LncRNA CASC2 inhibits lung adenocarcinoma progression through forming feedback loop with miR‐21/p53 axis

Abstract

Lung adenocarcinoma (LUAD) is the most common type of lung cancer. Currently, the survival rate of LUAD patients remains low due to heterogeneity and high invasiveness. The long non‐coding RNA (lncRNA) cancer susceptibility candidate 2 (CASC2) is reported to be related to LUAD development. Hence, we investigate the roles and regulatory mechanism of CASC2 in LUAD. The expression levels of CASC2, microRNA (miR)‐21, and p53 were quantified by quantitative real‐time polymerase chain reaction, and the protein levels of Bax, Bcl‐2, p53, and p21 were examined using western blotting. A dual‐luciferase reporter experiment was conducted to prove the molecular interactions between CASC2 and miR‐21 or p53. CCK‐8 and flow cytometry assays were conducted to assess cell proliferation and apoptosis, respectively. CASC2 was expressed at a low level in LUAD patients and LUAD cell lines. CASC2 overexpression markedly suppressed cell proliferation and enhanced apoptosis. Mechanistically, CASC2 overexpression dramatically inhibited miR‐21 expression and increased p53 expression by directly targeting miR‐21. Moreover, rescue experiments suggested that either miR‐21 overexpression or p53 silencing obviously weakened the biological effects of CASC2 overexpression. In addition, p53 was proven to be an upstream transcription factor of CASC2 and can activate CASC2 transcription. These results provide evidence that the lncRNA CASC2/miR‐21/p53 form a positive feedback loop to mediate cell proliferation and apoptosis in LUAD, which may provide a new insight into the pathological mechanisms of LUAD.

INTRODUCTION

Lung cancer is the most common malignant tumor worldwide with high morbidity, mortality, and invasiveness.¹ According to histopathology, lung cancer is divided into two subtypes: small cell lung cancer (SCLC) and non-small cell lung cancer (NSCLC).² NSCLC is the most common type of lung cancer, occurring in 85% of all lung cancer patients.² In addition, NSCLC mainly includes lung adenocarcinoma (LUAD) and lung squamous cell carcinoma (LUSC). LUAD has obvious ground-glass opacity (GGO) phenotypes, such as pulmonary interstitial fibrosis, inflammation, and bleeding. Seventy percent of patients with advanced LUAD are found to have tumors that metastasize to other parts of the body.³ Currently, LUAD is diagnosed with computed tomography examination, puncture biopsy, and surgical resection. Most LUAD patients are diagnosed at an advanced stage, which misses the window for surgical treatment, resulting in a high mortality rate for LUAD.^4,5 Thus, it is important to find effective biomarkers and investigate pathogenesis for the diagnosis and treatment of LUAD.

Non-coding RNAs are mainly classified as long non-coding RNA (lncRNAs) and microRNA (miRNAs).^6,7 lncRNAs are a class of single-stranded RNA with a transcript length greater than 200 nt, and they play the regulatory roles in many biological processes.^8,9 Studies have shown that lncRNAs are closely related to the occurrence of lung cancer.^10,11 For example, the expression of the MAFG-AS1 is upregulated in LUAD cells, which accelerates cell proliferation by targeting the miR-744-5p/MAFG axis.¹² Cancer susceptibility candidate 2 (CASC2) is generally considered to be a tumor suppressor gene.¹³ Previous reports have illustrated that CASC2 expression is downregulated in various tumors, including lung cancer, and CASC2 participates in regulating angiogenesis, cell proliferation, invasion, and drug resistance.^14–16 However, the biological effects and regulatory network of CASC2 in LUAD remain unclear.

It has been confirmed that transcription factors can combine with specific sequences in target gene promoters, resulting in transcriptional activation or repression. p53 is an early-identified tumor suppressor that affects downstream target genes and signals mainly via transcriptional regulation, thus modulating a variety of biological processes, such as apoptosis, proliferation, cell cycle, and metabolism.^17–19 In addition, several lncRNAs and miRNAs are confirmed as regulators of p53 signaling, modulating p53 expression and activity. For example, PIK3CD-AS2 inhibits the expression of p53 by sustaining the stability of Y-box binding protein 1 (YBX1).²⁰ However, the effects of CASC2 on p53 in LUAD and associated mechanism are still unknown.

By bioinformatic software prediction, the potential binding site between miR-21 and CASC2 was found. miR-21 is frequently upregulated in a variety of cancers such as ovarian cancer, and is widely used as a target miRNA for cancer therapy.²¹ Additionally, Zhang et al.'s study has showed that miR-21 restrains p53 expression in hepatocellular carcinoma by directly targeting HBP1 that is a transcriptional activator of p53.²² Likewise, Wei et al. has identified that miR-21 inhibitor facilitates the anti-tumor effects of doxorubicin in melanoma by triggering p53 signaling.²³ Whereas the expression and functional correlation between miR-21 and p53 in LUAD remain unclear. In the present study, we examined the expression levels of CASC2, miR-21, and p53 in LUAD tissue samples and analyzed their relevance at the clinical level. In addition, the regulatory roles and underlying network of CASC2 were also investigated. Ultimately, we identified that CASC2/miR-21/p53 formed a positive feedback loop to mediate cell proliferation and apoptosis in LUAD cells, which would provide a new insight and a deeper understanding of the pathological mechanisms of LUAD.

MATERIALS AND METHODS Clinical samples

All tissue samples (n = 24), including tumor and corresponding adjacent tissues, were collected from primary LUAD patients (Stage II–IV) at Zhuzhou Central Hospital. None of the patients enrolled in this study received any cancer treatment for 3 months before surgery. All patients who participated in this work were provided the relevant medical information and signed a consent form. This research was authorized by the Zhuzhou Central Hospital Ethical Research Committee. All tissue samples were stored at −80°C for subsequent analysis.

Cell culture

The 16HBE, A549, and PC-9 cell lines were purchased from American Type Culture Collection (ATCC, VA). Cells were cultured in dulbecco's modification of eagle's medium dulbecco (DMEM, Bioind, Haemek, Israel) supplemented with 10% fetal bovine serum (FBS, Gibco, MD) in a humidified atmosphere of 5% CO₂ at 37°C.

Quantitative real-time polymerase chain reaction assay

Total RNA was extracted by using TRIzol reagent (Thermo Fisher Scientific, CA). cDNA was synthesized by using reverse transcriptase kits in accordance with the manufacturer's instructions (Toyobo, Osaka, Japan). Then, the cDNA was used for a quantitative real-time polymerase chain reaction (qPCR) assay conducted on an Eppendorf MasterCycler RealPlex4 (Eppendorf, Wesseling-Berzdorf, Germany) using a SYBR kit (Toyobo). Relative expression levels were calculated by the 2^−ΔΔCT method, and the expression levels of GAPDH and U6 were used to normalize the expression of genes and a miRNA, respectively. The specific primer sequences used in this study are listed below (5′-3′):

CASC2 (F): -ATTGGACGGTGTTTCCGTGT-;

CASC2 (R): -ACCACCACTGACTGTCCTGT-;

miR-21 (F): -TGGGCTTATCAGACTGATGTTGA-;

miR-21 (R): -CTCAACTGGTGTCGTGGAGTC-;

TP53 (F): -CTTCGAGATGTTCCGAGAGC-;

TP53 (R): -CAGGCCCTTCTGTCTTGAAC-;

GAPDH (F): -CTGACTTCAACAGCGACACC-;

GAPDH (R): -GTGGTCCAGGGGTCTTACTC-;

U6 (F): -TGCGTTCCCTTTGTCATCCT-;

U6 (R): -AACGCTTCACGAATTTGCGT-.

Cell transfection assay

The overexpression plasmids carrying CASC2 or p53 (pcDNA-CASC2 and pcDNA-p53, respectively) and an inhibitor or mimics of miR-21 as well as their negative controls (pcDNA-NC, inhibitor NC and mimics NC) were purchased from GenePharma (Shanghai, China). A549 and PC-9 cells were cultured in 24-well plates, and then the plasmids were transfected into the cells by using Lipofectamine 3000 (Invitrogen, CA). After 24 h, the transfection efficiencies of the plasmids mentioned above were detected by qPCR.

Cell proliferation assay

Cell proliferation was detected with Cell Counting Kit-8 (CCK8, Dojindo Molecular Technologies, Tokyo, Japan). Cells were cultured at a density of 2000 cells per well in a 96-well plate. CCK-8 reagent (10 μl/well) was added, and the cells were incubated at 37°C for 1 h in the dark. The absorbance was measured at 450 nm with a microplate spectrophotometer (Thermo Fisher Scientific).

Apoptosis assay

Cell apoptosis was detected with the Annexin V-FITC/PI Apoptosis Detection Kit (Yeasen, Shanghai, China) according to the manufacturer's instructions. After treatment, cells were harvested and washed twice with pre-chilled PBS. Then, the cells were resuspended in 100 μl of 1X binding buffer. The cells were then incubated with 5 μl of Annexin V-FITC and 10 μl of PI staining solution protected from light at room temperature. After the 10-min incubation, the samples were analyzed using flow cytometry.

Dual-luciferase reporter assay

The sequence of CASC2 with/without the binding site for miR-21 (CASC2-wt and CASC2-mut) and the sequence of the CASC2 promoter containing p53 binding sites (pmirGLO-CASC2) were synthesized by GenePharma and then cloned into the pmirGLO vector (Promega, WI). Then, A549 cells were co-transfected with the CASC2-wt or CASC2-mut plasmid and mimics NC or miR-21 mimics using Lipofectamine 3000 (Invitrogen). The pmirGLO-CASC2 plasmids were co-transfected with the pcDNA-p53 or pcDNA-NC plasmid into A549 cells using Lipofectamine 3000 (Invitrogen). After transfection for 48 h, luciferase activity was measured with a dual-luciferase reporter assay system (Promega).

Protein extraction and western blotting

Proteins were isolated from cells by using RIPA lysis buffer (Beyotime, Shanghai, China) mixed with 1% protease inhibitor and phosphorylase inhibitor (Beyotime). Then, a BCA kit (Beyotime) was used to detect the protein concentrations. Proteins were separated by 6% and 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, the proteins were transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore, MA). According to the size of the target proteins, the membranes were cut at the corresponding position and blocked with a blocking solution (Beyotime) for 1 h at room temperature. Then, the membranes were incubated with primary antibodies including anti-p53 (1:1000, ab26, Abcam, Cambridge, UK), anti-Bax (1:1000, ab32503, Abcam), anti-p21 (1:1000, ab190520, Abcam), anti-Bcl-2 (1:1000, ab32124, Abcam), and anti-GADPH ((1:5000, sc-322233, Santa Cruz, CA), overnight at 4°C. After washing with TBST, the membranes were further incubated with an HRP-conjugated goat anti-rabbit IgG secondary antibody (1:5000, sc-2004, Santa Cruz) for 1 h at room temperature. The proteins were visualized with ECL WB Detection Reagents (Beyotime), and images were captured with a GEL imaging system (Bio-Rad, CA). Protein quantification was performed with ImageJ software.

Data analysis

Results are expressed as the mean ± standard error of the mean. Statistical analysis was performed with Starview. Differences in normally distributed data were analyzed by one-way analysis of variance and Student's t-tests. p values less than 0.05 were considered significant.

RESULTS CASC2 was downregulated and exerted tumor-suppressive function in LUAD

We detected the expression of CASC2 in LUAD patients and cell lines, and the result suggested that CASC2 expression in LUAD tissue samples was lower than that in tumor-adjacent tissue specimens (Figure 1(A)), which was consistent with the predictive data of the UALCAN online database (Figure 1(B)). Moreover, the expression of CASC2 in PC-9 and A549 cells was also downregulated compared to that in 16HBE cells, which further indicated that CASC2 was closely associated with LUAD development (Figure 1(C)). Next, we investigated the biological roles of CASC2 by transfecting the pcDNA-CASC2 vectors into PC-9 and A549 cells. qPCR assay results indicated that CASC2 expression was significantly upregulated in the pcDNA-CASC2 group (Figure 1(D)). Functional experiments showed that overexpression of CASC2 significantly inhibited cell proliferation (Figure 1(E)) and obviously enhanced cell apoptosis (Figure 1(F)). Moreover, the levels of p53, Bax, and p21 were upregulated, while the level of Bcl-2 was downregulated in LUAD cells following CASC2 overexpression (Figure 1(G)). The above results confirmed that CASC2 functioned as a tumor suppressor and acted as anti-proliferative and pro-apoptotic roles in LUAD cells.

FIGURE 1. CASC2 expression was downregulated in LUAD, which exerted a tumor-suppressive function. (A) The expression level of CASC2 in LUAD patients was determined using qPCR. (B) The expression of CASC2 in LUAD patients was analyzed with the UALCAN online database (http://ualcan.path.uab.edu/analysis.html). (C) The expression level of CASC2 in PC-9, A549 and 16HBE cells was examined using qPCR. (D–G) pcDNA-CASC2 or pcDNA-NC plasmids were transfected into PC-9 and A549 cells. (D) The expression of CASC2 was assessed using qPCR. (E) Cell proliferation was evaluated with a CCK8 assay. (F) Cell apoptosis was tested with a flow cytometry assay. (G) The protein levels of p53, Bax, p21 and Bcl-2 were quantified via western blotting. *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001. CASC2, cancer susceptibility candidate 2; LUAD, lung adenocarcinoma; qPCR, quantitative real-time polymerase chain reaction

miR-21 was upregulated in LUAD and interacted with CASC2

First, the binding site between miR-21 and CASC2 was predicted using bioinformatic software (Figure 2(A)). The result of dual-luciferase reporter assay indicated that co-treatment with CASC2-wt and miR-21 mimics markedly reduced luciferase activity, while the luciferase activity of cells co-transfected with CASC2-mut and miR-21 mimics was not significantly different from that of cells transfected with mimics NC (Figure 2(B)). Moreover, overexpression of CASC2 obviously restrained the expression of miR-21 (Figure 2(C)). Then, the expression level of miR-21 in LUAD tissues was detected, and the result revealed that miR-21 expression was significantly upregulated in LUAD tissues compared to adjacent tissues (Figure 2(D)). A similar trend was observed in the expression level of miR-21 in PC-9 and A549 cells (Figure 2(E)). Pearson correlation analysis showed a reverse correlation between CASC2 and miR-21 expression in clinical LUAD tissues (Figure 2(F)). These finding illustrated that miR-21 was overexpressed in LUAD and could be negatively regulated by CASC2.

FIGURE 2. miR-21 was upregulated in LUAD and interacted with CASC2. (A) The binding site between miR-21 and CASC2 was predicted with bioinformatic software (http://starbase.sysu.edu.cn/). (B) The binding relationship between miR-21 and CASC2 was detected with a dual-luciferase reporter assay. (C) PC-9 and A549 cells were transfected with the pcDNA-CASC2 or pcDNA-NC plasmid, and the expression of miR-21 was quantified using qPCR. (D, E) The expression level of miR-21 in LUAD patients and cell lines was examined using qPCR. (F) The correlation between miR-21 and CASC2 expression at the clinical level was analyzed by Pearson correlation analysis. *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001. CASC2, cancer susceptibility candidate 2; LUAD, lung adenocarcinoma; qPCR, quantitative real-time polymerase chain reaction

Overexpression of miR-21 reversed the regulation of cell proliferation and apoptosis mediated by CASC2 overexpression

Based on previous data, we next sought to explore the biological impacts of miR-21 on CASC2-mediated functions in A549 and PC-9 cells. First, we transfected miR-21 mimics or mimics NC into cells overexpressing CASC2, and transfection efficiency was measured by qPCR. The data indicated that miR-21 mimics significantly weakened the inhibitory effect of CASC2 overexpression on miR-21 expression, which increased the level of miR-21 (Figure 3(A)). Moreover, the results of CCK-8 and flow cytometry assays indicated that miR-21 overexpression markedly reversed the modulatory effects of CASC2 overexpression on cell proliferation and apoptosis (Figure 3(B), (C)). Similarly, the promoting effects on the protein levels of p53, Bax, and p21 and the inhibitory effect on the protein level of Bcl-2 induced by CASC2 overexpression were also impeded by miR-21 upregulation (Figure 3(D)). In summary, miR-21 negatively affected the biological effects of CASC2 on LUAD cells.

FIGURE 3. Overexpression of miR-21 reversed the regulatory effects of CASC2 upregulation on cell proliferation and apoptosis. A549 and PC-9 cells were co-transfected with pcDNA-NC or pcDNA-CASC2 and miR-21 mimics or mimics NC. (A) The expression of miR-21 was detected using qPCR. (B) Cell proliferation was analyzed using CCK-8 assay. (C) Cell apoptosis was evaluated via flow cytometry assay. (D) The protein levels of p53, Bax, p21 and Bcl-2 were quantified via western blotting. *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001. CASC2, cancer susceptibility candidate 2; qPCR, quantitative real-time polymerase chain reaction

CASC2 upregulated p53 expression by inhibiting miR-21, thus exerting an anti-tumor effect

Previous studies have shown that miR-21 can mediate the proliferation, apoptosis, and invasion of tumor cells by inhibiting p53.²⁴ Therefore, we examined the modulatory effects of miR-21 on the expression level of p53 in PC-9 and A549 cells. qPCR assay results revealed that in cells treated with miR-21 inhibitor, the expression of miR-21 was markedly decreased, while the mRNA level of p53 was markedly increased (Figure 4(A), (B)). Pearson analysis presented that the expression of miR-21 was negatively correlated with p53 in LUAD tissues (Figure 4(C)). Moreover, CASC2 overexpression notably increased the p53 mRNA level, while this promoting effect was impeded by miR-21 overexpression (Figure 4(D)), suggesting that CASC2 promoted p53 expression by targeting miR-21. Next, functional experiments revealed that treatment with pifithrin-μ (a p53 inhibitor) significantly weakened the regulatory effects of CASC2 overexpression on cell proliferation and apoptosis (Figure 4(E), (F)). Furthermore, pifithrin-μ treatment obviously reversed the promoting effects of CASC2 overexpression on the levels of p53, Bax, and p21 and the inhibitory effect on the level of Bcl-2 (Figure 4(G)). The results showed that p53 was a downstream functional mediator of CASC2 to modulate apoptosis and proliferation in LUAD cells.

FIGURE 4. CASC2 upregulated p53 expression by inhibiting miR-21, thus exerting anti-tumor effects. (A, B) A549 and PC-9 cells were treated with a miR-21 inhibitor or an inhibitor NC, and the expressions of miR-21 and p53 were determined using qPCR. (C) The correlation between p53 and miR-21 expression at the clinical level was assessed by Pearson correlation analysis. (D–G) A549 and PC-9 cells were co-transfected with pcDNA-NC or pcDNA-CASC2 and miR-21 mimics or mimics NC. (D) The expression of p53 was determined using qPCR. (E) Cell proliferation was assessed using CCK-8 assay. (F) Cell apoptosis was evaluated with flow cytometry assay. (G) The protein levels of p53, Bax, p21, and Bcl-2 were quantified by western blotting. *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001. CASC2, cancer susceptibility candidate 2; qPCR, quantitative real-time polymerase chain reaction

p53 promoted CASC2 expression and formed a positive feedback loop with the CASC2/miR-21 axis

We analyzed the promoter region of CASC2 by using the UCSC and PROMO databases, and the prediction data showed that p53 has a potential binding site in the CASC2 promoter. We first examined the expression of p53 in LUAD patients and found that p53 expression was significantly downregulated in tumor tissue samples compared to adjacent tissue samples from LUAD patients (Figure 5(A)). Moreover, p53 expression was positively correlated with CASC2 expression at the clinical level (Figure 5(B)). Next, we overexpressed p53 by transfecting pcDNA-p53 into PC-9 and A549 cells. As shown in Figure 5(C), the protein level of p53 was obviously elevated in the pcDNA-p53 group. Subsequently, the results of dual-luciferase reporter assay suggested that p53 overexpression remarkably increased the luciferase activity of cells transfected with pmirGLO-CASC2 plasmids compared with those transfected with pcDNA-NC, indicating that p53 could bind to the CASC2 promoter region (Figure 5(D)). Furthermore, overexpression of p53 significantly upregulated the expression of CASC2 and downregulated the expression of miR-21 (Figure 5(E), (F)). Overall, p53 activated the expression of CASC2 and formed a CASC2/miR-21/p53 positive feedback loop in LUAD cells.

FIGURE 5. p53 promoted CASC2 expression and formed a positive feedback loop with the CASC2/miR-21 axis. (A) The mRNA level of p53 in LUAD patients was evaluated by qPCR. (B) The correlation between p53 and CASC2 expression at the clinical level was analyzed by Pearson correlation analysis. (C) pcDNA-p53 or pcDNA-NC was transfected into cells, and the protein level of p53 was determined by western blotting. (D) The binding relationship between p53 and the CASC2 promoter was validated using dual-luciferase reporter assay. (E, F) The expression levels of miR-21 and CASC2 were examined using qPCR assay. *p [less than] 0.05, **p [less than] 0.01, ***p [less than] 0.001. CASC2, cancer susceptibility candidate 2; LUAD, lung adenocarcinoma; qPCR, quantitative real-time polymerase chain reaction

DISCUSSION

Recently, the malignancy of LUAD has been characterized by high incidence and mortality, a poor prognosis, and undirected metastasis, thereby resulting in a serious threat to global public health.⁵ Many features of LUAD, including pathology, molecular properties, clinical manifestations, radiology responsiveness, and surgical indications, are highly heterogenous.²⁵ Additionally, many defects exist in the diagnosis and treatment of LUAD, such as the lack of specific biomarkers and effective molecular targeted drugs. It has been shown that miRNAs and lncRNAs may be potential diagnostic biomarkers of LUAD, such as LINC00460, HOXA10-AS, and miR-1293.^26–28 In the present study, our results indicated that CASC2 and p53 expressions were downregulated and miR-21 expression was increased in LUAD patients and cell lines, and then we further explored the regulatory roles and network in vitro to confirm the potential of these molecules as diagnostic biomarkers.

CASC2, located on chromosome 10q26, has been identified as a tumor suppressor in a variety of tumors.²⁹ Studies showed that the expression of CASC2 was downregulated in NSCLC tissues, which was closely associated with an advanced TNM stage and large tumor volume.¹⁴ Wang et al. also found that CASC2 was related to tumor metastasis and the survival rate in lung adenocarcinoma patients and upregulation of CASC2 expression inhibited cell metastasis and epithelial-mesenchymal transition by inhibiting SOX4.³⁰ Consistent with previous findings, our results identified that CASC2 was expressed at low level in LUAD tissue samples and cell lines, which agreed with predictions made with data from the TCGA database. In addition, functional assays suggested that overexpression of CASC2 obviously suppressed cell proliferation and induced apoptosis, further confirming that CASC2 functioned as a tumor suppressor.

A growing number of studies have suggested that lncRNAs can affect post-transcriptional regulation by regulating miRNA expression by acting as competing endogenous RNAs (ceRNAs).³¹ It has reported that miRNAs are also abnormally expressed in multiple tumors and play critical regulatory roles in tumor migration, invasion, and metastasis.^32,33 A large amount of evidence revealed that miR-21 was commonly highly expressed in multiple cancers and worked as an oncogene.^34,35 In addition, it was reported that CASC2 directly targeted miR-21, inhibiting glioma growth, and metastasis.³¹ However, the effects and mechanism of the CASC2/miR-21 axis in LUAD have not been studied. In our study, the binding relationship between miR-21 and CASC2 was confirmed in A549 cells, and the expression of miR-21 was negatively regulated by CASC2. Subsequently, the negative correlation between miR-21 and CASC2 at the clinical level further verified the idea that miR-21 was a target of CASC2. In addition, miR-21 overexpression significantly impeded the effects of CASC2 on cell proliferation and apoptosis, revealing that miR-21 acted as a functional target in CASC2-mediated LUAD progression.

p53, encoded by the TP53 gene, has been proven to participate in many biological processes, such as cell repair, survival, immunity, and stem cell regeneration, and usually acts as a tumor suppressor.^33,36 Lei et al. suggested that p53 inhibited cell proliferation and migration in bladder cancer cells, which were regulated by miR-21.³⁷ Moreover, another report indicated that p53 transcriptionally activated the expressions of target genes, including the lncRNA Pvt1b and lncRNA PRECSIT, to suppress tumor growth and metastasis.^38,39 Similar to previous studies, our data suggested that p53 expression was downregulated in LUAD tissue specimens. Likewise, we also found that p53 acted as a target of miR-21 and was positively regulated by CASC2. In addition, the binding relationship between p53 and a sequence in the CASC2 promoter was verified, and overexpression of p53 markedly increased CASC2 expression, indicating that CASC2/miR-21/p53 could form a feedback loop to suppress LUAD progression.

Taken together, our research clarified the impacts and mechanism of CASC2 in LUAD, which was to form a feedback loop with the miR-21/p53 axis, thereby modulating cell proliferation and apoptosis. Thus, our research identified some new biomarkers for the diagnosis and treatment of LUAD.

CONFLICT OF INTEREST

All authors agree with the presented findings, have contributed to the work, and declare no conflict of interest.

Abbreviations
• ANOVA
• analysis of variance
• CASC2
• cancer susceptibility candidate 2
• CCK-8
• cell counting kit-8
• CT
• computed tomography
• DMEM
• DuIbecco’s modified eagIe’s medium Dulbecco modified Eagle medium
• FBS
• fetal bovine serun
• FACS
• fluorescence-activated cell sorting
• GAPDH
• glyceraldehyde 3-phosphate dehydrogenase
• GGO
• ground-glass opacity
• lncRNAs
• long non-coding RNAs
• LUAD
• lung adenocarcinoma
• LUSC
• lung squamous cell carcinoma
• miRNA
• microRNA
• ncRNA
• non-coding RNA
• NSCLC
• non-small cell lung cancer
• PVDF
• polyvinylidene fluoride
• qPCR
• quantitative real-time polymerase chain reaction
• SCLC
• small cell lung cancer
• SDS-PAGE
• sodium dodecyl sulfate-polyacrylamide gel electrophoresis
• SEM
• standard error of the mean
• ssRNA
• single-stranded RNA
• YBX1
• Y-box binding protein 1