNA regulation mechanism mediated by PITPNA-AS1 in LUSC is unknown. We found that the lncRNA PITPNA-AS1 expression was higher in LUSC and that this increased expression was associated with a poor prognosis.
LncRNAs share miRNA-binding regions known as ceRNA, which are used to indirectly regulate mRNAs by absorbing miR-NAs. 23,24 The importance of the lncRNA-miRNA-mRNA regulatory network in the development of LUSC has also been discovered. 25,26 Many ceRNA networks are involved in the LUSC progression.
The PTN was shown to be a downstream target gene. According to new findings, PTN is abnormally expressed and acts as a crucial regulator in a variety of malignancies. In osteosarcoma, for example, miR-627-3p inhibits cell growth and metastasis by targeting PTN. 33 T the circ-LDLRAD3/miR-137-3p/PTN axis slows the progression of pancreatic cancer. 34 Furthermore, the OIP5-AS1/miR-137-3p/ PTN axis affects doxorubicin resistance in osteosarcoma. 35 We further demonstrated that PITPNA-AS1 negatively controlled PTN expression by absorbing miR-223-3p, which is consistent with the preceding results. Furthermore, rescue experiments revealed that

AUTH O R CO NTR I B UTI O N S
YFJ and XJQ analyzed and interpreted data. BHP was a major contributor in writing the article. All authors read and approved the final work.

ACK N OWLED G M ENTS
Not applicable.

CO N FLI C T O F I NTE R E S T
The authors declared no potential conflicts of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data used to support the findings of this study are included within the article.